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Andrei Daniel MIHALCA

Textbook of Veterinary

Introduction to parasitology. Protozoology. AcademicPres Andrei D. MIHALCA

TEXTBOOK OF VETERINARY PARASITOLOGY

Introduction to parasitology Protozoology

AcademicPres Cluj-Napoca, 2013

© Copyright 2013

Toate drepturile rezervate. Nici o parte din această lucrare nu poate fi reprodusă sub nici o formă, prin nici un mijloc mecanic sau electronic, sau stocată într-o bază de date, fără acordul prealabil, în scris, al editurii.

Descrierea CIP a Bibliotecii Naţionale a României Mihalca Andrei Daniel Textbook of Veterinary Parasitology: Introduction to parasitology; Protozoology / Andrei Daniel Mihalca. Cluj-Napoca: AcademicPres, 2013 Bibliogr. Index ISBN 978-973-744-312-0

339.138

Director editură – Prof. dr. Carmen SOCACIU

Referenţi ştiinţifici:

Prof. Dr. Vasile COZMA Conf. Dr. Călin GHERMAN

Editura AcademicPres Universitatea de Ştiinţe Agricole şi Medicină Veterinară Cluj-Napoca Calea Mănăştur, nr. 3-5, 400372 Cluj-Napoca Tel. 0264-596384 Fax. 0264-593792 E-mail: [email protected]

Table of contents

1 INTRODUCTION TO PARASITOLOGY ...... 1

1.1 DEFINING PARASITOLOGY. DIVERSITY OF IN NATURE...... 1 1.2 PARASITISM AS AN INTERSPECIFIC INTERACTION ...... 2 1.3 AN ECOLOGICAL APPROACH TO PARASITOLOGY ...... 5 1.4 A BRIEF HISTORY OF PARASITOLOGY ...... 6 1.5 TYPES OF PARASITES ...... 10 1.6 TYPES OF HOSTS ...... 14 1.7 LIFE CYCLE OF PARASITES ...... 16 1.7.1 Types of parasitic life cycles ...... 16 1.7.2 Stages in the external environment ...... 19 1.7.3 Getting in/on the host ...... 22 1.7.4 Migration and development in the host ...... 24 1.7.5 Biological background for host specificity ...... 26 1.8 HOST-PARASITE INTERACTIONS ...... 27 1.8.1 Pathogenicity of parasites ...... 28 1.8.2 Immunity of host to parasites ...... 31 1.9 CLASSIFICATION OF PARASITES ...... 33 1.9.1 Principles of zoological ...... 33 1.9.2 Major parasitic taxa ...... 35 SELECTED REFERENCES AND FURTHER READING ...... 35 2 ...... 37

2.1 GENERAL CONSIDERATIONS ...... 37 2.2 DIVERSITY OF PARASITIC PROTOZOA ...... 38 2.3 THE FLAGELLATED PROTOZOA ...... 38 2.3.1 Kinetoplastids ...... 40 2.3.1.1 Equine dourine ...... 42 2.3.1.2 Vector-borne trypanosomoses in domestic ...... 47 2.3.1.3 Canine leishmaniosis ...... 60 2.3.1.4 Feline leishmaniosis ...... 69 2.3.2 Trichomonads ...... 69 2.3.2.1 Genital trichomonosis in cattle ...... 70 2.3.2.2 Buccal trichomonosis in dogs and cats ...... 75 2.3.2.3 Anterior digestive trichomonosis in birds ...... 76 2.3.2.4 Intestinal trichomonosis in birds ...... 81 2.3.2.5 Intestinal trichomonosis in mammals ...... 83 2.3.2.6 Histomonosis of poultry ...... 84 2.3.3 Diplomonadids ...... 88 2.3.3.1 Giardiosis ...... 88 2.4 ...... 93 2.4.1 ...... 96 2.4.1.1 Intestinal coccidiosis in mammals ...... 98 2.4.1.2 Intestinal eimeriosis in birds ...... 110 2.4.1.3 Hepatic eimeriosis in rabbits ...... 119

2.4.1.4 Renal eimeriosis in geese ...... 121 2.4.2 Cryptosporidiidae ...... 123 2.4.2.1 ...... 123 2.4.3 ...... 130 2.4.3.1 Sarcocystoses ...... 131 2.4.3.2 Toxoplasmosis ...... 141 2.4.3.3 Neosporosis...... 155 2.4.3.4 Other heteroxenous parasitic in domestic animals ...... 166 2.4.4 Hepatozoidae...... 167 2.4.4.1 Canine hepatozoonosis ...... 168 2.4.4.2 Feline hepatozoonosis ...... 173 2.4.5 ...... 173 2.4.5.1 Babesioses ...... 174 2.4.6 ...... 184 2.4.6.1 Theilerioses ...... 184 2.5 CILIOPHORA ...... 189 2.5.1 of domestic animals ...... 190 2.5.1.1 Balantidiosis ...... 190 2.5.1.2 Buxtonellosis ...... 193 SELECTED REFERENCES AND FURTHER READING ...... 193

M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y |

1 INTRODUCTION TO PARASITOLOGY

para (beside) and sitos (grain, food). If we 1.1 Defining parasitology. Diversity summarize different definitions given to of parasitism in nature. parasites, there are many similarities but The amount of information in the field of also notable differences (table 1.1). parasitology is huge, and more than 20 international peer-reviewed scientific Table 1.1 Different definitions given to the journals are currently being published in parasite this biomedical field. Parasitology is, An that lives completely at the expense of arguably, one of the most intriguing and plants, animals, or humans1 fascinating biological sciences and A plant or animal that lives upon or within another living organism at whose expense it certainly one of the most complex ones, obtains some advantage2 involving a broad interdisciplinary An organism which lives in or on another 3 approach. Knowledge from fields like organism and benefits at the other’s expense An organism that lives in or on and takes its taxonomy, phylogeny, ecology, nourishment from another organism4 biochemistry, genetics, molecular 1 - Mehlhorn (2008); 2 - Gosling (2005); 3 - Soanes biology, immunology, epidemiology, (2008); 4 - Webster's New World Medical pharmacology, pathology, etc. is required Dictionary (2008) for this complex approach. We will try to limit this chapter to the essentials.

When attempting to define parasitology Based on most of these definitions, many you get a snowball effect. To make it organisms could be considered parasitic. simple, parasitology represents the study Even if all viruses, many , many of parasitism. Parasitism is a very fungi, but even some plants or complex interaction between two species, vertebrates are ecologically parasitic one called parasite and the other called organisms, customarily, parasitology host. However, to give a more complex comprises the study of parasitic definition is trickier than expected, as and invertebrate animals (mostly suggestively emphasized by Bush et al. helminthes and , but also (2001): “if you assemble 10 scientists and some other small groups of metazoans). ask them to define parasitism, you would Parasitism is a relatively common way of obtain 10 different answers”. life, and practically every major phylum of Animalia includes parasitic Etymologically, the word parasite derives species, some of them exclusively, at least from the association of two Greek words: in one stage of their life cycle (table 1.2).

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Table 1.2 Animal phyla with parasitic 1.2 Parasitism as an interspecific representatives* interaction Percent of parasitic Phylum1 species2 Any interaction in which a species of Porifera <1 Placozoa 0 organism spends a part of or its entire life Myxozoa 100 in association with another species is Cnidaria <1 called symbiosis. The word symbiosis Ctenophora ~1 Priapula 0 (Greek: syn = along with, together; bios = Nematomorpha 100 life), as originally used by Heinrich Anton Nematoda ~50 de Bary (1831-1888) refers to organisms Loricifera 0 Kinorhyncha 0 living together. Some suggestions to use Onychophora 0 this term only for the bilateral positive 3 Arthropoda ~3 interspecific relationships (equal to Tardigrada4 0 Platyhelminthes ~80 mutualism) are confusing and will not be Mollusca ~1 considered herein. Annelida ~6 Sipuncula 0 Partner species that are involved in a Rotifera ~1 symbiosis may benefit from, be harmed Acanthocephala5 100 Nemertea <1 by, or not be affected by the association. Phoronida 0 Despite some expected overlaps, there Bryozoa 0 are five main types of symbiotic relations: Brachiopoda 0 Gastrotricha 0 phoresis, inquilinism, mutualism, Entoprocta 0 commensalism and parasitism. Phoresis Chaetognatha 0 Gnathostomulida 0 and inquilinism do not imply trophic Echinodermata <1 interactions. Hemichordata 0 Chordata <1 Phoresis (also called phoresy) (Greek: Cycliophora ? pherein = to bear) refers to interactions Rhombozoa 100 Orthonectida 100 where one partner (the host) mechanically carries one or more * Adapted from Bush et al. (2001) individuals from another species (the

1 Several classifications are available in the phoretic organism) (figure 1.1). literature. The phyla name used in this book are Nevertheless, some phoretic species can those listed on Animal Diversity Web (ADW): http://animaldiversity.ummz.umich.edu eventually cause harm to their host 2 At least in one stage of their life-cycle mainly because of overburdens. In the 3 Herbivorous are not included 4 Including pentastomes (according to ADW) pictured case, the interaction can 5 According to ADW, Acanthocephala is part of arguably be called parasitism, as the phylum Rotifera damage is purely mechanical and no physiological or trophic mechanisms are Some estimates state that about half of involved. the known species on Earth are parasitic at least in one of their life stage. Inquilinism (also called inquilism) is another type of pure mechanical interaction where two or more organisms

2 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | of different species share a dwelling used in this work refers to the broader place. concept, where commensalism includes all types of associations when one In a mutualistic symbiosis, both partners partner (the commensal) benefits and the benefit from the relationship (figure other (the host) is not harmed. Moreover, 1.2). The extent to which each partner some others consider phoresis and benefits, is difficult to assess, but it is inquilinism particular types of generally accepted that for any benefit commensalism. there is a certain biological cost.

Figure 1.2 One of the most common Figure 1.1 Phoretic on insects are mutualistic interactions, the lichen. extremely frequent in nature. (Photo (Photo Andrei D. Mihalca) Andrei D. Mihalca)

With the view of all above, parasitism The term commensal (literally meaning can be defined as a symbiosis (certainly “together at the same table”) was first the most common one) in which one of used by the Belgian parasitologist P.J. van the symbionts (the parasite) benefits at Beneden (1809-1894) to explain the the expense of the other (the host). All associations in which one animal shares the main aspects of parasite-host food obtained by another animal. There is interactions as well as the types of hosts some controversy on how broad this and parasites will be detailed in the meaning should be. Some authors following chapters. An interesting type of consider that commensalism do not parasitic-like interaction is known to involve any physiological interaction nor occur in echiurans, a small group of dependency between the partners, and vermiform, bottom-dwelling marine only the spatial proximity allows the organisms. In this case, males (1-3 mm in commensal to feed on nutrients captured length) are parasitic in the kidneys of or ingested by the host. Yet, the meaning

3 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | females (80 mm in length). It is highly With high probability, most mutualistic arguable if this particular association symbioses probably began as parasitic could be considered parasitism, as it is an ones, with one organism attempting to intraspecific interaction not an exploit another one. As brilliantly interspecific one. perceived by Paracer and Ahmadjian (2000) in their monograph on symbiosis, To include a certain symbiotic interaction if one considers parasitism as an (figure 1.3) in one of the types defined antagonistic relationship then mutualism above is more or less conventional. Many can be regarded as a standoff or a draw relationships are dynamic, and there may between the two antagonists. The widely be frequent transitions from one type to accepted theories of the origins of some another. Symbiotic associations may cell organelles like mitochondria or change because of external factors chloroplast consider that these are (environmental or host-dependent) or transformed bacteria that may have due to internal influences (symbionts- begun as parasitic symbionts in larger dependent). prokaryotic cells. On the contrary, a The evolutionary approach to symbiotic mutualistic or commensalistic association interactions is the most interesting one. may degenerate into a parasitic one if the For instance, a parasitic association can defense mechanisms of the host are evolve into mutualism or commensalism. decreased.

Figure 1.3 Main features of symbiotic associations which involve trophic interactions (phoresis and inquilinism are excluded).

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The first exhaustive attempt to classify of the parasite however, differs. During symbiotic interactions was made by M.P. the parasitic phase, as shown above, the Star in 1975. He used several criteria for environment of the parasite is the host’s his classification like location of symbiont body. However, the vast majority of within the host, persistence, dependence parasites have at least one stage in the and specificity of symbiosis. Parasitism external environment during their life basically fits to this system with some cycle, also very important in the peculiarities detailed in Chapter 1.5. understanding of this complex interaction.

Ecology of the host itself greatly 1.3 An ecological approach to influences the adaptations of the parasite parasitology in order that it could easily infect the host. The best examples with this view We can regard parasites from many refer to parasites with heteroxenous life- points of view, but certainly the medical cycles (see Chapter 1.7). aspects of parasitism, especially in humans and domestic animals are the An important question of an ecological most considered worldwide. However, approach to parasitology is what the role especially in wild animals, parasites do of parasites within the ecosystem is. not cause apparent disease, and the There are well-defined theories stating relative balance between parasites and that parasitism plays a major role in the hosts is the result of a complex and long evolution of species diversity and coevolution. regulation of host populations.

If we consider parasitology a branch of Another question is if and how parasites ecology, the habitat and environment of are able to regulate biodiversity in the parasite is provided by another general and host populations in organism, the host. Many ecological particular. Some consider parasites as a principles which apply to free-living threat to biodiversity, mainly in the case organisms can be applied to parasites. of endangered hosts. Nevertheless, The only particular situation is that the parasites are part of our biodiversity as environment of the parasite is alive and are their hosts. Parasites of endangered actively fights against it. Therefore, the species can sometimes play a significant main issues for such an approach to role in the conservation efforts. At least parasitology include adaptations of one documented case of parasite- parasites to their environment and life mediated extinction was described in the style, adaptations of hosts to antagonize snail Partula turgida, where the last- parasites and mainly the nature of host- known individual was killed in captivity parasite relationship. To put it simply, by a microsporidian parasite. However, in ecological parasitology studies the the case of highly host-specific parasites, complex relationships between parasites, the extinction of the host equals with the hosts and environment. The environment extinction of the parasite itself (host- parasite co-extinction). It might be the

5 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | case of the rhino ticks (i.e. Dermacentor helminthes of dogs, pigs and fish. Galen of rhinocerontis) from Africa. Pergamum (131-199), the Roman physician and philosopher of Greek origin One thing is for sure. A world without (figure 1.4), recognized three parasites would look completely different macroparasites of humans: Ascaris, than the world we know today, not Taenia and Enterobius. Human necessarily in a good way. hydatidosis was already known by Aretaeus of Cappadocia (81-138).

1.4 A brief history of parasitology

History of medical sciences is a fascinating topic and basic knowledge is required for a proper understanding of progress dynamics in certain fields. There are several reviews on history of parasitology, most of them focusing on human parasites. Some of these works focus also on general parasitology, and a great resource is the review of Cook GC (in Gillespie SH, Pearson RD. 2001. Principles and Practice of Clinical Parasitology. Wiley. 752 pp.). Early records. Since humans became Figure 1.4 Galen of Pergamum. aware of their social and ecological (reproduced from a lithograph by Pierre identity, they were also probably aware Roche Vigneron) of some macroparasites like larger helminthes or cutaneous arthropods living associated with Homo sapiens or . All these early animals nearby (domesticated or records and many other observations did hunted). However, the first written not have a real biological background. documentation on a parasitic organism is The general belief until the mid- found in the Papyrus Ebers (~1550 BC). nineteenth century was that parasites, In ancient Egypt, several other writers like all other living organisms, appear were aware of some major helminthic through spontaneous generation. This infections of humans, like theory, synthesized by Aristotle, was schistosomiasis, dracunculiasis or firstly doubted on by the Dutch biologist ascariasis. Dead female Dracunculus (1637-1680) and by worms have been found in Egyptian the Italian physician Franceso Redi mummies older than 3000 years. In (1626-1697). The later did not agree that ancient Greece, Aristotle (384-322 BC) flies arouse spontaneously from rotting mentioned in his writings parasitic meat. Despite his morphological proof on

6 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | the sexual reproduction of A. monograph on parasitic organisms lumbricoides, the British anatomist “Osservazioni intorno agli animali viventi Edward Tyson (1650-1708) was also an che si trovano negli animali viventi“ [Notes adept of spontaneous generation. A on living animals found in living animals] particular application of this theory was (figure 1.6). Five years later, Nicolas extrapolated to parasitology by Marcus Andry (1658-1742) was the first to Bloch (1723-1799) and Johan Göze illustrate the scolex of a human (1731-1793). The two famous European tapeworm, in his “De la parasitologists embraced the opinion that génération des vers dans le corps de parasites were “inborn in their host”. The l’homme” [On the generations of worms concept of spontaneous generation was inside the human body] (figure 1.7). finally abandoned after strong experimental proofs brought by Luis Pasteur (1822-1895).

Emergence of parasitology as a science (17th and 18th centuries). Although most historical records of parasites are related to humans, the birth of parasitology as a science is linked to .

Figure 1.6 Original drawing from Redi’s “Osservazioni intorno agli animali viventi che si trovano negli animali viventi” depicting: “a big worm found in the kidney of a marten” (in the middle); “a worm found under the skin of a lion” (right); “a worm very frequently found under the skin of martens and skunks”. (left)

Figure 1.5 Francesco Redi. (reproduced Eighteenth century brought three major from an engraving by Lodovico Pelli) contributions in parasitology. In 1760, Pierre Pallas (1741-1811) wrote a dissertation called “De infestis viventibus Francesco Redi (figure 1.5) is considered itraviventia”. Göze, who discovered the the father of parasitology, after he scolex of Echinococcus in hydatid cysts, published in 1694 the first scientific

7 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | published in 1787 his "Versuch einer parasitologists arose in the scientific Naturgeschichte der Eingeweidewürmer circles of the century. Probably the most tiericher Körper" [Natural history of prominent of them was Carl Rudolphi intestinal worms from the body of (1771-1832). The Swedish scientist animals]. Last but not least, Bloch, who wrote two monumental works was the first to note the hooklets on the “Entozoorum sive vermium intestinalium scolex of tapeworms, wrote in 1782 historia naturalis” from 1808 and “Abhandlung von der Erzeugung der “Entozoorum synopsis cui accedunt Eingeweidewürmer und den Mitteln wider mantissa duplex et indices locupletissima” dieselben“ [Treatise on the Generation of from 1819 which substantially increased Intestinal Worms and the means of their the number of known species of extermination], winning the gold medal parasites. for best essay at the Copenhagen The French parasitologist Félix Dujardin Academy of Sciences (figures 1.8 and (1801-1860) was the first to understand 1.9). that the life cycle of trematodes and cestodes involve an intermediate host. In 1845 he published his most important work: “Histoire naturelle des helminthes ou vers intestinaux” [Natural history of helminthes or intestinal worms] (figure 1.10).

As mentioned above, basically all published parasitological works of the time originated from mainland Europe. First English texts appeared as translations from German, French or Latin. The first book of parasitology written by a British scientist was “Entozoa, an Introduction to the Study of Helminthology” in 1864 by Thomas S. Cobbold (1828-1886).

The end of the nineteenth century marked a shift in the concepts of parasitology. Step by step, scientists Figure 1.7 Cover of Andry’s “De la understood the medical importance of génération des vers dans le corps de parasites in humans and animals. Thus, l’homme”, re-published in 1750. from a branch of zoology, parasitology was more and more viewed as a medical Parasitology in the nineteenth science. century. Several outstanding

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Figure 1.8 Cover of the first edition of Bloch’s gold medal essay.

Figure 1.10 Cover of the first edition of Dujardin’s major work.

The first journal entirely devoted to parasitology, “Archives de Parasitologie” had its first volume published in 1898 (figure 1.11) by Émil Raphaël Blanchard (1819-1900). However, after 16 volumes and a break during the WW1, the publication was suspended in 1919. Overseas, the father of American parasitology was a paleontologist, Joseph Leidy (1823-1891).

Modern parasitology. Definitely, the

most dramatic shift in modern Figure 1.9 One of the ten plates of parasitology as in many other natural Bloch’s book. sciences was the discovery of nucleic acids and of molecular biology

9 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | techniques. In the last decades, molecular interactions with the host. Moreover, the tools became almost ubiquitous in categories frequently overlap. Like in biological or medical parasitological many other areas of science, research. classifications are used mainly for scholastic purposes. The main criteria But there is still a long way to go. Many used for categorizing parasites are: parasitic diseases are becoming emergent. Some, otherwise harmless (1) Location of parasites within the parasites are killing immune host compromised hosts (i.e. HIV positive).  Ectoparasites are organisms living Despite enormous amount of research parasitically on the outside of their and money, there is still no vaccine for host. Although this broad definition malaria, the deadliest parasitic disease on seems clear enough, some comments Earth. And the list can continue…. are required. Certain parasites are normally living on the skin (integument), hair, feathers or scales of their hosts. These are the typical ectoparasites (i.e. ticks, some mites, fleas, lice, biting insects etc.). Other arthropods are living within the structures of the skin (i.e. Demodex in the hair follicles; mange-causing mites in the dermis etc.) but are customary considered ectoparasites. Larval forms of Hypoderma species migrate through various parts of the hosts’ body and finally they stop subcutaneously (figure 1.12), hardly being considered ectoparasites. Parasites also inhabit various external mucosae of their hosts (i.e. Thelazia in the conjunctiva; Oestrus Figure 1.11 First volume of Archives de the nasal cavities; monogeneans or Parasitologie. ciliates on the gills of fish (figure 1.13); some trichomonadids within the buccal or genital mucosa; Otodectes mites in the ear canal etc.) 1.5 Types of parasites Their classification as ectoparasites is debatable. Except the taxonomic approach, there are several other criteria to classify parasites,  Endoparasites are those which all of them conventional, as they do not inhabit the internal organs of their always properly reflect the complex hosts.

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brain, muscles, tendons, heart, blood vessels, serosae etc. Moreover, some unicellular organisms (i.e. protists) are intracellular parasites (intracytoplasmatic or intranuclear). As shown in the previous paragraph, some ectoparasites could be arguably considered endoparasites.

(2) Size of parasites

 Microparasites are those not visible by humans with naked eye. We include here protists, larval stages of most helminthes, most of the Figure 1.12 Larvae of Hypoderma diana monogeneans, mange causing mites are commonly found in the subcutaneous etc. tissue of roe deer. (Photo Andrei D. Mihalca)  Macroparasites are visible to the human eye without the aid of the

. Although some parasites like Dermanyssus mites of birds (figure 1.14) or Strongyloides nematodes from the intestine of various vertebrates are hardly visible macroscopically, they are considered macroparasites. On the other hand, there are enormous-sized parasites. For instance, many cestodes frequently reach lengths of several meters (i.e. Diphyllobothrium latum, a tapeworm of fish-eating mammals, including dogs and humans can reach up to 12 meters in length). The Figure 1.13 Ciliated protozoa largest known trematode, (Ichthyophthirius multifiliis) parasitic on Nematobibothrioides histoidii from the gills of common carp. These parasites the muscles of the sun fish can reach also infect the integument. (Photo Andrei a length of 12 meters. The largest D. Mihalca) known parasitic nematode is Placentonema gigantissima from the

placenta of female whales, Also called internal parasites, they can which can reach up to 8.5 meters in inhabit all organ systems: digestive tube, length. liver, respiratory tube, urinary system,

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(4) Number of hosts required for the completion of life cycle

 Homoxenous (Greek: homos = identical; xenos = host) parasites include those species which require a single host category for the completion of their life cycle. Homoxeny defines parasite transmission through hosts of same ontogenetic category. These hosts should not necessarily be one and the same species. A homoxenous

parasite, therefore, can be Figure 1.14 The red poultry , monoxenous, oligoxenous or Dermanyssus gallinae, parasitic on polyxenous. Homoxeny is also called chicken are visible with the naked eye, direct life cycle. Some examples of but a careful examination should be homoxenous parasites include: performed. (Photo Cristian Magdaș) apicomplexans from genera and ; monogeneans; certain nematodes of domestic animals or humans (Ascaris, (3) Host specificity Strongylus, Oxyuris, Strongyloides, etc.). The vast majority of parasitic  Monoxenous parasites are limited to arthropods also have homoxenous a single host species in certain life life cycles (mange causing mites, stages of their development. Many fleas, lice etc). A particular type of Eimeria species are strictly host- homoxenous development was specific and could be included here. described in gallotiae  Oligoxenous parasites have a small parasitizing lizards. For this species host range (2-5 species). Adults of of apicomplexan parasite, are typical transmission occurs by cannibalism, examples. However, larval forms of so both the definitive and the same parasite have a broad range intermediate host are different of hosts (polyxenous). individuals from the same species (dihomoxenous life cycle). In some  Polyxenous parasites have a broad parasites with typical homoxenous range of hosts (low or no host life cycle, occasionally, in order to specificity). For instance, Toxoplasma cross some trophic boundaries, they gondii is a polyxenous parasitic use an additional non-obligate host. apicomplexan. They are called facultative heteroxenous parasites and examples include Toxocara (an

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ascarid nematode of dogs and cats), cycle takes place as free-living stages; Syngamus (a respiratory strongyle of in other cases, adult females become galliforme birds) and many others. parasitic. Another example of facultative parasites is the case of  Heteroxenous (Greek: heteros = larval stages of Calliphoridae flies. different; xenos = host) parasites They are opportunistic and have the require transmission via alternation ability to exploit living tissue, of hosts of different ontogenetic although characteristically they are categories. This type of development carrion feeders. is also called indirect life cycle. According to the number of hosts, these parasites are called (6) Duration of parasitism diheteroxenous (i.e. ), triheteroxenous (i.e.  Temporary parasites are in contact ) or with their host for short periods tetraheteroxenous (i.e. Alaria alata). during a certain stage of their life The term polyheteroxenous is also cycle. Mosquitoes or leeches are used to refer to parasites requiring typical examples. If a temporary more than two hosts. parasite visits its host several times during a particular life stage it is

called a periodic parasite. (5) Obligativity of parasitic life  Permanent parasites infect their  Obligate parasites are those which host for longer times. All adult stages need a host for survival, development of trematodes and cestodes are and/or reproduction during at least associated with their definitive host one of their life stages. In some during their entire adulthood. species of parasites, all

developmental stages are found associated with only one host (i.e. (7) Parasitic life stage Trichinella, lice etc.). In some others,  Pre-imaginal parasites are parasitic only certain stages are obligatory only during their immature life parasitic. For instance, in many stages, while adults are free-living. nematodes, the first larval stages are All myiasis causing flies are pre- free-living, while the later stages and imaginal parasites. In these species, adults are obligatorily parasitic (i.e. usually the adult stage is short living Strongylus nematodes of horses). and many times it doesn’t even feed.  Facultative parasites are generally In the representatives of phylum free-living species, which may Nematomorpha, the larval stages are accidentally become parasitic. The always obligatory parasites while the nematodes of genus Strongyloides adults are free-living. can undergo two types of development. In certain cases, all life

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 Imaginal parasites infect their hosts particular. Basically, a host is any living only during their adult stage, while organism which harbors another one, immature stages are free-living. Fleas parasitic. The following terminology used of genus Ctenocephalides (figure for host classification is not based on 1.15) parasitic on dogs and cats are single criteria. imaginal parasites. Definitive host (or final host) is traditionally defined as the host where the parasite reaches sexual maturity or as the host which harbors the adult parasites. Even if this definition looks clear and simple, due to the complexity and diversity of parasites and parasitic interactions, some comments are required. Most homoxenous metazoan parasites reach sexual maturity in the host, thus this should be called definitive host. However, as this host is singular, and no other organism is required in the development of homoxenous parasites (i.e. intermediate host), it is easier to use

just the term “host”. Moreover, in some Figure 1.15 Heavy infestation with the homoxenous parasites, only the flea, Ctenocephalides canis on a dog. Only immature stages are parasitic, while the the adults are parasitic; larvae and adults are free-living, thus, the term nymphs are found in the dog’s “definitive host” would not fit to the environment. (Photo Andrei D. Mihalca) generally accepted definition. On the other hand, there are parasites with facultative heteroxenous life cycles. In  Note: in many parasites, immature this case, it would be useful to use the and adult stages are both parasitic in term “definitive host” to differentiate the same or in different hosts. The them from the eventual facultative term to include these cases is not intermediate or paratenic hosts. well-defined, but permanent parasite Moreover, in parasitic protozoa there is can be a feasible option, although it no such stage as “adult” or concept of overlaps with the previous criteria. “sexual maturity”. Conventionally a definitive host for heteroxenous parasitic protozoa is the host in which sexual 1.6 Types of hosts reproduction occurs. However, in some parasitic heteroxenous protozoans the The ecological concept of “host” would life cycle does not include any sexual not exist without the concepts of reproduction; hence the definition of symbiosis in general and parasitism in definitive host in this case is arguable.

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Asexual reproduction is also known in ecological or trophic. For examples, adult females of homoxenous nematodes humans are dead-end host for many of genus Strongyloides, so the traditional parasites (i.e. Echinococcus, Trichinella, definition should be reconsidered. etc) as it is unlikely that definitive carnivore hosts will prey on humans. Intermediate host is any host involved in heteroxenous life cycles which is not Notes: (1) In some cases, a single definitive and in which the parasites individual can be definitive and undergo some developmental and intermediate host for the same parasite. morphological change. In For example, a cat which harbors the polyheteroxenous life cycles, gametogonic (sexual) stages of intermediate hosts are customarily in its intestines is a numbered according to their definitive host. However, cats can also consecutiveness in the ontogeny of the develop systemic infection with parasite (i.e. intermediate host 1, merogonic stages of T. gondii making intermediate host 2, etc.). them intermediate hosts. Often, the same cat can harbor both stages, being in the Paratenic host (or transport host) is same time intermediate and definitive used by some parasites to bridge a host, but in different life cycles. (2) trophic gap. Ontogenetically, they are not Another unusual situation is encountered obligatory in the life cycle of parasites, in the life cycle of the nematodes of genus but ecologically they are very important. Trichinella (figure 1.16). The same The most accepted definition considers individual acts first as definitive and later the paratenic host as an organism which on as intermediate host in a particular serves to transfer a larval stage or stages heteroxenous life cycle. from one host to another but in which little or no development takes place. The term is rather appropriate in helminthology than in protozoology.

Reservoir hosts are those organisms which are responsible to maintain the parasite populations in certain ecosystems.

Vector hosts are defined as organisms which transmit certain pathogens from one host to another. However, definitions widely vary according to the vectored pathogen.

Dead-end hosts are usually intermediate or paratenic hosts which are not able to Figure 1.16 Larva of Trichinella britovi transmit the parasites to further hosts. parasitic in the skeletal muscles of red Limitations are most commonly fox. (Photo Călin M. Gherman)

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1.7 Life cycle of parasites In homoxenous life cycles parasites require a single host to complete their Every living organism has a life cycle development. This way of development is (also known as developmental cycle or encountered in some parasitic life history). Life cycle is a series of protozoans, in all monogeneans and in developmental stages through which an various nematode species. There are organism goes through. Always, the last three different possible situations: stage from a life cycle must be able to  In most homoxenous parasites, there produce the initial stage from the is an alternation of stages in the subsequent cycle. In parasitic organisms, environment with parasitic stages the life cycle is extremely complex and it (figure 1.17). The host acquires the is the results of coevolution with their parasite from the environment via hosts. Regardless of the taxonomic group, various routes, most commonly by development of parasitic species most ingestion. Typical examples for this often comprises an alternation of free- case are apicomplexan protozoans of living and parasitic stages. genus Eimeria. In some other cases, In order to complete their life cycle, infective stages from the parasites have to overcome three critical environment actively enter the host, steps: by penetration of skin or mucosae  the immune system of the host(s); (i.e. Ancylostoma, Bunostomum, etc.).

 the adverse environmental factors;

 the ecological requirements for host- to-host transmission.

Despite all odds, an impressive number of parasitic species succeeded through the caudine forks of evolution.

1.7.1 Types of parasitic life cycles

As shown above (Chapter 1.5), there are Figure 1.17 Typical homoxenous life- two main types of parasitic life cycles: cycle (E = external environment; H = homoxenous and heteroxenous. Each host). species has a characteristic life cycle which will be detailed in the corresponding section of this textbook,  Although not common, in certain but some general aspects should be homoxenous parasites the life cycle discussed further on. can be completed without stages in the external environment. This is

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possible due to autoinfection of the single host (figure 1.18). For instance, the parasitic females of the nematodes of genus Strongyloides typically lay embryonated eggs which pass through the host’s feces to the external environment where they hatch. Subsequently, infective larvae will penetrate through the skin of a new host. However, in some cases, eggs are able to hatch while still Figure 1.18 Homoxenous life-cycle in inside the host’s intestine, so larvae autoinfective parasites (E = external will be autoinfective to the same environment; H = host). host. Another possibility herein, involving two different individuals is the sexual transmission of some protozoans (Trypanosoma equiperdum, Tritrichomonas foetus).

 The third possibility for homoxenous life cycle was described for certain species of genus Sarcocystis parasitic in lizards. These parasites are transmitted from host to host by cannibalistic behavior during which, lizards from the very same species eat each other’s tails. This particular Figure 1.19 Dihomoxenous life-cycle (H life cycle (figure 1.19) was = host) denominated as dihomoxenous and

sometime even the same individual lizard can act as both definitive and Heteroxenous development comprises of intermediate host. a definitive hosts and one or more intermediate hosts. Although the Heteroxenous life cycles are very possibilities and variations in the complex and ecologically challenging alternation of hosts and stages in the pathways in the development and host- external environment are multiple, the to-host transmission of parasites. Several following situations will be considered: groups of parasitic organisms have exclusively heteroxenous development.  Most of the so called vector-borne All trematodes, all cestodes and all parasites (i.e. , , acanthocephalans are included here. Trypanosoma, Leishmania, Dirofilaria, Many protozoans and nematodes also etc.) have a typical diheteroxenous embrace heteroxeny. life cycle with direct host-to-host

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transmission and with no stages in the external environment (figure 1.20). Transmission occurs both ways through hematophagy. Another interesting diheteroxenous life cycle with no external stages has been described in hemoparasites of genus . The definitive hosts are ticks which acquire the infection by hematophagy from tortoise intermediate hosts. Remarkably, the

tortoises get the infection by ingesting infected ticks. Figure 1.21 Diheteroxenous life-cycle with single indirect transmission (E = external environment; DH = definitive host; IH = intermediate host)

 In other groups of diheteroxenous parasites there is no direct interaction between the definitive and the intermediate host, and transmission from one host to another always occurs through some external stages (figure 1.22). For instance, in the trematode Fasciola Figure 1.20 Diheteroxenous life-cycle hepatica, definitive hosts shed with no external stages (DH = definitive parasitic eggs to the pasture where host; IH = intermediate host) the larval stage (miracidium) hatches and penetrates a snail intermediate  Many diheteroxenous parasites - like host. After several asexual most of the cestodes and reproductions, cercariae actively acanthocephalans, but also some leave the snail and encyst as apicomplexan protozoans (i.e. metacercariae on vegetation. A new Sarcocystis, Toxoplasma, , definitive host will ingest them and a etc.) - are characterized by indirect new life cycle begins. transmission through the external Most trematodes have a typical environment from the definitive host triheteroxenous development, to the intermediate host and direct involving very diverse possibilities. transmission through predatorism Considering a single situation (figure from the intermediate host to the 1.23), we will illustrate the life cycle definitive host (figure 1.21). of Dicrocoelium dendriticum.

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Ruminant definitive hosts are known to occur in the cestode shedding the eggs of this parasitic Diphyllobothrium latum infecting humans liver trematode, through their feces and other piscivorous mammals. to the external environment.

Figure 1.23 Triheteroxenous life-cycle (E Figure 1.22 Diheteroxenous life-cycle = external environment; DH = definitive with double indirect transmission (E = host; IH = intermediate host) external environment; DH = definitive host; IH = intermediate host)

1.7.2 Stages in the external environment The first intermediate host (IH1), in this case a terrestrial snail will ingest these In the very previous chapter we showed eggs and inside its body the miracidium that in many parasitic life cycles, will hatch and multiply by asexual transmission from a host to another reproduction. When reaching to the requires passage through the external cercarial stage, they actively emerge from environment. For an organism which is the snail’s body and are ingested by a primarily adapted to a parasitic lifestyle, second intermediate host (IH2) which is the contact with environmental factors an ant. Inside the ant’s body, the can seriously affect its survival. On the cercariae will encyst into a other hand, parasitism as a way of life metacercariae. Further transmission to a probably evolved in previously free living new definitive host implies accidental organisms as an adaptation to avoid ingestion by ruminants of infected ants. these factors. The chances for this apparently An interesting situation of organism hazardous event are increased by the which is able to opt for a free-living or a pathogenic effect of the metacercarial parasitic lifestyle is known for nematodes stage on ants, causing them impaired of genus Strongyloides. In these motility. Another similar example of life nematodes, only females are parasitic in cycle but involving different hosts is various parts of the digestive tube of their

19 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | host. By parthenogenesis, parasitic complex developmental changes, females of Strongyloides produce eggs essential for their further transmission. which embryonate and first-stage larvae For instance, in coccidia, the cysts are hatch. In the external environment larvae called oocysts, which in most cases when either develop into female infective shed through the feces of the host are not larvae or grow and molt four times to sporulated, hence non-infective to a new produce a single free-living generation host. During its environmental life, if the consisting of both males and females. physical conditions are proper, oocysts What factors determine whether larvae sporulate and become infective. However, will develop into free-living stages or into in other protozoans (i.e. ) the cyst infective forms is still not fully known, is mainly important as a resistance stage, but it is believed they may depend on and excystation usually occurs when they environmental conditions such as pH, reach in their typical parasitic habitat in a pO2, pCO3, consistency of substrate, new host. More complex situations are temperature and level of nutrients. known for parasites with a certain degree of host-specificity, when only the contact Parasites use various strategies to with a suitable host triggers excystation. survive or to avoid environmental Mechanisms for excystation include conditions while being in their journey rehydration and action of host digestive from a host to another. enzymes on the cyst wall. Many protozoans are able to secrete a Encystment as a survival strategy is also resistant covering and enter a latent known for trematodes (flukes). Typically, stage called cyst. Encystment is very trematodes have a three-host life cycle. common among free-living protozoa The definitive host is always a vertebrate, during harsh environmental conditions the first intermediate host is always a but also in most parasitic protozoa when mollusk and second intermediate hosts they are outside their host, in the are various metazoans. In few cases, the environment. The encystment provides life cycle comprises only two hosts: the protection against unfavorable vertebrate and the mollusk. Nevertheless, environmental conditions but also regardless of the number of hosts, provides a biological background for trematodes always have two stages in the nuclear division. The triggers for environment. The definitive vertebrate encystment include lack of nutrients, host passes through the feces the eggs of water loss, decreased oxygen the fluke. If the life cycle involves aquatic concentration or changes in the mollusks, the first larval stage of the temperature or pH. During encystment in trematode (called miracidium) hatches protozoans, a cyst wall is produced, from the egg and it actively swims movement organelles (cilia or flagella) foraging for its mollusk host. As a survival are lost and food reserves (i.e. starch, strategy in terrestrial cycles, miracidia do glycogen) are stored within the cell. In not hatch but remain inside the egg. The certain types of parasitic protozoans, the next free living stages of trematodes in environmental cystic stages suffer ontogenetic order are cercariae. They

20 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | actively leave the mollusk’s body and larvae stay inside the eggs to avoid either forage for the second intermediate environmental factors (i.e. in ascarids or host or become encysted on vegetation. pinworms). In other cases, larvae hatch and are infective for the definitive host. In tapeworms (cestodes) the usually For instance, third stage larvae (L3) of complex life cycle requires at least one Strongylidae parasitic in horses are stage in the external environment. The swallowed together with the grass. They definitive host (always a vertebrate) hatch as L1 and remain in the which hosts the adult stages in its environment where they undergo two intestines sheds the eggs through the molts before turning into infective L3. feces. In certain groups of cestodes (i.e. During this time, they avoid drying out by Pseudophyllidea) the life cycle is related being active during cooler periods of the to aquatic environment; hence the eggs day (morning, dusk, dawn) and hiding in are adapted to float to enhance their shade over sunny days. In certain groups chance to be swallowed by a suitable of nematodes, after L1 hatch, they must intermediate host. In other species of immediately get into an intermediate “aquatic” tapeworms, larval stages called host to avoid the improper coracidia (singular, coracidium) hatch environmental factors (i.e. from the eggs and swim waiting to be Protostrongylus). ingested by a crustacean. These aquatic stages are usually not very resistant (i.e. Most arthropods are external parasites so coracidia can survive only 24-36 hours) they are exposed to environmental and normally do not feed. On the other factors all the times. Additionally, many hand, in other groups of cestodes (i.e. arthropods are temporary parasites, Cyclophyllidea), eggs containing embryos hence most of the time they spend away (oncospheres) are very resistant in the from the host. Biting insects like environment. They can easily survive mosquitoes or sandflies are parasitic only several months before being ingested by for very short time, when they are a suitable vertebrate host. feeding. Ticks spend a great part of their life in the search of a suitable host, In nematodes, environmental stages are dwelling in the vegetation of burrows. In known for most of the groups. Notable other species, only larvae are parasitic exceptions are species of genus and adults are typically free-living Trichinella which are transmitted from a creatures (i.e. myiasis-causing insects). host to another by predatorism or vector- Other insects (fleas for instance) are borne nematodes (i.e. Dirofilaria). parasitic only as adults while imago However, typical nematodes pass from a stages are free-living and relying on host to another through the external environmental food sources. In environment. The diversity of life cycles mosquitoes, larval stages develop in the in nematodes makes it very hard to water. This is why successful campaigns outline some general developmental against mosquito-borne diseases are patterns. Nevertheless, in most of the focused on desiccations. Other parasitic cases, the infective stage is a larva. Some

21 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | arthropods however, lack environmental Transmission strategies are extremely stages (lice). diverse in the world of parasites. Some of them are remarkable examples of

(co)evolution and natural selection of 1.7.3 Getting in/on the host successful traits.

One of the most crucial milestones in the life cycle of a parasitic organism is the successful detection of a suitable host. Parasites have evolved many different strategies to increase the chance of successfully finding a host. One of the most common strategies used by parasites is the production of an enormous number of offspring. Most of the offspring will be unsuccessful and only a limited number will encounter a suitable host.

Parasites can use passive dispersion (random chance to contact a host) or Figure 1.24 Many important tick species active host finding (when mobile parasite spend the majority of their lifetime in the stages are actively searching for the host wait of their host, on vegetation. This using various sensorial features). In host-finding behavior is called questing. active strategies host detection is (Photo Andrei D. Mihalca) essential. To detect the proximity of a potential host, parasites use complex sensory organs. The first larval stages of In homoxenous life cycles, transmission digenetic trematodes are called of parasites from a host to another is miracidia. They are able to swim at a rate direct. The host usually sheds stages to of about 2 mm per second and use their the environment. In most of the cases chemoreceptors to find a suitable snail these stages are not yet infective for a host. After locating it, the miracidium new host, but they spend some time and attaches to the snails integument and undergo some biological changes using cytolytic enzymes it embeds deeper becoming infective. They will enter the and deeper into the snail’s body. In new host passively (i.e. ingestion of questing ticks (figure 1.24), the infective eggs, cysts, oocysts, larvae) or detection of the host is based on complex actively (i.e. skin penetration by larval sensorial organs called Haller organs, hookworms or schistosome cercariae). which are able to perceive CO2 from the breath of hosts but also on temperature Particular situations of direct and movement detection. transmission with no stages in the external environment are known. Some

22 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | situations include direct transmission and complex. Many parasites are through sexual contact. Trichomonas transmitted from a host to another by vaginalis in humans or Trypanosoma predation. One of the typical models is equiperdum in equines are examples of the life cycle of cestodes in Taeniidae sexually transmitted parasitic diseases. family. Typical definitive hosts for these Skin contact between an infected and a tapeworms are carnivorous mammals healthy individual is the way how scabies which acquire the infection after preying mites (Sarcoptes) get to a new host on infected intermediate hosts. Larval (figure 1.25). taeniids which infect these intermediate hosts usually induce severe lesions to Transmission strategies for parasites make them more susceptible to be with heteroxenous life cycles are more predated. This adaptation enhances their complex. Transmission from a host to chance to get to the definitive host. another can be via the environment, Similar examples (figure 1.26) are where there is no direct contact between known for various nematodes definitive and intermediate hosts. (Eustrongylides in fish intermediate hosts) or trematodes (Dicrocoelium in ants).

Figure 1.25 Sarcoptic mange in a camel. Parasite transmission occurs by direct host-to-host contact. (Photo Andrei D. Figure 1.26 Fish infected with the Mihalca) nematode parasite Eustrongylides excisus are easier preys to dice snakes, Natrix In this case, detection of hosts follows the tessellata. (Photo Andrei D. Mihalca) same principles as for homoxenous There is a significant number of parasites parasites. When the transmission from which infect their host through the definitive host to the intermediate hematophagy by other parasites. These host requires direct contact between the are the so called vector-borne parasitic hosts, strategies are sometimes intriguing

23 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infections. The vectors are usually blood 1.7.4 Migration and development in eating arthropods (ticks, mites or insects) the host and they play the major role for inoculating various parasites to the Parasites are adapted to a multitude of vertebrate host. Probably the most well- habitats within the host. However, the known examples are among protozoans. site where parasites gain access to the species causing malaria are host is different than the target vectored by certain mosquitoes. organ/tissue. So the parasites have to move from the site of infection to the site Another tropical disease, the sleeping of predilection within the host’s sickness is caused by various species of organism. In some other cases, the site of Trypanosoma. Their transmission is done infection is the same as the typical site for by tsetse flies. Blood sucking insects are the final stage in that particular host, but also responsible for vectoring metazoan parasites need to undergo some parasites. Many filarial nematodes are development in order to survive there. injected as larvae to their vertebrate host For this development they might migrate by mosquitoes or flies (i.e. Dirofilaria, through various tissues before returning Loa, Onchocerca, Wuchereria) to the initial site. Except the aforementioned possibilities Most parasites enter the host via the of host infection, there are also some digestive tube after being ingested (see particular situations. A form of Chapter 1.7.3 for details). Some of these autoinfection known as retrofection has parasitic species will need to get to their been described for pinworms typical habitat within the host. As (Oxyuridae). The eggs of these nematodes situations are quite diverse, we will use hatch on the anal skin and mucosa and some examples instead of drawing a the larvae migrate up the bowel to the general picture. cecum. Another type of autoinfection is known in the hydatid disease where the Herbivores acquire the infection with the protoscolices of Echinococcus are able to liver fluke Fasciola hepatica after eating infect other tissues in the same host grass with encysted cercariae. In the individual by metastasis. intestine of the herbivores, cercariae excyst and start their journey towards Last but not least, vertical transmission of the bile ducts from the liver. Their parasites from the mother to the migration can follow three pathways: (1) offspring is another strategy used by some travel directly through the intestine some parasites. The zoonotic protozoan wall, penetrating the peritoneum, the Toxoplasma is one prominent model. liver capsule and hepatic tissue; (2) Roundworms of genus Toxocara are also others will use the common bile duct or known to transplacentally pass from (3) after penetrating the intestinal wall mothers to fetuses during pregnancy. they will enter the blood stream and through the hepatic portal venous system will reach the liver.

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If in Fasciola hepatica there are multiple dogs, the larvae use the same migration possible routes, most helminthes which route until they reach the lungs, but enter the host’s body through the instead of leaving the blood they continue digestive system use the blood or their journey back to the heart via the lymphatic stream for migration. The main pulmonary veins. After they reach the gate for parasites to enter the circulatory right atrium they pass into the right system from the intestine is the hepatic ventricle and from there, through the portal system. This remarkable venous aorta the get to various tissues (muscles, system drains the blood with nutrients brain, etc.). This migration pattern is absorbed by the intestinal mucosa and called entero-pneumo-somatic. transports it to the liver. Together with Another nematode, these molecules, microscopic parasites enters its carnivorous host after this make their way to the liver and further feeds on the infected meat of another on, through the posterior vena cava to the host. After completing its life cycle in the right atrium of the heart. From the right intestine, newborn larvae use several atrium they pass to the right ventricle pathways to get to the skeletal muscles. and continue their travel via the These include direct invasion of pulmonary arteries to the lungs. In the capillaries and lymphatic vessels in the lungs they either leave the blood stream intestine as well as migration through the entering the respiratory ducts or they intestinal serosa to the peritoneal cavity continue their blood adventure and or via the hepatic portal vein blood to the return to the heart through the general circulation. pulmonary vein to the left atrium and left ventricle. Among tapeworms (Cestoda) we take into discussion again the family The best group to illustrate the diversity Taeniidae. Their eggs, if ingested by a of migrations is represented by ascarid suitable intermediate host will hatch in nematodes. Ascarids of horses, pigs or its intestine. The newborn embryo will humans (genera Ascaris and Parascaris) migrate via the circulatory system to are within the first type, leaving the various organs, depending on the vascular system in the lungs. tapeworm species. Eggs of Subsequently they migrate through the from human feces, if ingested by a pig, bronchi and trachea until the pharynx will hatch in its intestine. The embryos from where they are swallowed and will migrate via the blood and will spread reach again the digestive tube. During systemically to skeletal muscles of pigs. this migration they undergo several Similar migration route is known for molts and they grow in size accordingly. Taenia saginata but intermediate hosts in This type of migration is called entero- this case are bovines. For both species, pneumo-tracheo-enteral. Ascarids of humans are the definitive hosts and they genus Toxocara (parasites of carnivores acquire the infection after eating raw or and cattle) use even more complex undercooked meat from the respective migration pathways. If larvae of Toxocara intermediate host. canis (parasitic in canids) infect adult

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In unicellular parasites, the mechanism of state that a parasite with a narrow host spreading in the host’s body is usually range depends on its host in a greater involving intracellular parasitism in degree than one with a broader host transport cells. Protozoans like range. Medically, the importance of host Toxoplasma gondii enter the host by the specificity resides in the possibility of the digestive route. In the intestine they generalist parasites to jump on to a new enter macrophage cells and move host species and the possibility of throughout the body spreading to various development of a new, emerging disease. tissues and inducing a systemic infection. Moreover, some of these parasites are transmissible from vertebrate animals to Other parasites use different entry routes humans often producing important to the body. Nematodes from the family conditions known generically as Ancylostomatidae penetrate the skin or zoonoses. oral mucosa of their host. However, the migration is hematogenous as well. Several hypotheses try to explain the mechanism for host specificity. However, It is beyond the scope of this general none of them is fully explaining the chapter to exemplify all the parasites and complexity of parasite-host interaction in their migration patterns. Though, one nature. All of them have limitation and idea is evident: most parasites infect the probably the mechanism is a combination host via the digestive route and use the of these factors. They were reviewed circulatory system of the host to get to recently by Schmid-Hempel in his the predilection tissue/organ. During this excellent monograph on Evolutionary migration, most of them undergo Parasitology. Below is a synthetic account complex changes, some of them aimed to of these theories. evade the host’s immune system. (1) Host range is limited by Development in the host is extremely phylogenetic constrains: some varied according to the taxonomic group parasites tend to have more host species and will be discussed in more detail in when the hosts belong to a species-rich the respective chapters. taxonomic group (many similar enough hosts to be infected); for instance, microsporidia are typical parasites of 1.7.5 Biological background for host invertebrates and rarely of worm- specificity blooded vertebrates; one reason is that microsporidia do not tolerate high Parasites can infect variable numbers of temperatures. host species. Specialized parasites infect a narrow spectrum of host species while (2) Host range depends on the generalist parasites infect a wide range of phylogenetic age of the parasite group: host species. Medically and ecologically, during the evolutionary history of a the degree of host specificity is one of the parasite group, the host range expands as most important characteristics of a the parasites evolve. For example in some parasite. An ecologic approach might genera of fleas parasitic in small

26 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | mammals, host specificity is low as (7) Host range depends on immune compared to other flea taxa. defenses: Although still not fully understood, the immune mediated host (3) Host range depends on specificity can be synthesized in the transmission mode: some parasites following sentence: the host range is with active transmission (i.e. with mobile, given by the parasite’s capacities to evade free-living stages) can be more selective the host’s immune system. regarding the host than parasites with passive transmission (i.e. transmitted by direct contact between hosts or by unspecific vectors). 1.8 Host-parasite interactions

(4) Host range depends on the stages Life-cycle of parasites is usually an of the parasitic life cycle: in many alternation of free living and parasitic heteroxenous parasites the larval stages developmental stages. During this have low specificity for the intermediate parasitic phase, stages of the parasites host while the adult parasites are more are located within various tissues of their specialized to a narrow range of host. The interactions between parasites definitive hosts. When the parasitic stage and their hosts are complex and not is a encysted form (hypobiotic) the always fully understood. As for all living selection is probably weaker on cysts beings, natural selection shapes the than on the active adult forms. A good evolution of both, the parasite and the example to support this theory is the host (independently or together), tapeworm Echinococcus granulosus. through the same general mechanisms. Larval stages (known as hydatids) infect virtually almost all mammal species Usually, parasite-host interactions are while adults infect only canids. long term relationships, resulting in a non-lethal coexistence of both partners. (5) Host range depends on the However, in certain cases, parasites can virulence of the parasites: if a parasite seriously impair the homeostasis of their is more virulent the host specificity hosts, sometimes resulting in the death of should be broader; if the host range of a the later. In other cases, the immune virulent parasite is narrow, the number system of the host is able to keep parasite of susceptible individuals in the receptive development under control and even population might be decreased by the eliminate it completely. parasite, leading to co-extinction. The parasite-host interaction has been (6) Host range depends on parasite commonly described as an antagonistic geographic distribution: parasites with relation, where both organisms are in a a wider geographical distribution tend to permanent struggle for survival. This encounter a larger range of likely hosts antagonistic state might be easily than parasites with a more territorially questionable from evolutionary point of restricted distribution. view, but medically, it eases the understanding of parasitism as such.

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In this short chapter we will summarize parasites on their host but rather to be the host-parasite interactions focused on its medical and veterinary independently. First approach will side. Hence, in the next paragraphs, we include the actions and effects of will approach the pathogenic effect of parasites on their host, mainly from parasites on their host, or, to put it in medical point of view (i.e. how parasites other words, how parasites are able to produce disease). In the second part, the produce diseases. reaction of host will be discussed, with Certainly there are many factors emphasis on its immunologic strategies. influencing the pathogenicity of parasites. Some of them are related to the host, some others to the parasite. The factors 1.8.1 Pathogenicity of parasites related to the host include: species, breed, age, sex and individual immunity. To the inexperienced reader, it seems The species is very important when strange to find out that most of the wild considering pathogenicity. Some species animals (invertebrates or vertebrates) are very prone to develop clinical signs harbor parasites. We can say with almost when infected by certain parasites, while no chance to be wrong, that each single others are infected but evident symptoms animal burdens at least one parasite at a are absent. For instance, humans are very certain time. The situation in domestic sensitive to the infection with the animals is not very much different. nematode Trichinella spiralis and develop However, the clinical effects are not a severe disease, often lethal if not present all the time; on the contrary, the treated. On the other hand, infected pigs onset of the disease is the exception for or carnivores can harbor immense most parasite-host associations. number of larvae in their muscles By bearing in mind the definition of without any sign of disease. Even within parasitism we can easily conclude that the same host species, there might be parasites are supposed to induce some variations between different breeds. pathology to their host. And this is true. Usually, highly specialized breeds are Nevertheless, these lesions are in most more sensitive to parasitic infections situations minor and not reflected in the than local breeds. Probably the most general health status of the infected host. prominent example is the existence of the In wild animals (maybe even in so called trypanotolerant breeds of cattle, domestic), the “non-clinical” parasitism is sheep and goat, very resistant to the likely to influence in a bigger or smaller infection with the otherwise deadly extent the overall fitness of the host. agents of Nagana in Africa (figure 1.27). Although “fitness” is hard to be evaluated, When colonists introduced highly there are multitudes of examples in this productive European cattle breeds in direction. Africa with the hope of huge profits, their efforts were soon vanished by massive The aim of this section is not to discuss die-offs due to the tsetse fly transmitted the ecological effect and influence of trypanosomoses. Another significant

28 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | host-related factor which influences mostly related to immunity is a key factor pathogenicity is the age. Parasites are when considering parasite pathogenicity. usually able to infect all age groups of For causes not so evident, in certain host their host. However, in many situations, groups with similar populational features only the young ones develop (severe) (same species, breed, age or sex), some clinical diseases. Coccidia of genus individuals develop a more severe Eimeria are able to produce epidemic parasitic diseases than others. mortality in chicken or young domestic The second group of factors influencing rabbits, but adults are usually infected the pathogenicity comprises those without showing clinical signs. This is related to the parasite. Maybe the most particularly important mainly from important in this category is the intensity epidemiologic point of view, when adults of the infection. Usually the higher the with undetected infections are the main number of parasites within a host source of infections for the young individual is, the more severe the clinical offspring. signs are. The ascarid nematodes infect a wide variety of hosts. When only few nematodes are present in the intestine, the clinical effect is usually absent. However, when the infection intensity is of tens or hundreds of individual parasites, severe symptoms or death due to intestinal obstruction may occur. Another important factor is the strain within the parasitic species. Some strains may be more pathogenic than others or may have different host affinities.

The mechanism by which parasites are pathogenic can be grouped in five main categories: physical damage, spoliation, Figure 1.27 Some local African cattle toxin production, inoculation effect and breeds are resistant to trypanosome interactions with the host’s defense infections. (Photo Andrei D. Mihalca) mechanism. Most parasites fit into several of these categories, if not in all together.

Sex of the host is also able to influence Physical damage can take various forms, the clinical course of the disease. The depending on the organ affected or the human genital parasitic protozoan parasite species or stage involved. Some Trichomonas vaginalis is commonly parasites, due their large size or high producing clinical infection in females but number in the host’s tissues and organs males are often asymptomatic carriers. can induce severe mechanical trauma. Last but not least, individual resistance, Bladder worms (vesicular structures of

29 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | larval cestodes) located in retarded growth. With effects similar to parenchymatous organs are compressing spoliation, the malabsorption caused by the surrounding tissues inducing intensive intestinal mucosal damage is atrophy. This is highly evident in case of also a common pathogenic feature of the “coenurus” type larvae of Taenia parasitic infections. multiceps located in the brain of small ruminants. For large parasites or large parasite groupings in luminal organs (i.e. intestine, bronchi etc.) one of the most common extreme effects is obstruction. The most common parasites responsible for intestinal obstruction in dogs, horses and humans are ascarid nematodes (figure 1.28). Parasites are also responsible for direct tissue destruction. Parasites can destroy the tissues by several ways. Migrating parasites (see Chapter 1.7.4) are responsible for important traumatic lesions in various organs. Other situations include direct tissue damage during feeding or Figure 1.28 Ascarid nematodes like attachment. Most parasites possess in dogs are often various adherence structures to avoid obstructing the host’s intestine. (Photo being eliminated by the host. These Andrei D. Mihalca) structures (i.e. hooks, spines, suckers etc.) are highly irritating to the host’s tissues, causing local destruction at the Toxin production by parasites can have site of parasite fixation. local or systemic effect. The toxins can result either as parasite waste products Spoliation (the act of plundering) or or due to massive destruction of nutritional robbing is common among parasites. The agents of human malaria intestinal parasites. The parasites utilize (apicomplexans of genus Plasmodium) the same food resources as their hosts do. are producing a toxin called hemozoin, Large parasites or large parasite responsible of an overall reduced groupings are able to use huge amounts phagocytic performance by host’s white of certain nutrients ingested by the host. blood cells. The saliva of ticks contain The fish-borne tapeworm various products, which, when injected Diphyllobothrium latum absorbs large into the host may induce general amounts of vitamin B12 from the host’s paralysis. Following the death of large intestine inducing systemic deficit which numbers of individuals of Toxocara canis, results in anemia. Other parasites are the post-mortem release of toxins responsible for unspecific spoliation, resulting in general malnutrition and

30 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | induces nervous signs similar to epilepsy This section can hardly be more detailed in puppies. than this, but pathogenesis will be discussed individually for each parasite The inoculation effect refers to in the chapters to follow. situations when parasites facilitate the invasion of other inside 1.8.2 Immunity of host to parasites the hosts body, tissues or organs. Several bacteria are commonly found in the Usually, the immune system of the host is intestine where they are harmless. able to eliminate or to stop the parasitic However, if they are carried by parasites invasion. Most hosts are hence resistant in other organs or tissues (liver, brain, to the majority of parasitic infections. peritoneum, etc.) they are able to Some parasites which are host specific produce severe infections. In other cases, are able to infect individuals from a single parasites induce lesions to the mucosa of host species, while all the other host the intestine or respiratory ducts, organisms are able to stop the parasite allowing pathogenic bacteria or viruses invasion. This gives the so called host to produce the infection which is susceptibility or resistance to certain otherwise unlikely through unharmed parasites. However, resistance is not epithelium. One of the most well-known synonym to immunity. Immunity refers inoculation effects of parasites is the case to those mechanisms by which of vector-borne infections, when specialized cells or tissues of an organism hematophagous arthropods are are able to recognize foreign (non-self) transmitting various pathogens to their structures and eventually protect against hosts (i.e. ticks, tsetse flies, mosquitoes, potential invasions. The immune system sand flies, biting midges etc.). is present in various degrees of The most severe and complex complexity in all animal organisms, pathogeneses in parasitic infections are invertebrate or vertebrate. caused by the altered immune response Most vertebrate animals possess in of the host. The host responds to the general two types of immunity: the innate presence of parasites by inflammation. immunity and the acquired immunity. Severe granulomatous lesions or strong inflammatory reactions are produced by The innate immunity (also known as migrating nematode larvae in various non-specific immunity) includes various tissues. Parasites are also able to induce inborn defense mechanisms known in all changes on the surface of various cells, plants and animals. There are certain cheating the host’s immune system and physical or chemical barriers which producing autoimmune responses. The prevent invasion by pathogens. In red blood cells infected with Babesia are vertebrates, the skin together with recognized as non-self and destroyed by mucosal layers lining the inner lumen of the host’s own immune system resulting respiratory ducts and digestive tube are in severe hemolytic anemia. the first obstacle for most pathogens, including parasites. Except these

31 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | anatomical barriers, the innate immune hypereosinophilia, increased system includes important components: immunoglobulin E (IgE) production, the cytokines, the complement system or mastocytosis and goblet cell hyperplasia. a range of specialized cells like mast cells, Immunity against biting saliva phagocytes (macrophages, neutrophils, has been also described in detail. dendritic cells), basophils, eosinophils, Despite the complex immune system natural killer cells or gamma-delta T cells. involved in the protection of host, The acquired immunity (also known as parasites are often able to evade all these adaptive or specific immunity) is known mechanisms and to produce infection or only in vertebrates. Nevertheless, the even severe disease or death. The specific immune response is activated by avoidance strategies are both complex the innate components. The adaptive and interesting. One common mechanism immunity is responsible for protecting is antigenic variation when the parasite the organism specifically against is one step ahead the immune system of pathogens and to “remember” specific the host. By the time the antibody is antigens (immune memory). The main produced, the surface of the parasite has factors of the acquired immunity are a completely new antigenic structure so antibodies, produced by B lymphocytes. T the initial antibodies are useless. This lymphocytes are also part of the acquired strategy is used by many important immunity. There are several types of T protozoan parasites (i.e. Plasmodium cells know, each of them with specific falciparum, ) or functions: helper, cytotoxic, memory, molting nematode larvae. Another regulatory, natural killer, gamma-delta interesting avoidance mechanism is etc. Acquired immunity can be active molecular mimicry, when the parasite is (post-infection or post vaccination) or able to pass undetected (i.e. Plasmodium passive (maternal transfer or after falciparum). Some parasites are able to immunoglobulin administration). produce immunoglobulin cleaving proteases (i.e. Dirofilaria immitis, Compared to prokaryotic pathogens Fasciola hepatica) which are destroying (viruses and bacteria), traditional all adherent antibodies. Others are parasites (protists, helminthes, producing prostaglandin E2 (i.e. Brugia arthropods) are much larger in size and malayi, Taenia taeniaeformis) which has a have a much more complex antigenic strong anti-inflammatory effect. Many surface. Moreover, some of the parasitic helminth or protozoan parasites are able antigens are excretory antigens, not to interfere with the complement surface antigens, and are produced cascade, blocking certain steps in its intermittently. There are several activation (Echinococcus granulosus, components of the immune system which Taenia solium, T. taeniaeformis, act in the defense against parasitic Trypanosoma brucei, T. cruzi, Entamoeba protozoa, mostly antibodies and T cells. histolytica, Leishmania spp.). These are In the case of helminthes, the infections only few of the extremely various known are usually associated with molecular mechanisms.

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Except these intimate mechanisms, there taxonomic position of each parasite. So are also some general strategies which the most important objective of this small allow parasites to avoid host’s immune section is to make scholars understand system. Most parasites are located in the the practical importance of taxonomy in intestinal lumen. This can be the process of teaching and learning. evolutionarily explained by the low All parasite species are grouped in amounts of immune effectors present at systematic assemblages called taxa based the surface of the intestinal mucosa. The on certain morphological and biological only immunoglobulin normally present in features. For instance, genera like the intestinal lumen is IgA, but this has Sarcocystis, Toxoplasma, Neospora, almost no effect against helminthes. and are all Other parasites simply hide from the included in the family Sarcocystidae. It immune effectors. Larval cestodes means that all members of these genera (bladder worms) are isolated within a share common characteristics, the ones cystic membrane and some unicellular of the family grouping all of them. One of parasites develop inside various cells of these characteristics is that all species in the host organisms (i.e. Toxoplasma, the family Sarcocystidae are Babesia). heteroxenous. So if one knows that any of Detailed studies of immunology are the genera above is part of Sarcocystidae, available for many parasites. A practical it also knows that its life cycle is use of this knowledge is the possibility of heteroxenous. immunodiagnostic of parasitic infections by detection of circulating antibodies or antigens using various laboratory or 1.9.1 Principles of zoological clinical tests: intradermal allergy test taxonomy (IDR), indirect hemagglutination (IHA), indirect fluorescent antibody (IFA), Zoological nomenclature is the system of complement fixation (CF), enzyme-linked scientific names applied to taxonomic immunosorbent assay (ELISA), western units of extant or extinct animals. These blot (WB) etc. units are called taxa (singular: taxon). Parasites, as all animal taxa, are classified 1.9 Classification of parasites according to the rules of the International Code of Zoological Nomenclature. The Definitely, taxonomy is not the most species is the basic unit and taxonomical important part of medical parasitology. rank in biological classification. Except Most veterinary students consider species, there are seven main taxonomic taxonomy boring and very difficult to ranks: Domain, Kingdom, Phylum, Class, learn and remember. Moreover, they do Order, Family and Genus. not understand why it is important to know basic taxonomy of parasites. On the The taxa are hierarchically arranged so contrary, others tend to overestimate its that always a higher taxon includes importance and learn by heart the usually several lower ones (i.e. one

33 | Introduction to Parasitology M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | kingdom includes several classes, one occurrences are abbreviated (i.e. H. family includes several genera, one genus sapiens, C. lupus). As a rule, the names of includes several species). To face the genus and species are written with italics. increasing diversity of described life There are also typical terminations for forms, taxonomists introduced various supraspecific taxonomical ranks intermediary ranks for further divisions. (table 1.3), although exceptions are For instance a class might contain several known. Biological taxonomy is probably subclasses, a subclass could include the most dynamic science. Entire taxa are several superorders or more families permanently reordered (reclassified) could be all included in the same according to molecular phylogenetic superfamily. studies. Hence, it is very difficult to put down on paper a kind of “officially” Each taxon above the rank of species gets recognized taxonomical hierarchy. a scientific name in one word Moreover, different authorities have (uninominal name), always spelled with different opinions. capital letter. A species always has a binomial name (composed of two words).

The first word is always spelled with Table 1.3 Scientific names of metazoan capital letter and represents the name of parasites the genus. The second word is called the Rank Termination specific epithet and is spelled with lower Order -ida case (i.e. Homo sapiens, Canis lupus). Superfamily -oidea Family -idae Sometimes, especially when in a text the Subfamily -inae species name was written in full, the next

Table 1.4 Main taxa of parasites

Phylum Class* Common name Euglenozoa Kinetoplastea Parabasalia Tritrichomonadea Trichomonadea Flagellates Hypotrichomonadea Fornicata Retortamonadea Trepomonadea Apicomplexa Coccidia Coccidia Cryptosporidea Cryptosporidia Haematozoea Piroplasms Ciliophora Ciliates Platyhelminthes Trematoda Flukes Cestoda Tapeworms Nematoda Secernentea Roundworms Adenophorea Acanthocephala Archiacanthocephala Thorny-headed worms Arthropoda Pentastomida Tongue-worms Insecta Insects Arachnida (Subclass Acari - order Ixodida) Ticks Arachnida (Subclass Acari - except Ixodida) Mites *Only classes of veterinary significance are included

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Parasitology. Caister Academic 1.9.2 Major parasitic taxa Press. 232 pp.

All species of parasites are included in 5. Gillespie S, Pearson RD (2001) domain Eukaryota. The unicellular Principles and Practice of Clinical heterotrophic mobile species are Parasitology. Wiley. 752 pp. included in a paraphyletic group referred 6. Gosling PJ (2005) Dictionary of to as Protista. Protista includes the Parasitology. Taylor & Francis heterotrophic Protozoa, which groups Group. 394 pp. animal-like unicellular organisms. They 7. Grove DA (1990) A History of include the following phyla with Human Helminthology. CAB representatives parasitic in domestic International. 856 pp. animals: Euglenozoa, Parabasalia, Fornicata, Apicomplexa and Ciliophora. A 8. Mehlhorn H (2008) Encyclopedia of group formerly regarded as protists are Parasitology, 3rd Edition. Springer- Microsporidia which now are classified Verlag. 1573 pp. within Fungi. All the other parasites are 9. Odening K (1976) Conception and members of Kingdom Animalia. Table Terminology of Hosts in 1.4 lists the major phyla and classes of Parasitology. Advances in parasites. The right column from the Parasitology 14:1-93. table lists the most widely used terms in English. 10. Paracer S, Ahmadjian V (2000) Symbiosis: An Introduction to

Biological Associations. Oxford University Press. 304 pp. Selected references and further reading 11. Roberts L, Janovy J (2008) Foundations of Parasitology, 8th 1. Bowman DD (2008) Georgis' Edition. McGraw-Hill. 728 pp. Parasitology for , 9th 12. Schmid-Hempel P (2011) Edition. Saunders. 464 pp. Evolutionary Parasitology: The 2. Bush AO, Fernández JC, Esch GW, Integrated Study of Infections, Seed JR (2001) Parasitism: The Immunology, Ecology, and Diversity and Ecology of Animal Genetics. Oxford University Press. Parasites. Cambridge University 496 pp. Press. 576 pp. 13. Soanes C (2008) Compact Oxford 3. Cox FEG (2002) History of Human English Dictionary of Current Parasitology. Clinical English. Oxford University Press. Reviews 15:595-612. 1210 pp.

4. Elsheikha HM, Khan NA (2011) 14. Taylor MA, Coop RL, Wall RL (2007) Essentials of Veterinary Veterinary Parasitology, 3rd Edition. Wiley-Blackwell. 600 pp.

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15. Thomas F, Renaud F, Guégan JF Parasitism. Oxford University Press. (2005) Parasitism and Ecosystems. 240 pp. Oxford University Press. 232 pp. 17. Webster's New World Medical 16. Thomas F, Guégan JF, Renaud F Dictionary (2008) 3rd Edition. (2009) Ecology and Evolution of Wiley. 480 pp.

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2 PROTOZOA

2.1 General considerations apparatus, lysosomes, ribosomes, rough and smooth endoplasmatic reticulum and Recent phylogenetic studies brought a nucleus with the genetic material apparent chaos to traditional systematics organized in chromosomes. The nucleus of living organisms. Through time, is separated by the rest of the cytoplasm unicellular eukaryotic organisms were by a nuclear membrane, hence the name included in various taxonomic groups. (Greek: eu = true; karyon = Currently, they are all referred to with nucleus). The number, structure and the name Protista. However, this term position of these organelles within each has no taxonomic ranking. Within cell are highly dependent on the protists we include autotrophic, taxonomic group. heterotrophic and phototrophic Parasitic protozoa can inhabit virtually all organisms. Traditionally, all organ systems and tissues of their host, heterotrophic protists are included in the with both intracellular (i.e. group known as Protozoa. apicomplexans) or extracellular (i.e. Protozoa are found in all possible flagellates, ciliates) locations. Some of the habitats and include free-living or parasitic protozoa cannot survive in the symbiotic forms. Among the later, environment (i.e. flagellates) and parasitic protozoa can be associated with therefore are transmitted from host to all types of hosts, from plants to animals. host by direct contact or using living Protozoans are a group of very old and vectors (i.e. arthropods). In some other divers unicellular organisms, groups (i.e. apicomplexans) when hypothetically originating from the long eliminated by the hosts to the coevolution of two or more symbiotic environment, they transform into prokaryotic cells. With this view, the resistant stages, like cysts or oocysts. mitochondria and the chloroplasts from Parasitic protozoans obtain their food the eukaryotic cells were originally from their hosts. Mechanisms used for prokaryotic endosymbionts of larger uptake of nutrients include phagocytosis, cells. All protozoans have a typical pinocytosis, osmosis or active ingestion eukaryotic structure of the cell. The cell via a cell “mouth” called cytostome. membrane may be naked or covered with Maybe the most heterogenic process in locomotion structures like cilia or protozoa is reproduction. Some groups flagella. Within the cytoplasm, the use only asexual reproduction (binary organelles include mitochondria, Golgi or cell division). All flagellates use

37 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | this multiplication mechanism. Some within the metazoans while other groups employ also sexual Microsporidia is within Fungi. reproduction. In apicomplexans, the fecundation of micro- and macrogametes is a typical phase of the life cycle. Ciliates 2.3 The flagellated protozoa use conjugation to exchange genetic information during multiplication. Flagellates are motile protozoans, many Protozoans are extremely important from of them free-living, but some pathogenic medical point of view. In humans, to humans and animals. The locomotory diseases like malaria, sleeping sickness or structures are known as kinetids. The Chagas’ disease are responsible for genera of medical importance are millions of fatalities every year. In currently included into three phyla (table domestic animals protozoans cause 2.1). massive economic losses especially in They infect almost all animal phyla tropical areas (i.e. Nagana of ) occupying various habitats within their but also worldwide (i.e. eimeriosis of host, with either extra- or intracellular poultry). Last but not least, several location. Most of the species feed by parasitic protozoans are transmissible osmosis and reproduce by binary fission. from animals to humans. These zoonotic Phylum Euglenozoa includes a single conditions include toxoplasmosis, class of medical importance, Class sarcocystoses or leishmaniosis. All major Kinetoplastea (table 2.1). The class groups will be detailed in the following comprises of organisms with a sections. kinetoplast, a cell organelle containing a particular type of mitochondrial DNA. The kinetoplast is located close to the 2.2 Diversity of parasitic Protozoa basal body of the flagellum, and is easily visible at Giemsa staining as a deep Several classifications of protozoans are purple dot. Several genera are included available in the literature, and apparently here, but only two will be considered in none is generally accepted. Several this book, as the others are parasitic complex phylogenetic approaches gave mostly in invertebrates. All diseases birth to new clades, but these will not be produced by members of this phylum are considered here. Herein, we chose an treated under Chapter 2.3.1 and the adaptation of the revised classification of members will be generically called by Adl et al 2012. kinetoplastids. The protozoans of veterinary importance Phylum Parabasalia includes organisms are grouped in five phyla (see text which have a parabasal apparatus. The below). Other higher ranked taxonomic kinetid consists of four kinetosomes. groups, previously included within Three classes of veterinary significance Protozoa were shown to be distinct are included here (table 2.1). Most groups. Myxozoa are currently included genera of medical importance in this

38 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | phylum were formerly known as trichomonoses and will be discussed in trichomonads. In general, they possess a Chapter 2.3.2. variable number of flagella, according to Phylum Fornicata includes species with genus. In most of the cases, they have a single or one pair each of kinetids and group of flagella at their apical pole and nuclei. They usually possess a feeding an additional recurrent flagellum which groove or a cytopharyngeal tube runs backward, along an undulating associated with each kinetid. The phylum membrane. Each flagellum originates in a includes two classes of medical basal body. Another common feature of importance (table 2.1). Although genus species from this group is the presence of Chilomastix is currently included here an axostyle. This structure plays the role and not considered to be related to of a cytoskeleton. Generally, this group trichomonads, it will be listed under the lacks mitochondria. All the species in this chapter referring to them (2.3.2). Genus group will be referred to as Giardia and giardiosis will be discussed trichomonads. They are responsible for a separately in chapter 2.3.3. group of diseases medically known as

Table 2.1 Current classification of flagellates parasitic in domestic animals (adapted after Adl et al. 2012)

Phylum Class Genera Disease (Chapter) Euglenozoa Kinetoplastea Trypanosoma Equine dourine (2.3.1.1) Vector-borne trypanosomoses in domestic animals (2.3.1.2) Leishmania Canine leishmaniosis (2.3.1.3) Feline leishmaniosis (2.3.1.4) Parabasalia Tritrichomonadea Tritrichomonas Genital trichomonosis in cattle (2.3.2.1) Intestinal trichomonosis in birds and mammals (2.3.2.4, 2.3.2.5) Histomonas Histomonosis of poultry (2.3.2.6) Trichomonadea Trichomonas Anterior digestive trichomonosis in birds (2.3.2.3) Tetratrichomonas Buccal trichomonosis in dogs and cats (2.3.2.2) Intestinal trichomonosis in birds and mammals (2.3.2.4, 2.3.2.5) Pentatrichomonas Intestinal trichomonosis in domestic mammals (2.3.2.5) Hypotrichomonadea Trichomitus Intestinal trichomonosis in domestic mammals (2.3.2.5) Fornicata Retortamonadea Chilomastix Intestinal trichomonosis in birds (2.3.2.4)

Trepomonadea Giardia Giardiosis in domestic animals (2.3.3.1)

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body and running forward along an 2.3.1 Kinetoplastids undulating membrane).

Introduction. Kinetoplastids include The trypomastigote (figure 2.2) is the several genera parasitic in invertebrates, most important stage from diagnostic vertebrates and plants. Only two genera point of view, as it is the most frequently (Trypanosoma and Leishmania) are of encountered in the blood of the veterinary importance. The main feature vertebrate host. Its trepan (= drill) shape of Trypanosomatidae is the presence of a gave the name to the genus. The single flagellum and a kinetoplast. Based trypomastigotes are usually lanceolate in on their life cycles, members of this shape, like a flat elongated blade, oval or family are either homoxenous or elliptic in transverse section and with heteroxenous. All species parasitic in tapering ends. Conventionally, the domestic animals are heteroxenous, with anterior end is considered the one stages alternating between the directed forwards during locomotion. invertebrate and the vertebrate host. The body surface of trypanosomes is However, a single species, namely covered with a periplast. The main cell Trypanosoma equiperdum is transmitted organelles are the nucleus, the directly by coitus and does not require an kinetoplast and the locomotion system additional host. (figure 2.1).

General morphology. The morphology The kinetoplast, defining structure of all of trypanosomatids is heterogenic during members of order Kinetoplastida (hence different stages of the life cycle. The main the name), is always located in the close feature distinguishing the morphological vicinity of the basal body. The locomotion stages of Trypanosomatidae is the system (also known as the mastigont position of the flagellum. The most system) is represented by the flagellum common morphological types (figure and the basal body. The single flagellum 2.1) are: amastigote (rounded or originates from the posterior end, then elongated forms lacking flagellum); runs forward along an undulating sphaeromastigote (rounded forms with membrane, and freely terminates at the a free flagellum); promastigote anterior end of the cell. At the starting (elongated forms with antenuclear point of the flagellum stays the basal kinetoplast and flagellum arising near it body (also called blepharoplast). Most but emerging from the cell at the anterior stages are mobile, but mobility is seldom end); epimastigote (elongated forms observed, as most morphological with juxtanuclear kinetoplast and examinations require fixation and flagellum arising near it but emerging staining. from the side of the body); Ecology and transmission. With the trypomastigote (elongated forms with exception of T. equiperdum, all species of postnuclear kinetoplast; flagellum arising Trypanosomatidae parasitic in near it, emerging from the side of the vertebrates have heteroxenous development. Vertebrates are considered

40 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | definitive hosts and invertebrates are the invertebrate host. This cyclic (indirect) intermediate hosts, also referred to as transmission is the typical one for vectors. For all species parasitic in heteroxenous trypanosomes. In some mammals, the intermediate hosts are cases, direct transmission from mammal insects from orders (true to mammal was reported, when blood bugs), Diptera (true flies) and stages are mechanically passed by Siphonaptera (fleas). The insects take up hematophagous insects or by syringe the bloodstream forms of the parasites inoculation of infected blood. However, when feeding on infected mammals. In the ability of vectors to mechanically the intermediate hosts, they transmit the disease is measured in undergo a cycle of development with the minutes, while the typical cyclic final production of special infective forms transmission equals sometimes the called metacyclic (Greek: meta = after) whole life duration of the insect. trypanosomes. These are transmitted to a Medical importance. Diseases caused by new definitive vertebrate host by various members of genus Trypanosoma have the ways, according to the location of the generic name of trypanosomoses final developmental stage within the (singular: trypanosomosis). Most of the vector. Transmission from vector to the infections occur in tropical areas of the mammal occurs only after the world where they cause severe, often trypanosomes have completed their lethal conditions in humans and animals entire cycle of development in the as well.

Figure 2.1 Main stages of trypanosomes: a - amastigote; b - sphaeromastigote; c - promastigote; d - epimastigote; e - trypomastigote.

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Figure 2.2 General structure of a trypomastigote, the blood stage of trypanosomes.

The only pathogenic species occurring in signs to what we know today as dourine. temperate areas seems to be T. Apparently, the first description belongs equiperdum, the agent of equine dourine. to the Byzantine Chiron in Many other trypanosomes are not his book on the diseases of horse, pathogenic, although infections are “Mulomedicina Chironis” (~ 400 AD). common worldwide. Genus Leishmania is Dourine is mentioned also in a treatise of responsible for several infectious veterinary medicine published in the 12th conditions in humans and animals century by the Arabian, Ibn-al-Awan in worldwide. Among domestic species, Seville. The first description of the dogs and cats are the only common hosts disease in Europe was done in a Prussian to these parasites. horse by Ammon and Dirkhausen in 1796. The causative agent was seen for

the first time by Rouget in 1894, who 2.3.1.1 Equine dourine demonstrated its presence in the blood of an Algerian horse. In 1900, Buffard and Dourine (Arabic: darina = mangy, dirty), Schneider reproduced dourine in a horse also known as the covering disease, is a after they subcutaneously injected the chronic protozoal disease, with venereal parasite isolated from a naturally transmission, naturally occurring in infected horse. One year later, in 1901, equids. It is eradicated in most of the Doflein described and named the countries but is still present in parts of causative agent Trypanosoma Africa and Asia. Main clinical signs equiperdum. include edematous lesions of the Etiology. In classical parasitology genitalia, typical skin plaques and textbooks, the agent of equine dourine is paralytic nervous signs, usually followed considered to be Trypanosoma by death. equiperdum. Recent molecular analysis of Historical notes. Ancient Arab texts different laboratory strains originating mention a disease in horses with similar from endemic areas, brought controversy

42 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | on the validity of this species. medial position. The length of T. Trypanosoma equiperdum is very closely equiperdum ranges from 15.6 to 31.3 m related to several subspecies of in Asian strains and from 22.3 to 29.0 m Trypanosoma brucei: T. brucei brucei, T. in Russian strains. brucei gambiense and T. brucei Life cycle. T. equiperdum is the only rhodesiense, all agents of African trypanosome not transmitted by an trypanosomoses in a variety of hosts, but invertebrate vector. Transmission occurs also to Trypanosoma evansi, which causes mainly during mating from stallions to a disease called Surra. Some authors mares and vice versa. There are some suggest considering the agent of dourine reports stating that foals from infected as a subspecies of T. brucei (T. brucei mothers can get the infection through equiperdum). Until the status of the their conjunctival mucosa during birth or species will be clarified, the conventional even through milk contaminated from name T. equiperdum will be used herein. lesions of the udder. Transmission by the Several natural strains were described means of unsterilized instruments used over time, varying mainly in for artificial insemination has also been pathogenicity. Even if in the last decades cited. no single strain was isolated from natural Unlike other species of the genus, T. infections, at least 12 laboratory strains equiperdum is primarily a tissue parasite are available worldwide (table 2.2). which rarely invades blood. They are extracellular parasites, typically

Table 2.2 Available laboratory strains of T. inhabiting the surface of the mucosa or equiperdum* between the epithelial cells. In stallions, they are found also in the seminal fluid. Code Origin Host BoTat 1.1 Morocco, Horse Later in the course of infection they 1924 invade also surrounding tissues or even STIB 818 China, 1979 Horse OVI South Africa, Horse blood. 1977 ATCC 30019 France, 1903 Horse Nutrition takes place by osmotic ATCC 30023 France, 1903 Horse absorption of dissolved substances from American America (?) Horse stabilate the host’s tissues, through the body Canadian Canada (?) Horse pellicle. stabilate Alfort (?), 1949 Horse Experimental infection of laboratory AnTat 4.1. (?) (?) Hamburg (?) (?) animals is possible, but difficult. In order SVP (?) (?) to establish the infection in murine TREU 2259 (?) (?) rodents, first inoculation using a natural * - modified from Claes et al. (2005) strain should be done in splenectomized animals. Once the strain becomes Morphology. T. equiperdum is a adapted, it can be subsequently passed monomorphic species of trypanosome, virtually for unlimited times. Wild strains morphologically indistinguishable from T. can be adapted after several passages to evansi. The nucleus is usually located in

43 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | laboratory animals (mice, guinea pigs, Pathogenesis. All the lesions and rabbits, dogs), but they change their symptoms of dourine are related to the pathogenic properties. However, first histotropism of T. equiperdum for the inoculation form naturally infected epithelial tissues of the genital mucosae horses typically fails. Attempts to infect or skin. After the onset of the infection, domestic ruminants or pigs resulted in the flagellates invade also surrounding inapparent disease and low or no tissues. It is questionable if they are able detectable parasitemia. to penetrate intact mucosae or they priory need some degree of abrasion. Epidemiology. Since the 19th century, However, it seems they invade local dourine has occurred sporadically in capillaries. Europe. Around 1918, the disease was reported only in Russia, Turkey, Hungary The local effect is considered to be and Spain. During World War II, the induced by a toxin secreted by the disease was spread by army into Western parasite which causes vasomotor Europe. After the war, dourine was disturbances with exudation of the eradicated from Western Europe by plasma and inflammatory reaction in the systematic screening and control, invaded tissues. The nervous damages including stamping out. are considered to be also the result of the toxin, carried systemically by the blood Currently, natural infections occur only in stream. If all the motor and sensory horses, donkeys and their hybrids in alteration can be attributed to nerve Africa, Asia and parts of Russia. damage, the emaciation is due to the Occasional outbreaks are known secondary atrophy of the muscles. sporadically from Europe, following However, the toxin was never isolated, international trade with horses. Official but other proof which sustains the toxin OIE reports state that the only countries hypothesis is the sudden death of where dourine occurred in the last years laboratory rodents infected with a high are: Botswana, Mongolia, Ethiopia, number of parasites. Kyrgyzstan, Namibia, Pakistan, Russia and South Africa. This officially reported Not all strains of T. equiperdum invade distribution of the disease may not be the blood stream of horses. Parasitemia is accurate because testing of horses in more common in laboratory rodents many countries is not being done where trypanosomes invade the blood 2- routinely. 3 days after the inoculation.

All equines are theoretically susceptible Immunology. As some species of equids to the infection with T. equiperdum. or some breeds of horses are naturally However, the donkeys and mules are resistant to infections, there is certainly more tolerant than horses. Even among an inborn immunity acquired through a horses, there is an evident difference in long parasite-host coevolution. The sensibility, with thoroughbred breeds immune factors responsible for the more susceptible than native ones. defense against the agent of dourine include both humoral and cellular types,

44 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | with an important role of phagocytosis. The chronic disease is conventionally There are some evidences that passive divided into three phases. During the immunity can be transmitted from first phase of the infection, the common immune mares to foals during pregnancy. lesions are localized within external genitalia. In mares, the first usual sign of Despite many other similarities, there is infection is a small amount of vaginal no cross immune protection between T. discharge. Swelling and edema of the equiperdum and T. evansi. Moreover, T. vulva develop later and extend along the equiperdum is able to periodically shift its perineum to the mammary glands and surface glycoprotein antigens, allowing ventral abdomen, accompanied by chronic, persistent infections. vulvitis, vaginitis, polyuria and elevated Clinical signs. Symptoms vary tail. These last signs mimic heats, and considerably, depending mostly on the usually mares are receptive to males. If infecting strain. Those originating in pregnant, abortion may take place when northern Africa, Europe and Asia seem to the infection is with more virulent strain. be more virulent than those from In stallions, the initial signs are edema of southern Africa and from the former the prepuce and glans penis, of variable American strains. The nutritional status degree which may spread to the scrotum, of the animal and other stress factors can perineum, ventral abdomen and thorax. influence the severity of symptoms. Paraphimosis may is not uncommon. In The incubation period is variable. Clinical both sexes, the swelling may resolve and signs usually appear within a few weeks reappear periodically. Following edema, of infection but may not be evident until vesicles and ulcers usually appear on the after several years. Although acute, genitalia. They may heal and leave asymptomatic or latent infections are permanent white scars, called known, the most common character of leukodermic patches. In some outbreaks, the dourine in horses is chronic. The conjunctivitis and keratitis are common duration of the disease in mild chronic ocular signs in infected animals. The cases may persist for 1-2 years, and second stage of chronic dourine is more occasionally up to 4-5 years. or less pathognomonic. Typical cutaneous Experimentally, horses infected with plaques or skin thicknesses can occur, laboratory have survived for up to 10 with sizes ranging from extremely small years after infection with these strains. In to several centimeters. Interestingly, more severe chronic cases, animals die these plaques have also been observed after several months. In acute forms, sporadically in animals infected with T. disease lasts for 1-2 months, or, evansi. To complicate the understanding exceptionally one week. As a rule, of the taxonomic status of the causative dourine is a fatal disease and the average agent, in the case of certain strains of T. mortality is about 50%. Recovery can equiperdum these typical skin lesions do occur spontaneously, especially in not occur. The third phase usually onsets stallions. after several month from the infection and is characterized by progressive

45 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | anemia and disorders of the nervous in North America and Europe. Also system. Initially these signs consist of xenodiagnosis is considered feasible. restlessness and the tendency to shift Direct parasitological diagnosis by weight from one leg to another followed observing the flagellates in samples is by progressive weakness and achievable in the first 4-5 days of the incoordination. Paralysis of the hind legs infection. Scrapings of the vaginal mucosa and paraplegia and ultimately in mares or urethral washings in stallions recumbency and death are the final are recommended in this case. In the later stages of infection. stages of the infection the parasites may All these symptoms are characterized by be found in aspiration of fluids from periods of exacerbation and relapse that edema and cutaneous plaques, especially may vary in duration and occur several shortly after eruption. times before death. Recovery is also possible, especially in infection with less virulent strains. The disease caused by more virulent strains is often acute and the mortality rate is higher. In other equids (i.e. zebras) animals can be positive and show no clinical signs.

Pathology. If the outcome of the disease is death, anemia and cachexia are the most evident lesions of gross necropsy. Signs of early disease like edema can be found as indurated areas on the genitalia or ventral parts of abdomen and thorax. Internal lymph nodes are hypertrophied.

If nervous signs occurred before the death of the animal, perineural Figure 2.3 Trypanosoma equiperdum in a connective tissue is infiltrated with blood smear from an artificially infected edematous fluid and a serous infiltrate rodent. (Photo Andrei D. Mihalca) may surround the spinal cord, especially in the lumbar or sacral regions. Detection in blood smears from naturally Diagnosis. Most commonly, the diagnosis infected horse is exceptional, but possible is based almost exclusively on clinical in laboratory rodents (figure 2.3). In signs. As the isolation of the parasite from countries where other trypanosomoses infected tissues or blood is rather than dourine occur in horses, the difficult, other laboratory methods were morphological differentiation of T. used through time for etiological equiperdum from other species (i.e. T. diagnosis. The use of complement evansi and T. brucei) is impossible. fixation test (CFT) was widely spread in implementation of eradication programs

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Xenodiagnosis (inoculation of suspicious solution injected im with a samples to laboratory animals) was second injection after 24 h at half successfully achieved in splenectomized dose; animals or after intratesticular injections  suramin: 10 mg/kg bw, given iv in rabbits. for two or three treatments at a Serology has been used in combination weekly interval; with clinical diagnosis. The complement  quinapyramine dimethylsulfate: fixation test (CFT) is the recommended 3-5 mg/kg bw in divided doses test for international trade. Uninfected injected sc. equids, mainly donkeys and mules, often show false positive results due to Control. As no immune prophylaxis anticomplementary effects in horse measures are available, the most effective serum. Indirect fluorescent may help to way for preventing and controlling the resolve these cases. Other serologic tests disease are systematic surveys for include ELISA, radioimmunoassay, identifying positive animals by serology. counter immunoelectrophoresis, agar CFT has been used successfully for gel immunodiffusion (AGID) and card eradication programs in North America agglutination. However, cross-reactions and Europe. Infected animals should be can occur, especially with T. brucei and T. euthanized or castrated to prevent evansi, but correlations with clinical signs further transmission. All equids in areas might be helpful. A test that can where dourine is diagnosed should be distinguish equine piroplasmosis, quarantined and breeding should be dourine and glanders by immunoblotting stopped for until no new positive cases has been developed in USA but it is not are found. To avoid accidental commercially available. transmissions in endemic areas, during artificial breeding, all instruments should Differential diagnosis is done against be sterilized properly. In dourine free coital exanthema, contagious equine countries, importation of horses from metritis, Surra, Nagana, anthrax, equine endemic countries should be prohibited. viral arteritis, equine infectious anemia, purpura hemorrhagica, malnutrition, helminth infestations etc. 2.3.1.2 Vector-borne Treatment. Although reported in some trypanosomoses in domestic animals endemic areas, treatment is not recommended as asymptomatic infected Vector-borne trypanosomoses include carriers may result. If attempted, mainly tropical infections of humans and treatment should be done with animals caused by several species of trypanocidal drugs. Suggested treatments genus Trypanosoma (table 2.3). Two include: severe diseases affect humans: the  diminazene aceturate (i.e. sleeping sickness in Africa and the Chagas Berenil): 7 mg/kg bw, as a 5% disease in South America.

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Table 2.3 Main vector-borne trypanosomoses of animals and men*

Main vertebrate (Sub)Species Disease Vector Transmission Distribution host T. theileri cattle, antelopes Nonpathogenic Tabanidae Feces Worldwide T. rangeli humans, wide Nonpathogenic Bite South range of domestic America and wild mammals T. lewisi rats, humans (?) Nonpathogenic Rat fleas Feces Worldwide (?) T. cruzi humans, virtually Chagas’ Disease Reduviidae Feces South all mammals America T. evansi camels, equines, Surra Tabanidae Mechanical Asia, Africa, bovines, goats, Stomoxys Australia, dogs and wild spp. South and animals Central America T. vivax ruminants, Nagana Glossina Bite Africa, South equines, camels (Souma) spp. Mechanical America Tabanidae T. congolense bovines, equines, Nagana Glossina Bite Africa sheep, goats, spp. camels, pigs, dogs T. simiae pigs, camels, Nagana Glossina Bite/Mechanical Africa horses, cattle spp. T. suis pigs Nagana Glossina Bite Africa spp. T. brucei brucei domestic animals, Nagana Glossina Bite Africa camels, antelopes, spp. carnivores T. brucei humans, pigs, Sleeping Glossina Bite Africa gambiense sheep sickness spp. T. brucei humans, cattle, Sleeping Glossina Bite Africa rhodesiense pigs, goats, dogs, sickness spp. primates, various wild animals, including antelopes * Compiled from Maudlin et al. (2004)

Trypanosomoses bear different names in in 1680. However, the first accurate animals, according to their geographical description of a blood stage was done in distribution and etiologic agent. 1841 by Gabriel Valentin (1810-1883) in However, some vector-borne blood smears from trouts. The genus trypanosomoses are also present in Trypanosoma was erected in 1843 by temperate areas (i.e. the T. theileri David Gruby (1810-1898) for asymptomatic infections of cattle hemoflagellates found in the blood of worldwide). frogs.

Historical notes. Probably the stages of The first major discovery from medical the first seen trypanosome were those of point of view came only in 1880, from T. theileri from the gut of horse flies, by Griffith Evans (1835-1935). While Antonie van (1632-1723) working as a veterinarian in India, he

48 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | discovered the agent of Surra, a disease of importance, T. theileri, T. rangeli, T. lewisi local horses and camels. Later, the and T. cruzi are included in this group. species was named in his honor, T. evansi. They always possess a free flagellum. The Another major milestone in the history of kinetoplast is large and it is never located trypanosome research was represented terminally. The posterior end of the body by the works of David Bruce (1855- is pointed. Reproduction in the 1931). Between 1894 and 1897 when he mammalian host is discontinuous, was working in southern Africa, he typically taking place in the amastigote or proved that the disease of livestock epimastigote stages. With the exception known as Nagana was also caused by a of T. cruzi, the other species are not trypanosome. As a tribute for him, this pathogenic. species was later named T. brucei. Bruce The salivarian trypanosomes include was the first scientist to prove the vector- those species in which the metacyclic borne nature of trypanosomoses. He trypomastigotes develop in the “anterior demonstrated that Nagana is transmitted station” (proboscis and salivary glands) from wild to domestic animals by the bite and the transmission is inoculative. of tsetse flies. Interestingly, the same Species from this group include: T. vivax, hypothesis, though not proved, was T. congolense, T. simiae, T. brucei, T. evansi suspected 40 years before Bruce’s and T. suis. The free flagellum may be demonstration by the famous British present or absent and the kinetoplast is explorer of equatorial Africa, David always located subterminally or Livingstone (1813-1873). Joseph Dutton terminally. The posterior end of the body (1879-1905) was the first to describe the is usually blunt. Reproduction in the agent of sleeping sickness in humans, T. mammalian host is continuous and takes gambiense. He died three years later by place in the trypomastigote stage. Most of the disease he had been studying. the species are pathogenic to mammals. Etiology. There are several species of Morphology. Specific morphological genus Trypanosoma involved in the details for the stages in the blood of etiology of vector-borne trypanosomoses vertebrates of each species were given of domestic and wild mammals (table above. Nevertheless, morphologic 2.3). Traditionally, members of genus identification to species level is not Trypanosoma parasitic in domestic generally feasible, and new molecular animals are divided into “Stercoraria” techniques tend to replace the traditional and “Salivaria”. for this goal. All The stercorarian trypanosomes (Latin: morphological characters given below stercoralis = living in feces) comprise refer to the stage from the blood of the species in which the development of the vertebrate host. T. theileri (figure 2.4) is metacyclic stages in the vector takes a common, non-pathogenic parasite place in the fecal medium of the hind gut inhabiting the blood of various domestic (“posterior station”) and transmission is and wild ruminants worldwide. It is one contaminative. Among species of medical of the largest mammalian trypanosome

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(60-70 m in length). The posterior end report from Malaysia, where the species of the body is pointed. The kinetoplast is has been isolated from a human clinical located near the nucleus, the later being case. positioned centrally in the cell. The T. cruzi has a wide host range. More than undulating membrane is well developed 150 species of mammals were reported and the free part of flagellum relatively to be infected with this species, and long. virtually all mammals are considered to be susceptible. It causes the Chagas Disease, a severe condition of humans from several South American countries. The species is a small, “C” shaped trypanosome, measuring 16.3-21.8 m in length. The large kinetoplast is located near the posterior end of the cell.

T. evansi is probably the widest distributed pathogenic trypanosome, causing a disease called Surra. Infection occurs in many hosts, but the most important include dromedaries, equines and dogs. The trypomastigotes (16.8-24.9 m) are usually slender, with a long free Figure 2.4 Trypanosoma theileri in a flagellum and a rounded posterior end. blood smear from a naturally infected However, the species is quite cow. This species does not seem to be polymorphic, and stumpy or intermediate pathogenic for their bovine or wild hosts forms have been described. (Photo Viorica Mircean) There are several species of the genus Trypanosoma listed as the agents of a group of diseases of livestock from Africa, T. rangeli is a non-pathogenic species collectively called Nagana: T. vivax, T. infecting humans and a multitude of wild congolense, T. simiae, T. suis and T. brucei. and domestic mammals. The trypomastigotes are slender and large T. vivax causes a disease called Souma in west, central, east and South Africa. Its (26-34 m). The kinetoplast is hosts are various ungulates (cattle, sheep, subterminal and relatively small. goats, horses, camels, antelopes). It was T. lewisi is parasitic in rats worldwide. also introduced to West Indies, Central The body is curved and pointed at the and South America together with cattle posterior end, with a mean length of 21- imported from Africa. 37 m. The well-developed free flagellum In the New World, the disease got the delimitates a more or less developed various names (Secadera, Huequera, undulating membrane. There is a single Cacho Hueco) and interestingly, became

50 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | transmitted mechanically by non-tsetse vectors (horse flies and stable flies). Mean length is between 21.0 and 25.4 m. There are two recognized subspecies: T. vivax vivax (in Africa) and T. vivax viennei (in the New World).

T. congolense infects a broad spectrum of domestic hosts. It is a small species, with a mean length between 11.5 and 14.0 m. There are several strains with no taxonomic status which differ in certain morphological feature but also virulence.

T. simiae is primary a parasite of pigs, Figure 2.5 Trypanosoma brucei from with high pathogenic importance, though laboratory culture. (Photo Andrei D. the name is misleading, meaning “of Mihalca) monkeys”. Morphologically, the species resembles T. congolense but it is more motile. The average length is 14.9-19.0 Life cycle. General aspects regarding m, with the kinetoplast typically development and biology of occupying a marginal position near the trypanosomatids were given above. Yet, posterior end of the body. each species has its own peculiarities.

T. suis parasitize suids in several regions The metacyclic trypanosomes of the of Africa, being relatively pathogenic. It stercorarian species (T. theileri, T. exhibits slow movements. The body is rangeli, T. lewisi, T. cruzi) develop in the short and stumpy (14.0-16.0 m), with a posterior part of the vector’s gut and are small kinetoplast in subterminal position. discharged through the feces of the T. brucei (figure 2.5) includes several insect. The metacyclic stages then invade strains, some of them assigned to the vertebrate host through the wound subspecies. T. brucei gambiense and T. caused by the insect bite, through skin brucei rhodesiense are responsible for abrasions or other wounds. If infected human sleeping sickness. insects are swallowed by vertebrates, trypanosomes may enter the Another subspecies, T. brucei brucei bloodstream through various contact completes the list of etiologic agents of mucosae. Inside the mammalian body, Nagana of livestock in Africa. The development of trypanosomes varies trypomastigotes of the later subspecies according to species. are polymorphic, with slender, intermediate and stumpy forms with In T. theileri, T. lewisi multiplication is by wide variation of the cell’s average binary or multiple fission of length, between 11.0 and 42.0 m. epimastigotes in the plasma of blood. In T. cruzi binary fission of amastigotes

51 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | takes place inside the reticuloendothelial Glossina (figure 2.6). Transmission from cells (i.e. macrophages) from the liver, the vector to the vertebrate host is spleen or bone marrow but also in inoculative, through the saliva. myocardial tissue. In T. vivax, trypomastigotes from the Various intermediate hosts were blood of the vertebrate host multiply by reported for stercorarian trypanosomes: binary fission. When ingested by tsetse flies, they become epimastigotes in the  For T. theileri the vectors are esophagus, than multiply and migrate to tabanids (horse flies). Several species the pharyngeal region where they were proven to transmit the transform to metacyclic trypanosomes. parasites to cattle: Tabanus These are the infective stages and are glaucopis, T. striatus and inoculated to a new vertebrate host. Haematopota pluvialis. Some authors There are several species of Glossina suggested that also hippoboscids are reported as vectors for T. vivax: G. feasible vectors for T. theileri. morsitans, G. pallidipes, G. longipalpis, G.  For T. rangeli the intermediate hosts swynnertoni, G. austeni, G. palpalis, G. are bugs of subfamily fuscipes, G. tachinoides, G. vanhoofi, etc. In (family Reduviidae). The main geographical areas where tsetse flies are vectors are Rhodnius prolixus, R. not present (parts of Africa, South pallescens, Triatoma infestans, T. America), tabanids are able to transmit dimidiata and many other the disease mechanically, by bite. experimental insect hosts.

 T. lewisi is transmitted by rat fleas. In

temperate areas, the main vector is Nosopsyllus fasciatus while in tropical and sub-tropical regions the natural vector is Xenopsylla cheopis. Many other flea species were infected experimentally and were able to transmit the trypanosomes.

 T. cruzi is using as vectors bugs of Triatominae subfamily. Many species were reported as insect hosts, the most important genera being Triatoma, Rhodnius, Panstrongylus and Eratyrus.

In the other group, Salivaria, the Figure 2.6 Glossina sp. feeding on human metacyclic trypanosomes develop in the host. (Photo Andrei D. Mihalca) salivary structures of insects. The main vectors are tsetse flies from genus

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In T. congolense the aflagellated parts of Africa free or cleared of tsetse, trypomastigotes multiply in the blood and parts of Central and South America. continuously and are transmitted to According to OIE, Nagana is spread over tsetse flies. After ingestion, they arrive to the territory of 37 countries (mostly sub- the midgut of the flies, change shape to Saharan African) on 10 million square longer and slender trypomastigote forms kilometers. It is the most important which migrate to the pharynx where they disease of cattle in Africa. At least 30 become epimastigotes. Finally the species of wild mammals are known as epimastigotes transform to aflagellate natural reservoirs for Nagana-causing infective metacyclic trypomastigotes. The trypanosomes, although they do not main vectors for T. congolense are: G. show clinical signs of infections. Nagana morsitans, G. palpalis, G. pallidipes, G. is widespread also because of the high longipalpis and G. austeni. number of competent biological vectors. About 23 species of tsetse flies (genus T. simiae has at least ten species of Glossina) are competent vectors for competent vectors in genus Glossina. trypanosomes. Moreover, an infected fly However, mechanical transmission by remains infective for all its life. other blood-sucking dipterans was reported commonly. Experimental work, showed that the complete cyclic development of T. simiae in G. brevipalpis takes 20 days.

Only two vector species are known for T. suis: G. brevipalpis and G. vanhoofi. The cycle in the tsetse fly takes about 28 days.

All subspecies of T. brucei develop in the midgut, proboscis and salivary glands of various Glossina species. In the case of T. b. brucei, mechanical transmission by tabanids (horse-flies) or Stomoxys has also been described.

Epidemiology. The geographical distribution of animal vector-borne Figure 2.7 Distribution of genus Glossina trypanosomoses is closely related to the is shown with red shading (redrawn, distribution of their vectors. African from Leak, 1999) animal trypanosomoses occur where the tsetse fly vector exists in Africa, between Virtually all domestic mammals are latitude 15°N and 29°S (figure 2.7). As susceptible to clinical infection with some species of Trypanosoma can also be African trypanosomoses. However, there transmitted mechanically by biting flies, are several livestock breeds considered the distribution range of Nagana is also in to be resistant (known as trypanotolerant

53 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | breeds). These include West African infection with the first peak at 14-21 days indigenous cattle breeds (N’Dama, of infection. This first peak corresponds Baoule, Muturu, Laguna, Somba and to the first presence of specific Dahomey), East African zebu breeds antibodies. (Orma Boran and Maasai zebu) and Lymphocyte sequestration and increased African indigenous breeds of small macrophage population are responsible ruminants (West African dwarf sheep for the splenomegaly. The same applies and goats, and East African goats). to the liver, which might be increased and The infection sources are blood, lymph congested due to increased phagocytic and other fluids of infected animals. activity by Kupffer cells. Trypanosome infection is also responsible for a more or Pathogenesis. After the transmission by less severe pancytopenia. The resulting tsetse flies, trypanosomes undergo a anemia is responsible for further pathology period of multiplication in the dermis and in the myocardium and blood vessels subdermis where they enter the afferent irrigating the heart, finally inducing cardiac lymphatic vessels. decompensation. In female cows, chronic At the inoculation site, a lesion called infection leads to infertility, endometritis chancre will develop 4-10 days after the and abortion. tsetse bite. This cutaneous lesion Blood biochemistry changes in infected precedes the detection of trypanosomes animals consist of: decreased cholesterol in the blood by 4-6 days. The and lipid concentration, reduced total development of the chancre is depended serum lipids, decreased serum albumins on the infective dose and the rhythm of and increased globulin. There is no parasite multiplication but generally its change in the level of total proteins, cellular population consists of calcium, iron or fibrinogen. During neutrophils, lymphocytes, macrophages parasitemic phases, the animals are and mast cells, associated with edema hypoglycemic. Hematology is and congestion. characterized by leucopenia, anemia, and After a period of multiplication in the thrombocytopenia. skin, trypanosomes appear in very high Immunology. The primary immune numbers in the afferent lymph that response is targeted mainly on the drains from the site of vector inoculation. variable surface glycoproteins (VSG), Subsequently, the draining lymph nodes which cover the surface of the parasites. become significantly enlarged because of Based on the type of VSG, trypanosomes B-cell proliferation and local migration of are grouped in (sero)demes. Reinfection other leucocytes. Through the lymphatic of the animals with trypanosomes from system, the trypanosomes finally gain the same deme is usually associated with access to the main blood stream and to a reduced chancre development. The the all the internal organs. Detection of antigenic variability in T. congolense and trypanosomes in the blood is possible T. b. brucei is huge; hence development of usually during the second week after the immunity against all demes is virtually

54 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | impossible. On the other hand, the painful swelling of the skin, of variable number of VSG types in T. evansi and T. size. The symptoms in the acute phase vivax is more limited. include high fever, which subsequently is correlated with the fluctuating Clinical signs. Despite the diversity of parasitemia, anemia of variable severity agents involved in the etiology of (depending on the breed and age of the Nagana, many clinical features are infected animal), enlarged lymph nodes, common to all domestic animals, enlarged spleen, weakness and lethargy. regardless the host or trypanosome Abortion or birth of weak offspring and species. However, there are particular high neonatal mortalities are also pathogenic characteristics, listed in table common. In 1-4 weeks after the infection, 2.4. the animals are unable to rise and death may occur. If infected animals survive, Table 2.4 Pathogenicity of Trypanosoma they usually go into the chronic phase. species causing Nagana in different hosts* This is characterized by a persistent anemia, stunted growth, decrease of Degree of severity

productions (i.e. milk) and infertility.

However, there is no loss of appetite. The (Sub)Species

Pigs chronic phase might last even for years,

Dogs

Cattle

Horses Camels

Donkeys before the death of the animals. Sheep, goats Sheep, T. vivax 3 2 2 1 2 0 0 There are various clinical differences of T. congolense 3 2 2 2 3 1 2 T. simiae 0 1 0 0 0 3 0 Nagana which are dependent on the host T. suis 0 0 0 0 0 3 0 species. In local cattle from Africa, T. brucei brucei 1 2 3 3 3 1 3 0 - nonpathogenic; 1 - mild; 2 - moderate; 3 - severe. Nagana is usually chronic, although * - from Maudlin et al. (2004) hyperacute forms are also known. The severe drop in the packed cell volume is As shown in table 2.3, Nagana is a group correlated with decrease in milk of diseases caused by various species of production. The anemia in this stage Trypanosoma, which affects a wide range must be differentiated from other types of domestic animals in Africa and South of infectious or parasitic anemia (i.e. America: large and small ruminants, babesiosis, anaplasmosis, strongyles or camels, horses, donkeys, pigs and heavy tick-infestations). Small carnivores. ruminants usually display fewer and less severe symptoms than bovines. However, There are many descriptions on the in both sheep and goats, the milk clinical course of the disease originating production is significantly decreased and in observation on natural or the infection is associated with low experimental infections. The prepatent reproductive performance and neonatal period is 1-3 weeks. In some (but not all) mortality. In camels, the disease is animals, at the site of the tsetse fly bite an usually chronic, rarely lethal. Horses and inflammatory reaction called chancre donkeys are very sensitive to T. b. brucei may develop. This might appear as Nagana and they usually develop an acute

55 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | or hyperacute disease, with subcutaneous Pathology. Four types of lesions are edema, keratoconjunctivitis, ataxia and associated with trypanosomoses in paralysis. If affected by T. vivax or T. animals: inflammatory, congestive, congolense, equids are more resistant, hemorrhagic and degenerative affective and they develop chronic or various organs: skin, lymph nodes, asymptomatic infections. In pigs, spleen, liver, heart, central nervous infections are rare, and clinical, severe, system, eyes, testes, ovaries etc. acute forms are likely to be caused by T. The characteristic skin lesion is called simiae. The other Nagana-causing species chancre, and if it is visible are responsible for reproductive macroscopically it appears as a 2-5 cm disorders in pigs. In dogs, the infection swelling at the site of vector bite. with T. b. brucei produces and acute Histological sections show edema and disease, with high fever, generalized mast cell degranulation at the site of edema, keratitis and rabies-like trypanosome initial development in the symptoms, followed by death. skin. The other animal trypanosomosis, Surra, The gross appearance of lymph nodes is a is more widely distributed (Africa, Asia, marked increase in size. This South America) than Nagana and its enlargement is evident mainly in the specific pathogenicity is different. The nodes draining the lymph from the site of agent, T. evansi is producing severe the infected tsetse bite. Histologically, syndromes in camels, horses and dogs, this enlargement corresponds to a with significant mortalities. In camels, proliferation of B cells, with expansion of Surra is usually acute in young camels lymphoid follicles in the cortical and and pregnant females and it evolves with medullar areas and reduced paracortex. high fever, anemia, extreme weight loss, subcutaneous edema, kerato- The spleen is increased in size during the conjunctivitis, hypertrophy of lymph acute phase. The associated nodes, neurological signs, abortions and histopathology consists of development death. In horses from Africa and Asia, the of secondary lymphoid follicles and disease is similar to Nagana. In South expansion of the red pulp. The American horses and donkeys, Surra is hepatomegaly and hepatic congestion are rather chronic, with less severe clinical also common lesions in the acute signs. Dogs in Asia and South America are trypanosomoses. sensitive and develop the acute disease In histological sections, the main lesions with signs similar to Nagana. Cattle are consist in hyperplasia of the Kupffer cells, more resistant to Surra. periportal mononuclear cell infiltration The cause of death in animal and centrolobular necrosis. Severe trypanosomoses is usually congestive lesions are found in the heart. The blood heart failure, caused by the persistent vessels irrigating the heart congested, anemia, myocardial damage and with swollen and vacuolated walls and alteration of vascular permeability.

56 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | perivascular edema. The most severe Direct methods include: cardiac lesion consists in myocardosis.  The simplest methods to confirm the Other lesions observed in animals diagnosis are parasitological suffering of trypanosomoses are: methods by which parasites can be pituitary necrosis, orchitis or testicular seen under a microscope. The most degeneration, cystic ovaries, useful sample to detect the parasites endometritis, meningoencephalitis, is blood. This can be examined directly (fresh or stained smears) or Diagnosis. Although laboratory diagnosis using concentration methods. Direct is more or less essential, in the field detection in smears is not conditions where vector-borne considered being sensitive enough, trypanosomoses occur are hardly mainly because of the low numbers available or quasi-absent. So of trypanosomes in the blood. This veterinarians and animal health officers can be partly overcome by collecting should rely to a great extent on blood early in the morning and from symptoms. Although clinical signs are not peripheral capillaries (i.e. tail or ear). considered to be characteristic, they Smear can be examined fresh (wet should not be disregarded. Cattle with blood films) enabling the detection of anemia, fever, weight loss, enlarged live, motile trypanosomes. Stained lymph nodes, lacrimation, abortion and smears (thick or thin) are also useful rough hair coat should be at least but with limited value, mainly in the suspected as acute Nagana. In hyperacute chronic phase. However, if no cases animals are found dead and centrifuge is available, this can be a systemic hemorrhage is dominating the simple option. pathological picture. If symptoms progressively remit following  Two blood concentration methods administration of trypanocidal drugs, the are available: the microhematocrit diagnosis is confirmed. centrifugation technique (Woo If animals die and necropsy is performed method) and dark-field/phase- the atrophy of fat, enlarged lymph nodes, contrast buffy-coat technique spleen and liver, subcutaneous edema (Murray method) (OIE, 2012). and hemorrhagic lesions can be also  The Woo method is based on the indicative of trypanosomoses. separation of the blood components However, laboratory techniques are the depending on their specific gravity. only ones which can confirm the After the blood is collected into a etiological diagnosis. They can be divided heparinised capillary tube and one in direct methods (identification of the end is sealed, the tubes are parasites, parasitic antigens or parasite centrifuged in a microhematocrit DNA) and indirect methods (serology). centrifuge at 9000 g for 5 minutes. After centrifugation, the tube is examined under the microscope at the separation level of the plasma-

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cell interface (buffy coat) using the molecular techniques to identify the 40 objective. The Woo method is parasite’s DNA. These include PCR, more sensitive than direct Real-Time PCR and Restriction examinations techniques. According Fragment Length Polymorphism to OIE (2012), in the case of T. vivax, (RFLP). the sensitivity of this method is Indirect methods (serological methods) 100% when the parasitemia is >700 include: trypanosomes/ml blood and it decreases to 50% when parasitemia  Indirect Fluorescent Antibody Test is between 60 and 300 (IFAT) trypanosomes/ml blood. When  Enzyme-Linked Immunosorbent parasitemia is lower than 60 Assay (ELISA), targeted on detection trypanosomes/ml blood the Woo of anti-Trypanosoma antibodies. method usually fails to detect the infection.  Card Agglutination Test (available for T. evansi).  The Murray method is similar to Woo method, but the buffy coat is  Immune Trypanolysis Test (available extracted from the tube on a only for T. evansi). microscope slide (after the tube is The main problem with highly specific cut) and examined under a dark-field and highly sensitive laboratory tests or contrast-phase microscope. (molecular methods or serology) is their  Other concentration techniques price and the need for sophisticated (used mostly in the diagnosis of equipment which is rarely available in human trypanosomoses) include the the countries which really need them. anion exchange method (using the Acute Nagana differential diagnosis in miniature anion-exchange cattle includes in the acute phase chromatography technique) or in babesiosis, anaplasmosis, theileriosis, vitro cultivation. anthrax and acute pasteurellosis. In the  Identification of the parasite can be chronic stage, various helminthosis and achieved also using animal malnutrition must be considered. In inoculation (using mice and rats) horses infected with T. evansi the followed by examination of their differential diagnosis is made with blood. African horse sickness (a vector-borne viral infection), equine viral arthritis,  Another direct method which is equine infectious anemia or dourine. In aiming this time parasitic antigens camels, the T. evansi infection shows but used with inconsistent results is similar signs anthrax and in dogs rabies ELISA. should be also considered, mainly  The most sensitive methods which because both diseases are endemic in are more and more routinely used in many tropical countries. laboratories worldwide are

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Treatment. The number of drugs used in treatments (with prophylactic drugs like the treatment of animal trypanosomoses isometamidium chloride), treatment of is limited and most of them are available infected animals (diminazene aceturate) or for almost 50 years (table 2.5). The treatment of clinical cases (any other treatment is carried out based on certain trypanocide drug). plans: routine or strategic block

Table 2.5 Drugs used in the treatment of animal trypanosomoses*

Dose Target Drug Trade name(s) Route Species (mg/kg) parasites Diminazene aceturate Berenil, many others 3.5-7 i.m T. congolense Cattle T. vivax Sheep T. brucei Goats T. evansi Dogs Horses Donkeys Homidium chloride Novidium 1 i.m. T. congolense Cattle Homidium bromide Ethidium T. vivax Sheep Goats Pigs Horses Donkeys Isometamidium chloride Samorin, 0.25-0.5 i.m. T. congolense Cattle Trypamidium, T. vivax Sheep Veridium T. brucei Goats T. evansi Horses Donkeys Camels Quinapyramine Trypacide sulphate 3-5 s.c. T. congolense Camels dimethylsulphate T. vivax Quinapyramine Trypacide Pro-salt 3-5 s.c. T. vivax Camels dimethylsulphate:chloride T. brucei Horses T. simiae Donkeys T. evansi Pigs Dogs Suramin Naganol 7-10 i.v. T. evansi Camels g/animal Horses Donkeys Melarsomine Cymelarsan 0.25 s.c./i.m. T. evansi Camels * compiled from Maudlin et al. (2004)

Usually only animals with severe acute prophylaxis are known to work. For the forms are treated. Otherwise they are left control of tsetse fly populations various to develop immune response which methods have been used over time but eventually protects them during most of them were aborted (i.e. spraying subsequent infections. of land with insecticides, clearing of bush). Nowadays, the commonly used Control. No vaccines are available. methods include application of synthetic However, general prevention measures insecticides on the animals or biological (i.e. control of the vector populations, control using sterile male flies (as animal husbandry practices, selection of females tsetse flies reproduce only once trypanotolerant breeds) and chemical

59 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | in their life) or fly traps baited with Donovan are credited with the discovery pheromones. A proper farming and description of the parasite. The management should reduce the contact vectors were identified to be the sandflies of animals with the vectors. Selection of in 1921 by two brothers, Edouard and trypanotolerant crossbreeds is an Etienne Sergent. important strategy. These include West The New World leishmaniosis is known African indigenous cattle breeds from, pre-Incan pottery as early as 1st (N’Dama, Baoule, Muturu, Laguna, Somba, century AD in Peru and Ecuador and from Dahomey), East African zebu breeds: record of Spanish missionaries from the (Orma Boran, Maasai zebu) and 16th century. The agents, which initially indigenous breeds of small ruminants: were considered to be similar to the Old (West African dwarf sheep and goats, and World, were accurately described as new East African goats) (OIE, 2012). species in 1911 by Gaspar Vianna and the Chemical prophylaxis is achieved using vectors were recognized as flies from mainly Isometamidium. genus Lutzomyia in 1922. The first case of canine leishmaniosis was described in

1903. In 1940, it is estimated that 40% of 2.3.1.3 Canine leishmaniosis the dogs in Rome were infected with Leishmania.

Canine leishmaniosis is a severe zoonotic Etiology. More than 30 species are disease, affecting primarily dogs, but also currently recognized in genus humans and other mammals, transmitted Leishmania, all parasitic in mammals. The by hematophagous vectors (sandflies). genus is divided in two subgenera Historical notes. All early notes and the Leishmania and Viannia, based on the site discovery of Leishmania are related to of development in the sandfly host. human diseases. The first written Subgenus Leishmania develops in the documents on the Old World cutaneous anterior alimentary tract of sandflies leishmaniosis (oriental sore) are known while Viannia develops in the midgut and from the tablets from the library of King hindgut. Subgenus Leishmania is Ashurbanipal from the 7th century BC. distributed in the Old World (L. Avicenna and other Arab physicians also aethiopica, L. donovani, L. infantum, L. mention the disease as early as the 10th major, L. tropica) and the New World (L. century by the name of Balkh. The Old amazonensis, L. infantum, L. mexicana, L. World visceral leishmaniosis (kala azar pifanoi, L. venezuelensis). Subgenus was first mentioned in India in 1824. Viannia is restricted the New World However, the first observation of the (Central and South America) and includes parasite came in 1885 when a Russian several medically significant species: L. military surgeon, Borovsky saw unknown braziliensis, L. guyanensis, L. panamensis forms in the blood. A Scottish army and L. peruviana. All of the species listed physician, William Leishman and a above cause infections in humans, often physiology professor from India, Charles

60 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | with severe syndromes, life-long Life cycle. The life cycle is heteroxenous disabilities or death. without free living stages. As there is no direct evidence of sexual reproduction in The most important species responsible Leishmania, defining the intermediate for canine leishmaniosis is L. infantum. and definitive host is rather arbitrary. However, several other species of Hence, as most parasitology resources Leishmania were reported to infect dogs consider the sandfly vectors are the in various geographical regions: L. intermediate hosts and vertebrates are donovani, L. tropica, L. braziliensis, L. the definitive hosts, we will also follow peruviana, L. panamensis, L. amazonensis. this concept. Morphology. Leishmania has two developmental forms: the motile extracellular promastigotes (in the sandfly) and the amastigotes (in the vertebrate host).

The promastigotes (15 µm in length) are elongated and have a conspicuous free flagellum (figure 2.8). The amastigotes (figure 2.9) are usually found in vacuoles within the infected macrophages of the dogs. They are round or oval, lack a free flagellum and they measure 2.5-5 x 1.5-2 µm. They typically have one large basophilic nucleus.

Figure 2.9 Amastigotes of Leishmania in the macrophages of an infected dog. (Photo Andrei D. Mihalca)

In the Old World (Europe, Asia, Africa) the vectors are represented by sandflies in genus (figure 2.10) while in the New World (the Americas) by genus Lutzomyia. Sandflies are small (cca. 3 mm) blood sucking insects, with crepuscular and nocturnal activities. From more than 500 known species of sandflies (Phlebotominae), around 30 have proven vectorial capacity and other Figure 2.8 Promastigotes of Leishmania more than 40 are suspected as probable from laboratory culture. (Photo Andrei D. vectors. Other hematophagous Mihalca)

61 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | arthropods (ticks, fleas) have been When the infected female sandfly feeds suspected to act as competent vectors for again, the promastigotes are injected into Leishmania, but experimental results are the vertebrate host. Here, the scarce and hence inconclusive. promastigotes are phagocytized by the macrophages, they lose their flagellum

and become amastigotes.

Amastigotes multiply intracellularly by binary fission and increase in number until they rupture the macrophage. Subsequently, they invade other cells, and are disseminated systemically.

Epidemiology. Canine leishmaniosis is endemic to several areas of the world, usually in Mediterranean, subtropical and tropical climates. The disease in endemic in the countries bordering the Mediterranean Sea (Southern Europe and Northern Africa), Asia Minor, parts of Figure 2.10 Phlebotomus papatasi, a Central Asia and Western China, South vector for Leishmania infantum. (Photo America (mainly Brazil), Eastern North David Modrý) America and parts of Sub-Saharan Africa. However, sporadic cases of canine leishmaniosis are diagnosed also in non- In the sandflies, Leishmania develops in endemic countries, mainly because of the extracellular environment while in intensive travelling and importation of mammals, development is intracellular. dogs.

When uninfected female sandflies feed on It is estimated that millions of dogs are the blood of an infected host, they acquire infected in the endemic areas. Dogs are the amastigotes. The amastigotes are important reservoirs for the human released from the host macrophage in the infection, mainly in the Old World. The insect’s gut and they undergo a series of main diagnosis puzzle in these areas, are transformations and become flagellated. dogs with subclinical infections which are This initial stage in the sandfly is called a natural source of infection for sandflies procyclic promastigote. They start to (and indirectly to humans). They remain replicate and ultimately they detach from undiagnosed, unless serological surveys the intestinal surface and migrate to are carried out. foregut and mouthparts of the vector. There are several factors associated with They are known as metacyclic a higher risk of clinical disease. Age has promastigotes and they are infective for been shown to be among the most the vertebrate host. important. Young (2-4 years) and old

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(more than 7 years) dogs seem to be the skin and lymph nodes. Otherwise, the more commonly with symptomatic disease becomes systemic (lymph nodes, infections. The breed has also been bone marrow, spleen, liver, etc). incriminated as a risk factor. Certain dog The incubation period is between 3 breeds are more susceptible to disease months and 7 years. The main (German shepherd, Rottweiler, boxer, pathogenetic mechanism is related to the cocker spaniel) while others are resistant immune response. Generally, L. infantum because of co-evolution with the is responsible for the depletion of T pathogen (Ibizan hound). lymphocytes and a proliferation of B cell Even in the endemic areas, the higher risk regions in the lymphoid organs. This, of infection is present in rural areas. This together with the proliferation of other is caused by various factors, including cellular populations (plasma cells, more suitable sandfly habitats and histiocytes, macrophages) results in a availability of other reservoir hosts that significant and systemic dogs. Various domestic or wild animals lymphadenomegaly, splenomegaly and are known to harbor the infection, hyperglobulinaemia. However, the clinically or not: cats, horses, pigs, wild increased immunoglobulin response does canids, rodents, bats, seals etc. However, not offer protection. Adversely, they are only few of these are able to transmit the associated with supplementary infection to sandfly vectors feeding on detrimental effects like immune-mediate them. thrombocytopenia and glomerulonephritis. In addition, the large Although the main route of infection is amount of circulating immune complexes via the bite of the vector sandfly, other (CIC) is responsible for vasculitis, uveitis mechanisms have been suspected or and glomerulonephritis. Hence, usually incriminated: transplacental, venereal the cause of death in dogs with clinical and blood transfusion. leishmaniosis is renal failure. The Pathogenesis. Not all infected dogs immune-mediated vasculitis is develop clinical signs. The clinical responsible for tissue necrosis in the skin, outcome is dependent on various factors. internal organs and in the eye. Another Some of them are related to the vector interesting pathogenic mechanism (sandfly species, number and duration of involves the formation of cryoglobulins the infective bites), some others to the which precipitate when exposed to cold pathogen (strain-dependent) and others and result in ischemic necroses of to the host (genetic, age, breed, immune extremities exposed to low temperatures. status). The genetic basis for susceptibility or After the infectious bite by the sandfly, resistance to canine leishmaniosis has when metacyclic promastigotes invade been shown to be related to certain the dog’s body, they typically start to mutations and polymorphisms in the multiply in the macrophages. If the dog is Slc11c1 gene. This gene is responsible for resistant, it is able to limit the infection to encoding an iron transporter protein,

63 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | involved in the macrophage activation but non-protective IgG response against and development of parasites inside the Leishmania. Overall, there are strong phagolysosomes. proofs towards a cell-mediated anti- Leishmania immunity in the so called Immunology. Both components of the resistant dogs and a rather humoral immune system are involved in the immune response in dogs with clinical defense against Leishmania in dogs. signs. For instance, resistant dogs However, in dogs susceptible for clinical develop a strong delayed-type infections, Leishmania is able to evade hypersensitivity response (indicative of them, or use it in its own favor. cell-mediated immunity) when There are several mechanisms by which inoculated intradermally with Leishmania Leishmania escapes the innate, non- antigens. specific immunity. The most important is Clinical signs. Infected dogs may display the ability of amastigotes to survive and a great variety of clinical signs, from even replicate inside the macrophages. asymptomatic infection to severe clinical This is possible due to syndrome. When the infection is clinical, lipophosphoglycans produced by the the diversity of symptoms might be high parasite which inhibit the maturation of (table 2.6). the phagolysosomes.

The role of the specific (acquired) immune system in leishmaniosis has Table 2.6 Frequency of symptoms in dogs with clinical leishmaniosis* been intensively studied mostly on experimental models in laboratory Symptom Percentage rodents. How and if the results could be Lymphadenopathy 93.5 Onychogryphosis 75.0 extrapolated to dogs remains uncertain. Cutaneous lesions 58.7 The protective immunity in canine Weight loss 26.1 leishmaniosis is based on the T Cachexia 23.9 Locomotory 22.8 lymphocytes and macrophages. Activated abnormalities T cells produce IFN-γ, IL-2 and TNF-α Somnolence 21.7 Conjunctivitis 18.5 which induce the anti-leishmanial activity Anorexia 16.3 of macrophages. The macrophages Polydipsia 13.0 Polyphagia 13.0 produce nitric oxide which is ultimately Onychorrhexis 10.9 responsible for the intracellular killing of Epistaxis 8.7 Diarrhea 6.5 the amastigotes. Another line of specific Sickness 2.2 immune defense are CD8+ and CD4+ Cough 1.1 cytotoxic T cells which are responsible * - from Semiao-Santos (1995) for the destruction of macrophages infected with L. infantum. Incubation period is considered to be All these cellular processes are inhibited between 2-8 months, but sporadic in dogs which develop clinical signs. records extend this period to 15 months Alternatively, they develop an increased, or even several years. There are several

64 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | classifications of canine leishmaniosis. alopecic patches covered by abundant Some authors divide the disease in squamous debris (figure 2.11), phases: acute, subacute, chronic and seborrhea and ulcers (figure 2.12). The latent. In other texts, three types of onset of the cutaneous signs is usually leishmaniosis are recognized: around the orifices of the head and asymptomatic, oligosymptomatic and subsequently spread all over the body. polysymptomatic. However, the most The most severe skin lesions are at the useful classification is the one based on level of prominent bones. The apparent the chronology of the clinical signs (early similarity with human cutaneous symptoms, patent period and final period), leishmaniosis is restricted to the location accurately described based on of lesions. In humans the pathological experimental infection trials. changes of the skin are localized, while in dogs, the cutaneous phase is also Early symptoms consist of significant accompanied by visceral pathology. Loss weight loss, asthenia and apathy. Three of hair on the tail (“rat tail”) (figure 2.13) month after the infection, cutaneous or nasal hyperkeratosis (figure 2.14) signs may be visible (periorbital and have also been reported in infected dogs. auricular alopecia), accompanied by conjunctivitis, and renal pain on Ocular involvement is frequent in palpation. Hemorrhagic signs may also be infected dogs with mucous or present in this early stage. mucopurulent conjunctivitis, keratitis, corneal ulceration and subsequent Patent period may vary according to blindness. Other symptoms include: nasal dog’s immune status and pathogen strain discharge, uni- or bilateral epistaxis, involved, but also to other factors. progressive muscular atrophy, Combinations of non-specific symptoms perionyxis, onychogryphosis (figure (39-40C hyperthermia, apathy, asthenia, 2.15), onychorhexis, tremor, paralysis of loss of appetite, polydipsia, loss of the hind limbs, arthritis etc. Prior to weight) may appear. One of the most death, dogs show severe cachexia (figure present signs is lymphadenopathy, easily 2.16). detectable by superficial palpation of popliteal, prescapular and submaxillar Pathology. As explained in the previous lymph nodes. Ultrasound may reveal in section, the lesions characteristic for this phase marked hepatomegaly and leishmaniosis are caused primarily by the splenomegaly. altered immune response. These include mainly necrotic lesions in the skin, eye In general, the principal symptoms of and internal organs. clinically ill dogs include: skin lesions, enlarged lymph nodes, weight loss, The skin lesions are usually generalized, lethargy, ocular lesions, digestive signs and microscopically they involve the (vomiting, diarrhea) or lameness. decrease of collagen type I and the decrease of collagen type III fibers. Skin lesions are also common in infected dogs. They can appear as non-prurigenic,

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Figure 2.11 Exfoliative dermatitis in a Figure 2.13 “Rat tail” lesion in a dog dog infected with L. infantum. (Photo infected with L. infantum. (Photo George George Popa) Popa)

Figure 2.12 Cutaneous ulcers and Figure 2.14 Nasal hyperkeratosis in a erosions in a dog infected with L. dog infected with L. infantum. (Photo infantum. (Photo George Popa) George Popa)

The ocular lesions include: conjunctivitis, Other lesions associated with the altered blepharitis and anterior uveitis. In some immune response and CIC include cases, because of the retention of lacrimal mononuclear myositis, neutrophilic secretion due to adjacent inflammation, vasculitis, hemorrhages in internal dogs exhibit keratoconjunctivitis sicca. organs, granulomatous rhinitis, epistaxis and anemia.

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dermatitis. The histopathologic lesions in the skin are pyogranulomatous with hyperkeratosis.

Gross lesions include: generalized lymphadenopathy, splenomegaly and hepatomegaly, granulomatous nodules in various internal organs.

Diagnosis. Clinical signs are not enough for a positive diagnosis of canine leishmaniosis. Basically, for a certain diagnosis, the presence of the parasite must be demonstrated directly (PCR, cytology, histology, culture) or indirectly Figure 2.15 Onychogryphosis in a dog (antibody detection). infected with L. infantum. (Photo George Popa) Direct evidence of the amastigotes should based on microscopic observation of the

parasitic stages in biopsy from lymph nodes, spleen, bone marrow or skin. Although this method is 100% specific, its sensitivity is maximum 80%, depending on the experience of the examiner and examination effort. However, direct identification of Leishmania amastigotes is not always achievable, even in dogs with clinical infection. Amastigotes can be observed also in tissue sections by histology or immunohistochemistry. Although not commonly used as routine diagnostic methods, cultivation (Novy- MacNeal-Nicolle medium) and Figure 2.16 Severe cachexia in a dog experimental infection (hamsters) are infected with L. infantum. (Photo George also possible. Popa) Probably the most sensitive methods for the detection of Leishmania infection are molecular biology techniques. PCR has Usually the cutaneous lesions are been used routinely for the detection of generalized. Several types are mentioned Leishmania DNA in various tissues (bone in the literature: exfoliative dermatitis marrow, lymph nodes, spleen, skin), with alopecia, ulcerative dermatitis, blood (whole blood or buffy coat) or body nodular dermatitis, mucocutaneous fluids (urine). proliferative dermatitis, or popular

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The easiest diagnosis methods however nucleoside in the structure of tRNA). This refer to the detection of anti-Leishmania analogue is incorporated into the antibodies in the serum of dogs. Various structure of the Leishmania RNA, causing methods have been developed, some of an altered translation and inhibiting the using whole parasite extracts (more parasite multiplication. Unlike the sensitive, less specific), some others previous drug, allopurinol is given orally recombinant protein antigens (more (10 mg/kg body weight), twice a day, for specific, less sensitive). Whole parasite month or even years. Despite the fact that extract can cross-react with other the drug has little adverse effects, the kinetoplastids. Usually, it is considered discontinuation of treatment often results that high antibody titers in dogs with in clinical relapses. Hence, dogs may compatible symptoms are indication of require life-long treatment. One of the clinical leishmaniosis. If clinical signs are most common side effects is present but antibody titers are low, hyperxanthinuria, resulting in additional methods are recommended urolithiasis. However, the most effective (cytology, histology, PCR). therapeutic protocol is the combination of meglumine antimoniate with Treatment. Treatment of leishmaniosis allopurinol. does not necessarily eliminate the parasite form the organism and this is Several other drugs have been used for one of the causes of the frequent clinical the treatment of canine leishmaniosis: relapses. There are several drugs used for miltefosine (2 mg/kg body weight, orally, the treatment of canine leishmaniosis. once per day, for four weeks), The most commonly used are pentavalent amphotericin B (causes renal toxicity). antimonials. Their mechanism of action is Except specific treatment, a symptomatic by inhibiting the enzymes responsible for therapy must be conducted. oxidation of fatty acids and glycolysis. The most commonly used antimonial is Control. None of the several control meglumine antimoniate which is given by measures used is fully effective. The subcutaneous injection, for 4-8 weeks, euthanasia of infected dogs is the most daily, at 75-100 mg/kg body weight. controversial and its efficacy is doubtful Besides the side effects (local reactions, because of the persistence of reservoirs nephrotoxicity), there are several reports in wildlife. Environmental control of of resistant Leishmania strains in Europe. sandflies (spraying, destruction of breeding habitats) has been also shown Another compound used in the treatment to be little effective. Moreover, there is no of canine leishmaniosis is allopurinol. prophylactic drug available. However, This drug was originally developed for owners can reduce the sandfly bites on the treatment of gout (hyperuricemia) in their dogs by avoiding outdoor access human patients. Its efficacy against during maximum activity of the vectors Leishmania is explained by its capacity of (overnight, warm season) or by using being metabolized by the parasite into an prophylactic insecticides (spot-on, analogue of inosine (a common collars, sprays).

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Currently, there is a commercial vaccine produce also cutaneous signs, mostly for dogs, but its availability is variable located on the ear pinna. from country to country. This commercial Treatment. Despite the lack of extensive vaccine contains a glycoprotein from L. clinical studies, the few reported treated donovani (GP63). cases suggest that the same protocols which is used in dog can be successfully applied also in cats. 2.3.1.4 Feline leishmaniosis

Unlike canine leishmanioses, the disease 2.3.2 Trichomonads in cats is reported only sporadically. As most aspects have been already Introduction. The trichomonads group presented in the chapter on canine several genera of medical and veterinary leishmaniosis, only specific aspects will importance (table 2.7). be given here. The morphological differences between Historical notes. The first case of feline species (and even genera) are not very leishmaniosis was described in Algeria in easy to discern, mainly if using regular 1912, in a kitten housed together with an direct examination. They are unicellular infected dog and an infected child. organisms, lacking mitochondria with a Etiology. Several species have been typical anaerobic metabolism. They reported from cats: L. infantum (Europe, include mostly symbiotic species, but also South America), L. mexicana (USA), L. few free-living organisms. venezuelensis, L. braziliensis and L. General morphology. Although there amazonensis (South America). are slight morphological differences Epidemiology. The disease in cats is between different genera and species, sporadic, and all cases were reported generally, all trichomonads have a group from areas endemic to canine of three (Tritrichomonas), four leishmaniosis: Mediterranean Europe, (Tetratrichomonas, Trichomonas) or five North Africa, Middle East, and the (Pentatrichomonas) anterior free flagella, Americas. an additional recurrent flagellum which delimitates an undulating membrane. The Clinical signs. In immunologically axostyle, a rigid rod-like structure runs competent cats, the predominant medially through the cell. The axostyle is symptoms of L. infantum infection are usually longer than the cell. Due to these cutaneous (ulcers, nodular dermatitis, structures, trichomonads show a very scaling, alopecia) with the lesions located characteristic movement, making them mainly on the head. The visceral easily recognizable in fresh preparations involvement seems to be limited to cats coinfected with FIV (Feline Ecology and transmission. The vast Immunodeficiency Virus) or FeLV (Feline majority of the Trichomonads are Leukemia Virus). American species parasitic, mutualistic or commensals.

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Table 2.7 Most important species of trichomonads

Genus Species Hosts Location T. gallinae birds anterior digestive system Trichomonas T. vaginalis humans genital cattle genital T. foetus cats large intestine T. suis (=T. pigs digestive, nasal foetus) T. enteritis cattle colon Tritrichomonas T. caviae cavies cecum T. muris rodents large intestine T. minut rodents large intestine T. wenyoni rodents large intestine T. eberthi birds cecum T. canistomae dogs mouth T. felistomae cats mouth T. ovis sheep cecum, rumen T. buttreyi ungulates large intestine Tetratrichomonas T. pavlovi cattle cecum T. microti rodents large intestine T. gallinarum birds, humans cecum T. anatis ducks intestine T. anseris geese cecum Pentatrichomonas P. hominis primates, dogs, cats, rodents large intestine Trichomitus T. rotunda pigs large intestine Chilomastix C. gallinarum birds cecum

They inhabit the digestive or responsible for digestive symptoms in reproductive tracts of vertebrates or animals, usually young ones. One single invertebrates. Few species are free-living. species (Tritrichomonas foetus in cattle) The life-cycle is always homoxenous and is producing genital infection. In humans, generally, they are considered to be host- several species are known, some of them specific. producing digestive signs, some others genital symptoms. Feeding occurs by phagocytosis of fluids, leukocytes or bacteria from the in-host habitats. Reproduction is by longitudinal binary fission, with the formation of large 2.3.2.1 Genital trichomonosis in numbers of trophozoites. cattle

As there are no cystic stages known, Introduction. Genital trichomonosis is a transmission between hosts is always by venereal disease of cattle, with long-term direct contact. impact on fertility in females and Medical importance. Certain asymptomatic carrier state in bulls, with trichomonads are important human and worldwide distribution, caused by veterinary pathogens. Most of the species Tritrichomonas foetus. The disease is are located in the digestive tract (mouth, mainly important from its economic gut), others in the genital system. Most perspective. veterinary important species are

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Historical notes. The disease was infected animals. Transmission by reported and described for the first time artificial insemination has also been in 1888 by Kunstler in France and later, reported. in 1890, by Mazzanti in Italy as isolated cases of infertility. Later reports from 1924-1929 are from Germany, where enzootic outbreaks were described. The first cases of bovine genital trichomonosis in the United States were found in 1932.

Etiology. The causative agent, Tritrichomonas foetus has been subject to several molecular taxonomy approaches, and its synonymy with T. suis has long been debated. Most recent taxonomical papers list them as synonyms.

Three serotypes have been found using seroagglutination. The “belfast” strain (common in Europe, Africa and USA), the “brisbane” strain (in Australia) and the “manley” strain (the most rare one).

Morphology. Tritrichomonas foetus Figure 2.17 Tritrichomonas foetus. (figure 2.17) is a spindle to pear shaped unicellular organism, 10-25 x 3-15 µm, Passive transmission has also been with four flagella. Three anterior flagella reported. This means that, if a non- are free and the fourth, called the infected bull had recent coitus with an recurrent flagellum, runs backward and infected female, he is able to transmit delimitates an undulating membrane, infect another cow in a subsequent with 3-5 waves. The axostyle is thick and intercourse. it protrudes in the posterior part of the cell. The gynecological examination of cows can also induce passive transmission, if Life cycle. The typical habitat for T. instruments are not sterilized after being foetus is at the level of genital mucosae of used before in an infected cow. cattle, in both sexes. In bulls, they are found in the preputial cavity, penis Epidemiology. The disease is distributed mucosa, distal urethra, epididymis, testes worldwide. Bovines are the only and seminal vesicles. In cows, they infect important infection reservoirs, with the vaginal mucosa, the uterus and the asymptomatic bulls being responsible for fetus. Transmission between hosts is by infection of cows mainly by natural sexual contact between infected and non mating. Nonetheless, experimental

71 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infection has been established in a great more prone to develop chronic infection variety of mammalian hosts (rabbits, and to become long-time, asymptomatic laboratory rodents, dogs, small carriers. This is associated with a ruminants, pigs), but their morphologic trait of epithelial crypts of epidemiological importance is practically the penis and prepuce, which become zero. Since the taxonomic debate on the deeper as bulls are aging. Deeper crypts identity of T. foetus with T. suis, it is still offer a better microaerophilic not certain if cross infection could occur, environment. Conversely, young bulls are and how. Moreover, the identification of only transient carriers. T. foetus from cats with diarrhea make In cows, immediately after the infection, this puzzle even more complicated. the parasite produces a mild infection of Both breed and age susceptibility has the vaginal mucosa, with vaginitis. During been reported in bulls. Bulls of some estrus, the flagellates are able to enter the breeds (Bragus, Simmental, Charolais, uterus through the cervix. Within 7-14 and Angus) seem to be more predisposed days, T. foetus is able to colonize the to infection than others (Braford). The entire female reproductive tract. The origin of the breed is likely to be the endometritis is responsible for the cause of different susceptibility. It has persistence of the corpus luteum which been estimated that Bos taurus bulls are induces pyometra. more prone to infection than B. indicus. The mechanism of fetal death is not fully The farm management system is also understood, but cytotoxic and hemolytic important. The prevalence in bulls tends effects have been incriminated. The to be higher in larger farms. Similarly, a adhesion of T. foetus to mammalian cells higher bull-cow ratio is directly is facilitated by a surface adhesin. correlated with an increased prevalence of the disease. After the experimental infection in heifers, the clearance of the parasite The resistance of T. foetus outside its host without medication is between 3 and 28 is very limited. Hence, contamination of months. new hosts is only by direct contact. However, the organism can remain Immunology. There is no evidence infective in frozen semen. showing that after infection, cows will develop long-term or life-long immunity. Pathogenesis. As most venereal diseases However, a maximum 15 month of domestic animals, bovine convalescent immunity has been trichomonosis is asymptomatic in males reported. Tritrichomonas foetus produces and symptomatic in females. extracellular proteases which are able to In bulls, the parasites live on the mucosal digest immunoglobulins, impairing even surface and do no invade the epithelial the local immunity. tissue. One interesting observation The antigenic structure of T. foetus following an experimental infection trial, includes 55-60 proteins was that bulls older than 3 years are

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The surface antigens of T. foetus are able caused by bovine trichomonosis are to activate the alternative pathway of significant, with losses estimated to 35% bovine complement. The bovine of the profit in infected farms. complement system is able to kill T. Pathology. Following abortion, the foetus in the presence of specific lesions are detectable in the placenta and antibodies. On the other hand, in the fetus. In late term abortions, the neutrophils are not efficient in killing T. placenta shows focal or diffuse invasion foetus, even in the presence of specific of the chorionic stroma by the parasitic antibodies. flagellates. The typical morphology of T. Studies have shown that there is no foetus is easily visible in histological cross-immunity between the three sections stained with Bodian’s silver serotypes of T. foetus. technique but is imperceptible at routine staining methods. Additionally, Clinical signs. The infection of bulls is monocytic infiltrate is present in the usually asymptomatic. placental layers. The aborted fetuses have In females, a mild vaginitis is the first sign pyogranulomatous bronchopneumonia, of infection. If the infective mating results interstitial pneumonia, hepatic and in gestation, the usual outcome is intestinal necrosis with the presence of abortion between days 50 and 70. About the parasites in the air ducts but also in one third of the abortions caused by the esophagus, abomasum and intestine. trichomonosis occur in the first trimester. Diagnosis. The clinical signs and features The death of the embryo or fetus during of the disease are neither characteristic this early stage is usually followed by a nor pathognomonic to trichomonosis. longer interestrous interval. As the Similar reproductive problems are abortion is very early, often it goes caused by bacterial agents (e.g. unobserved by farm owners. Usually, all Campylobacter foetus, Leptospira spp., fetal membranes are passed after Ureaplasma diversum) or by nutritional abortion and the cows recover quickly. conditions. Hence, the certain diagnosis Nevertheless, fetal membrane retention must be based on the identification of the leads to chronic endometritis and parasitic agent by various laboratory possibly permanent sterility. Most cows methods. are however able to bare a normal gestation and deliver normal calves. As a The following section is based on the result of abortion about 5% of the cows recommendations by OIE (The World develop pyometra. Detection of pyometra Organization for Animal Health). The is usually late, and by that time the samples which are used for the detection uterine mucosa is severely damaged. of T. foetus are: vaginal mucus, vaginal washing or scrapings, preputial washing Persistent infection with T. foetus in cows or scraping, uterine washing, pyometra results in temporary or permanent discharge, placental fluid, stomach infertility, irregular estrus, persistent content of the aborted fetus. abortions etc. The economical losses

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A correct technique and timing for the T. foetus. A PCR-based assay which is collection of samples are essential. It is used has certain advantages: increased important mainly to avoid fecal sensitivity, possibility to detect non- contamination, or if so, the examiner viable pathogens. Additionally, should be very experienced in order to immunohistochemistry on tissues differentiate intestinal flagellates from T. (placenta, fetal lungs) has been also used. foetus. Contamination can be avoided by Treatment. For the treatment of bulls, mechanically removing the dirty hair various drugs have been used in the past: around the preputial orifice or around dimetridazole, ipronidazole and the vulva. Chemical disinfectants are nor metronidazole. As all are nitroimidazoles, recommended, as may inactivate T. foetus their use in livestock is banned in most and reduce diagnosis sensitivity. countries. Hence, currently there are no Collection of preputial samples from bulls therapeutic options for treating bovine can be done using an artificial trichomonosis. Treatment of bulls can be insemination pipette, a brush, by performed only under certain conditions, preputial washing or by washing the mainly in the case of expensive, valuable artificial vagina after seminal material breeding animals. collection. Samples from cows are collected by vaginal washing or by Control. Prevention and control by herd scraping the cervix with a brush or management are the only reliable pipette. methods for reducing the economic impact of bovine genital trichomonosis. Samples must be examined as fast as General measures include: control of the possible. If they cannot be sent to the animal movement, avoid grazing on laboratory in maximum 24 hours, they common pastures where bulls from other should be included in a transport herds may have access, purchase only medium (thioglycollate broth media with virgin bulls and heifers for restocking, antibiotics) and kept at temperatures purchase the animals from T. foetus-free between 5 and 38°C, away from direct farms, ask for the preputial washing sunlight. result for any bull purchased, graze Samples can be examined immediately separately cows and bulls, keep the under the light microscope, or if the average age of bulls as young as possible sample is poor in trichomonads, after (maximum 3 years), use of artificial enrichment on cultivation media insemination with tested semen. (Diamond’s trichomonad medium or Commercial vaccines for cows are other commercial media). Tritrichomonas available in certain countries. They are foetus should be motile, with typical killed vaccines and initial vaccination movements and morphology (pear-shape, must be given twice, subcutaneously, at presence of free flagella and undulating 2-4 weeks apart. In the following years, membrane, presence of axostyle). all cows must be revaccinated, 4 weeks More recently, new, molecular techniques prior to the beginning of the breeding have been developed for the detection of season. Although the vaccine does not

74 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | prevent the infection, it significantly axostyle extends far beyond the posterior decreases the economic impact of the end of the cell. The size is 7-12 x 3-4 µm. disease by reducing the rate of abortions and the duration and severity of the disease.

2.3.2.2 Buccal trichomonosis in dogs and cats

Introduction. Buccal trichomonosis in carnivores are poorly known conditions, caused by flagellates in genus Tetratrichomonas and characterized by gingivitis usually in immunodeficient patients.

Historical notes. Both conditions (in cats and dogs) were described by Hegner and Ratcliffe in 1927 in United States.

Etiology. The species responsible for buccal trichomonosis in cats is Figure 2.18 Tetratrichomonas Tetratrichomonas felistomae and in dogs canistomae. it is T. canistomae. As the original description is very simple, and records of these parasites are scarce, the taxonomic Trophozoites of T. felistomae (figure status of T. felistomae and T. canistomae 2.19) have a similar morphology. Their is uncertain. Moreover, recent studies size is 6-11 x 3-4 µm. They have a well revealed the presence of flagellates with a visible axostyle and the undulating different morphology, puzzling even membrane has 3 waves. more the situation. Life cycle. Not too many aspects are Morphology. Species in genus known about the biology of these Tetratrichomonas have 4 free flagella and parasites. They are located in the mouth one more trailing free flagellum, running of dogs and cats, along the gums or backwards. Trophozoites of T. canistomae associated with dental calculus. (figure 2.18) have four anterior flagella, Transmission between hosts is probably that rise in pairs from a large by direct contact. blepharoplast at the anterior end of the Epidemiology. In cats, the parasite have body. The recurrent flagellum starts from been reported only in USA, Italy and the blepharoplast, runs backwards along Germany. In dogs, the parasite is known the edge of the undulating membrane and from United States and various European ends freely at the posterior end. The

75 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | countries. No data about resistance in the Pathology. Gingivitis in cats, dental environment are available. calculus in dogs.

Diagnosis. Fresh wet mounts from dogs and cat with buccal lesions might reveal the presence of flagellates. In dogs, scraping the dental calculi from the lower jaw and decayed teeth yielded better results.

Treatment. Unknown.

Control. Unknown, but a proper oral hygiene with removal of dental calculi and treatment of buccal inflammations might be useful.

2.3.2.3 Anterior digestive trichomonosis in birds

Introduction. It is a worldwide Figure 2.19 Tetratrichomonas felistomae. distributed parasitic disease of pigeons, doves, galliformes and birds of prey, with

possible severe buccal lesions mainly in Pathogenesis. In a recent study, the young birds. The disease in pigeons is trichomonads in cats were found only in also known as canker. In poultry, the individuals infected with name of the disease is “roup” and in birds immunosuppressive viruses: feline of prey “frounce”. immunodeficiency virus, feline leukemia Historical notes. The first description of virus and feline infectious peritonitis the disease was in pigeons, together the virus. etiological agent, by Rivolta in 1878 in Immunology. Unknown Italy.

Clinical signs. The presence of the Etiology. Only one species is involved, parasites in cats was associated with namely Trichomonas gallinae. This gingivitis, while in dogs with dental species must not be confused with calculus. Tetratrichomonas felistomae was Tetratrichomonas gallinarum, which never isolated from cats without lesions, infects the large intestines of birds. while it seems that in dogs, T. canistomae Different strains are know, some of them can be found also in healthy patients. more pathogenic than the others and Hence, some authors consider it to be some considered avirulent, part of the mostly non-pathogenic. normal buccal fauna. One of the most virulent strains is Jones’ Barn, which is

76 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | able to kill even adult pigeons at 8 days after the infection.

Morphology. Trophozoites (figure 2.20) are piriform to rounded, 6-19 x 2-9 µm, Four free flagella extend forward, and the fifth runs backward along the margin of the undulating membrane. Unlike in genus Tetratrichomonas, there is no free trailing flagellum.

Life cycle. The parasites inhabit the mucosal surface of the anterior digestive system (mouth, pharynx, esophagus, crop) and head sinuses in pigeons and doves. Although it seems that only Columbiformes are the natural host for T. gallinae, the infection was reported in many other groups of domestic or wild birds. Among domestic species, chicken and turkeys are susceptible to infections. Figure 2.20 Trichomonas gallinae. The infection was reported in captive finches and canaries as well as in quails. Among wild avian species, the most Epidemiology. The parasite is important clinical disease was described distributed globally, with virtually all in raptors. Moreover, many experimental domestic and wild pigeon populations trials succeeded transmitting T. gallinae positive for the infection. In domestic to many other host species, showing the fowl (chicken, turkey) the infection is parasites lacks specificity. rather sporadic. In birds of prey, the infection has been reported with variable Mammals are not susceptible to natural prevalence in several countries where infection. They multiply rapidly by binary studies were performed. Nevertheless, fission. Only a single stage is know, the the source if infection for the other birds trophozoite. are always pigeons or doves.

Transmission no new host is realized by Young pigeons are particularly direct contact between infected and non- susceptible for the diseases, while adults infected birds. In pigeons, the adult birds are usually asymptomatic carriers. are infecting their nestlings during the Certain breeds (i.e. tumbler) were shown regurgitation and feeding. Birds of prey to be more sensitive to the infection. The get the infection when feeding on other severity and clinical outcome of canker is infected birds. Other birds get the also influenced by certain environmental infection by drinking contaminated factors: poor ventilation, overcrowding, water. unbalanced diet, excessive humidity.

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Interestingly, the prevalence of infection extremely virulent strains can produce is usually higher in adults than in young. mortality rates up to 50%. The outbreaks and the prevalence are In the acute cases, the duration of the positively correlated with humid seasons. disease is 10-14 days. The typical clinical Stress is also a factor contributing to picture includes: partial or total loss of clinical outbreaks. appetite, weight loss, ruffled aspect and Although the resistance of T. gallinae in listlessness. Some pigeons which still the environment is very limited, it present appetite have difficulties in survives enough time in drinking water swallowing. Other birds may also show to facilitate transmission in birds which signs of respiratory distress. When do not feed their newly hatched offspring, examining the mouth, small, whitish or like chicken or turkey. yellowish adherent masses are seen on the surface of the buccal and pharyngeal Pathogenesis. The chronic infection is mucosa (figure 2.21). Sometimes, these usually accompanied by a low number of caseous nodules join and become large parasites which are not able to produce enough to completely obstruct the the disease. When certain factors are met pharynx (figure 2.22). These small (see above), the parasite multiplies very nodules disappear spontaneously within fast and invades the surface of the upper few days and the bird becomes healthy. digestive system, causing severe In other cases, the infection results in inflammation followed often by death in death. Interestingly, the severity of the acute form. In severe infection, T. nodular lesions is not correlated with the gallinae colonizes also the sinuses and severity of clinical form. the liver. The most virulent strains produce the Immunology. Despite the lack of first oral lesions after 1 week, as targeted studies on the immune response yellowish areas on the mucosa of mouth of birds to the infection with T. gallinae, it and the tissue bordering the nasal cleft or seems that if the original exposure is not palatal flaps. When nodules become big followed by death, sufficient immunity is enough to obstruct the food passage, the developed to protect the adults against clinical course becomes rapid, with clinical disease. Non-exposed adults extreme and fast weight loss and fluid introduced to infected colonies develop a accumulation in the crop which virtually severe form of trichomonosis and may can drown the bird. Death occurs in even die. It is not fully understood if there about 10-12 days from the first clinical is cross acquired immunity against the sign (around 20 days from infection). different strains of the parasite. Sometimes also diarrhea is present. Clinical signs. Infection may vary from The infection with less virulent strains asymptomatic cases to severe outbreaks can be asymptomatic or can cause with over 90% mortality, mainly in young maximum excessive salivation and mild birds. In adults mortality is rare, but stomatitis.

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whitish fluid. The crop may be covered with a diphteric membrane which lines the digestive mucosa down to the glandular stomach.

Histology shows congestion with mononuclear inflammatory infiltration in the lamina propria of the larynx and pharynx, surrounded by area of necrosis.

The liver may show signs of congestion or areas of yellow necrosis, with peritoneal adhesions. Microscopically these correspond to hyperemia, hepatocellular necrosis, hyperplasia of the bile duct, all Figure 2.21 Small, disseminated caseous with heterophils and mononuclear nodule on the surface of buccal mucosa in infiltration and presence of a pigeon naturally infected with T. multinucleated giant cells. gallinae. (Photo Andrei D. Mihalca) The heart may also be affected, with

caseous material deposited on the apex. In birds of prey and gallinaceous birds, Similar lesions are occasionally reported buccal trichomonosis is a severe in all organs found in direct contact with condition and the clinical expression is the liver: small intestine, spleen, somehow similar with the one described pancreas, air sacs, lungs, kidneys. in pigeons. Additionally, these hosts show moderate or severe dyspnea along with nasal and oral exudation. In exotic birds (i.e. budgerigars, finches) clinical sings include also wasting, vomiting and diarrhea.

Pathology. The lesions are usually limited to the anterior digestive system, liver and sometimes to the respiratory system. The less virulent strains usually produce only buccal and pharyngeal lesions while more virulent strains are able to cause damage to various internal organs. Yellowish to grayish necrotic lesions are found on the mucosa lining the mouth, pharynx, esophagus, crop and Figure 2.22 Large caseous nodule in the proventriculus. Sometimes, the crop and palatal flaps area in a pigeon naturally the proventriculus contain a greenish or infected with T. gallinae. (Photo Andrei D. Mihalca)

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Microscopic lesions in the respiratory organs of pigeons infected with virulent strains are predominantly congestive and inflammatory. Severe congestion might be present in the lungs, with extensive infiltration of heterophils and mononuclear cells in the mucosa of the trachea and hyperplasia of tracheal mucous cells. If kidneys are involved, the microscopic lesions consists of mild tubular necrosis with mononuclear infiltrate.

Diagnosis. The diagnosis might be done in healthy birds to estimate the Figure 2.23 Collection of the pharyngeal asymptomatic carrier status. This swab for the diagnosis of buccal approach is essential in pigeon housings, trichomonosis in pigeon. (Photo Andrei D. to detect the infection in adults, before Mihalca) the reproduction season. The etiological diagnosis is of course important also in clinically ill birds. Treatment. Several drugs are available for the treatment of trichomonosis in The most reliable diagnosis is by pigeons: metronidazole (60 mg/kg body demonstrating the presence of weight); dimetridazole (50 mg/kg body trichomonads by microscopic weight); ronidazole (30 mg/pigeon), all examination. Wet smears should be orally. As resistance to these drugs was prepared using cotton-tipped swabs reported in various strains of T. gallinae, soaked in warm sterile physiological commercial products containing saline (figure 2.23). The content must be combination of these (i.e. metronidazole immediately examined under the + ronidazole) have been developed. All microscope, using medium or large these products are soluble, and are given magnification in order to observe the in drinking water for 5-6 consecutive typical morphology. When the intensity days. The recommended concentration is of the infection is low, the small number 0.05%. of parasites might go undetected. In these cases, an initial enrichment by cultivation Control. General measures like a good is helpful. hygiene, avoiding overcrowding and stress should reduce the impact of the A variety of cultivation can be used, but disease. Treatment of the adult pigeons the most recommended is Diamond’s before the breeding season will certainly medium. For more sensitive diagnosis, reduce the parasite load and mortality in various PCR assays have been developed, squabs to come. but their use as routine examinations might be costly.

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2.3.2.4 Intestinal trichomonosis in gallinarum have been reported as causing birds caecal infection in chicken and turkeys.

Morphology. All species have the typical Introduction. Is a worldwide distributed morphology of trichomonads, but small parasitic disease of various birds species, differences in size, shape, number of with digestive clinical signs mainly in flagella or internal structure allows more young birds. specific identification. Historical notes. Tetratrichomonas Tetratrichomonas gallinarum (figure gallinarum was described in 1911 from 2.24) has a piriform body, 7-15 x 3-9 µm. chickens. Like all member of its genus, it has four Etiology. Several species have to be free anterior flagella, and a recurrent considered as etiologic agents of flagellum which runs along the edge of intestinal trichomonosis in domestic the undulating membrane and ending birds. However, their taxonomic status with a free part. The axostyle is long, (or even validity) and pathogenic slender and pointed. potential have been questioned by recent Tetratrichomonas anatis has an elongated molecular biology works. body, 13-27 x 8-18 µm, with four anterior Although Tetratrichomonas gallinarum is flagella, a long undulating membrane a still listed by most veterinary the recurrent flagellum with a free parasitology textbooks, its pathogenic termination. Tetratrichomonas anseris potential is uncertain, and recent has the morphology is similar to T. anatis genetical insights have shown that this but it is slightly smaller. species might be actually a complex of Tritrichomonas eberthi (figure 2.25) has cryptic species with more implications in an elongated body, 8-14 x 4-7 µm, three human health than in birds. Until more free anterior flagella and a well- clarification is available, we will follow developed undulating membrane, lined the classical concept and consider all by the recurrent flagellum which ends these species valid. Tetratrichomonas freely at the posterior end. The axostyle gallinae was reported from various is relatively thick and extends the domestic and wild species of birds posterior end of the body. (chicken, turkey, guinea fowl, quail, pheasant, partridge, ducks etc.) and has Chilomastix gallinarum (figure 2.26) is been recently incriminated in the etiology piriform, 11-20 x 5-12 µm, three anterior of severe lung disorders in humans. flagella, and an additional spiraling flagellum. The undulating membrane is Tetratrichomonas anatis is the cause of relatively narrow. In the case of this small and large intestine trichomonosis species, also cysts are known to be in ducks. Tetratrichomonas anseris is formed (7-9 x 4-6 µm). responsible for the disease in geese. Tritrichomonas eberthi and Chilomastix

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Figure 2.24 Tetratrichomonas gallinarum. Figure 2.25 Tritrichomonas eberthi.

Life cycle. All the species inhabit the mucosa of the large intestine or small intestine where they multiply by binary fission. Transmission from a host to another is via fecal-oral route, by fresh feces. In the case of C. gallinarum, transmission can be done through cysts.

Epidemiology. All the species have global distribution. The resistance in the environment is limited. Usually, young birds seem to be more affected by the clinical diseases.

Pathogenesis. There is serious debate on the pathogenicity of certain species. Generally, they are considered to be non- pathogenic. However, most of them have been isolated from birds with clinical signs or lesions of enteritis. Figure 2.26 Chilomastix gallinarum.

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It is a question if they are the primary should be rather identified and agents, or they just over multiply on eliminated. lesions primarily induced by bacteria or Control. Hygiene measures are essential, viruses. as contamination is usually via infected Immunology. Unknown. water.

Clinical signs. With the same mentions as stated above, clinical signs associated with the presence of intestinal 2.3.2.5 Intestinal trichomonosis in trichomonads vary from asymptomatic mammals infections (in the vast majority of cases) to mild, moderate or severe diarrhea, or Introduction. Intestinal trichomonads of even sudden death (in ducks). domestic mammals include a great variety of species, usually non- Pathology. The lesions from birds pathogenic. If disease occurs, it is a result infected with intestinal trichomonads are of multiple pathogen infections. usually fibrinous or necrotic enteritis, mainly in the cecum (typhlitis). Etiology. For a list of species and hosts Sometimes, necrotic lesions on the liver please refer to Table 2.7. have been described. In ducks, Morphology. Tritrichomonas enteritis extraintestinal lesions were also has a small body, 6-12 x 5-6 µm, with described: mucopurulent sinusitis, three free anterior flagella, and a catarrhal rhinitis and tracheitis. medium-sized undulating membrane.

Diagnosis. In vivo, the flagellated Tetratrichomonas buttreyi is ovoid or protozoans can be identified under the ellipsoidal, 4-7 x 2-5 µm, with four free microscope in wet mounts from cloacal anterior flagella, an undulating swabs. Their presence must be membrane with 3-5 waves and a interpreted with caution, as usually the recurrent flagellum. The axostyle is underlying cause of the symptoms are rather narrow. more likely to be bacteria, viruses or even Tetratrichomonas ovis has a piriform other protozoans (i.e. Histomonas body, 6-9 x 4-8 µm, with typical number meleagridis). In dead animals, if they are of flagella for the genus. fresh, a wet mount from the cecal lesions may reveal the presence of mobile Tetratrichomonas pavlovi has a pear- organisms with the typical trichomonads shaped body, 11-12 x 6-7 µm, an morphology. Otherwise, cytology or undulating membrane with 2-4 waves histopathology are other options. and otherwise a similar morphology with T. buttreyi. Treatment. Usually the infections should not be treated, as nitroimidazoles are Pentatrichomonas hominis is piriform, 5- forbidden in animals intended for human 14 x 7-10 µm. There are five free flagella, consumption. The underlying conditions four of which are grouped together and oriented forward, and the fifth is separate

83 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | and somehow recurrent. The sixth was a major decline of the disease due to flagellum, the true-recurrent one, runs the introduction of efficient drugs. First along the undulating membrane ending report in chicken came in 1900 in USA. with a free portion. Etiology. The causative agent of black- Trichomitus rotunda has wide piriform or head disease in turkeys and chicken is ovoid shape, 7-11 x 5-7 µm Histomonas meleagridis. The same species has been reported occasionally Life cycle. Direct. No cysts stages are from a great variety of birds, including known. ostriches and birds captive in zoos. Pathogenesis. Clinical signs. Pathology. Another species (Parahistomonas The pathogenic role of intestinal wenrichi) which is non-pathogenic was trichomonads from mammals has not described more recently from turkeys been clearly defined. All the species and other birds. mentioned above have been isolated from Morphology. Histomonas meleagridis clinical cases with diarrhea and/or (figure 2.27) has several developmental enteritis. forms (pleomorphism). However, all of Diagnosis. Treatment. Control. See them are trophic stages; no cyst is known. comments from Chapter 2.3.2.4. When they inhabit the lumen of the cecum or when they are cultured, the shape is irregular (or amoeboid), 5-30 2.3.2.6 Histomonosis of poultry µm in diameter and with a single flagellum. Sometimes, during cell Introduction. Known also as the black- division, two flagella can be observed. head disease, histomonosis is a major Even during this stage they are able of disease causing extensive economic moving by pseudopodia to invade tissues. losses mainly in turkey farms with a very The tissue stages lack flagellum and unusual way of transmission. resemble amoebae. Like trichomonads, they lack mitochondria. Historical notes. The disease was described for the first time in Rhode Life cycle. The life cycle is considered Island, USA in 1893, in turkeys. Two heteroxenous, with poultry as definitive years later, Smith described the agent. hosts and nematodes from genus Immediately after its discovery, Heterakis as intermediate hosts. Heterakis Histomonas decimated turkey gallinae are nematodes parasitizing the populations of USA from 11 million birds ceca of domestic poultry. When they feed, in 1890 to 3.7 million in 1920, accounting they accidentally ingest the trophozoites for one third of all the mortality cases in of Histomonas meleagridis. In the this species. The mechanism of nematodes gut, they multiply and transmission was fully understood only subsequently they invade the in 1920, by Tyzzer. In 1926, the disease germinative area of the female ovaries. was already known also in Europe, Asia They continue to feed and multiply and and Australia. In the late 1960s, there

84 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | gradually they are incorporated in the Epidemiology. The distribution of forming eggs of the nematode. histomonosis is global. It is distributed virtually everywhere where large turkey

or chicken communities exist. Although the main clinical importance of Histomonas is in turkeys, the principal reservoirs for the infections are chicken, as asymptomatic carriers.

Infected Heterakis gallinae eggs can survive in the environment at least two years. However, if free trophozoites are eliminated in the environment directly with the feces of the birds they survive for very short periods of time. Hence, most transmission from host to host is Figure 2.27 Histomonas meleagridis: relying on Heterakis gallinae. form in the lumen of the cecum (upper Nevertheless, direct contamination is also left); intermediate form (upper right); possible and it is assumed that this tissue form (lower). situation is responsible for sudden epizootics in dense flocks of turkeys.

More interestingly, it continues Young birds are the most susceptible and multiplication even after the new the most infected age group is in turkeys nematode embryo starts in ovo up to 14 weeks, with the higher development, and H. meleagridis invades frequency of clinical cases between 3 and the newly formed nematode larvae, still 12 weeks. Mortality can reach 100%. The inside its egg. The larvated egg offers a main reservoirs for infection (directly for perfect shelter to Histomonas. turkeys if kept together, or indirectly, by infecting the vector nematodes) are When the infective nematode eggs of chicken, which are often asymptomatic Heterakis gallinae are ingested by a bird, carriers. nematode larvae hatch, and Histomonas meleagridis leaves its “Trojan horse”, Pathogenesis. After Histomonas invading the avian host. To make things trophozoites emerge from the carrier even more complicated, earthworms nematode hosts, they invade the ceca and commonly act as paratenic hosts to from there, via blood they are carried to Heterakis, and indirectly to Histomonas. various other internal organs (liver, Parahistomonas wenrichi has the same kidney, bursa of Fabricius). During the way of transmission, through Heterakis infection, the xanthophylls from the gallinae. blood decrease and methemoglobinemia increases, resulting in a dark coloration Histomonas multiplies by binary fission. of the skin (hence the name black head No sexual stages are known. disease). The symptoms are very

85 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | commonly exacerbated by the concurrent infection with Escherichia coli or Clostridium perfringens.

Immunology. As soon as histomonosis has been identified as a severe economical problem in farmed turkeys, researchers have tried to figure out a way for making them immune to the infection. Infection does not confer immunity. Moreover, reinfections can be as severe as any infection and may even cause death. Nevertheless, young turkeys are more sensitive than adult ones, but it was not shown if immunity plays a certain Figure 2.28 Liver of a turkey naturally role in this. An interesting observation is infected with Histomonas meleagridis that birds with some degree of immunity showing typical circular necrotic lesions. against Heterakis gallinae are more (Photo Cristian Magdaș) resistant to the infection with Histomonas meleagridis. On gross section, they extend also into Clinical signs. The most common form of the internal parts of the hepatic tissue. In the disease, mainly in young turkeys is chronic cases, these lesions might extent acute. Infected turkeys have ruffled to contiguous organs like kidneys or feathers, hanging wings and tail with lungs. These hepatic lesions may be small yellowish (sulfur-colored) diarrhea. The and multiple or large and confluent. In skin of the head might become dark other cases, they have a tumour like colored, almost black, hence the name of appearance (differential diagnosis with the diseases. If not treated, most birds die leucosis). Histology shows lymphocytic in 1-2 weeks. In older turkeys, the disease and macrophage infiltration with the is more often asymptomatic or chronic, presence of multinucleated giant cells in but virulent strains can induce even in these. The parasitic organisms appear in them an acute form. In chicken, the clusters. The cecal lesions in turkeys are infection is usually mild or asymptomatic. also necrotic, with abundant white Pathology. The main sites of lesions are fibrinous material filling the entire lumen the liver and the ceca. The hepatic lesions (figure 2.29). The mucosa shows also in turkeys are considered to be very small, hemorrhagic ulcers (figure 2.30). characteristic or even pathognomonic. The ceca are often enlarged. Cecal They consist of round, 1-2 cm areas of perforation may occur. In chicken, the necrosis on the liver surface, yellowish cecal lesions can be necrotic, like in and crater-like, with well-defined turkeys or hemorrhagic, resembling margins (figure 2.28). Eimeria tenella coccidiosis.

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From the hepatic and cecal lesions, the direct microscopic examination (wet mount) can demonstrate the presence of live, highly motile Histomonas.

Alternatively, in cytological examination or histological section the parasite is also well visible and identifiable.

There are techniques available for the in vitro cultivation of Histomonas meleagridis. Several cultivation media can be successfully used: Egg slant medium, Laidlaw and Devolt medium or Dwyer medium. Figure 2.29 Cecum of a turkey naturally infected with Histomonas meleagridis Recently, primers for the PCR diagnosis showing necrosis of the mucosa and were designed and employed for fibrinous content in the lumen. (Photo research, but their use in the routine tests Cristian Magdaș) is not yet widely introduced.

Treatment. Despite of many successfully in vitro tested compounds, no single drug is available for in vivo use.

Most of them have high toxicity and their use is forbidden due to human health risks. Such products include: arsenical compounds, nitroheterocyclic compounds etc. The only drugs which are allowed are those against the vector host, the nematode Heterakis gallinae (albendazole, fenbendazole).

Control. As no drugs for the treatment are available, the only real control

measure is prevention. In general, Figure 2.30 Cecum of a turkey naturally avoiding housing together turkeys and infected with Histomonas meleagridis chicken, pasture rotation if grazing showing multiple ulcerations of the systems are used, avoiding of mucosa. (Photo Cristian Magdaș) overcrowding are some measure which are somehow effective.

Diagnosis. The most common diagnosis Periodic treatment and is based on gross lesions. Circular liver chemoprophylaxis against nematodes are necrosis are considered pathognomonic. also very important.

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Several attempts have been made to Medical importance. Genus Giardia is an obtain a commercial vaccine. Live important pathogen of domestic animals attenuated strains used during the early and humans, with zoonotic potential, days offer some protection. However, causing digestive problems mainly in they cannot be used, as prolonged culture young animals and children. Genus induces the loss in their colonizing Hexamita is responsible for enteric capacity. After the discovery of the lesions in colonies of laboratory rodents, second, non-pathogenic species, in turkeys and pigeons. Both genera are Parahistomonas wenrichi, it was believed considered opportunistic. that it will induce cross immunity.

However, the results were inconsistent. There were some moderate results 2.3.3.1 Giardiosis following chemo-immunization (infection followed by treatment to stop the disease Introduction. Giardiosis is a worldwide before serious lesions appear). distributed zoonotic disease caused by , affecting hundreds of mammal species, including domestic 2.3.3 Diplomonadids animals and humans, responsible for diarrheal outbreaks and malabsorption Introduction. The family Hexamitidae is mainly in children and young or a member of order Diplomonadida. The immunosuppressed animals. order includes flagellates with two Historical notes. Giardia duodenalis has separate nuclei, a simple cytoskeleton, no a very important place in history. It was plasmids and no mitochondria. one of the first microscopic organisms to The family Hexamitidae includes five be ever seen by the human eye. It was genera. There are two medically observed for the first time by the Antonie important genera, Giardia and Hexamita, van Leeuwenhoek with one of the first the later not detailed in this textbook due , built by himself. He saw to its limited importance in domestic Giardia in his own feces in 1681. For animals. more than 200 years the parasite was forgotten. In 1902, Stiles associated the General morphology. The most striking presence of Giardia with diarrhea in aspect is the apparent bilateral symmetry humans. His assumptions were correct, (two symmetric nuclei, the position of the as in the World War 1, many soldiers even number of flagella). More aspects suffering from diarrhea were passing in will be discussed for members of genus their stools Giardia parasites. Giardia. Etiology. The taxonomy of genus Giardia Ecology and transmission. Some is still a controversial issue in systematic authors regard Hexamitidae as parasitology. We will follow the opinion commensals inhabiting the digestive tract of the latest reviews which list 6 valid of a variety of vertebrate species. species: G. agilis (from amphibians), G. Transmission is via fecal-oral route.

88 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | ardeae and G. psittaci (from birds), G. end and pointed posterior end. The body muris and G. microti (from rodents) and is flattened dorsoventrally, and has a G. duodenalis (from various mammals). convex shape towards the dorsal part (spoon-like aspect). On the ventral side of The only species with veterinary and its surface, Giardia possesses an adhesive zoonotic importance is G. duodenalis. All disk. The adhesive disk (which is rigid previous names used for this species in due to the presence microtubules) is used the past, some of them extensively (i.e. G. for adherence to the host cells. lamblia, G. intestinalis) must be regarded now as invalid and considered synonyms. The trophozoite bears four pairs of Today, G. duodenalis is divided into 7 flagella. One pair (known as ventral assemblages, named from A to G: flagella) is located in the ventral groove. Each flagellum originates from an  assemblage A (zoonotic): primates organelle called kinetosome. Posterior to (including humans), livestock, cats, the adhesive disk, Giardia has two dogs, beavers; structures with unknown function, called  assemblage B (zoonotic): primates median bodies. Giardia has no (including humans), dogs, beavers; mitochondria, no Golgi apparatus and no axostyle.  assemblage C (non-zoonotic): dogs; The cysts (figure 2.32) are 8-12 x 7-10  assemblage D (non-zoonotic): dogs; µm, with 2-4 nuclei and inner structures  assemblage E (non-zoonotic): cattle, corresponding to axonemes of the flagella sheep, pigs; and median bodies.

 assemblage F (non-zoonotic): cats; Life cycle. The infective elements are cysts from the environment (usually from  assemblage G (non-zoonotic): contaminated drinking water). rodents After ingestion, the excystation is There are some attempts to use binomial triggered by the low gastric pH and specific names for each of these pancreatic enzymes. From each cyst, assemblages: G. canis (for C and D), G. usually two trophozoites are emerging in simondi (for G), G. cati (for F), G. bovis (for the duodenum. They swim by rotational E), G. enterica (for B) and G. duodenalis movements towards the mucosal surface (for A). As they are not widely accepted, where they attach using the adhesive their further use in this textbook will be disks. Trophozoites inhabit the surface of avoided. the intestinal epithelium. Despite the Morphology. Species of genus Giardia trophozoites of Giardia are highly mobile, have two developmental stages: the they prefer to stay attached rather than endogenous stage called trophozoite and swim. the exogenous stage, known as cyst. The They multiply by binary fission. Three trophozoites of Giardia (figure 2.31) are cellular divisions take place before the piriform, with round and broad anterior trophozoites are mature.

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The speed and amount of multiplication in Giardia is amazing. Estimates say that diarrheic stools from infected humans can contain around 14 billion Giardia individuals.

As trophozoites move to the colon and the intestinal content becomes dehydrated, Giardia begins the encystation process. They are eliminated through the feces of the infected host in the environment where they are immediately infective to a new host.

The metabolism of Giardia is anaerobe. However, it tolerates well the presence of oxygen. Trophozoites feed on the mucus produced by the host cell.

Epidemiology. Although not a severe or life threatening disease, giardiosis is among the most widespread infection of humans and animals. The geographical

wide distribution, the large spectrum of Figure 2.31 Trophozoite of Giardia hosts, the high prevalence in certain duodenalis. areas makes giardiosis an important public health problem. This large scale importance is probably caused by the relatively high resistance of cysts in the environment and by the great variety and ease of transmission mechanisms. The transmission route is always fecal-oral, and the possible mechanisms, direct or indirect are: human to human, animal to animal, animal to human or human to animal. The infection sources are represented in all cases by: contaminated water (drinking or recreational), contaminated food (from water used in its preparation or from food handlers).

The most common source of infection for humans is drinking water from various Figure 2.32 Cyst of Giardia duodenalis. sources, including public systems. They

90 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | are usually caused by severe deficiencies malabsorption of other nutrients in the water filtration process. The together with dehydration induces contamination of water is through the weight loss. discharge of human sewage or drainage Immunology. Most of the knowledge on of animal feces, mainly from farms (cattle, the immune response in giardiosis is sheep, goats, pigs and horses). Another either from humans or laboratory animal very common source of infection are models. Infection with Giardia induces a aquatic mammals (muskrats, beavers, strong humoral immune response. The otters and nutria). Consumption of fresh most important antibodies are IgA, which vegetables, fruits or shellfish has also are secreted in the intestinal lumen. They been reported as infection sources. have good in vitro effect, but the Cysts of Giardia can survive in the water antigenic variation of Giardia reduces or soil for several months. Freezing their efficacy in vivo. The role of the during winter destroys the cysts. Cysts immune system in the defense against are sensitive to UV light. Exposure to Giardia is evident mainly in the light of chlorine and chloramines during the several clinical signs in immunodeficient water disinfection is ineffective against hosts. The role of cellular immunity is not Giardia cysts. Cysts are sensitive to ozone fully understood, but it seems it plays a treatment. less important role in protection.

Pathogenesis. The pathogenicity Clinical signs. Most of the infections in depends on the strain (assemblage, animals and humans are inapparent. In genotypes etc.) and on the host. Most of domestic animals, the clinical disease is the cases are asymptomatic. reported occasionally in dogs, cats, ruminants and pigs. Symptoms are more Several mechanisms have been common in young animals. incriminated in the pathogenesis of giardiosis in humans and animals. These In dogs and cats, the symptoms vary from include: production of toxins, intestinal subclinical to moderate or severe dysmicrobism, inhibition of normal abdominal discomfort and pain. Diarrhea enzymatic activity of the enterocytes, is not constant, but when it is present it is intestinal motility disorders. The soft, watery and coated with mucus and permanent attachment of trophozoites to often with steatorrhea and strong odor. the enterocytes induces mucus The diarrhea is self-limiting in dogs and hypersecretion and destruction of cats with normal immune status. microvilli. As a result of long-term malabsorption The anaerobic metabolism is responsible and diarrhea, chronic, persistent for gas production resulting in flatulence infections are accompanied by and intestinal distension with pain. The dehydration and weigh loss. Diarrhea can results of all these is diarrhea and be continuous or intermittent. Flatulence malabsorption. Impaired absorption of may be present, mainly in humans. fats leads to fatty stools, while the Typically, the blood is not present in feces

91 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | of animals suffering from giardiosis. If blood is present it might be the results of concurrent bacterial or coccidian infections. Some dogs and cats may vomit. Fever is usually absent. Differential diagnosis in dogs and cats include pancreatic insufficiency or other malabsorption syndromes.

In calves and other livestock, the clinical picture is more or less similar to the symptoms described above for dogs and cats. Diarrhea which is not responsive to antibiotic or anticoccidial treatment in young animals (1-6 months) might be an Figure 2.33 Cyst of Giardia duodenalis indication of giardiosis or crypto- from the feces of a dog. (Photo Viorica sporidiosis (see Chapter 2.4.2.1). Mircean)

In such cases, confirmation must be using laboratory methods. In livestock, the economic impact of the disease is also important, as infection results in a decreased feed efficiency and weight gain.

Hematology and biochemistry laboratory values are usually within physiological limits. They can reflect only dehydration with loss of electrolytes if diarrhea is severe.

Pathology. Gross intestinal lesions are rarely evident. Microscopic lesions consist in the villous atrophy of the enterocytes. Figure 2.34 Trophozoites of Giardia duodenalis from culture. (Photo Andrei D. Diagnosis. The preferred method for the Mihalca) diagnosis of Giardia cysts in feces is ELISA, employed for detection of coproantigens. Direct detection of cysts Other diagnosis methods include (figure 2.33) or even trophozoites immunofluorescence and phase contrast (figure 2.34) by microscopic microscopy. Identification of assemblages examination is possible. Various methods and genotypes is done by molecular are used, including flotation and staining techniques (e.g. PCR). methods (iodine).

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Treatment. Various drugs have been dogs and cats. Side effects include used for the treatment of giardiosis in anorexia and vomiting. animals. Most effective group of drugs are  Furazolidone, 4 mg/kg body benzimidazoles. weight/day, orally, for one week is There are no licensed drugs to be used in effective in dogs and cats. farm animals. In (dogs and cats),  Paromomycin, 50–75 mg/kg body several protocols are approved and weight/day, orally, for 5 consecutive licensed. The following protocols can be days is used in calves. used:

 Fenbendazole, 50 mg/kg body weight/day, orally for three These drugs may not eliminate consecutive days. It is recommended completely the infection, but they reduce for treatment of dogs, including significantly the oocyst shed and severity pregnant and lactating females. It is of symptoms. not approved for cats. In ruminants, Control. The most effective prevention is the dose is 5-20 mg/kg body by avoiding ingestion of cysts. In humans, weight/day, orally for three drinking pure water (boiled, filtered) is consecutive days. Fenbendazole has the key. In animals, these general also been used in birds. preventive measures are more difficult to  Oxfendazole, 11.3 mg/kg body be applied. However, a good waste weight/day, orally for three management in farms greatly reduce the consecutive days is used also in dogs. environmental pollution. It is not approved, but it is effective. Specific prevention is available for dogs  Albendazole, 25 mg/kg body and cats as vaccination. The commercial weight/day, orally for 4-5 product is a killed vaccine, efficient for consecutive days in dogs and cats. It both, prevention and treatment. is not approved but it is effective. Bone marrow toxicity of albendazole has been reported. In calves, the 2.4 Apicomplexa same dose given for fenbendazole is recommended. Phylum Apicomplexa includes exclusively parasitic organisms, some of them with  Pyrantel and febantel combination huge medical importance in both human (each at 30 mg/kg body weight/day, and veterinary medicine. Prominent orally, single dose, are effective in examples of medically important canine giardiosis. apicomplexans are the agents of human  Metronidazole, 25 mg/kg body malaria (genus Plasmodium) which are weight/twice a day, orally, for 5-7 responsible for more than one million consecutive days is 65% effective in deaths annually. In animals, genus Eimeria has a huge economic impact on

93 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | poultry farming. Other species have subpellicular microtubules radiate major zoonotic impact (e.g. Toxoplasma, posteriorly, parallel to the body axis. Cryptosporidium).  the conoid, consists of a conic spiral The name Apicomplexa is derived from of fibrillar structures. It is not the presence of a series of ultrastructural present in all apicomplexans. Based cell organelles, known as the apical on its presence or absence, Phylum complex (figure 2.35). The apical Apicomplexa is divided in two complex is present only in “zoite”-like classes: (with conoid) stage (e.g. sporozoites, merozoites). and (without conoid).

The apical complex includes several  two or more rhoptries, under the structures: shape of elongated bodies.

 one or two polar rings, which  micronemes, like smaller elongated surround the apical tip of the cell; bodies. from the polar rings, two

Table 2.8 Taxa of veterinary importance in phylum Apicomplexa

Most Class Order Family important Disease (Chapter) genera Intestinal coccidiosis in mammals (2.4.1.1) Intestinal eimeriosis in birds (2.4.1.2) Eimeria Hepatic eimeriosis in rabbits (2.4.1.3) Eimeriidae Renal eimeriosis in geese (2.4.1.4) Isosporosis in pigs (2.4.1.5) Coccidia Isosporosis in carnivores (2.4.1.6) Sarcocystosis in domestic Sarcocystis animals (2.4.3.1) Toxoplasmosis in Toxoplasma domestic animals Sarcocystidae (2.4.3.2) Neospora Neosporosis (2.4.3.3) Hammondia Hammondiosis (2.4.3.4) Besnoitia Besnoitiosis (2.4.3.5) Hepatozoidae Hepatozoonosis (2.4.4.1) Cryptosporidiosis in Cryptosporidea Cryptosporidiidae Cryptosporidium domestic animals (2.4.2.1) Babesiosis in domestic Babesiidae Babesia animals (2.4.6.1) Haematozoea Theileriosis in domestic Theileriidae Theileria animals (2.4.7.1)

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Micropores located along the body  Some groups (Eimeriidae, margins of the “zoites” have role in the Cryptosporidiidae) have ingestion of food by the parasitic homoxenous life cycle (black organism. Movement cell organelles pathway in figure 2.36), with (flagella, cilia, pseudopodia) are absent in endogenous and exogenous stages. apicomplexans, with the exception of Infection of the host is made with very few developmental stages (like some infectious stages from the gametes). environment.

Additionally, the apicomplexan cell  Some others (Babesia, Theileria, contains various cell organelles and Hepatozoon) are heteroxenous, with structures typical to eukaryote cells: no stages in the external nucleus, mitochondria, Golgi body etc. environment (red pathway in figure Apicomplexans have a very complex life 2.36). These include vector-borne cycle (figure 2.36), with three main species which are transmitted to the types of pathways. vector by blood meal and to the host by blood meal or ingestion of vector.  The third group includes members of Sarcocystidae family (Sarcocystis, Toxoplasma, Neospora) with heteroxenous development (blue pathway in figure 2.36). Transmission from the definitive host (which is usually a carnivorous species) to the intermediate host is through the environment, and from the intermediate host to the definitive host by predatorism.

The multiplication in Apicomplexa takes place in both ways: asexually (binary fission, multiple fission or endopolyogeny) and sexually (by male and female gametes). The sexual reproduction takes place always in the definitive host. The vast majority of species are intracellular parasites. All domestic species are affected by diseases produced by apicomplexans. Figure 2.35 General structure of the cell The apicomplexan species of veterinary in Apicomplexa. importance are included in three classes

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A more detailed list of genera and merogony once again. Merogony is associated diseases is shown in table 2.8. repeated several times, depending on several factors. The last generation of

merozoites will transform into gamonts, initiating the gametogony. There are two types of gamonts (gametocytes): microgametocytes (corresponding to male cells) and macrogametocytes (female). Each macrogametocyte develops into one macrogamete. Each microgametocyte divides by multiple fission producing numerous biflagellated microgametes. The macrogamete is fertilized by a microgamete and this produces the zygote. Zygotes develop into oocysts. By sporogony, inside each oocyst the sporozoites will be formed.

Figure 2.36 General life cycle paths in Apicomplexa of domestic animals. H = 2.4.1 Eimeriidae Host; DH = Definitive Host; IH = Intermediate Host; E = Environment. For Introduction. Family Eimeriidae include detailed explanations please refer to the thousands of species parasitic in text. vertebrates and invertebrates. There are three genera of veterinary importance: Eimeria, Isospora and Tyzzeria. Coccidia and Cryptosporidea inhabit the epithelium of digestive tube, the liver, General morphology. Each kidney, blood cells and various other developmental stage has a typical tissues of vertebrates and invertebrates. morphology. Nevertheless, the most important stage from diagnostic point of The typical life cycle of coccidia has three view and for specific identification is the phases: merogony, gametogony and oocyst. The internal structure of the sporogony. The infective stages are sporulated oocysts is different in the called sporozoites. These sausage-shaped various genera of the family (figure (or banana-shaped) cells infect the host 2.37). via various routes and after entering the host cell they become trophozoites and In genus Eimeria the sporulated oocysts start the merogonic development. By contains 4 sporocysts, each with two asexual multiplication, during merogony sporozoites. In genus Isospora, there are numerous merozoites are produce which two sporocysts, each with four escape from the host cell and infect other sporozoites. In genus Tyzzeria, there are susceptible cells, re-starting the eight sporozoites, free in the oocyst and

96 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | not within a sporocyst as mentioned for the previous two genera. The fine structure will be detailed, as an example for genus Eimeria (figure 2.38).

The oocyst wall has two layers. In many species there is a small opening in the oocysts wall called micropyle. This opening is used for the emergence of Figure 2.37 General morphology of the sporozoites when the oocyst reaches the most representative species of digestive tube of the host. The micropyle, Eimeriidae: left - Eimeria (four if present is covered by a cap. A refractile sporocysts, each with two sporozoites); structure called polar granule may be center - Isospora (two sporocysts, each also present. The internal structure of with four sporozoites); right - Tyzzeria typical sporulated oocysts comprises (asporocystic oocyst, eight free sporocysts in different numbers. sporozoites).

Figure 2.38 Diagrammatic representation of a typical sporulated oocyst of Eimeria.

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Between sporocysts, an oocyst residuum other livestock, affecting mainly the may be present, as a result of young animals. Their economic impact is unincorporated cytoplasmic material huge and losses are due to mortalities or during sporogony. expenses for prophylaxis and treatment.

Similarly, inside each sporocyst, as sporocyst residuum is evident. Each sporocyst is delimited by a sporocyst wall. At the anterior end of the sporocysts a small plug-like structure known as may be visible. Less typical is the general morphology of the unsporulated oocyst. Unsporulated oocysts (figure 2.39) are present in fresh fecal material and they have similar morphology in all Eimeriidae genera. It is impossible to identify the species based on the morphology of unsporulated oocysts. They contain one single round central body called sporont, which has a fine granular structure.

Ecology and transmission. All species inhabit the intracellular environment of epithelial cells, usually enterocytes. They Figure 2.39 Diagrammatic can be parasitic intracytoplasmatically or representation of an unsporulated oocyst intranuclearly. As a rule, all species are of Eimeriidae. homoxenous, although exceptions are documented. Merogony and gametogony take place within the host, while 2.4.1.1 Intestinal coccidiosis in sporogony occurs in the external mammals environment. The infective stage is the sporulated oocyst. Transmission is by Introduction. Intestinal coccidiosis in ingestion of infective oocysts. Usually domestic mammals are globally they are highly specific parasites, and distributed in infections responsible for interspecific transmission is very limited. hemorrhagic diarrhea in young Eimeriidae are not zoonotic. Moreover, in individuals, with possible mortality and certain host species (like chicken for with important economic losses. Under instance) there is also a very pronounced the name of “eimeriosis” we include organ specificity. diseases caused by species of genera Eimeria and Isospora. Medical importance. Members of Eimeriidae are responsible for severe Historical notes. The first oocysts of intestinal infections in poultry but also in Eimeria were seen in the liver of an

98 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infected rabbit, by Antonie van carnivores harbor only genus Isospora. Leeuwenhoek in 1674 but it was not Pigs are the only domestic species to be described as a new species during those infected with both genera. Previous times. The first description of a species in reports of genus Eimeria in carnivores genus Eimeria came in 1870, when Eimer, are probably pseudoparasitic species a German zoologist, named a small from their prey which are passed organism found in house mice as passively through the feces. Gregarina falciformis. Five years later

Johann Gottlob Schneider, another German naturalist erected a new genus in Table 2.9 Species of genus Eimeria parasitic in domestic ruminants the memory of Eimer and named it Eimeria. In the following years many Species Host other species parasitic in domestic and E. alabamensis Cattle, Buffalo E. auburnensis Cattle, Buffalo wild animals have been described. Genus E. bovis Cattle Isospora was proposed by the same E. brasiliensis Cattle E. bukidnonensis Cattle Schneider, in 1881. E. canadensis Cattle E. cylindrica Cattle Etiology. The diversity of the species in E. ellipsoidalis Cattle genus Eimeria parasitic in domestic E. illinoisensis Cattle E. pellita Cattle mammals is huge. The quasi-complete list E. subspherica Cattle of them is shown below for ruminants E. thianethi Cattle, Buffalo (table 2.9), equids and camelids (table E. wyomingensis Cattle E. zuernii Cattle, Buffalo 2.10), pigs (table 2.11), rabbits (table E. ankarensis Buffalo 2.12) and laboratory rodents (table E. bareillyi Buffalo E. gokaki Buffalo 2.13). E. ovoidalis Buffalo E. ahsata Sheep In domestic animals, the species of genus E. crandallis Sheep Isospora are parasitic only in pigs and E. danielle Sheep E. faurei Sheep carnivores (table 2.14). E. gilruthi Sheep E. gonzalezi Sheep The taxonomic value of genus Isospora is E. granulosa Sheep being under debate for some years. In E. intricata Sheep 1977, a new genus was proposed E. marsica Sheep E. ovina Sheep () to include species E. ovinoidalis Sheep parasitic in carnivores with facultative E. pallida Sheep, Goat E. parva Sheep heteroxenous life cycle. For educational E. punctata Sheep, Goat purposes, these taxonomic debates are E. absheronae Goat E. africiensis Goat more confusing, hence we will use in this E. alijevi Goat textbook the name Isospora for all E. arloingi Goat species. E. caprovina Goat E. christenseni Goat One should note that in ruminants, E. gilruthi Goat E. hirci Goat horses and rabbits the only parasitic E. jolchijevi Goat genus of Eimeriidae is Eimeria while E. kocharii Goat E. ninakohlyakimovae Goat

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Morphology. The general morphology Table 2.13 Species of genus Eimeria parasitic in laboratory rodents for Eimeriidae was described previously.

Species Host E. caviae Guinea pig Table 2.10 Species of genus Eimeria parasitic E. amburdariana Golden hamster in domestic equids and camelids E. aurata Golden hamster E. razgovica Golden hamster E. arasinaensis House mouse Species Host E. baghdadensis House mouse E. leuckarti Horse E. falciformis House mouse E. solipedum Horse E. ferrisi House mouse E. uniungulati Horse E. hansonorum House mouse E. bactriani Camels E. hindlei House mouse E. cameli Camels E. keilini House mouse E. dromedari Camels E. krijgsmanni House mouse E. pellerdyi Camels E. musculi House mouse E. rajasthani Camels E. musculoidei House mouse E. alpacae Llama, Alpaca E. papillata House mouse E. lamae Llama, Alpaca E. paragachaica House mouse E. macusaniensis Llama, Alpaca E. schueffneri House mouse E. peruviana Llama E. vermiformis House mouse E. punoensis Llama, Alpaca

Table 2.11 Species of genus Eimeria parasitic in domestic pigs Table 2.14 Species of genus Isospora parasitic in domestic mammals Species Host E. almaataensis Swine Species Host E. betica Swine I. suis Swine E. debliecki Swine I. burrowsi Dog E. guevarai Swine I. canis Dog E. ibrahimovae Swine I. neorivolta Dog E. neodebliecki Swine I. ohioensis Dog E. perminuta Swine I. felis Cat E. polita Swine I. rivolta Cat E. porci Swine E. residualis Swine E. scabra Swine E. spinosa Swine Specific morphology for sporulated E. suis Swine oocysts is given for selected species in tables 2.15, 2.16, 2.17, 2.18 and 2.19. Table 2.12 Species of genus Eimeria parasitic in domestic rabbits Identification of species can be done solely if the oocysts are sporulated. Species Host Measurements are necessary for such a E. coecicola Rabbit E. elongata Rabbit purpose not only for the oocyst but also E. exigua Rabbit for internal structures (sporocyst, E. flavescens Rabbit E. intestinalis Rabbit sporozoites). Other specific E. irresidua Rabbit characteristics must be also observed: E. magna Rabbit E. matsubayashii Rabbit presence or absence of the micropyle, E. media Rabbit Stieda body, polar granule, aspect of E. neoleporis Rabbit oocyst and sporocyst residuum etc. In E. perforans Rabbit E. piriformis Rabbit fresh feces, the oocysts are not

100 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | sporulated and their specific Table 2.15 Basic morphology of Eimeria parasitic in domestic ruminants identification is not achievable.

Life cycle. The common aspects of the Species Oocyst shape Size (µm) life cycle were described under general E. alabamensis subellipsoidal 13-24 x considerations about Eimeriidae 11-16 E. auburnensis elongated- 32-46 x (Chapter 2.4.1). Intestinal Eimeria species ovoidal 20-25 develop intracellularly in epithelial cells E. bovis ovoidal 23-34 x 17-23 of the intestine (enterocytes) (figure E. brasiliensis ovoidal 34-42 x 2.40). Each species has a specific habitat. 24-29 E. bukidnonensis piriform 33-41 x Some species inhabit the small intestine, 24-28 some other the large intestine. Other E. canadensis ellipsoidal 28-37 x 20-27 species are very limited (only duodenum) E. cylindrica cylindrical 16-27 x others are more generalist and can infect 12-15 E. ellipsoidalis ellipsoidal 12-27 x portion of the intestine. Hosts acquire the 10-18 infection when ingesting sporulated E. pellita ovoidal 32-42 x oocysts (figure 2.40 - 1). Sporozoites 22-27 E. subspherica subspherical 9-13 x escape (figure 2.40 - 2) through the 8-12 opened micropyle. In the species which E. wyomingensis ovoidal 37-45 x 26-30 lack micropyle, the oocyst wall ruptures E. zuernii spherical 15-22 x in order to release the sporozoites. 13-18 E. ankarensis elongated- 32-43 x Sporozoites will enter a host cell (figure ovoidal 25-29 2.40 - 3) and start the merogony. They E. bareillyi piriform 23-29 x 16-22 soon become enlarged trophozoites E. gokaki ovoidal 22-32 x (figure 2.40 - 4), divide their nucleus and 18-25 E. ovoidalis ovoidal 32-40 x become multinucleated cells (called 20-28 ellipsoidal 29-37 x meronts (figure 2.40 - 5). Each nucleus E. ahsata will be incorporated in the structures of 17-28 ellipsoidal 17-23 x E. crandallis the merozoites (figure 2.40 - 6). 17-22 ovoidal 25-36 x Merozoites than rupture the host cell E. faurei 19-28 (figure 2.40 - 7) and actively search for a ellipsoidal 26-38 x E. gonzalezi new cell which they infect, repeating the 20-26 ellipsoidal 22-35 x E. granulosa merogony (figure 2.40 - 8). The last 17-25 ellipsoidal 39-53 x generation of merozoites (figure 2.40 - 9) E. intricata 27-34 will enter enterocytes (figure 2.40 - 10 ellipsoidal 23-36 x E. ovina and 11) and become macrogametes 16-24 subspherical 12-23 x E. parva (figure 2.40 - 12) and biflagellated 10-19 ellipsoidal 18-25 x microgametes (figure 2.40 - 13). The E. punctata 16-21 microgametes will fertilize the E. arloingi ellipsoidal 17-42 x macrogamete (figure 2.40 - 14) forming 13-27 E. christenseni ovoidal 34-41 x the zygote (figure 2.40 - 15) which 23-28 eventually becomes an oocyst (figure E. hirci round 18-23 x 2.40 - 16). 14-19

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Table 2.16 Basic morphology of Eimeria Table 2.18 Basic morphology of Eimeria parasitic in domestic equids and camelids parasitic in domestic rabbits

Species Oocyst shape Size Species Oocyst shape Size (µm) (µm) E. leuckarti piriform 75-88 x E. exigua subspherical 10-18 x 50-59 9-16 E. solipedum spherical 15-28 x E. intestinalis piriform 27-32 x 15-28 17-20 E. uniungulati oval-ellipsoidal 15-24 x E. irresidua ovoidal 31-43 x 12-17 22-27 E. bactriani subspherical 32 x 25- E. magna ovoidal 31-40 x 27 22-26 E. cameli oval 81-99 x E. matsubayashii ovoidal 22-29 x 63-94 16-22 E. dromedari ovoidal 23-33 x E. media ovoidal 27-36 x 20-25 15-22 E. pellerdyi ellipsoidal 22-24 x E. neoleporis subcylindrical 33-44 x 12-13 16-23 E. rajasthani ellipsoidal 34-39 x E. perforans ellipsoidal 15-29 x 25-27 11-17 E. alpacae ellipsoidal 22-26 x E. piriformis piriform 26-32 x 18-21 17-21 E. lamae ovoidal 30-40 x 21-30 E. macusaniensis ovoidal 81-99 x Table 2.19 Basic morphology of Isospora 61-80 parasitic in domestic mammals E. peruviana ovoidal 28-37 x 18-22 Species Oocyst shape Size (µm) E. punoensis ellipsoidal 17-22 x I. suis subspherical 20-24 x 14-18 18-21 I. burrowsi ellipsoidal 17-22 x 16-19 Table 2.17 Basic morphology of Eimeria I. canis ellipsoidal 34-42 x parasitic in domestic pigs 27-33 I. neorivolta oval 15-19 x Species Oocyst shape Size (µm) 13-16 E. debliecki ellipsoidal 18-24 x I. ohioensis oval 23-25 x 15-20 19-20 E. guevarai piriform 26-32 x I. felis oval 39-48 x 15-19 26-37 E. neodebliecki ellipsoidal 17-26 x I. rivolta oval 20-25 x 13-20 15-20 E. perminuta ovoidal 11-16 x 10-13 E. polita ellipsoidal 22-31 x The time needed for sporulation is 17-22 E. porci ovoidal 18-27 x variable, from species to species and it 13-18 depends a lot on various environmental E. scabra ovoidal 22-36 x 16-26 factors like temperature and humidity. E. spinosa ellipsoidal 16-22 x The average sporulation times for 13-16 selected species of Eimeria and Isospora

are given in tables 2.20, 2.21, 2.22, 2.23 Oocysts are eliminated into the and 2.24. environment through the feces of the In genus Isospora, the general life cycle is host (figure 2.40 - 17) where they similar with the one described for sporulate (figure 2.40 - 18).

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Eimeria. However, in several species Table 2.21 Sporulation times of Eimeria parasitic in domestic equids and camelids infecting carnivores, the presence of paratenic hosts (facultative heteroxenous Species Sporulation time life cycle) has been reported. In this case, (days) E. leuckarti 14-21 the final hosts (dogs, cats) become E. bactriani 10 infected after preying on infected hosts. E. cameli 10-15 E. dromedari 15-17 Moreover, in species of genus Isospora E. pellerdyi 5 parasitic in carnivores, extraintestinal E. rajasthani 7 replication can take place in mesenteric lymph nodes, spleen or liver. Table 2.22 Sporulation times of Eimeria parasitic in domestic pigs

Species Sporulation time Table 2.20 Sporulation times of Eimeria (days) parasitic in domestic ruminants E. debliecki 7 E. guevarai 10 Species Sporulation time E. neodebliecki 13 (days) E. perminuta 9-11 E. alabamensis 4-5 E. polita 8-9 E. auburnensis 2-3 E. porci 9 E. bovis 2-3 E. scabra 9-12 E. brasiliensis 6 E. spinosa 10-12 E. bukidnonensis 4-7 E. canadensis 3-4 E. cylindrica 2 Table 2.23 Sporulation times of Eimeria E. ellipsoidalis 2-3 parasitic in domestic rabbits E. pellita 10-12 E. subspherica 4-5 Species Sporulation time E. wyomingensis 5-7 (days) E. zuernii 2-3 E. intestinalis 1-2 E. ankarensis 3-4 E. irresidua 2 E. bareillyi 3-4 E. magna 2-3 E. gokaki 7 E. matsubayashii 1-2 E. ovoidalis 4-5 E. media 2 E. ahsata 2-3 E. neoleporis 2-3 E. crandallis 1-3 E. perforans 3-5 E. faurei 1-2 E. piriformis 1-2 E. gonzalezi 5-6 E. granulosa 3-4 E. intricata 3-5 Table 2.24 Sporulation times of Isospora E. ovina 2-4 parasitic in domestic animals E. parva 2 E. punctata 1-2 E. arloingi 1-2 Species Sporulation time E. christenseni 6 (days) E. hirci 2-3 I. suis 3-5 E. ninakohlyakimovae 1-4 I. canis 2 I. neorivolta 2 I. ohioensis 4 I. felis 2 Epidemiology. In general, intestinal I. rivolta 4 eimeriosis in mammals is distributed worldwide. However, some species are It is beyond the scope of this textbook to known only from certain parts of the provide detailed information on the world.

103 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | distribution of each mammalian Eimeria Intestinal Eimeria of domestic rabbits is or Isospora species. Generally, they are extremely common. Virtually all rabbits more prevalent in areas with warmer and are infected. Coprophagia has been humid climate (i.e tropics, subtropics). incriminated as a cause for such a high Usually, infection is present in all age prevalence. The rabbits are susceptible groups, at lower intensities in adult from the age of 16 days. animals which are asymptomatic carriers In dogs and cats, younger individuals are and a permanent source of infection for more commonly affected. Cats under four young animals. Severity of the disease is years are at increased risk. Purebred exacerbated by various stress factors like animals are more susceptible. overcrowding, poor nutrition, post- weaning stress, transportation etc. The The resistance of oocysts in the disease is hence more common and more environment is normally high, with severe in farmed animals than in specific limits for each species. In general, backyard ones. Oocysts survive up to one year at temperatures between -30°C and +40°C. In cattle the disease occurs all over the Prolonged freezing and direct sun light world. It causes huge economic losses. kill most oocysts on the pastures. Mortality can reach up to 50%. Infection is rare in suckling calves. The most Pathogenesis. It is believed that the affected age group is 9-12 months. severity of symptoms is correlated with the relative length of the intestine and the In sheep and goats, the disease is very potential number of host cells. This is common, with moderate to high mortality why species affecting the large intestine in young animals. It is distributed or specific portions of the small intestine worldwide and responsible for important (which have rather low lengths) are economic losses. The most susceptible usually more pathogenic than those with age is 6-12 weeks, usually in the first few less selective histotropism. Moreover, the weeks after the first contact with the large intestine has a much lower turn- pasture. Outbreaks are acute, and they over rate of the epithelial cell population usually affect a substantial part of the and as consequence, a lower regenerative flock. potential. In horses, the infection with Eimeria is The pathogenic effect is mostly caused by sporadic, and it has been reported so far the direct damage of the enterocytes only Europe and parts of Asia. All species during the merogonic phase of the life can infect horses, donkeys and their cycle. Hence, the acute form of the hybrids. disease is during parasitic merogony. The infection in pigs is very common, This has a practical impact on the although rarely symptomatic. Suckling diagnosis, as during the acute diarrhea piglets acquire the infection from the skin the typical oocysts are absent from the around the mammary glands. feces.

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Figure 2.40 Life cycle of genus Eimeria. For the meaning of numbers, please refer to the text.

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Denudation of the intestine with the diarrhea is catarrhal in the beginning and destruction of the epithelial lining results hemorrhagic during the merogonic in intestinal hemorrhage, reduced water development of the coccidia. resorption with consecutive diarrhea. In bovines the most common species Chronic infections with catarrhal involved in clinical cases are E. zuernii inflammation are responsible for and E. bovis. The first two days of clinical malabsorption and various consequent infection are characterized by catarrhal nutritional deficiencies. diarrhea, followed by hemorrhagic Immunology. As only young animals are discharges. Other symptoms reported in susceptible and stress is a favoring factor cattle include: anorexia, accelerated it is evident that the immune system has respiration, convulsions, emaciation and a well-established role in the active tremors. Fever is rarely present. In dairy protection against coccidial infection. The cows, milk production might be affected. main problem with post-infective Cows with chronic infection display immune protection is the lack of cross- intermittent/recurrent diarrhea. immunity between the different species Eimeriosis of sheep is a very severe of Eimeria infecting certain hosts. parasitic problem, with significant There are no extensive studies on all mortality in lambs and less severe in species of Eimeria. However, some goats (kids). Lambs with acute infections aspects can be concluded based on show a profuse diarrhea, often several experimental studies. It is hemorrhagic, with almost liquid feces, thought that certain species are highly which lasts several days. Debilitated immunogenic and the infection with very lambs become weak, they cease eating few oocysts (less than 10) can induce a and some die. strong immunity. In horses, the infection is usually Both components of the immune system asymptomatic. If clinical cases occur in are involved in the anti-eimerial foal, they are generally mild, with protection. IgG antibodies and moderate and self-limiting diarrhea. lymphocytes are responsible for Coccidiosis in pigs is rarely a clinical protection. A strong immune response problem. Diarrhea is not usually can be detected at around 14 days after hemorrhagic. More severe cases show the initial infection. loss of appetite, emaciation and retarded Clinical signs. Despite the diversity of growth. Mortality is very rare. etiological agents in the various domestic The main symptoms in domestic rabbits hosts, the clinical signs of coccidiosis are are diarrhea and bloating. Mortality is rather uniform. In adult animals the high, sometimes even superacute, infection is usually asymptomatic. Clinical without any prodromal signs. signs in young animals are predominantly digestive, with diarrhea, In dogs and cats, diarrhea is uncommon, dehydration, anemia and weight loss. The unless associated with

106 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | immunosuppressive condition or petechial hemorrhages and necrotic concurrent infection. In kennels, enteritis with edema of the intestinal outbreaks can be enzootic. wall.

Pathology. The main lesions are located The lesions in horses were described in the intestine. Generally, they consist in after experimental infection. They consist an inflammation of the intestinal in catarrhal enteritis, with whitish foci epithelium (enteritis). Their severity and easily visible at gross examination. location depends on the age of the animal In pigs, the gross lesions consist in and the species of coccidia involved. catarrhal enteritis mainly in the large Some species are located deeply, under intestine. the lamina propria of the enterocyte lining and are responsible for Domestic rabbits show catarrhal hemorrhagic lesions. Some other are enteritis, with whitish deposits. more superficial and the lesions are In dogs and cats, lesions are usually catarrhal. Often, whitish spots are easily catarrhal, rarely hemorrhagic. visible on the intestinal surface. They represent the merogonic stages of the Diagnosis. Based on clinical signs, eimerids. diagnosis must be confirmed microscopically. The presence in the In cattle, the body of animals which died samples of any of the developmental of acute eimeriosis is weak, with the stages of Eimeria or Isospora has posterior parts (perianal area, ventral diagnostic value. During the acute stage surface of the tail) soiled with red- of infection, when the life cycle has barely colored feces. In the abdominal cavity reached its merogonic or gametogonic there are small amounts of reddish phases, the oocysts (which appear after exudate. The body is generally anemic, the gametogony) are absent from the with pale colored organs. Mesenteric feces. During this clinical stage, other blood vessels are hyperemic. The most developmental forms can be detected in characteristic lesions are at the level on the feces (e.g. meronts). Examination of intestinal mucosa. The luminal surface of large number of samples from the same the intestines is coated with reddish flock can be helpful. For the detection of mucus, congestion is present and the most Eimeria and Isospora oocysts, mucosa is congested, mainly in the flotation methods are recommended. In terminal part of the ileum and in the large certain Eimeria species (i.e. E. leuckarti intestine (cecum and colon). The from horses) better results are obtain intestinal content is liquid and using sedimentation. malodorous. The hemorrhages on the intestinal mucosa can be punctiform or The oocysts from feces are normally not confluent. sporulated (figure 2.41), thus their specific identification is impossible at this In sheep and goats, the lesions are stage. For detailed morphological studies different depending on the causative and specific diagnosis, they can be species. In general they consist of

107 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | sporulated in the laboratory at room temperature, using a 2.5% solution of potassium dichromate in the presence of oxygen. In older fecal samples, the oocyst can begin the sporulation process and they are found in various stages (figure 2.42).

The presence of oocysts in the feces of ruminants, horses, and rabbits normally means genus Eimeria. In swine, both genera can be present (Eimeria and Isospora). The most controversial coproscopic diagnosis is in dogs and cats, as domestic carnivores pass in feces Figure 2.42 Unsporulated oocyst of oocysts of several species of intestinal Isospora sp. from the feces of a cat. Note coccidia: Isospora, Toxoplasma, Neospora, the oocyst beginning the sporulation Hammondia and Besnoitia. Usually, the process (lower right). (Photo Andrei D. differentiation should be made on the Mihalca) basis of size. In dogs, “Isospora”-like oocysts which are larger than 15 µm are with high probability members of genus In cats, oocysts larger than 20 µm are Isospora. If they are smaller, they belong likely to be Isospora, while those smaller to (10-12 µm) or than 12 µm are one of the heteroxenous Hammondia heydorni (11-12 µm). species (Toxoplasma gondii, , Besnoitia darlingi or Besnoitia

oryctofelisi).

However, Besnoitia wallacei also reported in cats has slightly larger oocysts than its congeners (17 x 12 µm).

Post-mortem diagnosis is achieved by direct examination of the intestinal content from the parts with lesions or by histopathology, when all developmental stages can be identified. For dogs and cats, differentiation of small oocysts can be achieved also by PCR.

Treatment. The number of available drug used for the treatment of coccidial Figure 2.41 Unsporulated oocyst of infections in mammals is huge. They are Eimeria sp. from the feces of a domestic known generically as anticoccidial drugs. rabbit. (Photo Andrei D. Mihalca)

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Table 2.25 Drugs used for the treatment of coccidiosis in domestic mammals

Drug Species Dose (mg/kg) Route Duration (days) cats, dogs 50-60 PO 5-20 Sulfadimetoxine sheep 50-100 PO 5 cattle 140 PO 3 Sulfadimidine sheep 25-50 PO 3 cats, dogs 100-200 PO 5 Sulfaguanidine sheep 250 PO 7 swine 200 PO 3-4 cattle 110 PO 5 Sulfamethazine sheep 60 PO 3-5 cattle 6 PO 3-5 Sulfaquinoxaline sheep 60 PO 3-5 dogs 30-60 PO, SC 5 Trimethoprim-sulfonamide cats, dogs 15-30 PO, SC 5 Ormetoprim-sulfadimethoxide dogs 66 PO 7-23 Furazolidone cats, dogs 8-20 PO 5 cats, dogs 300-400 PO 5 cats, dogs 110-200 PO 7-12 cats 60-100 PO 7 cattle 10 PO 5 Amprolium cattle 65 PO 1 sheep 50-65 PO 4-7 swine 25-65 PO 3-4 rabbits 25 PO 4-5 Quinacrine cats, dogs 10 PO 5 Clindamycin cats 10 PO, SC, IM 7-28 dogs 15-30 PO 1-6 cattle 15 PO 1 Toltrazuril sheep 20-40 PO 1 rabbits 20 PO 1-3 Diclazuril cats 25 PO 1 dogs 30-50 PO 1-7 Ponazuril cats 15 PO 7 cattle 1 PO 10 Monensin sheep 1.5 PO 21

Treatment must be implemented for exposed to risk, chemoprophylaxis. The treating animals with clinical symptoms most important factor for avoiding the or to eliminate asymptomatic carriers, as onset of clinical disease is to maintain a a control measure. Drugs can be used as good overall health status in young chemoprophylaxis. animals. Neonates should be given colostrum. Proper environmental A check-list of available drugs and their conditions, microclimate, a balanced dosage for various domestic mammals is nutrition and avoidance of overcrowding given in table 2.25. are essential factors. Moreover, Control. The most efficient way for sanitation, disinfections, use of clean controlling coccidiosis in mammals is water and watering devices are also prevention. This can be achieved by using important. general measures and in the animals

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Unlike in birds, the use of Not only they are host specific, but they chemoprophylaxis is not compulsory only have different tropism for various when outbreaks are imminent (i.e. segments of the intestine (figure 2.43). previous history of disease, introduction Other species of Eimeria parasitic in birds of new animals). In principle, the same are shown in tables 2.27, 2.28 and 2.29. molecules which are used for treatment Morphology. General morphology of can be used for chemoprophylaxis, but in Eimeria parasitic in poultry is concordant lower doses and long-term with genus characteristics, discussed administration. In ruminants, the use of above. Specific morphology for selected decoquinate and ionophores was shown species parasitic in chicken is given in to be effective. During the first month in table 2.30. No data will be provided for calves exposed to infection, lasalocid, species parasitic in other domestic bird monensin or amprolium are efficient. In species. pigs, sulfonamides and amprolium were successfully used.

Table 2.26 Species of genus Eimeria parasitic in chicken

2.4.1.2 Intestinal eimeriosis in birds Species Intestinal segment E. acervulina Duodenum Introduction. Eimeria infections in E. brunetti Large intestine E. dispersa Small intestine poultry, mainly in chicken, are among the E. hagani Duodenum most important diseases in industrial E. maxima Small intestine E. mitis Duodenum farms. They have a huge economical E. mivati Duodenum dimension, mainly by expenses with the E. necatrix Small intestine E. praecox Duodenum prophylaxis. The estimated annual cost E. tenella Cecum related to poultry eimeriosis is situated around 2 billion euros.

Historical notes. The first description of Life cycle. The life cycle follows the same a coccidian life cycle from birds came in phases and developmental stages as for 1910, when Fantham published his work species parasitic in mammals. In birds, on red grouse. In 1929, Tyzzer described the life cycle is somehow faster that in the life cycle for several species of species parasitic in mammals. For some Eimeria parasitic in chicken (E. species (i.e. E. tenella) the entire acervulina, E. mitis, E. maxima, E. tenella). development takes 4-6 days. The first anticoccidial drugs have been introduced in 1948, when FDA approved Epidemiology. The disease is present in USA the use of sulphaquinoxaline and everywhere where the hosts are nitrofurazone. The first commercial presents. Their distribution is hence vaccine was introduced in 1952. global. As the parasitism is strictly host- specific, the only sources of infection are Etiology. There are ten species of conspecific birds. coccidia parasitic in chicken (table 2.26).

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Table 2.27 Species of genus Eimeria parasitic in poultry farms is mechanical, by in other galliformes personnel.

Species Host E. adenoeides Turkey E. dispersa Turkey Table 2.28 Species of genus Eimeria parasitic E. gallopavonis Turkey in ducks and geese E. innocua Turkey E. meleagridis Turkey Species Host E. meleagrimitis Turkey E. abramovi Duck E. subrotunda Turkey E. anatis Duck E. gorakhpuri Guinea fowl E. battakhi Duck E. grenieri Guinea fowl E. boschadis Duck E. numidae Guinea fowl E. danailovi Duck E. colchici Pheasant E. mulardi Duck E. dispersa Pheasant E. nottion Duck E. duodenalis Pheasant E. saitamae Duck E. langeroni Pheasant E. schachdagica Duck E. megalostomata Pheasant E. anseris Goose E. pacifica Pheasant E. fulva Goose E. phasiani Pheasant E. hermani Goose E. mandali Peacock E. kotlani Goose E. mayurai Peacock E. magnalabia Goose E. patnaiki Peacock E. nocens Goose E. pavonina Peacock E. stigmosa Goose E. pavonis Peacock E. striata Goose

Table 2.29 Species of genus Eimeria parasitic in pigeons

Species Host E. columbae Pigeon E. columbarum Pigeon E. kapotei Pigeon E. labbeana Pigeon

Table 2.30 Basic morphology of Eimeria parasitic in chicken

Species Oocyst shape Size (µm) E. acervulina ovoidal 17.7-20.2 x 13.7-16.3 E. brunetti ovoidal 20.7-30.3 x 18.1-24.2 E. maxima ovoidal 21.5-42.5 x 16.5-29.8 Figure 2.43 Location of Eimeria species E. mitis subspherical 11.7-18.7 x in chicken. 11.0-18.0 E. mivati ellipsoidal 11.1-19.9 x 10.5-16.2 E. necatrix ovoidal 13.2-22.7 x 11.3-18.3 As a consequence, unlike in other E. praecox ovoidal 19.8-34.7 x bacterial or viral disease of poultry, wild 15.7-19.8 E. tenella ovoidal 19.5-26.0 x birds are not infection sources. The most 16.5-22.8 important way for spreading the coccidia

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There is a clear age predisposition. The and lesions below). The pathogenicity is most sensitive age is variable in each also host-dependent (age, immune status, Eimeria species. For instance in the case species) and is influenced by the of the most commonly occurring species presence of concurrent infections. in chicken, E. tenella (cecal eimeriosis), Although the lesions are generally local, the most affected age is 5-7 days in birds they affect the whole organism. are exposed to high infective doses The most important pathogenic effect is immediately after hatching. Normally, due to destruction of enterocytes and cecal eimeriosis appears at 21-25 days. other associated tissues (e.g. lamina Cecal coccidiosis is also among the most propria, submucosal layers, blood severe forms of disease, with an acute vessels) by the merogonic development. onset and high mortalities (50-100%) in Massive destructions are responsible for few days, if not treated. Infection with E. intestinal hemorrhages. Intestinal tissue necatrix produces clinical infection in 5-7 damage results in motility disorders, weeks old chicken, and the onset and altered absorption of nutrients, evolution is slower. Mortality is much decreased water resorption, malnutrition lower. Infections with E. brunetti are rare etc. Their severity is variable and but the onset is very fast. Eimeria depends on the surface of damaged acervulina affects mainly older chicken epithelium. Epithelial destruction allows and even adults, while E. maxima is undisturbed access of other pathogens responsible for coccidiosis in laying hens. (mainly bacteria) to the blood stream and Turkeys are less sensitive to clinical tissues. Chronic infections, although not eimeriosis, and outbreaks are less severe life-threatening produce long-term than in chicken. The most susceptible age debilities (i.e. rickets). is 6-8 weeks. In ducks and geese, the In order to assess the severity of infection is sporadic. In pigeons, the infection, mostly in experimental trials infection is common and mortality is high for testing anticoccidial drugs or (15-70%) at the age of 3-4 months. vaccines, a scoring system has been Resistance of oocysts in the environment developed (please refer for details to is relatively high. Alternation of freezing- Conway and McKenzie (2007). defreezing or direct sunlight exposure Immunology. The knowledge on the kills them rapidly. However, the most immunology of eimeriosis in chicken is important aspect regarding oocyst very extensive, as the quest for safer and resistance is within the more ecological preventive measures (i.e. microenvironment of the farm. Common vaccination) is permanent. There is an disinfectants have limited efficacy on the evident acquired immunity, as adult birds oocysts. Most of them inhibit sporulation; are non-receptive to clinical infection and hence unsporulated oocysts are more the development in their intestine is self- sensitive. limiting. Local intestinal cellular Pathogenesis. Some species are more immunity from the associated lymphoid pathogenic than others (see symptoms tissues are responsible for the post-

112 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infective resistance. B cells produce anti- The most pathogenic species in turkeys Eimeria antibodies shortly after the debut is E. adenoides. The feces are liquid and of infection. However, their protective may be coated with hemorrhagic mucus. role is limited. The most important Mortality can be present if infective doses components seem to be intraepithelial are high. Chronic infections are and lamina propria cytotoxic T responsible for significant weight loss. lymphocytes. In ducks and geese, the signs of infection Clinical signs. Clinical signs are mainly with E. anseris are: anorexia, weight loss, digestive and certainly not characteristic. general weakness, distress, diarrhea and even mortality. Clinical signs in chicken range from asymptomatic infections in adult birds to In pigeons, the common signs of infection mild, moderate or severe disease in are greenish diarrhea, anorexia, young birds. Clinical eimeriosis in dehydration and extreme weight loss. chicken varies from chronic forms with The feces may be hemorrhagic. Chronic decreased growth rate to severe diarrhea, eimeriosis can cause mineral deficiencies often with high and fast mortality. There (figure 2.44). are some differences in the clinical signs between Eimeria species involved. In the case of cecal eimeriosis (caused by E. tenella), the diarrhea is watery and contains often hemorrhagic droppings. As a consequence, chicken are anemic and may exhibit even nervous signs. Hemorrhagic feces can be present also in infections caused by other species of Eimeria, but they are not as severe as in the case of E. tenella. In the case of E. brunetti, E. necatrix, E. acervulina, E. maxima or E. mivati, the hemorrhage in the feces is weaker and it appears as discrete streaking on the droppings. Moreover, in these later species, the feces Figure 2.44 Typical “S” shaped keel as a are rarely watery, or even with normal result of secondary calcium deficiency in consistence and soaked with slightly a pigeon suffering from chronic hemorrhagic mucus. eimeriosis. (Photo Andrei D. Mihalca)

Sometimes the infection with Eimeria in chicken does not result in changes in the Pathology. The lesions in poultry consistence of feces, nor other evident coccidiosis are different for the various symptoms. However, even low levels of species of Eimeria involved. Their infection can induce low weight gain or location and gross aspect are of great decreased feed conversion rate.

113 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | diagnostic value, some almost pathognomonic. In cecal coccidiosis (E. tenella) the most prominent finding is a severe hemorrhagic typhlitis (figure 2.45), with a dilated cecum (figure 2.46) containing liquid or partially clotted blood.

The extent of the lesion might be reduced to some patches which in severe forms are confluent and affect both ceca, totally. Sometimes the lesions consist in hemorrhagic-necrotic typhlitis or even with the presence of fibrinous material. Figure 2.46 Dilated ceca with focal

hemorrhagic typhlitis caused by Eimeria tenella in chicken. (Photo Adriana Györke)

The same situation is encountered in the infection with E. brunetti, but the punctiform hemorrhages are located in the rectum and cloaca. Necrosis of the rectal wall may be present.

Figure 2.45 Hemorrhagic typhlitis caused by Eimeria tenella in chicken. (Photo Adriana Györke)

The infection with E. necatrix is responsible for intestinal congestion, with pinhead-sized hemorrhagic spots (figure 2.47). In severe cases, these spots become confluent and the middle part of the small intestine becomes entirely hemorrhagic (figure 2.48). Sometimes Figure 2.47 Small intestine enteritis the lesions are fibrinous or necrotic caused by the infection with E. necatrix. (figure 2.49). (Photo Adriana Györke)

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The lesions in other bird species vary from catarrhal enteritis to various degrees of hemorrhagic enteritis (focal or generalized) or fibrinous-necrotic inflammations.

Diagnosis. Based on clinical signs and necropsy, the etiology must be confirmed Figure 2.48 Severe hemorrhagic enteritis in the laboratory. This is done by direct caused by the infection with E. necatrix. identification of various stages of Eimeria (Photo Adriana Györke) in the feces of living birds or in the lesions in the case of dead birds (figures 2.50, 2.51, 2.52, 2.53). Eimeria maxima is responsible for hemorrhagic enteritis in the anterior and Please refer to the diagnosis section from medial segment of the small intestine, the previous Chapter (2.4.1.1) on very clearly delimited from the healthy mammal coccidiosis. contiguous intestinal segments.

Eimeria acervulina and E. mivati produce more or less similar lesions, with focal (petechial) hemorrhagic enteritis in the small intestine with the presence of whitish colonies.

Figure 2.50 Meronts of Eimeria sp. in wet mount from intestinal lesions of chicken. (Photo Adriana Györke)

Treatment. The disease in chicken is very severe. Treatment in large bird

communities (farms) is most often Figure 2.49 Fibrinous-necrotic enteritis impossible, mainly because of logistic caused by the infection with E. necatrix. reasons. (Photo Adriana Györke)

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Figure 2.51 Merozoites of Eimeria sp. in Figure 2.53 Various developmental wet mount from intestinal lesions of stages of Eimeria sp. in histological chicken. (Photo Adriana Györke) section from intestinal lesions of chicken. (Photo Andrei D. Mihalca)

Several drugs have been used in chicken in traditional (backyard), free-range systems. Sulfaquinoxaline (125 mg/kg) or Sulfadimidine (150-200 mg/kg) for 3- 5 days are effective against E. tenella. In pigeons, sulfonamides and clazuril are used in the drinking water.

Control. Prevention of eimeriosis in chicken is crucial in the poultry industry. It represents the only viable solution for controlling outbreaks. Otherwise, any clinical outbreaks are equivalent with economic disaster. Figure 2.52 Merozoites of Eimeria sp. in General hygiene methods and specific stained smear from intestinal lesions of methods are used for controlling chicken. (Photo Adriana Györke) eimeriosis in birds.

General measures include: maintaining Even if the parasites are successfully the general health status of birds at eliminated using specific anticoccidial optimal levels, decreasing the level of medication, the lesions do not heal fast stress, use of proper and balanced diet, enough in order to allow full recovery. optimal environment, avoiding of overcrowding, good lighting and

116 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | ventilation and general biosecurity  robenidine (inhibits development of measures (strict access control, cleaning, meronts). disinfections). (2) Polyether antibiotics or ionophores Specific measures include Their mechanism of action is by chemoprophylaxis (with low and long interfering with the metabolism of term/continuous administration of sodium and potassium. Three types are anticoccidial drugs) and immune known: prophylaxis (using vaccines) or an alternation of them.  monovalent ionophores (monensin, narasin, salinomycin); Various anticoccidial drugs are currently being used in the  monovalent glycosidic ionophores chemoprophylaxis of eimeriosis in (maduramicin, semduramycin); chicken. They are grouped in two  bivalent ionophores (lasalocid). categories, based on their chemical structure (classification based on Peek Ionophores cannot be associated with and Landman, 2011). certain other antibiotics (e.g. tiamulin, chloramphenicol, erythromycin,

oleandromycin, sulphonamides). (1) Synthetic compounds Additionally, there are various  amprolium (competes for the commercial products where a

absorption of vitamin B1 by the combination of the aforementioned parasites); products is used. The poultry category and the concentration to be given to  aprinocid (interferes with DNA chicken in food for each drug is listed in synthesis); table 2.31.  clopidol (inhibits development of The main problem when using sporozoites); chemoprophylaxis is the emergence of  decoquinate (inhibits development of resistant strains. In the case of chicken sporozoites); eimeriosis, resistance is known for all compounds and it has been reported  diclazuril (blocks the excretion of worldwide. Despite this, severe oocysts); outbreaks are occasional, as the insidious  dinitolmide (inhibits development of presence of Eimeria due to resistance meronts); stimulates immunity.

 halofuginone (inhibits maturation of To reduce the impact and occurrence of merozoites); resistance, rotation of anticoccidial drugs is recommended. Each drug should be  nequinate; used for maximum 2 months, or in  nicarbazin (inhibits development of broilers, for maximum two fattening meronts) cycles.

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Table 2.31 Anticoccidials used in the expressed from DNA) of different prophylactic treatment of eimeriosis in chicken (after Peek and Landman, 2011) developmental stages (sporozoites, merozoites, gametogonic stages). There is Concentration Poultry a single subunit commercial vaccine Drug in fodder category (ppm) available (CoxAbic). It is used for Amprolium Broiler, 125-250 vaccination of mother hens and immunity rearing Amprolium+ Broiler, 125-250+ is transmitted to broiler chicken via ethopabate rearing 4 trans-vitelline route. Aprinocid Broiler 60 Clopidol Broiler, 125 (2) Live vaccines contain non-attenuated rearing Decoquinate Broiler 30 or attenuated sporulated oocysts of Diclazuril Broiler, 1 various species of Eimeria. Two rearing Dinitolmide Broiler, 125 vaccination strategies are employed: rearing single shot (high dose) or multiples shot Halofuginone Broiler, 3 rearing (low dose). The vaccinal strains might be Nequinate Broiler, 20 sensitive to anticoccidial drugs. Thus, rearing discontinuation of these is essential in Nicarbazin Broiler 125 Robenidine Broiler 33 vaccination strategies are to be Lasalocid Broiler 75-125 implemented. Maduramicin Broiler 5-6 Monensin Broiler, 100-120 Non-attenuated vaccines have been used rearing Narasin Broiler 60-80 for long time (CocciVac, Inovocox, Salinomycin Broiler, 44-66 Advent, Imunocox), but their rearing Semduramycin Broiler 25 administration is risky, as they can induce clinical infection if protocols are not followed strictly. Shuttle programs (simultaneous use of two or more drugs with different Attenuated vaccines (with decreased mechanism of action during the same virulence obtained by repeated in vitro technologic cycle) are also recommended. passages on chicken embryos) are safer but they lose immunogenicity over time Before implementing chemoprophylaxis and their production is more costly. programs, it is highly recommended to Examples of attenuated vaccines include perform in vivo Anticoccidial Sensitivity Livacox or Paracox. Tests, using local strains. However, antigenic variability between New approaches to the management of various Eimeria species restrict the anticoccidial drug resistance include the commercial value of live vaccines. Some rotation of chemoprophylaxis with of the vaccines are also available for immune-prophylaxis (vaccination). turkeys (e.g. Coccivac, Immunocox). There are several types of vaccines available today.

(1) Subunit vaccines contain antigens (either native or recombinant proteins

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2.4.1.3 Hepatic eimeriosis in rabbits via blood through the portal, free or in macrophages. The sporozoites reach the Introduction. Hepatic eimeriosis is a sinusoid veins between liver cells 1-6 severe condition of rabbits caused by E. days after the infection. They stiedai, with high mortality in young subsequently migrate to the biliary ducts animals. and penetrate the epithelial cells where Historical notes. E. stiedai is probably they start the merogony. Merogony takes the first ever unicellular “animal”-like around 12 days. Gametogony lasts for 4 organism ever observed (by Antonie van more days. Unsporulated oocysts appear Leeuwenhoek in 1674). The species was in feces as early as 16 days after the described as Monocystis stiedae by infection. In the environment, the Lindemann, in 1865. The first study on its sporulation takes 2-3 days. life cycle date from 1903 (Metzner). The Epidemiology. Eimeria stiedai is present first reports of disease date back from the all over the world, affecting various mid-19th century. species of rabbits and hares. Adult Etiology. The causative agent of hepatic rabbits are the source of infection for eimeriosis in rabbits is Eimeria stiedai. young rabbits. Wild rabbits can also Except domestic rabbits, E. stiedai is harbor the infection and contaminate the parasitic in various wild rabbit and hare environment. Contaminated grass fed to species (Lagomorpha): European rabbit rabbits can bring the infection. After (Oryctolagus cuniculus), European brown elimination through the feces, they hare (Lepus europaeus), Snowshoe hare become infected in 2-3 days. Rabbits up (Lepus americanus), mountain hare to two weeks old are resistant to (Lepus timidus) and Eastern cottontail infection. The rabbits are normally (Sylvilagus floridanus). receptive to the infection with E. stiedai after 16-18 days of life up to 4 months. Morphology. The oocysts are oval or After this age, they become completely narrow oval, sometimes asymmetrical resistant, and clinical cases are normally and they have a micropyle. The size of absent. oocysts is 28-40 x16-25 µm. Pathogenesis. As parasites are invading Life cycle. Eimeria stiedai follows the the biliary ducts and merogonic stages typical life cycle of genus Eimeria, as are developing, the epithelial cells are described in Chapter 2.4.1.1. However, being destroyed. The biliary ducts there are few particular aspects which become distended and filled with cellular require some attention. First of all, their debris and nodules appear in the liver typical habitat is the epithelial lining of parenchyma. These changes are most biliary ducts. After sporulated oocysts are prominent by day 16, when merogonic ingested by rabbits they typically excyst gametogonic phases are already complete in the duodenum. Sporozoites leave the (figure 2.54). If rabbits survive and no intestine by penetrating the mucosa and massive reinfection takes place, all these reach the liver via lymphatic system or lesions heal completely. These lesions are

119 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | able to produce significant functional the liver is severely affected, jaundice, disorders. The main alteration seems to ascites and fever may be present. be in the metabolism of vitamin E. Mortality can be present.

Pathology. The gross lesions in rabbits which died of coccidiosis consist in hepatomegaly with the presence of numerous, disseminated white or grey- white nodules (figure 2.55) even in the more profound regions of the liver (figure 2.56).

There are various degrees of lesion severity. In mild forms, the nodules are few and small, while in the most severe cases nodules are confluent and normal liver tissue is almost absent. The bile is full of debris. Bile ducts are fibrous and enlarged.

Figure 2.54 Various developmental Liver lesions must be differentiated from stages of Eimeria stiedai in histological the white and fibrous migratory routes or section rabbit liver. (Photo Andrei D. the vesicle-like cysts of the larval stages Mihalca) of Taenia pisiformis (i.e. “Cysticercus pisiformis”).

Immunology. All infections, regardless they produce clinical disease or not are ultimately inducing a strong immunity. Experimental data suggest that development of protective immunity requires around 21-30 days. It seems the most important component of this post- infective resistance is humoral immunity. Cross immunity with intestinal Eimeria have been suspected (i.e. with E. magna).

Clinical signs. Infection with E. stiedai may be asymptomatic, may cause various clinical signs or may even result in Figure 2.55 Typical gross lesions in the sudden death without any prodromal liver of rabbits suffering of hepatic signs. Symptoms can vary from only eimeriosis: hepatomegaly with the moderate weight loss to more evident presence of multiple whitish nodules. signs like bloating, loss of appetite, (Photo Andrei D. Mihalca) diarrhea or constipation. If function of

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Figure 2.56 More detailed view of the Figure 2.57 Wet mounts from the lesion pictured previously. (Photo Andrei hepatic lesions reveal usually high D. Mihalca) number of oocysts. (Photo Andrei D. Mihalca)

Diagnosis. In living rabbits, the positive diagnosis is based on the demonstration Control. Asymptomatic carriers can be of oocysts in the feces. By the time clinical treated to eliminate the infections signs are present, the oocysts are already sources for the young susceptible rabbits. present in stools. They must be Sulfaquinoxaline (0.04% in drinking differentiated from intestinal Eimeria. water for 30 days or 0.025% in food for 20 days), sulfadimetoxine (0.06% in In dead animals, the lesions are almost drinking water), sulfadimerazine (0.2% pathognomonic. Fresh preparations (wet in drinking water) or amprolium 9.6% (in mounts) done directly from the whitish drinking water) are all effective for nodules from the liver or from the bile chemoprophylaxis. Withdrawal time for reveal the presence of various sulfaquinoxaline is 10 days. developmental stages but mainly of unsporulated oocysts in impressive General hygiene measures (frequent numbers (figure 2.57). removal of feces from pens and disinfection with 10% ammonia help in Treatment. Suffering rabbits are treated reducing the infective preasure. using oral medication. The anticoccidial drugs recommended for treating clinical cases in rabbits is toltrazuril (25 ppm in drinking water for two days). 2.4.1.4 Renal eimeriosis in geese

Other anticoccidials may be used. Introduction. It is a locally relatively common protozoal infection of domestic

121 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | geese which is responsible for acute of debris and possible micro-obstructions symptoms and high mortality in goslings. of the tubules. This results in significant increase of the kidneys’ size and Historical notes. The etiological agent, deposition of urates. Eimeria truncata was described as Coccidium truncatum by Railliet and Immunity. No studies are available, but Lucet in 1891 from domestic geese. birds which survive the infection become refractory to new infections. Etiology. The only known species able to infect the epithelial layer of the renal Clinical signs. The usual form of the tubules in domestic geese is Eimeria disease is acute. Renal coccidiosis may be truncata. The species was reported a very serious disease of goslings, with subsequently from various other wild severe depression, general weakness, avian hosts. whitish diarrhea and anorexia. During more chronic forms, birds have Morphology. The oocysts of E. truncata polydipsia and nervous signs may be are oval but with a truncation of the present (gait). Neurologic sequelae narrow end. The size is 20-22 x 13-16 include vertigo and torticollis. µm. A micropyle is present. Pathology. The most characteristic Life cycle. All stages develop in the lesion is the enlarged aspect of kidneys tubular epithelial cells of the kidneys. As which have a greyish-yellow to yellowish experimental infection is difficult, it is not red colored surface (normal color is yet fully understood how the infective reddish brown). Kidneys often protrude sporozoites reach from the intestine to from their sacral bed. The surface is the kidneys. The contamination route is covered with small pinhead-sized oral, although some authors have grayish-white foci. Hemorrhagic lesions questioned this. Oocysts can be recovered can be occasionally present. from the feces 5-6 days after the infection. Sporulation takes 1-2 days. Diagnosis is based on clinical sign and lesions, followed by demonstration of Epidemiology. The disease seems to be oocysts in the droppings or in the kidney spread worldwide. Outbreaks are lesions. sporadic, but when they occur, they are able to affect a significant number of Treatment and control. No reliable animals. The most susceptible age groups experimental studies are available. are goslings between 3 weeks and 3 Anecdotal reports suggest sulfonamides months old, in which mortality can reach as being effective. Keeping goslings high values. The prepatent period is 5-6 separated from the adults reduces the days. infective pressure.

Pathogenesis. Development of the various stages in the epithelial cell of the renal tubules is responsible for desquamation followed by the presence

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2.4.2 Cryptosporidiidae 2.4.2.1 Cryptosporidiosis

Introduction. The family includes a Introduction. Cryptosporidiosis is a single genus, genus Cryptosporidium, with widespread zoonotic parasitic infection 20 species parasitic in all vertebrate of all vertebrate groups, produced by classes. small apicomplexan parasites from genus Cryptosporidium. It affects mainly very The name (Greek: kryptos = hidden; spora young or immunosuppressed individuals, = seed) suggests its very small size and producing severe diarrhea and the difficulty in detecting it. dehydration. General morphology. The morphology Historical notes. The first observations is discussed in more detail in the of these parasites came in 1907 by corresponding section from Chapter Tyzzer, when he described 2.4.2.1. Cryptosporidium muris from mice. Ecology and transmission. All species The first association of Cryptosporidium are primarily parasitic in the digestive with clinical cases of diarrhea in turkeys tract of all vertebrates groups, but some has been reported in 1955 and in cattle may inhabit also other organ systems came only more than half century after its (e.g. respiratory). discovery, in 1971. The first human cases They have a homoxenous life cycle and were reported in 1976 and two years are transmitted from host to host via later, Cryptosporidium parvum was fecal-oral contamination. designated as a zoonotic species. In 2004, Medical importance. Species of genus the complete genome of a couple of Cryptosporidium are distributed Cryptosporidium species was published. worldwide and are able to produce Etiology. Although the taxonomy of clinical infection in a large variety of genus Cryptosporidium is still debated, hosts. Some species are host specific, generally 20 named species are currently some others not. recognized (valid) (table 2.32). In general, there is a relative host class Additionally, there are several other specificity. This means that species genotypes which do not have assigned a parasitic in reptiles are not able for specific epithet. instance to infect birds or mammals. A new genus (Piscicrytposporidium) was On the other hand, some species seem to erected to designated two species lack host specificity and infect a wide parasitic in fish: P. cichlidis and P. range of species within a class. One reichenbachklinkei. prominent example is Cryptosporidium Morphology. The number of studies on parvum which has been reported from the morphology of various species of more than 150 mammal species. Cryptosporidium produced huge amount of information.

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Table 2.32 The species of genus environment by infected hosts (figure Cryptosporidium 2.58 - 1). Infection of the host takes place

Species Hosts by ingestion of these sporulated oocysts C. molnari fish (figure 2.58 - 2). Immediately after C. scophthalmi fish C. fragile amphibians ingestion by a suitable host, the oocysts C. serpentis reptiles will start excystation (figure 2.58 - 3) and C. varanii reptiles the free motile sporozoites approach the C. meleagridis birds, mammals C. bailey birds apical end of the potential host-cells C. galli birds (figure 2.58 - 4) and invade them (figure C. andersoni cattle, camels, humans 2.58 - 5). After attachment, the C. bovis cattle sporozoites become oval or spherical and C. canis canids, humans C. fayeri marsupials, sheep vacuoles form inside them (figure 2.58 - C. felis cats, humans 6). This stage is called trophozoite. The C. hominis humans, cattle, goats C. macropodum marsupials newly formed parasitophorous vacuoles C. muris rodents, camels, are located intracellularly in the host cell, goats, primates, pigs, but as they are not in direct contact with dogs C. parvum various mammals its cytoplasm their location is called C. ryanae cattle epicellular. The next stage is the C. suis pigs, cattle, humans C. wrairii guinea pigs merogony, which is the phase of intense asexual multiplication. This phase is different in the various species of Nevertheless, the identification of species Cryptosporidium. In general, the nucleus is more routinely based nowadays on of the trophozoite will divide several molecular data. times (figure 2.58 - 7), resulting in The most important stage from multinucleated structures called meronts. diagnostic point of view is the oocyst. In Each nucleus will eventually be general, oocysts are very small (2-6 µm in incorporated in the structure of the diameter). The shape is usually spherical. forming merozoites (figure 2.58 - 8). Under direct light, they are highly The mature merozoites will leave the refractile. Each sporulated oocyst surface of the infected host cells (figure contains 4 free, slender or fusiform 2.58 - 9) and will re-infect other cells sporozoites. Unlike most other coccidian (figure 2.58 - 10). The number of parasites (Eimeria, Isospora, Sarcocystis, merozoites per meronts is different from Toxoplasma etc.) oocysts of species to species, but it is usually eight. Cryptosporidium lack a sporocyst. These meronts are called type I meronts. Life cycle is homoxenous (figure 2.58), The last generations of meronts (type II) typically with three phases: merogony, always contain only four merozoites and gametogony and sporogony. The only these will be responsible for the initiation exogenous stages known are sporulated of the next phase of the life cycle, the oocysts, which are eliminated to the gametogony (figure 2.58 - 11).

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Figure 2.58 Life cycle of Cryptosporidium. For the meaning of numbers, please refer to the text.

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Hence, the last generation merozoites days for calves infected with C. bovis, 2-9 will differentiate into female days in pigs infected with C. suis and 5-6 macrogamonts (syn. macrogametocytes) days for cats infected with C. felis. (figure 2.58 - 12) and male microgamonts The sources of infection are oocysts from (syn. microgametocytes) (figure 2.58 - the environment and the route of 13). Microgamonts will develop into infections is ingestion. Oocysts of microgametes and will penetrate the Cryptosporidium are sporulated when female gametes (figure 2.58 - 14) in order they are eliminated so they are to produce fertilization with formation of immediately infective. The sources of the egg cell (zygote) (figure 2.58 - 15) and environmental pollution are infected ultimately unsporulated oocysts (figure animals (wild or domestic) or humans. 2.58 - 16). Farm husbandry practices which enhance Oocyst will sporulate in situ (figure 2.58 - the transmission cycles include shared 17), hence in Cryptosporidium the feeding of neonates with older animals sporulation is typically endogenous. This not necessarily conspecific. Improper important from epidemiological point of disposal of manure and other fecal view, as the oocysts from the feces of wastes is contaminating water the infected animals will be immediately sources. Raw sea food (i.e. oysters, clams) infectious to new hosts. Sporulated have been incriminated is several human oocysts are released from the host-cell outbreaks. (figure 2.58 - 18) and are eliminated in the feces (figure 2.58 - 1). Susceptibility is higher in very young animals and decreases with the age. Some sporulated oocysts might excyst Immunodeficient individuals are before being eliminated in the feces, and particularly sensitive. Calves are sensitive the released sporozoites are responsible between 1 and 4 weeks of age for the for autoinfection (figure 2.58 - 19). infection with intestinal species. Cryptosporidium species are responsible Cryptosporidium andersoni infects only for the infection of various parts of the cattle older than 5 months. In sheep and digestive system. In mammals, some goats, young animals can be infected and species inhabit the distal small intestine, seriously affected from the first days of cecum and colon. Cryptosporidium life. In pigs, the highest susceptibility is andersoni is found in the digestive glands between weaning time up to the age of 2 of the abomasum. In birds, some species months. In horses, the susceptible age are able to infect also the respiratory group is 5-8 weeks. system, conjunctival mucosa or the bursa of Fabricius. Cryptosporidium oocysts are relatively resistant in the environment. At optimal Epidemiology. The distribution of temperatures (5-15°C) and humidity they Cryptosporidium in domestic animals is remain infective for 6 months. Deep global. Prepatent periods are 2-7 days in freezing destroys them in 24 hours. calves infected with C. parvum, 10-12

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Pathogenesis. Unlike other Immunology. The role of immunity is apicomplexans, Cryptosporidium is not an very important in the case of intracellular parasite. They are typically cryptosporidiosis. It is very evident that located on the epithelial surface (figure immunocompromised hosts are severely 2.59), and their pathogenicity is related affected by the infection. In humans with to the villous atrophy (loss and AIDS, cryptosporidiosis can be a shortening of microvilli) or even potentially lethal complication. The first detachment of enterocytes. This results in line of defense is the innate immunity. diarrhea, intestinal malabsorption and Toll-like receptors on the intestinal hypersecretion of chloride and water. epithelial cells are enabling the pathogen After attachment to the cell, C. parvum is recognition. In the case of C. parvum, the able to use the host cell’s membrane recognition activates the nuclear factor transport systems for its own metabolic kappa-light-chain-enhancer of activated processes. Abomasal infection with C. B cells pathway and expression of pro- andersoni causes inhibition of proteolytic inflammatory cytokines. Interferon-γ and enzymes. Moreover, the epicellular natural killer (NK) cells have a significant location (between the cell membrane and effect against C. parvum. The phagocytic cell cytoplasm) makes them very system also plays an important role in refractory to chemotherapy. Significant cryptosporidiosis. The production of the part of the pathogenetic process is free radicals of the nitric oxide by dependent on the host’s immune system neutrophils and macrophages is and will be detailed in the section on increased during the infection with C. immunity. parvum. The complement system is activated by C. parvum on the classical

and lectin pathways.

The acquired immunity plays crucial role in the defense against the parasite but also in the pathogenesis. T lymphocytes are mediating the microvillus atrophy.

Clinical signs. The most important symptom in clinical cryptosporidiosis is diarrhea in unweaned animals, mainly livestock (calves, lambs, goat kids). In adult animals the infection is regularly asymptomatic, while in very young ones the diarrhea is severe (scouring). Mortality is uncommon and it if is Figure 2.59 Oocysts of Cryptosporidium present, it is usually because of parvum covering the intestinal coinfections with other pathogens. epithelium in a naturally-infected goat Infected calves may cause various kid. (Photo Alexandru Bejan) associated symptoms: dehydration,

127 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | anorexia, dullness, weight loss and even The main lesion in calves infected with C. fever. parvum is enteritis in the small intestine, atrophy of villi with the presence of Cryptosporidiosis of pigs is a rare various developmental stages on the condition. The clinical signs described surface of the epithelial cells. Histological after experimental infections are: lesions consist of cellular infiltrates in inappetence, depression, vomiting, lamina propria and hyperplasia of diarrhea. Clinical cryptosporidiosis has epithelial cells of the intestinal crypts. been reported also in other livestock, but The infection with C. andersoni invades rarely. the peptic and pyloric glands in the In dogs and cats the clinical infection is stomach, producing their dilatation with rare, but even inapparent infections pose hypertrophy of the gastric mucosa. an increased zoonotic risk. Clinical Similar intestinal lesions are found in symptoms in pets include: diarrhea, small ruminants and pigs. In dogs and anorexia, weight loss, tenesmus, etc. Cats cats, lesions can be found also in the large infected with FeLV or FIV are more prone intestine. to develop clinical cryptosporidiosis. In Diagnosis. Clinical signs of diarrhea in horses the infection is relatively common young animals is an indication for but rarely causes serious problems. cryptosporidiosis, mainly if the usual In birds, the infection with C. baileyi is treatments are not efficient. Diagnosis rarely responsible for digestive signs. should be based on the identification of More commonly it produces respiratory oocysts in the feces of animals. As oocysts symptoms: sneezing, coughing, are very small, their detection in feces is orthopneic position. The respiratory not always an easy task. Regular signs can last up to 4 weeks. coproscopic methods (i.e. salt flotation) Cryptosporidium meleagridis infects the are not very sensitive. Moreover, when ileum of turkeys and other birds the number of oocysts in the feces is very producing severe diarrhea. low their detection is even harder. Cryptosporidium galli infects the There are three main method of choice proventriculus and the clinical infection for the detection of Cryptosporidium in in chicken (even adults) results in puffed the feces, or other sample types. plumage and decreased growth. (1) Direct detection (visualization) of In quails, unnamed Cryptosporidium oocysts in the feces is based either on species produce similar digestive and their concentration from fecal material, respiratory signs. or special staining methods. The most Pathology. Lesions are located at the site commonly used concentration methods of infection. Usually no other systemic are: sucrose flotation or formalin-ether lesions are found, except situations method. For stools which have large fat where bacterial or viral pathogens contents, the formalin-ether method is complicate the diseases. recommended. As oocysts are very small and conventional light microscopy does

128 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | not allow accurate identification, staining Usually animals recover if only given methods have been developed. The most symptomatic treatment (i.e. preventing commonly used staining method for fecal dehydration and correcting electrolyte smears is the Ziehl-Neelsen modified by balance). The minimum duration of Henriksen and Pohlenz. Using this chemical treatment should be at least 5 method, the oocysts appear bright red on days. a green background (figure 2.60).

Table 2.33 Drugs used in the treatment of cryptosporidiosis in domestic animals (after Stockdale et al., 2008; Scorza and Lappin, 2012)

Drug Animal Dose (/kg) Paromomycin calf 25-100 mg goat kid 100 mg dogs, cats 125-165 mg Halofuginone calf 30-500 µm lactate Lasalocide calf 6-15 mg Sulfadimidine calf 200 mg Sulfaquinoxaline goat kid 100 mg β-Cyclodextrin lamb 500 mg Nitazoxanide dogs, cats 100 mg Azithromycin dogs 5-10 mg cats 7-15 mg

Figure 2.60 Oocysts of Cryptosporidium In birds, anticoccidial drugs can be used, sp. in a fecal smear as they appear at Ziehl but with low moderate success. Neelsen modified by Henriksen and Control. Controlling the disease is not Pohlenz method. (Photo Andrei D. easy. Although there are some Mihalca) chemoprophylaxis protocols, they are rarely used. General hygiene methods are recommended. As infection is potentially (2) Immunological methods include dangerous during the first days of life, the direct immunofluorescence, ELISA or maternity area of the farms should be other immunochromatographic methods, kept as clean as possible. Colostrum must all used to identify antigens in fecal be fed to newborns, mainly in calves. In material. ELISA tests are available also farms where imminent risk exists (i.e. for detection of serum antibodies. history of recent cryptosporidiosis) (3) Detection of DNA are highly sensitive newborn calves must be kept separated, and they are used mainly for genotyping. with no calf-to-calf contact during the first two weeks of life. Sick animals with Treatment. Various chemical diarrhea must be isolated from the compounds are used for the treatment of healthy ones. All in-all out system with cryptosporidiosis in animals (table 2.33) thorough disinfection between animal but their efficacy is often moderate. series must be strictly followed. As host

129 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | barrier for some species is low, the farm hosts are usually carnivorous species must be kept free of free ranging dogs, which acquire the infection after cats and rodents. Water sources must be ingesting infected intermediate hosts. In clean. the definitive host, the gametogony typically takes place in the intestinal As zoonotic risk is high, rules and epithelial cells. regulations for waste management must be strictly followed. The differentiation between the two subfamilies is based on the nature of

stages found in the intestine of their 2.4.3 Sarcocystidae definitive hosts and the type of cysts from the tissues of intermediate hosts. In Introduction. Family Sarcocystidae subfamily Sarcocystinae gametogony and includes several genera of great sporogony are both endogenous while in veterinary and public health importance. Toxoplasmatinae the sporogony is The family currently includes two exogenous. subfamilies: Sarcocystinae (with genera The infective stage for the intermediate Sarcocystis, ) and host is the sporulated oocyst. After it is Toxoplasmatinae (with genera ingestion, the sporozoites infect various Toxoplasma, Neospora, Hammondia, tissues and rapidly produce the first Besnoitia and others). generation of merozoites known as General morphology. All species have tachyzoites (Greek: tachy- = swift, fast, Isospora-like oocysts. This means, that speed). They subsequently infect other sporulated oocysts contain two tissues of the intermediate hosts and sporocysts, each with four sporozoites. continue merogony by a slow asexual Members of genera Toxoplasma, multiplication resulting in the production Neospora, Besnoitia and Hammondia have of later generations of merozoites known relatively small oocysts. Their as bradyzoites (Greek: brady- = slow). differentiation by morphological criteria They typically remain in this stage is virtually unachievable. Except oocysts, (known as tissue cysts) until a new the morphology of the other suitable definitive host preys on the developmental stages is also important infected intermediate host. In the for diagnosis purposes, as some of them intestine of the definitive host, the are commonly found in various tissues of “zoites” (tachyzoites or bradyzoites) are their intermediate hosts. released and invade the enterocytes Ecology and transmission. All species where they finish their merogonic are intracellular parasite in various development and undergo gametogony tissues and organs of animal and human with the formation of unsporulated hosts. They are parasitic in a great variety oocysts. In genera Toxoplasma, Neospora, of vertebrate species, including Besnoitia and Hammondia, the amphibians, reptiles, birds and mammals. unsporulated oocysts are shed with the Life cycle is heteroxenous. Definitive feces and they undergo sporulation in the

130 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | environment, becoming infective for a skeletal muscle of their intermediate new intermediate host. hosts and the intestinal epithelium of the definitive host with little clinical impact. In genus Sarcocystis, gametogony is However, their zoonotic transmission followed by endogenous (intraintestinal) confers them a public health importance. sporogony with the formation of typical sporulate Isospora-like oocysts. The Historical notes. In 1843, Miescher oocyst wall of Sarcocystis is very thin and found some white thread-like structure in fragile and it ruptures releasing the the skeletal muscle of house mice. He did sporocysts. The definitive hosts in this not realize they are parasites, and for case shed through their feces the more than 20 years, they were known as infective sporocysts (each of them with the “Meischer’s tubules”. Similar four sporozoites). structures were found in the muscle of domestic pigs in 1865 and described as For some genera (i.e. Toxoplasma), other Synchytrium miescheriana by Kühn. In complex transmission mechanisms are 1892, Lankester erected the genus described, like for instance the Sarcocystis and in 1899, Labbé transmission from an intermediate host transferred the species earlier described to another intermediate host, without the in pigs to this new genus and named it presence of definitive hosts. The life cycle Sarcocystis miescheriana. In the beginning for each genus will be detailed in the they were considered to be fungi. Their following chapters. correct taxonomic position was Medical importance. The members of established in 1967. The heteroxenous Sarcocystidae are important human and life cycle was discovered only in 1970. animal pathogens. Some of them are First clinical cases of equine protozoal responsible for systemic disease myeloencephalitis were described in (Toxoplasma, ), others 1970, but its etiology was clarified only in produce mild intestinal infections 1974, when Dubey and his team (Hammondia, Sarcocystis) or concluded the agent was a Sarcocystis reproductive and congenital disorders species. The name S. neurona was (Toxoplasma, Neospora). Toxoplasma proposed in 1991 by the same American gondii and certain species of genus author, Dubey. Its life cycle was Sarcocystis are of zoonotic importance. elucidated only at the end of the 1990s.

Etiology. The diversity of species parasitic in domestic animals is shown in 2.4.3.1 Sarcocystoses tables 2.34 (dogs as definitive hosts), 2.35 (cats as definitive hosts), 2.36 Introduction. Sarcocystoses are (humans as definitive hosts) and 2.37 extremely common parasitic infections (wild or unknown definitive hosts). produced by member of genus Sarcocystis Morphology. Two developmental stages (Greek: sarx- = meat, flesh; kystis = are important from morphologic point of bladder, pouch). They generally infect the view.

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Table 2.34 Species of genus Sarcocystis with Table 2.37 Species of genus Sarcocystis with domestic dogs as definitive host domestic animals as intermediate hosts and wild or unknown definitive hosts Species Intermediate host Species Intermediate S. bertrami, S. fayeri Equids host S. cruzi, S. levinei Cattle S. neurona Horse S. arieticanis, S. micros, S. Sheep S. novaki Cattle mihoensis, S. tenella S. ippeni Dromedary S. capracanis, S. hircicanis Goats S. asinus Donkeys S. miescheriana Swine S. canis Dog S. alceslatrans, S. Cervids S. felis Cats, Dogs capreolicanis, S. gracilis, S. cervicanis, S. sybillensis, S. wapiti, S. grueneri, S. In the intestine and feces of the definitive hemionilatrantis, S. odocoileocanis hosts (carnivorous mammals) two S. aucheniae Llamas, Alpaca developmental stages can be found: S. cameli Camels S. poephagicanis Yaks oocysts and sporocysts. S. baibacinacanis Squirrels S. erdmanae Skunks The sporulated oocyst contains two S. wenzeli Chicken sporocysts, each of them with four S. peckai Pheasants sporozoites (“Isospora”-like). The oocysts

are thin-walled and they normally Table 2.35 Species of genus Sarcocystis with rupture while still inside the intestine domestic cats as definitive host and release the two sporocysts. Species Intermediate Sporocysts (figure 2.61) found in feces host S. buffalonis, S. fusiformis, Cattle are small stages, and their morphology is S. hirsuta relatively similar, regardless the species. S. gigantea, S. medusiformis Sheep They contain four sporozoites and well- S. moulei Goats S. porcifelis Swine visible sporocyst residuum. S. cuniculorum, S. leporum Rabbits S. muris, S. rodentifelis, S. Rodents The size of sporocyst found in the feces is neotomafelis, S. cymruensis variable from species to species (table S. odoi Cervids S. wenzeli Chicken 2.38).

The stages found in the muscle of the Table 2.36 Species of genus Sarcocystis with intermediate hosts are called muscular humans as definitive host and domestic animals as intermediate hosts cysts or sarcocysts. Biologically they are the last generation of meronts. Their size, Species Intermediate shape and location is variable, but as a host S. hominis Cattle rule all are whitish, cyst-like structures S. suihominis Swine located in the skeletal muscles of various

animals (figure 2.62). A practical These are the stages in the striated classification based on their size classifies muscles of intermediate hosts and the them in macrocysts (visible with the stage in the feces of the definitive hosts. naked eye) and microcysts (visible only under the microscope) (figure 2.63).

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Figure 2.62 Muscular cysts of Sarcocystis sp. from a naturally infected pig are visible during meat inspection. (Photo Andrei D. Mihalca)

Figure 2.61 Sporocyst of Sarcocystis sp.

Table 2.38 Sporocyst size of selected species of Sarcocystis found in feces of dogs, cats and humans

Species Size (µm) S. cruzi 10.8 x 16.3 S. hominis 9.3-14.7 S. tenella 9.9 x 14.8 S. fusiformis 7.8 x 12.5 S. gigantea 8.1 x 12.4 S. miescheriana 9.6 x 12.6 S. suihominis 10.5-13.5 S. bertrami 10.0 x 15.2 S. neurona 10 x 8 Figure 2.63 Microcysts of Sarcocystis sp. are visible only under the microscope. Sometimes, sarcocysts are very long and (Photo Andrei D. Mihalca) thin, like for instance certain species found in rodents (figure 2.64).Internally, muscular cysts contain variable number Meronts and S. cruzi (dog-cattle cycle) of bradyzoites (figure 2.65). from the endothelial cells are very small, The sarcocysts are usually located 2-8 µm. The cyst in the muscle is fairly longitudinally, along the length and in large (0.5-5 mm) and often visible with between of the muscle fibers. the naked eye.

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bigger (37 x 22 µm with 100 tachyzoites first generation meronts; 14 x 6.5 µm with 35 tachyzoites the second generation meronts). Muscular cysts of Sarcocystis hominis (human-cattle cycle) are microscopic, with a thin wall (6 µm) with radial striations.

First generation meronts of S. tenella (dog-sheep cycle) are 19-29 x 7.5-24 µm, with 120-280 tachyzoites. The muscular cysts are relatively small (0.1-0.6 mm), have a thick and radially striated wall.

Endothelial meronts of S. gigantea (cat- Figure 2.64 In some hosts, the muscular sheep cycle) are small (2-8 µm) but their cysts of Sarcocystis are very thin and long, muscular cyst is very large (hence the like in this laboratory mouse. (Photo name) and measure up to 1.5 x 0.5 cm. Andrei D. Mihalca) Sarcocystis capracanis (dog-goat cycle) develops small muscular cysts (130-800 x 50-70 µm) with a thick (2.5 µm) and radially striated wall. On the other hand, the second species with dog-goat cycle, S. hircicanis produces larger muscular cysts, of around 2.5 µm but with thinner and smooth wall. The muscular cysts of S. moulei (cat-goat cycle) are even bigger (12 mm) with a thick and striated wall.

Muscular cysts of S. miescheriana (dog- pig cycle) are large, 0.5-2.2 mm x 160- 260 µm. In S. suihominis the muscular cysts are thin-walled (4-9 µm) and visible with the naked eye (1.5 mm).

Figure 2.65 Content of a squashed In S. bertrami (dog-horse cycle), the muscular cyst of Sarcocystis reveals the muscular cysts are large, up to 10 mm in presence of the sausage-like bradyzoites size, with a smooth and very thin wall. (Photo Andrei D. Mihalca) Sarcocystis fayeri (dog-horse cycle) has smaller muscular cysts (0.9 mm x 70 µm) and a thin, striated cyst wall. Sarcocystis The cyst wall is thin and smooth. neurona develops small meronts (5-20 x Muscular cysts of S. hirsuta (cat-cattle 4-40 µm) in various tissues of naturally cycle) are relatively big (8 mm) and have or experimentally infected hosts a striated wall. Endothelial meronts are

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Life cycle. The life cycle of Sarcocystis is hours after the infection. They fuse heteroxenous (figure 2.66). Each species (figure 2.66 - 7) resulting in the sexual is relatively host specific. formation of the zygote (figure 2.66 - 8). Each zygote will become on oocysts The definitive hosts (dogs, cats, humans, (figure 2.66 - 9). wild carnivores) eliminate through their feces the already infective sporocysts. If The oocysts will sporulate in the intestine they are ingested by a suitable of the host (figure 2.66 - 10) with the intermediate hosts (figure 2.66 - 1), in formation of two sporocysts each with their intestine the sporocyst wall four sporozoites. The very fine oocyst ruptures and the free sporozoites migrate wall ruptures (figure 2.66 - 11) and the through the epithelium of the gut and two sporocysts are freed in the intestinal invade the endothelial cell of blood lumen. They are subsequently eliminated vessels in various internal organs (figure in the environment together with the 2.66 - 2) host’s feces (figure 2.66 - 12).

Sarcocystis miescheriana will be used as Sarcocystis neurona is one of the most example in this section. Inside the interesting species of the genus. Its endothelial cells, they undergo usually natural life cycle includes two species of two merogonic developments. opossums (Didelphis virginiana and D. albiventris) in North and South America. The first merogony (figure 2.66 - 3) takes The natural intermediate host is not place at 5-6 days. The first generation of known, but the infection was found in a merozoites (tachyzoites) invade other great variety of mammal and bird species. endothelial cells (figure 2.66 - 4) and Among domestic animals, the natural undergo the second merogony at 12-17 infection was reported in horses, cats, days after infection. and dogs. All are however considered The second generation of merozoites aberrant (accidental) intermediate hosts. (tachyzoites) travels via the blood stream The life cycle is not yet fully understood. and when they reach the striated muscles Asexual merogonic stages develop in the they undergo the final merogony and central nervous system (brain and spinal become tissue (muscular) cysts with cord) of horses and other intermediate bradyzoites (figure 2.66 - 5). They remain hosts. A single neuron can harbor as in this stage for a long time, until the many as 13 meronts with altogether hosts or its flesh (raw meat) are several hundred merozoites. In consumed by a suitable carnivorous experimental infection in cats, cysts definitive host (figure 2.66 - 6). developed also in the skeletal muscles. After being ingested by the definitive Epidemiology. All species parasitic with host, muscular cysts are broken, the domestic life cycles (both, the definitive bradyzoites invade intestinal epithelial host and the intermediate host are cell and start the gametogonic domestic animals) have a global development with the formation of distribution. micro- and macrogametes as early as 14

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Figure 2.66 Life cycle of genus Sarcocystis. For the meaning of numbers, please refer to the text.

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Sarcocystis neurona has been reported encephalomyelitis. In pregnant females, only in the Americas, its distribution heavy infections with certain species (S. being determined by its strict specificity cruzi, S. tenella) are able to induce to the definitive hosts. abortions. Sarcocystis suihominis develops in the endothelial cells of blood The data regarding prevalence of vessels from the liver. Sever muscular infection in intermediate hosts is very destruction is responsible for gait and abundant in the literature. However, myalgia. most studies did not attempt to identify the species; hence their zoonotic Sarcocystis neurona was found in potential is not fully known. The neurons, mononuclear cells and glial cells prevalence of muscular cysts in but not peripheral nerves. Although not slaughtered cattle is very high in certain too many information are available on areas, but the most common species is S. the migration route, experimental cruzi which is infective for dogs. infection in ponies by Elitsur et al. (2007) Intestinal sarcocystosis in humans is the revealed the presence of S. neurona in most common in Europe (between 7.3 lymph nodes, liver and lungs. At 9-21 and 10.4%). The main source of infection days after the infection, parasites reach for humans is raw meat from cattle and the central nervous system and infect pigs. various parts: cerebellum (with depression, seizures, behavioral In the case of S. neurona, the disease is changes), brainstem and spinal cord most commonly reported in racing (abnormal gait) or damage of the cranial animals aged between 3 and 6 years. In nerve nuclei (various paralysis). USA, the seroprevalence of anti-S. neurona is between 30 and 50%. The intestinal development of gametogonic and sporogonic stages of Resistance of sporocysts is high. Sarcocystis in the definitive host is usually Sporocysts of S. cruzi remain infective if non-pathogenic. The main difference kept at -22°C for 10 days or at 56°C for 10 from the severe impact of Eimeria and minutes. Sporocysts of S. miescheriana Isospora on the intestinal mucosa are destroyed in the meat after 20 consists in the different phase of the life minutes at 60°C, after 15 minutes at 70°C cycle taking place here. In Eimeria and and in 5 minutes at 100°C. Freezing does Isospora, the repeated merogony is not inactivate sarcocysts. responsible for the epithelial destruction, Pathogenesis. In the intermediate hosts while in Sarcocystis only the gametogony the most important pathogenic effect is and sporogony take place here, both of caused by the first and the second them being non-repetitive. merogonic development which takes Immunology. In intermediate hosts, the place in the vascular endothelial cells. In immune reaction during the muscular certain species (i.e. S. tenella in sheep), phase of the life cycle is predominantly merogonic development can be also cellular. In time, the organism is able to responsible for severe myositis eliminate the infection. Definitive hosts

137 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | are easily susceptible to reinfections, Sarcocystis miescheriana produces demonstrating a decreased immune enteritis with diarrhea, myositis with response. impaired movement and lameness, fever, anorexia and weight loss. Infections wirh Clinical signs. With the exception of S. S. suihominis can be acute, with two fever neurona, sarcocystoses are benign peaks (days 5-9 and 11-15), apathy, muscular infections in intermediate hosts dyspnea, anemia, cyanosis, muscles and asymptomatic or light intestinal spasms and tremors, hyperexcitability, infections in their definitive host. Clinical, prostration and abortion. signs are very rare, and they are reported mostly after very high infective doses. In horses, two forms of sarcocystoses The pathogenicity and symptoms in such have been described. As in all other cases are variable from species to species. domestic animals, horses usually develop asymptomatic muscular sarcocystosis In cattle heavily infected with S. cruzi (with S. bertrami or S. fayeri, both from anorexia, fever, weight loss, anemia and dogs). However, S. fayeri may be difficulty in movements have been responsible for myalgia (associated with reported. Loss of hair on the tip of the myositis). tail, submandibular edema, exophthalmia, lymphadenopathy and The second type of sarcocystosis in horse abortion are also possible. This particular is the very severe infection with S. condition has been known also as the neurona causing a distinct parasitic Dalmeny disease. Infection of cattle with disease known as equine protozoal the species of cat-origin (S. hirsuta) is myeloencephalitis. This is a debilitating usually asymptomatic, but heavy disease, with progressive clinical infective disease may induce anorexia, evolution, responsible for a variety of weight loss, anemia, fever and even nervous signs. The disease may be acute diarrhea. Sarcocystis hominis infection is or chronic. In general, the first observed asymptomatic. symptoms are lameness which gradually increases in intensity leading to ataxia. Massive infection with S. tenella in sheep One of the most typical clinical signs are can be responsible for anorexia, loss of the asymmetrical gait disorders with weight, fever, anemia, recumbency and focal muscular atrophy. Some horses abortions in pregnant ewes. The cat- have abnormal upper respiratory origin species parasitic in sheep (S. function, difficulties in standing or gigantea, S. medusiformis) are rarely walking, dysphagia. Neurologic responsible for clinical infections. In examination reveals asymmetric signs of goats, S. capracanis and S. hircicanis can weakness, spasticity, hypoalgesia or produce similar clinical signs, including complete loss of sensitivity. If the abortions. Infection with S. hircifelis is function of cranial nerves is affected asymptomatic. horses have tilted head and facial nerve From the three species parasitic in pigs, paralysis. During all the course of the only two seem to be pathogenic. disease, the animals are bright and alert.

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Affected horses usually die during the inflammatory cells, mainly macrophages acute stage of the disease. Dogs and cats and lymphocytes. Older cysts are can be also infected with S. neurona and surrounded by thick capsules and may they develop similar signs of fatal degenerate. Muscular cysts of S. hirsuta myeloencephalitis. are easily visible and are located mainly in the striated muscle fibers from the In dogs infected with S. canis the parasite esophagus. produces a systemic disease involving the central nervous system and liver necrosis.

In the definitive hosts, heavy infections are responsible for mild diarrhea. Humans infected with S. miescheriana after eating raw infected pig meat can develop bloat, diarrhea, stomach ache, nausea, vomiting or loss of appetite. In dogs and cats with intestinal sarcocystosis, the infection is normally asymptomatic. However, the author of this book has found sporocysts in dogs with diarrhea.

Pathology. In the intermediate hosts, the merogonic stages produce lesions of Figure 2.67 Histological aspect of a different degrees, dependent mainly on muscular cyst of Sarcocystis. Bradyzoites the infective dose. The most striking are clearly visible. (Photo Andrei D. aspect during the gross necropsy is the Mihalca) disseminated presence of white nodules in the skeletal muscles, in the striated muscle of the esophagus in ruminants In sheep, goats and pigs, the lesions are and even in the cardiac muscle. Histology similar to those described in cattle. Cysts reveals the presence of intramuscular of some species (S. tenella, S. capracanis, parasitic cysts (figure 2.67) associated S. hircicanis) can be found also in the with myositis, myodystrophy or cardiac muscles. myocarditis. Cellular inflammatory Diagnosis. The diagnosis in the definitive infiltration around the cyst can be host is based on the detection of present. sporocysts in the feces (figure 2.68) by In bovines, meronts of S. cruzi are flotation methods. Differentiation of responsible for destructions of the species based on sporocyst morphology endothelial lining of capillaries. The is not easy. muscular stage produces myositis, with Diagnosis of muscular cysticercosis in the parasitic cysts surrounded by intermediate hosts is usually post-

139 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | mortem, during the compulsory meat Diagnostic of equine protozoal inspection in slaughterhouses. The myeloencephalitis caused by S. neurona is detection of large white cysts in the done by serological confirmation striated or cardiac muscles has diagnostic (immunoblotting) of cases clinically value. corresponding to the disease. The presence of the parasite can be They have to be differentiated from other demonstrated also in the cerebrospinal cystic structure like for instance from the fluid by western blot. bladder-like cysts of “Cysticercus bovis” (the larval stage of Taenia saginata) in Treatment. Animals infected with cattle or “Cysticercus cellulosae” (the muscular sarcocysts are not usually larval stage of Taenia solium) in swine. treated, as the diagnosis is most However, the detection of microcysts is commonly done post-mortem. There are impossible when examining the meat by several therapeutic studies done in naked eye. In pigs microcysts can be experimental infections. In acute accidentally detected during the sarcocystosis of lambs, amprolium (50- microscopical examination of meat for 100 mg/kg) decreased the intensity of Trichinella. clinical signs. Salinomycin (1-2 mg/kg) and halofuginone (0.67 mg/kg) had the In vivo, the infection (asymptomatic or same protective value in sheep and goats. not) can be detected using serological Monensin was found to be effective for tools (immuno-fluorescence, ELISA) or by the treatment of acute bovine muscular correlating the clinical signs (if present) sarcocystosis. with increased serum levels of bilirubin, creatine phosphokinase and lactic acid. The treatment of equine protozoal myeloencephalitis is an emergency, and if

it is applied in time and correctly, the success rate is up to 75%. The most common therapeutic protocol for horses is the use of sulfadiazine (20 mg/kg, PO) once or twice per day combined with pyrimethamine (1 mg/kg, PO) once per day. The duration of treatment is long (84-120 days) and it must be discontinued only when the cerebrospinal fluid is negative by western blot. In the case that this treatment fails to yield good results, the drugs of choice are diclazuril, toltrazuril or nitazoxanide.

The treatment of intestinal sarcocystosis Figure 2.68 Sporocyst of Sarcocystis sp. in the definitive host is similar to the from the feces of a grey wolf. (Photo Călin treatment of other intestinal coccidiosis M. Gherman) (see Chapter 2.4.1.1).

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Control. The most important means for toxoplasmosis in humans was reported in prevention are the general hygiene 1938 in an infant girl who died at the age measures which interrupt the life cycle of of one month in New York. However, the parasite. Free-ranging dogs and cats previous reports of human congenital must be excluded from farms. The chorioretinitis and encephalomyelitis, infected meat should not be given to initially attributed to some other agents, carnivores. There is no specific were later shown to have been caused by prophylactic method yet, but vaccines are Toxoplasma. The first case of human being developed for immune-prophylaxis acquired toxoplasmosis has been of the infection with S. neurona in horses. identified in 1940. Nevertheless, first reports of animal toxoplasmosis date

back to 1910, when Mello described an 2.4.3.2 Toxoplasmosis acute case in a dog from Italy. Although recognized as a widespread and Introduction. Toxoplasmosis is one of sometimes severe zoonotic infection, the the most widely distributed parasitic full life-history and the role of the cats in infections on Earth, affecting humans and the biology and transmission of T. gondii animals as well. Its highly zoonotic was not fully understood until the 1970s, potential and the severity of infection in when Dubey and Frenkel described the certain host categories make it one of the entire developmental cycle. most intensively studies parasites. The Etiology. The only known species is most important aspect of toxoplasmosis Toxoplasma gondii. It has a worldwide is probably its congenital transmission, distribution and an immense host the subsequent clinical problems in spectrum. Virtually it can undergo its children and the resulting social impact. asexual development in any warm- Historical notes. The description of the blooded host, mammal or bird. parasite came in 1908, when Nicolle and The genetic analysis of various strains of Manceaux, two researchers from Pasteur Toxoplasma gondii from Europe and Institute in Tunis have noticed protozoan North America revealed the presence of stages in the tissues of a laboratory kept three major genotypes (I, II and III). rodent known as the common gundi, Subsequently, new genetic variants were Ctenodactylus gundi. Initially, they identified worldwide and they were misidentified the parasite as Leishmania classified into 12 haplogroups. All these but subsequently, in 1909, they described genetic variants differ in their it as a new species, Toxoplasma gondii. pathogenicity on various hosts. The generic name was given according to the morphology of the stages they found Morphology. Toxoplasma gondii is a very (Lat. toxo = arc or bow; plasma = life) and common parasite of humans and animals the specific epithet is an erroneous and its diagnosis in the definitive and spelling of the host’s name. The first case intermediate hosts is based on the of correctly diagnosed congenital detection of various developmental stages.

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Figure 2.69 Sporulated oocysts of Figure 2.70 Tissue cyst of Toxoplasma Toxoplasma gondii from the feces of a cat. gondii in the nervous system of an (Photo Jana Juránková) infected mouse. (Photo Jana Juránková)

The typical stage found in the intestine of cats as definitive hosts is the unsporulated oocysts. In older feces, the oocysts are sporulated and they are ”Isospora”-like (two sporocysts, each with four sporozoites) (figure 2.69).

The tachyzoites are crescent-shaped (Greek: toxon = arc or bow), 2-6 µm with pointed anterior and rounded posterior end. The nucleus is in central position. They are found intracellularly in almost any cell type, except red blood cells. The group of tachyzoites resulting after repeated multiplication by endodyogeny Figure 2.71 Tissue cyst of Toxoplasma inside a cell is also known as pseudocyst. gondii freed from its host tissue. Numerous bradyzoites are visible inside Bradyzoites (5-8.5 x 1-3 µm, posterior (Photo Břetislav Koudela) nucleus, more slender than tachyzoites) are found in tissue cysts. Tissue cysts are also intracellular. They multiply by Small tissue cysts (~5 µm) contain two endodyogeny within the tissue cysts bradyzoites while larger cysts (70-100 (figure 2.70). The size of the tissue cysts µm) contain thousands of them (figure is variable and it depends on the number 2.71). The shape and size of the tissue of contained bradyzoites. cysts depend also on the host tissue.

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Tissues in the brain are usually smaller (4) Diversity of infected tissues is great, (~70 µm) and round while those in the and as the parasite has systemic muscles for instance are larger (~100 distribution, virtually any organ can µm) and elongated. The tissue cyst wall is be infected and infective. However, elastic and thin (<0.5 µm). In older tissue they are more common in the brain, cysts, the wall may be hardly visible. eyes, skeletal muscles and myocardium. Life cycle. The biology of Toxoplasma gondii typically includes two obligatory (5) Infected pregnant females (including host, the cats and other felids as women) are able to pass the infection definitive hosts and rodents as natural to the fetus via transplacentary intermediate hosts (figure 2.72). In route. many aspects, this typical life cycle is Toxoplasma gondii is an obligatory similar to Sarcocystis spp. However, intracellular parasite, with tropism several particular biology aspects make mainly for nervous and muscular tissues. Toxoplasma gondii a unique parasite: However, it can be found occasionally in (1) Extremely broad host specificity for any other organs. It is usually located in its asexual stage (merogony). the cytoplasm of the infected cells, but Toxoplasma gondii can use virtually sometimes it can invade also the nucleus. any mammal and any bird for its Typically, intermediate hosts are infected merogonic development. after ingesting sporulated oocysts from (2) Natural transmission can be done the cat’s feces (figure 2.72 - 1). After directly from an intermediate host ingestion, the oocyst wall ruptures to another intermediate host, releasing the sporozoites which, after without the presence of the definitive passing through the intestinal wall will host. For instance, humans (which penetrate into various types of cells are intermediate host) can get the (macrophages, endothelial cells, infection after eating infected tissues fibroblasts etc.) (figure 2.72 - 2) where from livestock (also intermediate they multiply by endodyogeny (figure hosts). 2.72 - 3) producing the tachyzoites. Tachyzoites may rupture the host cell and (3) All “zoite” stages are infective to invade other cells (figure 2.72 - 4) or any susceptible host. Sporozoites migrate through the host’s body, reaching (from the feces of cat definitive various tissues (including the fetus) hosts), and tachyzoites or where they continue their multiplication bradyzoites (from the tissues of in a slower rate. The result is the intermediate hosts) are infective to formation of tissue cysts with bradyzoites any other intermediate host. Cats are (figure 2.72 - 5). If cats or other felids also susceptible to infection with any feed on tissue cysts originating from an of these stages, but depending on infected intermediate host (figure 2.72 - which stage is ingested, they act as B2), they become infected (2.72-6). intermediate or definitive hosts.

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Figure 2.72 Life cycle of Toxoplasma gondii. For the meaning of numbers and letters, please refer to the text.

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The tissue cyst wall is dissolved by oocysts will ultimately shed oocysts in proteolytic enzymes from the feline their feces. The prepatent period in cats stomach and intestine, realizing the is different according to the infection bradyzoites. The bradyzoites invade the source. After ingesting tissue cysts epithelial cells of the small intestine of (bradyzoites), cats shed oocysts after 3- cats and undergo repeated asexual 10 days. After being infected with oocysts multiplication (figure 2.72 - 7), passing or tachyzoites, the prepatent period is through multiple generations of meronts, longer (18 days). classified in five types (type A to E). After One of the most important life cycle this supplementary asexual merogony pathways is the possibility of takes place in the intestine, the sexual transmission form an intermediate host gametogonic development starts (figure to another intermediate host by 2.72 - 8) with the formation of the carnivorism (figure 2.72 - C). According zygotes (figure 2.72 - 9) and ultimately to numerous opinions, this is the most the unsporulated oocysts (figure 2.72 - common way of infection for humans (i.e. 10). Unsporulated oocysts are eliminated following eating raw or undercooked through the feline feces (figure 2.72 - 11) meat from infected animals). This intro the environment where they bradyzoites-induced cycle in the sporulate in 1-5 days (figure 2.72 - 12), intermediate host is following similar becoming infective. patterns to that of the oocyst-induced Except this typical heteroxenous life cycle infection. However, the infectivity of (figure 2.72 - A and B), there are several bradyzoites to intermediate hosts is other pathways which can be followed by lower than the infectivity of sporozoites. Toxoplasma gondii. If cats are infected This is why oocyst and their hosts (cats) following ingestion of tachyzoites (figure are an absolutely essential link in the 2.72 - B1) the life cycle in the cat is complex transmission chains of slower. It is considered that tachyzoites Toxoplasma gondii. are less acid-resistant than bradyzoites From clinical point of view, the most but they can be still infective. important contamination route is the If cats ingest oocysts (figure 2.72 - A1) vertical transmission of tachyzoites the course of infection is different. In this (figure 2.72 - D), mainly in humans. case, in the first stage the cat acts as an Various studies have shown that the intermediate host with the formation of transplacental infection occurs only if the tachyzoites and bradyzoites in the tissue. mother is primo-infected during the In some of the cats infected with oocysts, pregnancy. The chances of transplacental after almost three weeks, oocysts can be transmission are low if the women found again in the feces. The acquire the infection just before the hypothesized scenario is that some pregnancy or during older, pre-natal bradyzoites reach back the intestine and chronic infections. Nevertheless, in start gametogony in the enterocytes. immunosuppressed pregnant women However, not all cats infected with with chronic infection, transplacental

145 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infection is possible. Transplacental sporulation success at lower infection was also reported in domestic temperatures. animals. The risk of congenital infection The infections sources are various. The is lowest when maternal infection is in direct sources of infective oocysts are the first trimester (10–15%) and highest always cats. However, cat owners are not when infection occurs during the third more exposed to infective oocysts trimester (60–90%). If maternal infection compared to people who never had a cat, occurs early in pregnancy, it results in as oocysts from cat feces can contaminate fewer infected babies, but they are more fruits and vegetables. Dogs can also shed severely affected than the greater T. gondii oocysts following coprophagia number of infected babies born when of cat feces. Dogs which rolled over infection is acquired later in pregnancy. infected cat feces pose also a risk for The highest risk to the fetus is when humans. Defecation sites in public parks infection is acquired between the 10th are important and oocysts may be and 24th week of gestation. Fortunately mechanically carried on shoes. about 60–70% of babies born of infected mothers escape infection. Another source of infective oocysts is represented by wild felids which acquire In certain species (i.e. laboratory the infection from their wild prey. Hence, rodents) transplacental transmission the wildlife reservoirs can be locally occurs even if the infection is chronic and important also. took place before the pregnancy. In domestic animals (i.e. sheep), various The main sources of infection with observation suggest that the situation is Toxoplasma for humans are not the same similar to humans, and only tachyzoites everywhere. In the areas where cats are can pass transplacentally (i.e. infection abundant and live in close contact with must be during pregnancy). people the oocysts are probably the Transplacental transmission was not principal infection sources. In developed documented in all domestic species. countries, where cats are mostly indoors There are no confirmed reports from and they feed on commercial food, the dogs, horses or cattle. most probable source for human infection is raw or undercooked meat. Epidemiology. As stated before, Raw goat milk was reported as a source Toxoplasma gondii is probably the most of infection for humans. Raw cow milk or common parasite on Earth. It is present uncooked chicken eggs are not wherever cats are present which means considered dangerous for the everywhere where humans are present. transmission of Toxoplasma. Despite its global distribution, Toxoplasma is more common in warmer Other sources of infection are climates than in colder ones and in uncommon. Tachyzoites accidentally lowland compared to highlands. This is reaching the cornea during laboratory most probably related to the lower manipulation can induce the infection. survival of oocysts and their lower Transfusion of whole blood is not

146 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infective as the number of circulating less aversion to cat odor. This host- tachyzoites is very low and the period of manipulation makes infected mice easier parasitemia is low. However, transfusion preys to cats. of packed leukocytes can be a risk. Organ The values for the infection prevalence in transplants are also incriminated. The cats are variable. If we assess this presence of tachyzoites has been epidemiologic parameter on the basis of demonstrated in semen and saliva but no the presence of oocysts in the feces the venereal or salivary transmission were prevalence is very low (average less than reported. 1%). However, if we assess the The prevalence in humans is not prevalence of anti-Toxoplasma antibodies necessarily related to the prevalence in (seroprevalence) it can reach an cats, but rather to cultural habits. In astonishing 100% in certain cat nations where eating raw or population. This can be explained if we undercooked meat is a common practice consider the biology of T. gondii in its (i.e. France) the seroprevalence of definitive host, as the time for oocysts toxoplasmosis in humans is higher. elimination in cat’s feces is very short (1- Eating raw meat seems to be a more 2 weeks) and the number of oocysts can common habit in more developed be low, under the coproscopic detection countries. In third-world countries, meat threshold (<1000 oocysts per gram). is usually well cooked because of other Nevertheless, PCR detection of health risks. This explains the lower Toxoplasma DNA in feline feces can yield prevalence in humans from Africa and higher prevalences (up to 11%) and is Asia. The prevalence in humans is also able to differentiate between other small influenced by the species they most intestinal coccidia like Hammondia. commonly eat. In countries where Another very important epidemiological mutton is a common dish, the prevalence question is if cats shed oocysts more than of human toxoplasmosis is higher. Viable once in their lifetime or they acquire kind Toxoplasma cysts are commonly found in of immunity. Data from experimental pigs and sheep; in cattle, viable cysts are studies are controversial. Even though, very rare. Reported values for probably the number of oocysts seroprevalence in humans are very eliminated by cats during their first heterogeneous and they depend on infection is much higher than during various factors. The higher values have subsequent infection. More details are been reported in South America, and the given in the immunity section. lowest in Eastern Asia. Quantitative assessments found The prevalence of infection in cats is impressive number of oocysts in cats dependent on various factors, but the with primary infection (up to 13 million most important factor seems to be the oocysts per gram of feces). The average presence and availability of infected values are of course lower, but still rodents. Interestingly, mice infected with impressive (around 10 million oocysts T. gondii are less neophobic and show per cat).

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The seropositivity in cats increases with Seropositivity greatly depends on the the age and it is higher in feral cats than farming system. The infection is virtually in indoor cats. Prevalence of cats with absent from intensive farms and more tissue cysts is between 5 and 70% in common in free-ranging birds. various countries. Surprisingly, the resistance of T. gondii In sheep the infection was also found oocysts in the environment is not too well worldwide. Except cats, seroprevalence known under natural conditions. Under in sheep is among the highest from all experimental conditions they proved to domestic animals. In certain adult be relatively resistant. At -21°C in water populations, it can easily reach 95%. In they were not killed. They also resisted intensive farming system the 4.5 years in water at 4°C and more than 1 seroprevalence is lower than in semi- year in water at 22°C. Warmer aquatic intensive systems. Risk factors include medium kills them faster (1 month at mostly the age, presence of free-ranging 40°C; 2 minutes at 55°C; 1 minute at cats in the farm and history of abortions. 60°C). If kept in cat feces, they resist It is estimated that between 10 and 23% several years at temperature between 15 of abortions in sheep are caused by T. and 35°C. In dry condition they die faster gondii. Toxoplasmosis in goats is also (11 days in air at 11% relative humidity globally distributed, with variable and 2 days at 0% relative humidity). seroprevalence which is generally lower Unsporulated oocysts are generally more than in sheep. In cattle the global sensitive to environmental factors than seroprevalence is generally lower than in sporulated oocysts. Oocysts are also very sheep. resistant to disinfectants. Kept in 10% formalin they resist 48 hours. The 5% The same worldwide distribution was ammonium hydroxide kills them in 30 reported in pigs. The seroprevalence is minutes but not in 10 minutes. Iodine much higher in backyard and free-range tincture (2%) kills oocysts in 3 hours but pigs and almost absent in pigs from not in 10 minutes. Oocysts survive intensive farm system. There is also a standard water chlorination for at least clear age related distribution of 24 hours. seropositivity values. Adult saws show higher prevalences than market-age pigs. Tissue cyst resistance is a key epidemiologic factor. Treatment and In dogs the distribution is global. injection with salt of meat-based Seropositivity is variable and higher in products kills bradyzoites from tissue older dogs and in rural dogs. In general, cysts. Tissue cysts are sensitive to the seroprevalence in dog is high (up to conventional cooking. They are killed as almost 90%). In horses the the internal temperature is over 60°C. seroprevalence is generally low. Freezing also kills tissue cysts (at -12°C in Multiple seroprevalence studies are 1 day). In decomposing carcasses available for chicken, as their meat forms bradyzoites survive for several days. a significant part of human diet. Resistance of tachyzoites is very low

148 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | compared to the resistance of cattle). Other determinant factors for the bradyzoites or oocysts. pathogenicity are the route of infection, the source of infection and the parasite Pathogenesis. Despite the huge attention strain. Oocyst-induced infections are given to T. gondii, clinical infections are more severe that bradyzoites-induced rare. After the infection with “zoites” infections. Type I genotype is pathogenic (bradyzoites from tissue cysts, for mice, but not type II and III. In tachyzoites from pseudocysts, humans, the most pathogenic genotypes sporozoites from oocysts in cat feces), are I and III, but in France, severe cases they penetrate the intestinal wall and were reported also with type II. Certainly multiply locally in adjacent tissues the immune system of the host plays also including mesenteric lymph nodes. They a great role (see next section). cause here local necrosis. Similar necrotic lesions are produced in various other Parasitemia with tachyzoites during organs by the asexual development of pregnancy is a key factor for tachyzoites. Necrosis is the result of transplacental transmission. After intracellular development cellular death invading the placenta, they produce and not the result of toxins. No toxin has placental necrosis associated with been so far detected in T. gondii. The embryonic death, fetal resorption, initial necrosis may kill the host if it is mummification, abortion or stillbirth. If extensive enough and affects vital organs. the fetus survives, tachyzoites invading Otherwise, the necrosis is gradually the fetal tissue are able to cause necrotic replaced by local chronic inflammation lesions. As some cases of severe placental and the host develops immunity. necrosis did not result in abortions, also Tachyzoites usually disappear from the hormonal imbalances have been internal organs in three weeks after the incriminated in the pathogenesis of infection and afterwards, bradyzoites are toxoplasmic abortion. located in cysts mainly in the muscular Behavioral alterations in laboratory tissue (striated and cardiac) and central rodents and humans are linked to nervous system. perturbations in dopamine production. Tissue cysts remain arrested awaiting a Immunology. Immunity against carnivorous predator to prey on the host. Toxoplasma is very complex. It involves However, in certain situations (mainly both types of immunity, innate and related to a sudden decrease in acquired. The intracellular location immunity) the tissue cysts can rupture protects the parasite from the direct and the released bradyzoites invade contact with the host’s immune effectors. other tissues forming new cysts. The fact that clinical cases are rare Pathogenicity is dependent on various suggests that the immune system work factors. Certain host species (New World effectively against Toxoplasma in most of monkeys, Australian marsupials) are the cases. This is supported by the more susceptible to develop clinical signs extremely high and widespread than others (Old World cattle, horses, seroprevalence values reported in

149 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | various hosts worldwide. However, the oocysts formation is lost after several ubiquity of this parasite and also high years if no reinfections take place. An prevalence values for the presence of interesting immunologic interaction with infective viable tissue cysts suggest that other feline coccidia was reported in cats despite not being able to produce clinical with latent toxoplasmosis. When infected infection, Toxoplasma is capable of with Isospora felis, the bradyzoites from surviving in the host and remaining the cat’s tissue cyst become active again infective. and start to shed Toxoplasma oocysts in feces. On the other hand, another Cellular response is also very strong. coccidian parasite of cats, I. rivolta is not CD4+ and CD8+ T-cells are crucial in the able to induce the relapse in oocyst recovery form the primary infection. The shedding. protective role in subsequent infection is probably held by antibodies. This theory Clinical signs. Despite most of the of cellular-mediate immunity is also infections are asymptomatic, sustained by the increased susceptibility toxoplasmosis still remains a major of human patients suffering from AIDS, a disease. condition caused by the HIV virus which Symptoms are not characteristic and this causes depletion of CD4+ cells. may account for false negative diagnosis In general, the humoral response is and subsequent limitation of its clinical strong. In cats infected with tissue cysts, importance. Moreover, certain signs of the seroconversion appears after 10 days infection in humans are not considered to and is very persistent. Antibodies can be be real symptoms of a disease (decreased detected even years after the infection. reaction times, tendency for accidents, Seropositive mother cats transfer personality changes, lower guilt protective antibodies to kitten. This proneness, higher chance for more situation is probably valid also for other promiscuous lifestyle) and their animal species but it has not been correlation with toxoplasmosis is quasi- investigated in detail. This is why post- impossible in practice. In animals, these natal infection in newborns is rare, and “hidden” signs are even more difficult (if most animal become susceptible to oral not impossible) to trace. In mice infected infection after several weeks of life. In to T. gondii, various behavioral changes cats for instance, the passive maternally - which enhance the chance of being acquired immunity disappears by the age predated by cats have been recorded: of 3 month. decreased learning capacity, higher activity levels, lower ability to Various experimental trials showed that differentiate familiar and novel cats shed massive number of oocysts only surroundings or reduced predator during the first (primary) infection. All avoidance. subsequent infection of already immune cats result in no or very low levels of fecal Clinical signs and severity of disease vary oocyst elimination. The immune with the host species, age and immune protection responsible for inhibition of status.

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In cats with tissular infections, the resorption are also reported in infected clinical signs consist in one or more of the sheep. Sterility is also possible in following symptoms: fever, anorexia, Toxoplasma-infected ewes. In sheep respiratory abnormalities (dyspnea, flocks in which toxoplasmic abortions are polypnea), abdominal pain (due to present, a great number of lambs born- hepatitis or pancreatitis), icterus, alive can suffer of subclinical congenital neurologic signs (blindness, anisocoria, toxoplasmosis. Except abortion, other slow pupillary light reflex, ear twitch, symptoms associated with post-natal circling, torticollis, seizures, infection in sheep are fever and diarrhea. incoordination, increased affection, In goats, clinical toxoplasmosis is similar stupor, atypical cry, central with the situation described for sheep. hypothermia), cutaneous signs (nodules, Abortions and neonatal mortality are not ulcerations), locomotory problems uncommon. Goats are more susceptible (lameness, articular pain), and ocular to clinical toxoplasmosis than sheep. signs (iritis, mydriasis, hyphema, retinal Mortality following natural infection was hemorrhages). A statistical analysis on reported even in adult goats. 100 feline cases showed that 36% had systemic infection, 26% showed Clinical toxoplasmosis in pigs is a rare pulmonary involvement, 16% abdominal condition. Clinical signs of the acute form lesions, 12% hepatic involvement, 7% (postnatal) include fever, anorexia, neurologic involvement, 4% ocular dyspnea, weakness of the limbs, involvement while other location neurologic signs and abortions. Generally, (cutaneous, pancreatic, cardiac) were less pigs recover after 3 weeks. Mortality is common. Clinical tissue infections in cats possible, but rare. Congenital (neonatal) might result in sporadic cases of toxoplasmosis is associated with gait mortality. Cats infected with FIV might abnormalities, dyspnea, diarrhea and have aggravated symptoms and chronic mortality up to the second week of life. asymptomatic cases may become acute. In dogs clinical cases are usually Congenital toxoplasmosis results in associated with lower immunologic significant mortalities in kitten. status, mainly after surviving canine No clinical signs were described in cats distemper. Reported symptoms include: with intestinal infection with oocysts. orchitis, respiratory signs, nervous signs Lesions of severe enteritis were and death. Toxoplasmosis usually is described in cats but they were caused by diagnosed post-mortem and it seems to tissue cysts. complicate canine distemper cases in puppies. Rare cases of acute In sheep, the most important clinical sign toxoplasmosis in adult dogs included is abortion. Toxoplasma gondii is one of ocular and hepatic involvement. No the main causes of infective abortion congenital cases are known. worldwide. Toxoplasmic abortions in sheep are in the mid or last term of Except very few cases of abortion, in gestation. Mummified fetuses or fetal cattle clinical toxoplasmosis is a rare

151 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | condition. Clinical toxoplasmosis in Focal myocarditis and encephalitis with horses is virtually absent. However, in the presence of bradyzoites in tissue both these cattle and horses, any cysts are the main lesions in chronic reported case of toxoplasmosis must be toxoplasmosis. regarded suspicious. Instead, the other Diagnosis. Diagnosis has different related conditions are definitely more approach in the feces of the definitive prevalent: neosporosis in cattle (see host or in the samples from intermediate Chapter 2.4.3.3) and equine protozoal hosts. myeloencephalitis (see Chapter 2.4.3.1). Detection of oocysts in the feces of cats is In chicken, there are very few known done by classical flotation methods. cases of clinical toxoplasmosis. These However, finding non-sporulated or even scarce reports include sudden death and sporulated oocysts with Toxoplasma-like nervous signs (torticollis, lateral morphology is not enough to say it is recumbency). Toxoplasma. As shown in other sections Pathology. The lesions associated with of this textbook (see Chapters 2.4.1.1 and Toxoplasma infection are located in 2.4.3.4), various coccidia are parasitic in various organs and tissues. During acute the small intestine of cats. Their toxoplasmosis, tachyzoites are differentiation cannot be done relying responsible for producing necrotic strictly on morphological features (table lesions in various tissues (mesenteric 2.39). lymph nodes, liver, intestinal lamina propria, spleen, pancreas, lungs, adrenal glands, kidneys). Same tachyzoites- Table 2.39 Oocyst size of coccidia found in feces of cats (after Dubey and Greene, 2012) induced lesions are present in internal organs of early aborted or stillborn Species Average size fetuses or in newborns suffering of (µm) Isospora felis 40 x 30 congenital toxoplasmosis. Isospora rivolta 22 x 20 Toxoplasma gondii 12 x 10 Additionally, necrotic or degenerative Hammondia hammondi 12 x 11 encephalitis, endocarditis and retinitis Besnoitia wallacei 17 x 12 Besnoitia darlingi 12 x 11 have been described in congenital Besnoitia oryctofelisi 12 x 11 toxoplasmosis in goat kids, lambs, kittens, Sarcocystis spp.* 11 x 9 *sporocysts puppies and piglets. Histologically, in all the affected organs tachyzoites are visible. For safety reasons, all small oocysts Tachyzoite infection in pregnant females found in the feces of cats must be results in degenerative lesions of the regarded as Toxoplasma. Specific placenta. The main lesion is necrotizing identification can be done only by placentitis with the presence of large molecular biology (copro-PCR) or after amounts of tachyzoites in the sporulation by ultrastructural studies or trophoblastic layer. xenodiagnosis.

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The diagnosis in systemic infections of and peribronchial markings. Abdominal intermediate hosts is more complex. As radiology shows the enlarged mesenteric clinical signs are not characteristic, lymph-nodes. Other imagery techniques diagnosis must rely on detection of the used mainly for detection of lesions in the organism or its DNA in various tissues or central nervous system include the detection of antibodies. The most myelography, computer tomography (CT) difficult task is the differentiation of and magnetic resonance imaging (MRI). subclinical from clinical toxoplasmosis Clinical laboratory findings are not and to correlate the presence of specific but they may help in the Toxoplasma with the symptoms. diagnosis. They include: anemia, leukocytosis with neutrophilia, For diagnosing clinical toxoplasmosis, eosinophilia, lymphocytosis and various symptoms must be correlated monocytosis. Terminally ill cats present with imagery and clinical laboratory leukopenia (with absolute lympho- followed by identification of the organism cytopenia and neutropenia). Usually the from various samples, including biopsies leukocytosis is indicative of recovery and by cytology, histology, PCR, a better prognosis. xenodiagnosis (artificial infection in rodents) or serology. Clinical biochemistry shows hypoproteinemia, hypoalbumin-emia, In vivo diagnosis of clinical increase of alanine amino-transferase, toxoplasmosis is important for acute aspartate aminotransferase and alkaline post-natal cases and congenital phosphatase associated with hepatic and infections. It is mainly of interest in pets. muscular necrosis. The serum level of Fever in cats, correlated with creatine kinase is increased when unresponsiveness to antibiotic treatment muscular necrosis is present. In animals is one of the most common findings of with acute hepatic necrosis, the levels of acute toxoplasmosis. The procedure in serum bilirubin are also increased. this case is to continue with further Increased serum amylase and lipase clinical procedures. Fundic ocular activities are indication of pancreatic examination reveals multifocal involvement. All these clinical laboratory iridocyclochoroiditis. Cytology during the findings are not enough for a positive acute disease can reveal the presence of diagnosis of toxoplasmosis. free tachyzoites in various tissue and body fluids. Tachyzoites are more Isolation of the agent from aborted common in thoracic and peritoneal fetuses and fetal membranes is routinely pathologic effusions but they can be done by inoculation to laboratory mice. rarely detected also in other samples For this scope, the fetal brain and (blood, cerebrospinal fluid, tracheal placental cotyledons yield optimal washings). These samples should be results. further analyzed by PCR for confirmation. Identification in tissue section is done by Thoracic radiology can also help, showing histology and more specifically by a diffuse interstitial to alveolar pattern immunohistochemistry. Histopathology

153 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | is essential for concluding on the cause- In cats with systemic infection the effect association. treatment is done by combining two drugs. Sulfonamides (20-30 mg/kg, Serologic diagnosis include detection of orally, every 6-24 hours, for 2-4 weeks) humoral antibodies: Dye test known also in combination with pyrimethamine (0.5- as Sabin-Feldman test, indirect 1 mg/kg, orally, every 24 hours, for 2-4 hemagglutination test, complement weeks) are the drugs of choice. fixation test, modified agglutination test, Pyrimethamine can be given parenterally, latex agglutination test, indirect after dilution. Sulfonamides can be also fluorescent antibody test, ELISA, used in combination with trimethoprim. immunoglobulin M immunosorbent The use of these drugs may induce agglutination assay test and Western thrombocytopenia and/or leukopenia. In blotting. such a case, treatment should not be An ELISA method has been developed for discontinued but the cats should receive detection of Toxoplasma antigens in cats. folinic acid and yeast supplement. Molecular techniques are employed for Clindamycin (8-17 mg/kg, orally or the detection of parasite DNA. PCR is intramuscularly, every 8-12 hours, four 4 widely used on various samples mainly weeks) is also highly effective in feline for diagnosing abortions and for toxoplasmosis. molecular epidemiology surveys. The treatment of choice in dogs is similar For details on the technique of isolation, with the one in cats, but doses are slightly cultivation and serologic procedures different: clindamycin (3-13 mg/kg, refer to Dubey (2010) and to OIE (2012). orally or intramuscularly, every 8 hours or 10-20 mg/kg, same routes, every 12 Differential diagnosis is variable in each hours) for 4 weeks. species. Because of the great variety of clinical signs in cats, the list of diseases to As in the other domestic animal species be considered for the differential acute toxoplasmosis is rare, no diagnosis is too long to be given here. therapeutic protocols are recommended. Readers should refer to a more general In the case of toxoplasmic abortion, feline medicine book. In the case of chemoprophylaxis must be performed in toxoplasmic abortion, differential pregnant animals. diagnosis must include all the other Treatment of cats which shed oocysts is causes (infectious and non-infectious). In done by: clindamycin (25-50 mg/kg, dogs the differential diagnosis must be orally or intramuscularly, every 12-24 done against neosporosis. hours, for 1-2 weeks); a combination of Treatment. Treatment has two major sulfonamides (100 mg/kg, orally, every indications: to treat the clinical systemic 24 hours, for 1-2 weeks) with cases and two treat the cats with pyrimethamine (1 mg/kg, orally, every intestinal infection to stop the oocyst 24 hours, for 1-2 weeks); toltrazuril (5- elimination. 10 mg/kg, orally, every 24 hours, for 2 weeks). Monensin given in food for one

154 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | two weeks is also effective and stops Chemical prevention is done mainly in oocysts shedding. humans in certain cases when patients are exposed to risk of clinical infection or Control. Preventing infection with the risk of congenital toxoplasmosis is Toxoplasma is essential in all host high. If a human patient is serological categories (humans, cats, livestock). negative, meaning there is no protection Prevention in cats is easy if they are kept against Toxoplasma, and an indoors and never receive uncooked immunosuppressive treatment must be meat, bones or organs from animals, even applied (i.e. before transplants), the if these are bought from grocery stores. prophylactic therapy is recommended. In the case cats do not eat dry or canned Prophylactic treatment has been used food or cooked food, the option of choice also in sheep, in flocks with history of is to give them raw meat which was deep- toxoplasmic abortion. The drug of choice frozen before or beef which is usually in this case is monensin (17-28 mg/kg, free of infective cysts. Raw liver is an daily, for five days), given in the last half essential part of feline diet, as it is a of gestation. Other drugs (sulfamethazine, perfect dietary supplement of vitamin A. pyrimethamine, decoquinate) have been As infective Toxoplasma cysts are very shown to have similar effects in sheep. common in the liver, in such cases the organs must be deep frozen before fed to One commercial vaccine (Toxovax) is cats. Owners must avoid letting cats to available for prevention of toxoplasmic hunt outside or to scavenge in the trash abortions in sheep. can. In farms, cat populations must be controlled. In farms where abortions occur, fetuses and fetal membranes must 2.4.3.3 Neosporosis be eliminated promptly to avoid placentophagia by cats or other animals. Introduction. Neosporosis is a disease of Outdoor cats must be under permanent cattle and dogs with huge economic surveillance and treated if they are impact in dairy and beef cow farms. It is shedding small coccidian oocysts. considered one of the most important Prevention of infection in humans infectious causes of abortion in cattle, include: washing the hands after handling worldwide. It has been estimated that meat or after petting cats and dogs. Fruits annual global losses due to neosporosis and vegetables must be washed are between 1.2 and 2.3 billion US thoroughly. Any consumed meat must be dollars. Additionally, another Neospora properly cooked. Tasting of food during species is responsible for a neurologic cooking is not recommended. Microwave disease in horses. cooking does not kill the bradyzoites. Historical notes. The history of Freezing meat at -12°C kills the tissue neosporosis is very recent. Until 1988 cysts in few days. Pregnant women when the etiological agent was should avoid contact with cats, raw meat discovered, it was confused with and unwashed fruits or vegetables. Toxoplasma gondii. The first case of

155 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | neosporosis was described in 1984, in intermediate hosts (see life-cycle). Their dogs from Norway by Bjerkas et al. Three typical shape is crescent-like (figure years later, in 1987, O’Toole and Jeffrey 2.73). Bradyzoites (8 x 2 µm) are found described a clinical case in a newborn in tissue cysts surrounded by a thick-wall calf. Both reports were designated to be (4 µm), located in muscular and nervous caused by an unidentified protozoan, tissues. similar to Toxoplasma and Sarcocystis.

The agent was described only in 1988 by Dubey and named Neospora caninum. Table 2.40 Oocyst size of coccidia found in feces of dogs (modified after Dubey and This does not mean the disease is new, Greene, 2012) but that until 1988, all cases (except the Species Average size two listed above) were considered to be (µm) toxoplasmosis. Isospora canis 38 x 30 Isospora ohioensis 24 x 20 More recently, in 1998, a new species, Isospora neorivolta 17 x 15 was described from the Isospora burrowsi 20 x 17 Neospora caninum 12 x 10 central nervous system of a horse in Hammondia heydorni 12 x 11 California by Marsh et al. Sarcocystis spp.* 11 x 9 *sporocysts Etiology. Two species are known in genus Neospora. Neospora caninum is typically parasitic in dogs (which act as definitive hosts) and cattle (intermediate hosts). Several other species have been found naturally infected with viable N. caninum (sheep, buffaloes, horses, bison, deer). The second species, Neospora hughesi is responsible for a form of equine protozoal myeloencephalitis.

Morphology. Oocysts are small (10.6- 12.4 x 10.6-12.0 µm) and morphologically not differentiable from other small oocysts found in canine feces (see table

2.40). Oocyst wall is colorless. Sporulated oocysts contain two sporocysts (7.4-9.4 x Figure 2.73 Tachyzoites of Neospora 5.6-6.4 µm), each with four sporozoites caninum in cell culture. (Photo Ovidiu (5.8-7.0 x 1.8-2.2 µm). Oocysts Șuteu) (“Isospora”-like) can be found in the feces of dogs and other canids. Although many genetic strains of N. Tachyzoites (3-7 x 1-5 µm) are found in caninum have been described, little is intracellular parasitophorous vacuoles in known on their variation in virulence. In the cytoplasm of various cells in the the case of N. hughesi, only the asexual

156 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | stages in the intermediate host (horses) muscle cells where they can remain for are known. Tachyzoites are located in a all the life of the animal. parasitophorous vacuole inside host cell The typical life cycle continues when cytoplasm. Bradyzoites (2-3 x 4-7 µm) tissue cysts with bradyzoites are ingested are within a tissue cyst (7-16 x 10-19 by a canine definitive host. What happens µm), surrounded by a wall (0.1-1 µm here is still largely unknown, but it is thick). presumed that part of the bradyzoites Life cycle. In many aspects, the life cycle reconvert into tachyzoites and produce of Neospora caninum is similar to that of systemic infection which ultimately result Toxoplasma gondii. The life cycle of N. in the formation of tissue cysts. caninum is heteroxenous (figure 2.74). Systemic infection with tachyzoites can Domestic dogs and other canids (coyotes, result in the transplacental transmission grey wolves, dingoes) are definitive to puppies. Other bradyzoites will invade hosts. The typical intermediate hosts are the epithelial cells of the dog’s intestine cattle, but various other warm-blooded and they probably follow a similar vertebrates can serve as hosts for the development as Toxoplasma gondii does asexual stages. However, the spectrum of in cats, continuing merogony, and then recorded intermediate hosts is not as gametogony with oocysts formation. diverse as in the case of T. gondii. One Infected dogs shed through their feces very important aspect is the lack of unsporulated oocysts 5 days after human infections reported. So far, ingestion of tissue cysts. neosporosis is not considered a zoonotic disease. The oocyst elimination by dogs is at a much lower rate than in cats infected Dogs shed in their feces unsporulated with T. gondii. It has been estimated that oocysts which eventually undergo dogs shed around 500,000 oocysts exogenous sporogony (24-72 hours) and (compared to 1 billion oocysts excreted become infectious (sporulated) oocysts. If by one infected cat). In dogs, the time sporulated oocysts are ingested by a they shed oocysts extends up to 4 months suitable intermediate hosts (e.g. cattle), (unlike in cats which shed T. gondii only they excyst and the freed sporozoites will for 1-2 weeks). Considering the lower penetrate the intestinal wall and invade overall number of eliminated oocysts and various cell types: macrophages, neural the longer period, the chance of finding cells, fibroblasts, endothelial cells, muscle Neospora caninum oocysts in dog feces is cells and hepatocytes where the multiply very low. Another difference from by endodyogeny and producing Toxoplasma gondii is that dogs are not tachyzoites. Tachyzoites divide around susceptible for the infection with N. twenty times before becoming caninum oocysts or tachyzoites (like cats bradyzoites in tissue cysts (in cca. three are for T. gondii). Dogs can be infected weeks after the infection). Tissue cysts only with tissue cysts with bradyzoites with bradyzoites are found typically in from intermediate hosts. the central nervous system and striated

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Figure 2.74 Life cycle of Neospora caninum. Contamination of dogs as definitive hosts takes place after ingestion of tissue cysts from meat or organs of infected intermediate hosts (including aborted fetuses and fetal membranes). Intermediate hosts acquire the infection after eating sporulated oocysts passed by dogs in the feces. Infected intermediate host females are able to pass the infection to their offspring which will develop lifelong infection. Subsequently they are able to transmit the infection transplacentally again and again (dotted contour arrow). After ingesting tissues cysts, dogs develop both enteroepithelial and systemic infection and are able to pass tachyzoites transplacentally to puppies.

Additionally, the only known way of Except this typical life cycle involving horizontal transmission to intermediate alternation of the definitive and hosts is by ingestion of oocysts. intermediate hosts, Neospora caninum can be transmitted vertically, from Although placentophagia in cows was infected pregnant females to the fetus. suggested as a mode of transmission There are two types of transplacental (horizontal, intermediate to intermediate transmission described for N. caninum in host), no experimental trials support this cattle: (1) The exogenous transplacental theory yet.

158 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | transmission occurs when non-infected breeds. The highest seroprevalence in pregnant cows ingest infective oocysts. In cattle has been found in those farms this case, the tachyzoites will invade the where dogs were more abundant. fetus, transported probably by the Seroprevalence data in dogs is reported mononuclear phagocytes via the blood worldwide. In random populations it is stream. (2) The endogenous usually less than 20%. Purebred dogs are transplacental transmission happens in more likely to develop seropositivity than cows which are already infected before mixed breeds. Stray dogs with access to pregnancy. In such a case, bradyzoites raw organs and meat have significantly from tissue cyst are reactivated and they higher prevalence than indoor dogs fed differentiate into tachyzoites which exclusively with commercial diet. ultimately infect the fetus. The Serologic prevalence is higher in cattle endogenous transplacental transmission farm dogs than in dogs living in cities. explains why female calves with congenital neosporosis are able to pass The most important epidemiological the infection to their offspring when they factor from clinical point of view is the become reproductive females. prevalence of abortions caused by N. caninum in cows. Two epidemiologic It is not known if congenital transmission patterns of Neospora-induced abortions follows the same rule in dogs or other are known: endemic abortion and hosts. Infected female dogs can give birth epidemic abortions. The endemic pattern to infected puppies during several consists of persistent abortion rate pregnancies. (around 5-10%, all year around). The Transmission is not possible through most dramatic situation (epidemic milk or venereal route. pattern) is the so called “abortion storm”, when more than 10% of the The life cycle of N. hughesi is not known, pregnant cows from a single farm abort as only the asexual stages in the within a time frame on 12 weeks. intermediate hosts were described. Abortions can persist from several Probably the definitive host is a months to several years. carnivorous mammal. As dogs shed a very low number of Epidemiology. Neosporosis is oocysts, the main source of infection for distributed worldwide in cows and dogs, cows is the transplacental transmission as numerous seroprevalence and from their mothers. However, not all molecular epidemiology studies show. infected cows transmit the infection to Antibodies were found also in small their offspring. The transplacental ruminants, but the clinical importance in transmission rate varies roughly from 30 ovine abortion is still debated. The to 60%. It is not known if the prevalence of neosporosis is highly transmission rate is different between variable between countries, between endogenous and exogenous regions of the same country and between transplacental transmission routes. Some beef and dairy cows and between cattle authors suggest that post-natal infection

159 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | is equally important, as seropositivity tachyzoites is thought to be related to may increase dramatically in certain hormonal and immune factors of the flocks at a given time. In the recent years, pregnant cow. wildlife reservoirs increased in their The pathogenesis of abortion is related to epidemiologic importance. Abortion the damage of the placenta by rapidly storms are likely caused by exogenous multiplying tachyzoites. They initially transplacental transmission following invade the maternal caruncular septum exposure to oocysts. and subsequently the fetal placental villi. Resistance of oocysts of N. caninum in the The abortion occurs in two situations. environment is considered to be more or When the placenta is severely damaged, less similar with T. gondii. High the fetus receives insufficient oxygen temperatures (100°C) inactivate them in supply and nutrition. Other situation is one minute and treatment with 10% when tachyzoites destroy directly the sodium hypochlorite in 1 hour. fetal tissues. Immune-mediated fetal Bradyzoites in tissue cysts remain expulsion has been also suggested, infective in tissues for 7-10 days. associated with maternal pro- inflammatory cytokines (IL-10, gamma Pathogenesis. It is not fully understood interferon). why some infected cows are able to give birth normally while others abort. Pathogenesis in dogs is caused by the Various factors have been incriminated, rapid multiplication of tachyzoites in including the infective oocyst dose. It is various tissues. This can occur mainly also not known if there are any during primary infections but also if correlations between the clinical bradyzoites are reconverted to outcome and the mode of transplacental tachyzoites during persistent infection infection (endogenous or exogenous). (e.g. stress, pregnancy, immuno- Other hypothesized factors include the suppressive diseases). Development in existence of N. caninum strain with the brain of dogs causes monocyte- different virulence or the susceptibility of mediated lesions with altered function of cows under metabolic stress (e.g. dairy the nervous centers involved. Lower cows). The factors affecting the risk of motor neuron damage and severe abortion were summarized by Goodswen myositis are the causes of gradual hind et al. (2013). limb paralysis in puppies with congenital neosporosis. Destruction of the muscular For the exogenous transmission cycle, the layers of the esophagus can result in risk factors are the number of oocysts megaesophagus with dysphagia. ingested and the gestation stage. In the case of endogenous transmission cycle, Immunology. Immunity plays a crucial cows with higher antibody titers are role in the development of neosporal more likely to abort. infection. Weather the infected cows abort or they transmit the infection Reactivation of bradyzoites from tissue transplacentally is greatly dependent on cysts and their reconversion to the immune response. Moreover, some

160 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | offspring which are born alive display Post-abortion sterility is not considered a signs of disease, some others not. The problem. immune response in cows infected with

Neospora caninum is associated with high levels of IFN-γ, IgG-2 antibodies and Th1 cells. IFN-γ inhibits the growth of tachyzoites. IFN-γ activates the response of macrophages which play a major role in the differentiation of tachyzoites to bradyzoites and vice-versa (during reactivation of infection).

The immunological maturity of the fetus when it is infected determines probably its survival chance. As most organs involved in fetal immunity (thymus spleen, lymph nodes) mature in the last period of gestation, the risk of fetal death and abortion is higher during the early Figure 2.75 Six months old bovine gestation. The immunity can be aborted fetus caused by Neospora protective as seropositive cows are less caninum. (Photo Ovidiu Șuteu) likely to abort. Based on this, vaccination has been developed. Congenital neosporosis in calves (<2 Clinical signs. Most infections are months old) include variable clinical asymptomatic. In cows, the characteristic signs: inability to rise, ataxia, flexed or clinical sign is abortion (figure 2.75). hyperextended for or hind limbs, The outcome of gestation resulting from decreased patellar reflex, loss of infected cow can be: fetal resorption, fetal proprioception, exophthalmia and mummification, fetal autolysis, stillbirth, decreased weight. Calves can be born born alive with clinical signs, born with alive, but with severe congenital no clinical signs but with persistent life- deformities (like hydrocephalus) which long infection and born without infection. are incompatible with life. Other calves About 80-90% of the infected cows are born with neonatal encephalomyelitis produce apparently normal calves, part of and are paralyzed. them infected. The infection in dogs is usually Abortion can be present in cows of any asymptomatic. It can affect dogs of all age, from the age of gestation of three ages (post natal infection) but also month to term. However, most abortions neonates (congenital neosporosis). are reported to be between 4 and 6 Though, most cases are congenital. months of gestation. Repeated abortion is Puppies born from infected mothers do uncommon, and usually it is encountered not necessarily develop clinical signs. If in less than 5% of the aborting cows.

161 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | they do, anyhow they are usually born cerebrospinal fluid. There are no with no evident sign of infection. recorded clinical cases of canine intestinal neosporosis caused by the First clinical signs of congenital enteroepithelial cycle. neosporosis in puppies appear at 3-9 weeks of age. The most common Clinical signs caused by N. caninum in symptom is paralysis of the hind legs, other domestic hosts are rare and they often with spastic character include abortions in sheep, goats and (arthrogryposis) and gradual muscle South American camelids. The infection atrophy and stiffness. Usually the with N. hughesi in horses is responsible paralysis is gradual and ascending. for a similar syndrome with the equine Forelimbs may be also affected but less protozoal myeloencephalitis produced by severely. S. neurona. Neospora hughesi is not pathogenic for dogs. Other clinical symptoms in puppies include: joint deformations, cervical Pathology. Lesions caused by N. caninum weakness, dysphagia (caused by are very important mainly from megaesophagus). During all this time diagnostic point of view, as the presence dogs are fully conscious and alert. They of histopathological changes can can survive in this state for several represent a reliable cause-effect months until finally death occur. Not all correlation. They are different in each puppies from the same litter show host type. clinical signs. Lesions in aborted fetuses include In dogs older than six month, clinical serosanguinolent fluid accumulation in neosporosis can be caused by the the body cavities (figure 2.76). primary infection or by reactivation of a Sometimes, the fetal tissues are in chronic infection due to various factors incipient or moderate autolysis (figure (see pathogenesis). 2.77). Other gross lesions are more difficult to be detected (pale white foci in The clinical signs are related to the the muscles). Histopathology from fetal multifocal nervous lesions or to tissues reveals generalized non- polymyositis. Other clinical signs suppurative infiltrates. The brain is the recorded in adult dogs are: coughing, site of somehow more characteristic progressive ataxia, dysphagia, cutaneous lesions. They consist of scattered foci of ulcers. Dogs with severe multifocal non-suppurative cellular infiltrates, central nervous involvement usually die. sometimes necrotic. Neospora caninum Clinical biochemistry findings in dogs developmental stages can be visible or include increased levels of creatine not in these histological sections. Other kinase and aspartate aminotransferase lesions in aborted bovine fetus include: (due to severe myositis and hepatitis). epicarditis, myocarditis, myositis, The cerebrospinal fluid has an increased hepatitis, all focal, with non-suppurative level of proteins and pleocytosis. cellular infiltrates and even focal Tachyzoites may be present in the necrosis.

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include mainly non-suppurative encephalomyelitis.

Gross lesions in dogs include: multifocal areas of necrosis and/or fibrosis with or without mineralization in the striated muscles (mainly diaphragm), hepatitis with hepatomegaly, pneumonia and discoloration of certain areas of the central nervous system (visible on gross tissue sections). Histological sections in puppies which died of congenital neosporosis reveal the presence of

lesions and parasitic stages in the Figure 2.76 Serosanguinolent fluid following organs: muscles, myocardium, accumulation in the abdominal cavity of retina, nerve roots, thymus, kidney, liver, an aborted bovine fetus caused by adrenal gland, brain, spinal cord, stomach Neospora caninum. (Photo Ovidiu Șuteu) wall or dermis. The characteristic lesions are non-suppurative myeloencephalitis, myositis, polyradiculoneuritis (mainly in puppies), lymphonoditis, myositis, cerebral cortical necrosis etc.

Diagnosis. The most difficult task in aborting cows is to correlate the presence of Neospora caninum with the abortion. Finding antibodies in a cow which aborted or in the cow population from a farm with endemic or epidemic abortions is not enough. Moreover, finding the parasite in the tissues of an aborted fetus (by PCR or immunohistochemistry) is not always enough. Figure 2.77 Autolysis of the central nervous system of an aborted bovine The etiologic confirmation of abortions fetus caused by Neospora caninum. (Photo must be done in specialized laboratories. Ovidiu Șuteu) All of them have to be correlated with the presence of Neospora caninum in

histological lesions from the fetus or from Lesions in congenital neosporosis of the placenta and with other calves are more or less similar with epidemiological data, including the age of lesions from aborted fetuses. They fetus and the immune status of the cow. The lesions in fetuses aborted because of

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Neospora usually have disseminated inflammatory lesions in most of their internal organs (brain, heart, kidneys, liver, lungs, muscles). Additionally, other abortion causes must be excluded.

Various serologic tests are available for the detection of anti-Neospora antibodies (IFAT, ELISA, direct agglutination test, Western blot). They are widely used for seroepidemiologic surveys. However, their use in clinical diagnosis has many limitations. Most of them are not able to discern between chronic and acute infection. Moreover, the antibody titers in Figure 2.79 Cyst of N. caninum in the seropositive cows are fluctuant. cardiac muscles (IHC) (Photo Ovidiu Șuteu) Some seropositive cows may become seronegative (antibody titers below the cut-off value). Serologic testing can be Immunohistochemistry (IHC) is a very used also for fetal samples. Serologic useful tool for differentiating Neospora testing in calves must be done after the tissue cysts from other protozoal cysts age of 6 months, to eliminate the (figures 2.78 and 2.79). PCR is also positivity due to maternally transferred highly sensitive and specific. antibodies. Serologic diagnosis using recombinant proteins from tachyzoites (NcGRA7) and bradyzoites (NcSAG4) allows differentiating between chronic and acute infections. If both proteins yield positive results, this might be indicative of a reactivated infection during pregnancy.

The same diagnostic principles as for the abortion apply for diagnosis of congenital neosporosis in calves.

Diagnosis in dogs relies greatly on the correlation of symptoms, clinical laboratory (blood and CSF) and results of various serological assays (IF, ELISA, immunoprecipitation). The definitive diagnosis can be based on the Figure 2.78 Cyst of N. caninum in the demonstration of the presence of brain (IHC) (Photo Ovidiu Șuteu) Neospora caninum in the CSF. However,

164 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | this is a difficult task, as the number of 12 hours, for at least 4 weeks) with tachyzoites is very low. In dead animals, pyrimethamine (1 mg/kg, orally, the presence of the parasite can be made every 24 hours, for at least 4 weeks); also in histological sections from brain,  clindamycin (10 mg/kg, orally, every heart or muscles. 8 hours, for 4 weeks) in adult dogs; Differentiation from other cyst-forming  clindamycin (75-150 mg/dog, orally, coccidia is based on PCR or every 12 hour, for 6 months) for 13 immunohistochemistry. Differential months old puppies with congenital diagnosis in dogs must be done from neosporosis; many diseases which produce similar symptoms and lesions: infection with  sequential treatment with Hepatozoon canis and Hepatozoon clindamycin hydrochloride, americanum¸ leishmaniosis, trimethoprim-sulfadiazine and sarcocystosis, clostridial myositis, pyrimethamine leptospirosis, ehrlichiosis, trichinellosis, Treatment of dogs does not always result trypanosomosis etc. in the complete recovery or in the full Serological tests in horses and possibly elimination of the parasite. However, also in other animals can be difficult to clinical signs may improve. Sometimes interpret because of suspected cross- the treatment must be done for very long reaction between antibodies against N. time (up to 18 months) until caninum and N. hughesi. improvement is seen.

For the isolation of the Neospora caninum Control. It is essential mainly in farms. In in the lab and for experimental trials, uninfected bovine herds, preventing the laboratory animals such as mice, gerbils introduction of neosporosis is the main and fat-tailed dunnarts are widely used. assignment. Newly introduced cows must be purchased from Neospora-free farms Treatment is not routinely done in and they should be serologically tested bovine neosporosis. Experimental before. administration of toltrazuril to calves can delay the tachyzoite multiplication and Prevention of horizontal transmission is spread. There is no data on the effect of achieved by restricting the access of toltrazuril on bradyzoites. freely moving dogs in cattle farms. If they are present, their direct access to the The treatment of canine neosporosis animals or their food must be avoided. must be approached in all animals with This is more difficult (or virtually clinical signs and with a positive impossible) if cattle are grazed on diagnosis of neosporosis. Otherwise, the pastures where dogs or wild canids have condition is often fatal. The treatments of access. Monitoring programs must be choice in dogs are: introduced (serological testing of cows,  a combination of trimethoprim- laboratory examination of each aborted sulfadiazine (15 mg/kg, orally, every fetus).

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In infected herds the primary goals are to 2.4.3.4 Other heteroxenous coccidia prevent abortions, to prevent spreading parasitic in domestic animals the disease (both horizontally and vertically), to avoid introduction of new Except the three major heteroxenous infected animals and ultimately the coccidia presented before (Sarcocystis, elimination of infection. Toxoplasma, Neospora), several other genera have been identified in domestic Permanent serologic monitoring of the animals. Their importance is rather for herd is recommended, and negative the differential diagnosis than for their animals must be selected for breeding on clinical importance. long term. Animals with high antibody titers or history of repeated neosporal Genus Hammondia is represented by abortions must be considered for culling. two species. Both are heteroxenous. Embryo transfer from seropositive Hammondia hammondi has felids as donors implanted to seronegative definitive hosts and goats and mice as recipients produces non-infected natural intermediate hosts. Many others offspring. mammal species serve as experimental intermediate hosts. Cats are infected only Proper disposal of fetal membranes after eating tissue cysts and develop only (placenta) and aborted fetuses must be intestinal infection. strictly followed. Cats eliminate unsporulated oocysts, A killed anti-Neospora caninum vaccine is morphologically similar to those of commercially available in United States. Toxoplasma. Oocysts of Hammondia The vaccine must be used twice in early sporulate in the environment. gestation, mainly in farms where the Intermediate hosts are infected after infection is present. Vaccination was ingesting sporulated oocysts. Sporozoites shown to reduce the rate of abortions. invade the mesenteric lymph nodes and The main disadvantage of the vaccine is other abdominal organs where the production of seropositive cows; tachyzoites develop. Ultimately, they post-vaccinal seropositivity is not encyst as bradyzoites in the skeletal differentiable from the post-infective muscles. The second species, H. heydorni seropositivity. uses dogs as definitive hosts and various To prevent infection of dogs, domestic and wild mammals as administration of raw meat or organs, intermediate hosts. The life cycle is mainly of bovine origin must be avoided. similar to H. hammondi. The reports of In bitches known to be infected with the clinical signs associated with Hammondia parasite, birth control programs infection are scarce. Mild diarrhea has (including spaying) are to be considered been occasionally reported in dogs. In for prevention of vertical transmission. intermediate hosts the symptoms are There is no vaccine available against also normally absent. In experimentally canine neosporosis. infected goats, fever has been reported.

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Genus Besnoitia includes nine species signs include initial fever, anorexia, parasitic in a great variety of hosts. The tachycardia and tachypnea, life cycle is known, although not in great corresponding to the rapid multiplication details, only for few species. It seems the of tachyzoites in various tissues and life cycle is more similar to Toxoplasma internal organs. The next stage of the gondii than to Hammondia, as disease consists in cutaneous signs: skin extraintestinal development takes place congestions with increased sensitivity in also in the definitive host. various body areas and anasarca (generalized edema). In the next stage of Four of the species have cats as definitive the disease, the skin becomes hosts. These species and the respective sclerodermic, with complete loss of hair, intermediate hosts for each are: B. severe lichenification and wallacei (rodents), B. darlingi (opossums, hyperpigmentation. Sexual organs are lizards), B. oryctofelisi (rabbits) and B. also affected. In this chronic stage, the neotomofelis (woodrats). After cats ingest affected areas are full of tissue cysts, tissue cysts from the connective tissues of which are large enough to be seen at intermediate hosts, the bradyzoites gross necropsy. There is no treatment for follow enteroepithelial merogony and besnoitiosis. gametogony, followed by extraction of unsporulated oocysts. Part of the bradyzoites will invade also extraintestinal sites in the cat host, as 2.4.4 Hepatozoidae Toxoplasma does. Introduction. The family has a single Dogs are not known to harbor any genus, Hepatozoon. The genus includes species of Besnoitia as definitive hosts. around 300 species parasitic in all groups The life cycles for the other 5 species of of tetrapod vertebrates. the genus are not known only from few Ecology and transmission. The variety studies on the asexual development in of life cycles is very high within genus the intermediate hosts. Among these five Hepatozoon. The full development and species, three are parasitic in domestic complete life cycle is not known only for animals: (bovines), B. few species. The life cycle is bennetti (equids) and B. tarandi heteroxenous, at least for those species (raindeers). where it is known, but probably for all. The most important species of veterinary One of the hosts is always a vertebrate importance is Besnoitia besnoiti, (amphibian, reptile, bird or mammal) and responsible for the bovine besnoitiosis. the other is always an invertebrate. The Its life cycle is not fully understood. merogonic and gametogonic Bovines are intermediate hosts; the developments take place in various definitive hosts are not known. Not all tissues of the vertebrate host. The infections in cattle become clinically invertebrate is hosting the sporogonic evident. If they do, the principal clinical development with the oocyst formation.

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More details are given in the section of various leukocytes. The meronts of H. canine hepatozoonosis. americanum are described as multi- layered “onion skin” cysts, located Medical importance. Most species are between the muscular fibers, having 250– parasitic in cold-blooded vertebrates 500 micrometer in diameter and (amphibians and reptiles) where they containing a central nucleus surrounded usually cause asymptomatic infection. by concentric rings of membranes. The only two species of veterinary importance are H. canis and H. americanum.

2.4.4.1 Canine hepatozoonosis

Definition. Hepatozoonosis is a tick- borne disease of dogs, occurring worldwide with usually sub-clinical infection. The data in this chapter is mostly based on the excellent review of Baneth (2011).

Etiology. Genus Hepatozoon contains more than 300 species, parasitic in a variety of vertebrate hosts. Two species are responsible for canine Figure 2.80 Gamont of Hepatozoon canis hepatozoonosis: H. canis and H. in a neutrophil of an infected dog. (Photo americanum. Barbora Mitková) Morphology. The complexity of the life cycle results in a great diversity of developmental stages. However, two Life cycle. Hepatozoon canis has a stages are practically important for the heteroxenous life cycle (figure 2.81). diagnosis of the infection in dogs: the Dogs are the intermediate hosts and gamonts within the neutrophils (figure certain tick species are the definitive 2.80) and the meronts within the tissues. hosts. Although hepatozoonosis is a tick- Gamonts of H. canis are typically located borne disease, its transmission from ticks within the cytoplasm of the circulating to dogs is not via the tick bite. Though, neutrophils. They have an ellipsoidal the dogs have to ingest infected ticks to shape and the average size is 4 x 11 µm. acquire the infection with H. canis (figure The meronts of H. canis found in infected 2.81 - 1). After an infected tick or parts of tissues contain elongated it are ingested by a dog, the sporozoites micromerozoites arranged in a circle of H. canis leave the body of the tick, around a clear central centre forming the penetrate the epithelial lining of the typical “wheel spoke” aspect. The intestine and invade mononuclear cells gamonts of H. americanum are located in (figure 2.81 - 2). They are transported by

168 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | the blood and lymph to the target tissues canis. The main difference consists in the and organs: bone marrow, spleen, lymph target tissues, which in the case of H. nodes, liver, kidneys and lungs. americanum are skeletal and cardiac Immediately after they reach these muscles. The parasitic stages are organs, the merogonic development transported to the muscle fibers by starts, resulting in asexual multiplication macrophages. The merogonic stage with the formation of merozoites within produces a meront, which finally tissue meronts. Two types of meronts are becomes a cyst surrounded by formed: type 1, containing up to four mucopolysaccharide layers. When the larger merozoites (macromerozoites, cyst ruptures, the free merozoites invade figure 2.81 - 3) and type 2, containing 20 the surrounding tissues. Merozoites enter to 30 small merozoites (micromerozoites, into leukocytes where they may undergo figure 2.81 - 4). It is believed that an additional merogonic multiplication. macromerozoites are released and are Within the leukocytes, the last responsible for the production of generations of merozoites become secondary meronts in the same target gamonts. Ticks feeding on the blood of tissues. Eventually, the micromerozoites infected dogs take the leukocytes infected will invade monocytes and neutrophils with gamonts. In the body of the tick, (figure 2.81 - 5) and become gamonts gamonts continue to develop into (figure 2.81 - 6). The formation of gametocytes. Gametogony is followed by gamonts corresponds to the beginning of sexual reproduction with the formation the next stage of the life cycle, the of the zygote and sporogony with the gametogony. formation of polysporocystic oocysts in the tick’s hemocoel. If blood of parasitemic dogs is ingested by ticks (figure 2.81 - 7), the gamonts will Transmission of H. americanum to dogs is be released in the tick’s intestine. If the similar to H. canis, via the ingestion of tick is a suitable host for H. canis, male infected vector ticks. and female gamonts will develop into Several tick species have been shown to male and female gametes and will act as suitable definitive hosts for H. associate (figure 2.81 - 8) for the canis. The most widely distributed vector formation of the zygote within the tick’s is Rhipicephalus sanguineus, but other gut. The zygote begins the last phase of ticks species were also shown to transmit the life cycle, the sporogony with the the infection under experimental or ultimate formation of the oocysts in the natural conditions: Amblyomma ovale in tick’s hemocoel (figure 2.81 - 9). Each Brazil and Haemaphysalis longicornis and oocyst contains hundreds of sporozoites H. flava in Japan. The only know tick- which are infective for dogs if ingested. vector for H. americanum is Amblyomma The overall duration of the life cycle of H. maculatum. Transstadial transmission in canis is almost 3 month. ticks occurs in both Hepatozoon species. The life cycle of H. americanum is more or However, there is no evidence for less similar with the one described for H. transovarial transmission in ticks.

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Figure 2.81 Life cycle of Hepatozoon canis. For the meaning of numbers, please refer to the text.

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Transplacental transmission has also in young animals influence the been demonstrated for H. canis. Naturally pathogenesis of new H. canis infections or infected pregnant bitches gave birth to favors the reactivation of preexisting infected puppies. Although not proven, ones. The role of the immune system in another suspected route of infection for the development of clinical infections has dogs is via carnivorism by predation on been also demonstrated by the other infected intermediate hosts. appearance of parasitemia in infected dogs following immunosuppressive doses Epidemiology. Natural (autochthonous) of prednisolone. infections with H. canis generally overlap with the distribution of its main vector, In the infection with H. americanum, the the tick R. sanguineus. Thus, canine main pathogenesis is caused by the hepatozoonosis with H. canis occurs in merogonic development in the muscular most tropical and subtropical (including tissues. The release of merozoites from Mediterranean climate) regions, but the muscular cysts induces an intense cases are known also from countries with local inflammatory response associated warmer temperate climate. Nevertheless, with severe musculoskeletal pain. imported cases have been reported in Symptoms. Several studies suggest that many other countries. Although the main the severity of clinical signs in dogs vertebrate host is probably the domestic infected with H. canis is positively dog, several other wild canids are correlated with the level of parasitemia. suspected to act as natural reservoirs. The most common appearance of H. The infection with H. canis has been canis-hepatozoonosis is subclinical or detected in red foxes (Vulpes vulpes), mild diseases (moderate fever, lethargy, crab-eating fox (Cerocyon thous), jackals muscle pains). These cases are associated (Canis aureus, C. mesomelas), African wild with low levels of the parasitemia (1-5% dogs (Lycaon pictus) and spotted hyenas neutrophils infected with gamonts). The (Crocuta crocuta). severe cases can evolve with life- Amblyomma maculatum is found along threatening symptoms (extreme lethargy, the US Gulf Coast and Southern Atlantic, cachexia and anemia) and are usually hence the distribution of H. americanum associated with high parasitemia levels infection in dogs is limited to this area. (even 100% of the neutrophils infected with gamonts). The hematology of these Pathogenesis. There is an evident cases reveals anemia, extreme difference in pathogenicity between H. neutrophilia (as high as 150,000 canis and H. americanum. The dogs leukocytes/µl blood). Blood biochemistry infected with H. canis are usually findings include hyperproteinemia with asymptomatic. Severe symptoms are polyclonal hyperglobulinaemia and more common in young dogs or are hypoalbuminaemia, and elevated creatine frequently associated with concurrent kinase and alkaline phosphatase infections. It seems that the immune activities. Often, symptoms of suppression induced by other infectious hepatozoonosis are aggravated by other agents or the immature immune system

171 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | overlapping vector-borne disease: examination of blood smears from dogs ehrlichiosis (Ehrlichia canis), with clinical infection might reveal the anaplasmosis (Anaplasma presence of gamonts. They are present in phagocytophilum), leishmaniosis the cytoplasm of the infected neutrophils. (Leishmania infantum) and babesiosis Histopathology from target tissues (liver, (Babesia canis, B. vogeli). Concurrent lymph nodes, spleen etc.) reveals the infections with parvovirus or Toxoplasma presents of typical meronts with the gondii have also been reported. “wheel spoke” aspect.

The dogs infected with H. americanum In the case of H. americanum, the usually show fever, abnormal gait (limb parasitemia is generally lower than in stiffness, inability to rise), muscular pain dogs infected with H. canis. Therefore, the and generalized muscular atrophy. Often, usual direct confirmation of the disease is a mucopurulent ocular discharge is by showing the presence of parasites in present, as a result of decreased tear muscle biopsies. Additionally, production caused by with inflammation radiography of long bones showing of the external ocular muscles. American periostitis is another indication for H. canine hepatozoonosis evolves as an americanum-hepatozoonosis. acute infection but can also become Several serological methods (IFAT, chronic. In either situation, the muscular ELISA) are available for the diagnosis of pain is present and this can be canine hepatozoonosis. The most generalized or localized, usually at the sensitive method for the detection of level of lumbar and cervical spine. The Hepatozoon spp. in the blood of dogs is chronic infections can be complicated by PCR and quantitative evaluation is immune-mediated glomerulonephritis possible by real-time PCR. and uveitis. Clinical hematology and blood biochemistry are similar to those Treatment. The treatment of the dogs described for the infection with H. canis. with clinical infection with H. canis is done using imidocarb dipropionate, 5-6 Lesions. Following death subsequent to mg/kg every 14 days until gamonts severe hepatozoonosis, the meronts of H. disappear from the blood smears. canis can be found in various tissues: However, the absence of gamonts from liver, lungs, kidneys, spleen, bone the blood smears is not equal to zero marrow or lymph nodes. Associated parasitemia. Studies using more sensitive lesions include hepatitis, pneumonia and detection methods (i.e. PCR), showed that glomerulonephritis. In the case of H. no treatment can completely eliminate americanum, the most important lesion is the infection. As dogs with low the myositis. parasitemia usually show no clinical Diagnosis. Direct detection of H. canis signs, the prognosis of such cases is gamonts in the neutrophils of infected generally good. dogs is dependent on the level of The treatment of dogs infected with H. parasitemia. As symptomatic dogs americanum must follow a combination usually have significant parasitemia,

172 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | of oral therapy with trimethoprim- disease. The symptoms are not specific sulfadiazine (15 mg/kg every 12 h), and highly diverse: weakness, lethargy, pyrimethamine (0.25 mg/kg every 24 h) anorexia, weight loss, fever, and clindamycin (10 mg/kg every 8 h) for hypersalivation, mucosal ulcers, enlarged 14 days. lymph nodes, anemia. The disease can be successfully treated using doxycycline (5 In order to avoid clinical relapses, it is mg/kg, orally, every 12 hours), recommended that after relief of acute oxytetracycline (50 mg/kg, every 12 forms, an anti-coccidial drug to be given hours) until recovery signs or with a orally for long term. The treatment single dose of primaquine (2 mg/kg, suggested is decoquinate at 15 mg/kg orally). mixed in food every 12 h for two years. Supportive therapy might be considered for pain relief. 2.4.5 Babesiidae Control. Although canine hepatozoonosis is a vector-borne disease, its unusual Introduction. Includes several genera transmission route makes its prevention parasitic mostly in warm-blooded different than for the other arthropod- vertebrates. transmitted disease. It is recommended to avoid the oral ingestion of ticks by Ecology and transmission. All the dogs, both from the environment and members of the family undergo the while grooming. Hence, the use of topical merogonic development only in and environmental acaricides is erythrocytes with no preliminary encouraged. No vaccine is currently development in white leukocytes or other available. types of cells. The transmission of the species for which the life cycle is known is by the bite of ticks.

2.4.4.2 Feline hepatozoonosis Medical importance. Species of genus Babesia are very important veterinary Feline hepatozoonosis is by far less parasites, causing severe clinical common than the canine infection. It has diseases, mortality and economic losses. been reported from several countries in Some species are zoonotic, but clinical Asia, Africa, North America and Europe. signs in humans are present with few The species of Hepatozoon responsible exceptions only in splenectomized for the feline infection are not well people. defined, nor its life cycle known. The The diseases caused by Babesiidae and development of Hepatozoon meronts in Theileriidae are generically known as cats takes place in the skeletal muscles piroplasmosis. As the life cycle and and the myocardium causing elevated pathogenesis is different, more accurate levels of creatine kinase. designation babesiosis and theileriosis or Decreased immunity caused by FIV and their plural forms (babesioses, FeLV favor the development of clinical theilerioses) will be used herein.

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2.4.5.1 Babesioses Table 2.41 Named species of Babesia parasitic in domestic animals (modified and updated after Schnittger et al. 2012) Introduction. Babesioses are tick-borne diseases found in all species of domestic Host Species Distribution B. bovis Worldwide mammals and in many wild mammals, B. bigemina Worldwide with great economic impact but also B. divergens Europe Cattle medical and veterinary importance. The B. major Asia, Europe B. occultans Africa disease is clinically severe in non- B. ovata Asia immune hosts, causing anemia, fever, B. bovis America, Asia Buffalo B. bigemina America, Asia jaundice, hemoglobinuria and sometimes B. orientalis Asia Horse, Africa, America, death. B. caballi donkey Asia, Europe Historical notes. Two distinct Pig B. trautmanni Africa, Europe Sheep, Asia B. crassa geographical areas (Eastern Europe and goat Africa, Asia, North America) are particularly B. motasi Europe important from historical point of view. Africa, Asia, B. ovis At the end of the 19th century (1888), Europe Victor Babeş, a Romanian microbiologist, Dog B. canis Europe B. conradae North America discovered the agent of the disease in Asia, Africa, B. gibsoni cattle. One year later, Theobald Smith, a America, Europe B. rossi Africa medical doctor from New York, described B. vogeli Worldwide the agent of Texas fever in USA and was Cat B. felis Africa B. presentii Asia the first to elucidate the tick-borne nature. It was the first time ever when arthropods were shown to transmit a Certain species of Babesia have been disease. Babeș originally named the recorded in several mammal species, species Hematococcus bovis. Smith and raising the question if they are really host Killborne named the Texas fever agent specific. For instance, Babesia caballi is Pyrosoma bigeminum. In 1893, Starcovici commonly found in dogs and Babesia erects genus Babesia and includes both canis is also present in cats. Molecular species there. In the years to follow, many surveys have shown also other such new species have been described from cross-infections, but their clinical and various domestic and wildlife hosts. epidemiological importance is not yet fully understood. Etiology. The taxonomy and species status of genus Babesia is highly dynamic. Morphology. Among the various As most species descriptions were based developmental forms of Babesia in the mostly on morphology of the erythrocytic definitive (ticks) and intermediate stages and host specificity, the validity of (vertebrate) hosts, the most important some still needs molecular confirmation. stages from morphologic (diagnostic) The list of valid Babesia species parasitic point of view are those found in the in domestic animals is shown in table erythrocytes of the domestic mammals. 2.41. The most common intraerythrocytic

174 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | stages are the merozoites. They are usually found in pairs, forming a typical “V” shape (figure 2.82). In some of the species of Babesia, multiple divisions result in the formation of groups of 4 or 8 intraerythrocytic merozoites.

Based on the size of the intraerythrocytic stages, the species of Babesia are divided in two types: small Babesia (1.0-2.5 µm) and large Babesia (2.5-5.0 µm). Merozoites of small species usually form obtuse angles, while the larger ones form acute angles to each other. Sometimes, atypical forms (amoeboid, ring-shapes, Figure 2.83 Ring-shaped aspect of round) can be found in the erythrocytes Babesia canis in an erythrocyte from (figure 2.83). infected dog. (Photo Andrei D. Mihalca)

In other species of Babesia, 4 or 10 merozoites can be seen inside a single erythrocyte. Detection of free stages in the plasma is possible but very rare Life cycle. The life cycle of all species is event. heteroxenous (figure 2.84). As the sexual development takes place in ticks, they are considered definitive hosts. Mammals are intermediate hosts as they are harboring the asexual stages.

Mammals acquire the infection after a bite of an infected tick. Through the saliva, ticks inject into the blood stream of the vertebrate the infective sporozoites (figure 2.84 - 1).

They attach to erythrocytes (figure 2.84 - 2) and by endocytosis they enter inside them (figure 2.84 - 3). Inside the red blood cells, sporozoites start to feed, becoming trophozoites (figure 2.84 - 4) which subsequently divide by binary Figure 2.82 “V”-shaped disposition of fission resulting in the formation of two merozoites of Babesia canis in an merozoites in each erythrocyte (figure erythrocyte from infected dog. (Photo 2.84 - 5). Andrei D. Mihalca)

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Figure 2.84 Life cycle of Babesia spp. For the meaning of numbers and letters, please refer to the text.

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Sometimes, one additional cell division present some brief insights into the tick results in the formation of four biology and mechanism of vectorial merozoites in each erythrocyte (figure transmission. Ticks have three feeding 2.84 - 6). Merozoites rupture the infected developmental stages: larvae, nymphs erythrocytes (figure 2.84 - 7) and invade and adults (male and females). Most ticks new ones (figure 2.84 - 8), repeating the follow a so-called three host life cycle, merogony several times until many red meaning that each stage feeds on a blood cells are destroyed. During the different host. After larvae hatch from the course of infection, some merozoites are eggs, they attach to a host, feed with transformed into gamonts while still in blood and detach. After detachment, the erythrocytes of the intermediate larvae fall-off in the environment where hosts. When a new tick (figure 2.84 - 9, they undergo the first molting and 10, 11) feeds on the infected blood of the become nymphs. Nymphs search for a intermediate host, the erythrocytes with new host, they attach to it and feed again. gamonts reach its intestine. They will After fully engorged they detach, fall into invade the epithelial cells (figure 2.84 - the environment and molt for the second 12) and start the gametogony process. It time, becoming adults. Part of the seems that the other stages (i.e. nymphs become males, the others merozoites, sporozoites, trophozoites) become females. Adult ticks attach to the ingested by the vector are not able to third host, the feed and reproduce produce the intestinal infection in the sexually. Fully engorged and fertilized tick. After gamonts sexually join, the females fall-off the host. After some time zygote is formed. Each zygote spent in the environment, they lay differentiates into a mobile oocyst-like thousands of eggs and die. Males usually structure called kinete (figure 2.84 - 13). do not feed, and they are attached to the Through the hemolymph, the kinetes will host only for finding the females. invade all the tick’s tissue, including Considering all these events, it is evident ovaries (figure 2.84 - 15) and salivary that each stage of the tick feeds only once glands (figure 2.84 - 14). When reaching and only on one host. The next time it will the salivary glands, the kinetes start the feed it will be already as another sporogony with the formation of developmental stage. If a larvae (figure sporozoites. Kinetes from the ovaries will 2.84 - L) feeds on a host infected with be responsible for the transovarial Babesia, it will acquire the infection transmission to the eggs produced by the (figure 2.84 - 9). The next time it will female tick and eventually part of the feed, it will be as a nymph, on another next generation larvae will be already host. Hence, the maintenance of the infected when hatch. The sporozoites infection from a stage to another (figure from the salivary gland will infect a new 2.84 - 9’) is a sine qua non prerequisite host when the tick feeds again. for the existence and transmission for any tick-borne disease. This essential In order to understand the ecological event is known as transstadial passage rationale behind the transmission of (or less accurately, transstadial pathogens by ticks, it is essential to

177 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | transmission). The same situation it to nymphs and then to adults. So the happens if a nymph (figure 2.84 - N) next adult generation will be ultimately feeds on an infected host (figure 2.84 - responsible for infecting a new dog. This 10). It will pass the infection to adults means sometimes even years. (figure 2.84 - 10’) which will eventually An interesting mechanism of infect a new host. In the case the stage transmission for those species vectored which is infected is an adult female by one-host ticks (i.e. all stages feed on (figure 2.84 - F) (figure 2.84 - 11), for the the same host individual) is by males infection to be ecologically continuous, which move from an animal to the other. the female must be able to pass the acquired pathogen to the offspring One more essential factor in the (figure 2.84 - 11’). This is possible in transmission mechanism of tick-borne certain tick-borne pathogens (but not in disease is the infectivity of the tick saliva all) because of the presence of anatomical in the first hours or days after structures which connect the digestive attachment. It was said before that tube of the tick with the ovaries. This Babesia moves from the intestine of the feature is known as transovarial tick to the salivary glands where transmission. Not all species of Babesia sporozoites will be formed. This in-tick are able to pass by this transovarial migration takes place for most of the tick- route. It seems that B. felis from cats or B. borne pathogens only after the ticks microti from rodents are such species. attaches to a host which is suitable for the pathogen’s development. Factors from Given the relatively strict host specificity the vertebrate’s blood will activate the of Babesia, one more extremely migration of the pathogens to the salivary important barrier is not only the gland and eventually its transmission to alternation of ecological hosts but also the host. This key aspect is essential from the host species. Not all the three hosts practical point of view. If a tick dies from a complete life cycle of a tick are because of an antiparasitic treatment or it belonging to the same species. For is mechanically removed from the host instance in the case of Dermacentor before the salivary migration occurred, reticulatus the larvae and nymphs feed the risk for pathogen transmission is usually on micromammals and only adult limited. ticks use dogs. It is known that D. reticulatus is the vector of Babesia canis. The vectors for Babesia species of If Babesia canis infects only dogs, it is domestic animals are always hard-ticks evident that the only stages which (Ixodidae). Not all tick species transmit acquire the infection from dogs are all Babesia species. The spectrum of adults. It logically means that infected vectors for each Babesia species is shown adults females pass the infection to the in table 2.42. eggs. Infected larvae which hatch will A possible but rather unusual route of feed on small mammals, possibly not transmission for Babesia is the vertical infecting them. However, the larvae transmission in vertebrates, from an maintain the infection in their body, pass

178 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infected mother to the fetus. shown in table 2.41. Certain species are Transplacental transmission has been distributed worldwide (of course except reported in humans, cattle, horses and the polar regions), some others are more dogs. Transmission through infected or less limited. As domestic animals are needles or blood transfusion is also everywhere where people are present, possible. the spatial distribution of Babesia is largely influenced by the degree of

specificity to the tick-host and the Table 2.42 Tick vectors for selected Babesia distribution of those ticks. species of domestic animals As with most other vector-borne Babesia Tick vector species parasites, the occurrence of Babesia is Rhipicephalus microplus, R. influenced by the ecology of the vectors. annulatus, R. decoloratus, R. B. bigemina geigyi, R. evertsi, Transmission to the vertebrate host is Haemaphysalis punctata seasonal and correlated with the ticks’ Rhipicephalus microplus, R. B. bovis annulatus, R. geigyi dynamics. The outbreaks of acute Rhipicephalus evertsi, R. bursa, babesiosis typically take place during the R. sanguineus, Dermacentor albipictus, D. variabilis, D. warm seasons. nitens, D. marginatus, D. B. caballi reticulatus, D. silvarum, The newly introduced animals are more Hyalomma anatolicum, H. susceptible to acute infection. Long-term dromedary, H. marginatum, H. scupense, H. truncatum parasite-host associations result in Dermacentor reticulatus, B. canis acquired resistance, but with the Rhipicephalus sanguineus (?) B. conradae Rhipicephalus sanguineus (?) presence of parasites in the blood of B. crassa Unknown clinically healthy animals. This results in B. divergens Ixodes ricinus, I. persulcatus a permanent infection source for ticks B. felis Unknown B. gibsoni Haemaphysalis longicornis and through them to Babesia-free Haemaphysalis punctata, H. B. major individuals. The local breeds are more longicornis Haemaphysalis punctata, resistant than imported breeds. Bos Rhipicephalus bursa, H. B. motasi indicus breeds are more unlikely to qinghaiensis (?), Amblyomma variegatum (?) develop clinical babesiosis. Hyalomma marginatum, H. B. occultans rufipes, H. anatolicum, H. The concept of endemic stability is very truncatum important in the epidemiology of Rhipicephalus B. orientalis haemaphysaloides babesiosis, mainly in large animal B. ovata Haemaphysalis longicornis communities as livestock (cattle, small Rhipicephalus bursa, R. B. ovis sanguineus (?), R. turanicus (?) ruminants). The endemic stability means B. presentii Unknown that the pathogen is present in the host B. rossi Haemaphysalis elliptica population but the clinical disease occurs B. trautmanni Rhipicephalus turanicus B. vogeli Rhipicephalus sanguineus rarely.

Although the disease can be transmitted Epidemiology. The geographical by a single infected tick, the prevalence of distribution of various Babesia species is

179 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | infection in the tick population is usually Other mechanisms are also involved in very low. the destruction of infected erythrocytes. They become osmotically fragile. The susceptibility to develop clinical Moreover, during penetration by babesiosis is higher in adult animals than sporozoites or merozoites, direct injury in young ones. This rather unusual of the cellular membrane can result in situation is possibly related to immune mechanical destruction. factors, most probably to the acquired transplacental or colostral passive The altered erythrocyte metabolisms and immunity. membranary processes slow down their circulation speed in capillaries and favors Babesia, as most vector-borne parasites sludging. The most intense erythrocyte spends all their life inside a host. No sludging occurs in the central nervous exogenous stages exist. Therefore, we system and in the lungs. cannot discuss about the environmental resistance of Babesia. Babesia can survive The excessive production of cytokines in ticks for several years, until the life and other pharmacologically active cycle of that individual tick ends. compounds cause vasodilatation, hypotension, increased capillary Pathogenesis and immunology. Most of permeability and endothelial damage. the pathogenesis of babesiosis is related These are ultimately responsible for the to the altered immune response of the disseminated intravascular coagulation, hosts. This is the reason why the sections which is usually lethal. The increased are discussed together. capillary permeability together with the The most important pathogenetic resulting edema are responsible for the mechanism is related to the destruction severe respiratory distress in certain of erythrocytes. Two mechanisms are species. involved in this process: immune- The erythrocyte depletion causes tissular mediated erythrolysis and non-immune hypoxia and subsequent failure of various erythrolysis. internal organs, including kidneys. As a After the erythrocytes are infected they result of hypoxia, there is an over display on their outer surface modified production of lactic acid, resulting in antigenic structures. Therefore, they metabolic acidosis. Hyperventilation, as a target the synthesis of opsonizing result of anemia is also considered to be a antibodies and as a consequence, the compensatory attempt to fight acidosis. infected erythrocytes are destroyed by Overall, certain species of Babesia are the mononuclear-phagocyte system. Even responsible for the systemic uninfected erythrocytes can be the target inflammatory response syndrome which of autoimmunity, as soluble parasitic results in multiple organ failure: acute antigens adhere to their surface. renal failure (typically in dogs), hepatopathy or pulmonary edema.

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Massive destruction of erythrocytes is monocytes, macrophages and followed by hemoglobinuria and neutrophils. jaundice. However, these seem to be the The role of the specific immunity is feature of moderately pathogenic species. related to the production of antibodies. Interestingly, the level of parasitemia is They will mediate the cytotoxic killing of not correlated with the severity of clinical parasites during re-infections. signs. In cattle infected with B. bovis and Clinical signs. Most infections are at a with severe acute signs, the parasitemia very low level and they are clinically is normally less than 1%, while asymptomatic. Clinical signs usually subclinical or moderate cases with B. occur in individuals which have never bigemina are associated with 10-30% been in contact with the pathogen (see infected erythrocytes. This could be immunology). The clinical infection has explained also by the higher rate of been documented in most domestic erythrocytic destruction in the case of species, in humans as well as in wildlife. more pathogenic species, resulting in Because of the associated mortality, false “low” intensity, as most of the babesioses of livestock are economically infected erythrocytes could have been important. Although the clinical course already destroyed. and symptoms vary in each host species The low infective doses in immune and also between Babesia species, there animals are responsible for life-long is a general picture which can be resistance. This non-sterile immunity, considered. also known as premunition is the most In all species, the infection with Babesia prominent immunologic feature in can be acute, sub-acute or chronic. persistent, subclinical Babesia infection. Both components of the immune system Common features of acute babesiosis are important in the anti-Babesia include fever, anemia, jaundice and resistance. The innate immunity is the hemoglobinuria. The general status of the first line defining the host specificity of animals with acute signs can be severely Babesia species. Experimental altered, with malaise, lethargy, splenectomy allows the infection of tachypnea, muscle tremors, anorexia and otherwise resistant host species. The weight loss. An alternation of diarrhea involvement of innate immunity has been with constipation can be present. The suspected to be responsible for the duration of the acute form is usually resistance of young animals to infection. around one week. One key circumstantial proof for such a Sub-acute and chronic babesioses are hypothesis is the resistance to infection similar but less severe that acute forms. of calves born from non-immune Hyperacute forms are reported, but mothers. However, the colostrally passed rarely. They usually consist in sudden antibodies also play a role. The most death, within few hours from the onset of important components of the innate symptoms. immunity active against Babesia are

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As there are certain specific features for associated with a great diversity of various Babesia species-host species clinical signs: anorexia, lethargy, fever, associations, the most common of them weakness, weight loss, hemolytic anemia, will be discussed. icterus, splenomegaly, lymphadenopathy, vomiting, ascites, diarrhea, buccal ulcers, In bovines the most pathogenic species is seizures, ataxia, paresis etc. B. bovis. Other species (B. bigemina, B. divergens) are moderately pathogenic In cats the published reports are few. The while others (B. occultans, B. major) are main sign of the infection with B. felis is most often non-pathogenic. Babesiosis fever associated with depression, loss of produced by B. bovis (incubation period appetite, weight loss, weakness, 10-12 days) is characterized by high tachycardia, tachypnea, vomiting, and fever, ataxia, incoordination, anorexia, diarrhea. Jaundice is uncommon. hemoglobinuria, nervous signs, Pathology. Gross pathology of babesiosis circulatory shock and death. The is as diverse as the clinical picture is, both infections with B. divergens or B. reflecting its complex pathophysiology. bigemina (incubation period 4-5 days) produce fever, hemoglobinuria and In cattle infected with B. bovis, the acute anemia, but nervous signs are absent. lesions include: generalized congestion of abdominal organs (soft and pulpy spleen, In sheep and goats, the most pathogenic hepatomegaly, congested kidneys), species are B. motasi and B. ovis. It is generalized anemia and jaundice, responsible for fever, anorexia, presence of dark-red urine in the urinary listlessness, anemia, jaundice. bladder, distended gall-bladder with Hemoglobinuria is not constant. thick and granular content or pulmonary Acute cases caused by B. caballi in horses edema. (incubation period 10-30 days) are The lesions in dogs which died of acute associated with high fever, dyspnea, babesiosis (usually caused by small mucosal congestion, edemas, and anemia. Babesia) consist of staining of tissues The icterus and hemoglobinuria may be with hemoglobin, hepatomegaly, present, but are less severe that in the splenomegaly, lymphadenopathy, infection with Theileria equi. nephrosis, signs of disseminated The clinical course in dogs is often very intravascular coagulation. Histologic severe, but symptoms greatly vary. They findings are consistent with the depend on various factors, one of them pathophysiology: capillary congestion, being the species. Certain Babesia species thrombosis in various organs, erythroid (i.e. B. vogeli, B. gibsoni) have lower hyperplasia in the bone marrow and pathogenicity and they typically produce vasculitis. mild or subclinical infection. Others (B. The lesions in other host species are canis, B. rossi, B. conradae) are similar with those described in cattle and responsible for acute forms, with more dogs. severe outcome. The acute form in dogs is

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Diagnosis. The clinical signs and lesions Real Time PCR or Loop Mediated in babesiosis are characteristic but not Isothermal Amplification (LAMP). pathognomonic. Hence, the diagnosis Treatment. The efficacy of various drugs must always be confirmed in the is different against the various species of laboratory. The most reliable methods Babesia. Usually, small Babesia species are the microscopic detection methods are more resistant than large Babesia when the stages of the parasite are seen species. Two drugs are currently widely directly by the observer. used for the treatment of babesiosis in Detection of the parasite is possible in animals: imidocarb and diminazene blood smears, when the characteristic aceturate. Treatment does not always parasitic forms are seen inside the eliminate the infection but it can erythrocytes. From dead animals, other significantly reduce the clinical impact. In cytological examination can be severe cases, supportive therapy is performed if blood is not available required. It is forbidden to use the anymore (i.e. brain smears, blood from treatment together with anti-Babesia internal organs). Although the method is vaccination, as the drugs kill the vaccinal highly specific, its sensitivity is reduced, strain and interfere with a proper as usually during the chronic phase the installation of immunity. parasitemia is relatively low, under the In cattle, diminazene aceturate is detection threshold. Another effective at 3.5 mg/kg, intramuscularly, 1- disadvantage is the relatively long time 2 administrations. The injection protects necessary for the thorough examination the cows against reinfection with B. bovis of a blood smear. Specific identification is and B. bigemina for 2-4 weeks. Imidocarb not always possible, mainly in hosts at 1.2 mg/kg, single dose, given where more Babesia species are known. subcutaneously is also effective. Higher Various immunological methods are doses (3 mg/kg) have even protective available for the detection of anti-Babesia effect for 1-2 months. antibodies in the sera of infected animals: In dogs infected with large piroplasms the Indirect Fluorescent Antibody Test (i.e. Babesia canis), the treatment of (IFAT), ELISA, Immunochromatography choice is imidocarb (5-6 mg/kg, Test (ICT). The disadvantage of intramuscularly, single dose and then serological methods consist in failure to repeated in two to three weeks). detect acute infection (the antibodies are Imidocarb is not effective against the not yet produced) and difficulty in infection with small Babesia (i.e. B. interpreting the seropositivity (post- gibsoni). vaccinal versus post-infective). Diminazene aceturate has been used in The most sensitive and specific methods the past for the treatment of B. gibsoni are those targeting the parasite’s DNA. infection, but currently, the most effective The most widely used is the PCR, but also treatment is considered to be the Reverse Line Blot Hybridization (RLB),

183 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | combination between atovaquone and 2.4.6 Theileriidae azithromycin. Introduction. All Theileriidae are tick- In cats infected with small Babesia the borne intracellular parasites of wild and only effective treatment is with domestic animals with significant primaquine phosphate (0.5-1.0 mg/kg economic importance. Two genera are intramuscularly, orally or intravenously, particularly important in domestic single dose or daily for 3 consecutive animals: Theileria and . days). Diminazene aceturate (3.5 mg/kg, intramuscularly, single dose) is effective Morphology. The morphological against large Babesia of cats. There are no appearance of Theileria is more reliable clinical studies to show the heterogeneous than Babesia. Intracellular efficacy of imidocarb in cats. stages in the mammalian host includes round, ovoid, rod-like or irregular forms. Diminazene aceturate is recommended also for sheep, goats, swine and equines. Ecology and transmission. The biology The dose is between 3.5-5 mg/kg, of Theileriidae is comparable to intramuscularly, twice at 24 hours Babesiidae, but with two main interval. differences. In the vertebrate host, species of Theileria undergo the first Control. There are several approaches in multiplication in white blood cells and preventing and controlling babesiosis in only later in the course of infection they domestic animals. One of the most are able to infect the erythrocytes. In the successful approaches is the control of tick host, there is no transovarial vectors. Long-term strategic programs to transmission. reduce the tick parasitism in livestock result in great improvements. Medical importance. Theilerioses are important diseases of cattle, sheep, goats This can be achieved by the use of and horses in regions with tropical acaricide drugs. They are expensive and climate, mainly in Africa but also in very toxic to the environment so their warmer regions of Asia and Europe. They large scale use is often limited. Selecting are not zoonotic. Recently, the infection resistant breeds is another option. Bos with Theileriidae gained importance also indicus breeds are more resistant than in dogs and cats. Bos taurus breeds. The areas with endemic babesiosis foci should be populated with breeds known to have natural resistance. 2.4.6.1 Theilerioses

The most modern approach for Introduction. Species of genus Theileria controlling babesiosis is the use of are responsible for several nominal immunoprophylaxis. Commercial diseases mainly in cattle, small vaccines are available for bovines and ruminants, horses and dogs from tropical dogs. countries (table 2.43).

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Historical notes. The first to observe Etiology. The species of Theileria Theileria parasites in the blood of cattle parasitic in domestic animals are shown was Robert Koch. In 1897, he found rod- in table 2.44. Certain species of Theileria like parasites inside the red blood cells of have been divided into subspecies, but cows from the current Tanzania. He their taxonomic status is still debated. considered them to be morphs of Babesia Moreover, these subspecies are bigemina. In 1901, when Australian cows serologically and morphologically were introduced to Zimbabwe, many died indistinguishable. of babesiosis. The surviving cows were The main difference between them is in moved to another location. Three weeks the epidemiological features and later they died of another, more acute geographical distribution. However, as and unknown diseases. Again, Koch they are commonly used in various realized it is a different disease than textbooks, they will be briefly mentioned babesiosis, Theiler described in 1904 the herein. Theileria parva has been divided new agent as Piroplasma parvum. In the into three subspecies: T. parva parva (the same year, Dschunkowsky and Luhs typical agent of East Coast Fever), T. described the second species, Piroplasma parva lawrencei (causing the Corridor annulatum in Transcaucasia. Genus disease) and T. parva bovis (only mildly Theileria was erected in 1907 by pathogenic). Bettencourt, Franca & Borges.

Table 2.43 Species of Theileria and Cytauxzoon parasitic in domestic animals

Host Species Vector genus Disease Distribution Mediterranean Coast Europe, Africa, Asia T. annulata Hyalomma Fever T. buffeli Haemaphysalis Benign theileriosis Worldwide T. mutans Amblyomma Benign theileriosis Africa East Coast Fever, Africa T. parva Rhipicephalus Cattle Corridor Disease Oriental theileriosis Asia, Europe, Australia, T. sergenti Haemaphysalis Africa T. sinensis Haemaphysalis Benign theileriosis Asia T. taurotragi Rhipicephalus Benign theileriosis Africa T. velifera Amblyomma Benign theileriosis Africa T. lestoquardi (syn. T. Ovine and caprine Europe, Asia, Africa Hyalomma hirci) theileriosis T. luwenshuni Haemaphysalis Ovine theileriosis Asia Sheep, Rhipicephalus Benign theileriosis Africa, Europe Goats T. ovis Hyalomma T. separata Rhipicephalus Benign theileriosis Africa T. uilenbergi Haemaphysalis Ovine theileriosis Asia Dermacentor, Equine theileriosis Worldwide Horses T. equi Hyalomma, Rhipicephalus Dogs T. annae Ixodes Canine theileriosis Europe, America Dermacentor, Cytauxzoonosis America Cats C. felis Amblyomma

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Morphology. From diagnostic point of macromerozoites (2-2.5 µm) and view, the stages in the mammalian host microschizonts with 80-90 are important. In the white blood cells, micromerozoites (0.7-1 µm). After the the schizonts of T. parva appear as merozoites rupture the lymphocytes, circular or irregularly shaped structures, they can follow two ways. They can either also known as Koch’s blue bodies. They infect other lymphocytes and maintain measure between 2 and 12 µm (average 8 the lymphocytic infection or penetrate µm). The macroschizonts have around 8 into erythrocytes, where they usually nuclei and the microschizonts have 50- remain until engorgement by vector ticks. 120 nuclei. In the tick’s gut, they undergo gametogony and sexual reproduction, In the red blood cells, the merozoites are resulting in the formation of the zygote usually single (there is no which ultimately becomes motile and intraerythrocytic division), they have bears the name of kinete. They remain in rod-shape and measure 1.5 x 0.5-1.0 µm. this stage in the intestinal cells of the Sometimes, other forms can be seen in ticks until they molt. After the the erythrocytes: round, oval, ring etc. subsequent stages (nymphs or adults) The morphology of other species is more start to feed on the next host, kinetes or less similar. The main differences invade the salivary glands of the ticks and consist in morphometric values. produce thousands of sporozoites. Life cycle. As a model of life cycle for Sporozoites from the salivary gland will Theileria, the development of T. parva infect a new host. There is no transovarial will be described in the following transmission in Theileria. paragraphs. In other species, there are The life cycle in genus Cytauxzoon is small differences in the intensity and similar to genus Theileria. ration of multiplication in erythrocytes and lymphocytes. For instance, if in T. Transplacental transmission from parva there is no intraerythrocytic infected mother to offspring is suspected division, in T. mutans, the predominant for certain species, including T. annae in multiplication takes place in the red dogs. blood cells. Epidemiology. The spread and Transmission of the infection to the distribution of the vector is responsible mammalian host is by the bite of nymph for the distribution of the diseases. and adult ticks. As the infection is not Although some species of Theileria have a transmitted by vertical (transovarial) more broad distribution, with different route, larvae are not infective. After the vectors in each area they occur, they are sporozoites are injected into the blood causing endemic diseases only on few stream, they infect the lymphocytes regions. For instance, T. parva causing the where they start the merogonic East Coast Fever is distributed in South, development producing merozoites. Two East and Central Africa. The group of T. types of schizonts (meronts) are known: parva causing the corridor disease is macroschizonts, containing around 90 endemic to East and Central Africa, as the

186 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | main reservoir is the African buffalo. changes in the structure of the Theileria annulata is responsible for the membranary proteins of the erythrocyte. Mediterranean Coast Fever and its Like in the case of babesiosis, the severity distribution ranges from Northern Africa of disease is not correlated with the (Morocco) to Middle East and Southern intensity of parasitemia. Russia. Immunology. The primo-infection is The host specificity of Theileria is an always severe. If animals survive they are important epidemiological feature. As for usually resistant to clinical infection. The instance species parasitic in cattle infect infection with one species does not also buffaloes or various wild ruminants, confer cross immunity against the the diversity of reservoir hosts creates infection with another species. permanent risks of outbreaks. Often, the pathogenicity in wild ruminants is almost The main immunological mechanism absent, but when the infection occurs in involves cellular immunity. In the domestic cattle it has devastating impact. infection with T. parva, the bovine major histocompatibility complex is mediating The mortality is also variable. Theileria the immune response through cytotoxic T lestoquardi causes between 50 and 100% lymphocytes. The infection with T. mortality in sheep and goats. When East annulata activates the release of Coast Fever infects previously non- cytokines by CD4+ T cells. They produce exposed groups, the mortality can reach IFN-γ which triggers the production of α 90% or more of the animals. tumor necrosis factor and nitric oxide by Young animals are more resistant than non-infected macrophages. These active adults. Local breeds from endemic areas compounds will destroy the infected are usually resistant to the infection. cells. The extracellular merozoites are killed by antibodies produced by B Pathogenesis. The pathogenicity is lymphocytes. different from Theileria species to Theileria species and even within the Like in babesiosis, the altered immune same species, between hosts. response is ultimately responsible for the pathogenesis of the diseases. The In cattle, the infection of lymphocytes increased production of cytokines results with T. parva induces blastogenesis in increased vascular fragility and resulting in an increased production of permeability. lymphoblasts which invade various tissues (“leukemia”-like infection). The Clinical signs. Despite the great species pathogenetic effect is dominated by the diversity, the disease has several massive alteration of the structure and common clinical features. Usually the functions of the lymphoid tissue and by adult animals are more susceptible and the subsequent anemia caused by signs of disease are more severe. The erythrolysis. The oxidative stress has presence of fever and generalized been incriminated as the main lymphadenopathy are characteristic. mechanism of erythrolysis. These induce

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The East Coast Fever is characterized by cattle. Symptoms include high fever, very high mortality in susceptible cattle listlessness, frothy nasal discharge, populations. Incubation is 1-4 weeks. The jaundice and enlargement of superficial main clinical signs include: high fever, lymph nodes. lymphadenopathy, severe dyspnea, Equine theileriosis produced by T. equi is tachycardia, and extreme weight loss. The a severe disease. Infected horses have fever is high persists until recovery or high fever, listlessness, hyperlacrimation, death. Infected animals have aural and palpebral edema, severe anemia, palpebral edema and hyperlacrimation. hemoglobinuria and icterus (signs typical The severe respiratory distress and the to babesiosis). excessive frothy nasal discharge are indicative of the severe pulmonary Canine theileriosis produced by T. annae edema which usually precedes death. is a severe disease causing acute fever, Some animals show hemorrhagic weakness, lethargy, tachypnea, diarrhea. The clinical hematology tachycardia, anemia and hemoglobinuria. findings include leucopenia, Clinical hematology reveals regenerative anemia, reticulocytosis, and The clinical signs and course of the thrombocytopenia. Corridor Disease are similar to the East Coast Fever but outbreaks are associated Feline cytauxzoonosis is characterized with the presence of wild buffaloes. by fever, acute onset of anorexia and lethargy. Following these acute signs, The Mediterranean Coast Fever has a increased the cats show increased much lower mortality rate and occurs vocalization, weakness, jaundice, after 1-4 weeks of incubation with fever, dyspnea, seizures, delayed capillary refill depression, hyperlacrimation, superficial time, lymphadenopathy. The outcome of lymph node swelling, nasal discharge and the disease is often death, preceded by weight loss. Except these signs which are deep coma. similar but slightly less severe than in the case of African theileriosis, the infection Pathology. The lesions of animals which with T. annulata produces also anemia died of theilerioses consist in severe with hemoglobinuria. pulmonary edema, generalized lymphadenopathy with enlarged lymph Benign bovine theilerioses (produced by nodes and multifocal lymphoid T. buffeli, T. mutans, T. sinensis, T. hyperplasia in various parenchymatous taurotragi and T. velifera) and Oriental organs (i.e. kidneys, brain). The spleen is theileriosis (T. sergenti) are either enlarged and congested. In species subacute or chronic and if symptoms are producing marked anemia, the tissues are present they are typically mild. Death is icteric and the urinary bladder may uncommon. contain hemoglobinuric urine. Ovine and caprine theileriosis produced Diagnosis. Acute clinical signs by T. lestoquardi is a severe disease, insusceptible animals correlated with the clinically similar to East Coast Fever of lesions and the history of the local

188 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | endemicity are good diagnosis indicators. mg/kg, daily for 2-5 days). Imidocarb or The confirmation of the diagnosis is diminazene diaceturate (see babesiosis) based on demonstration of the Theileria is also effective in horses. in various samples. The presence of Dogs infected with T. annae can be schizonts (meronts) in the infected white treated with limited success with blood cells or merozoites in the babesicidal drugs. Feline cytauxzoonosis erythrocytes can be observed under the is treated with a combination of microscope in blood smears or lymph atovaquone (15 mg/kg, orally, every 8 node biopsies. hours) and azithromycin (10 mg/kg, The most widely used serological test for orally, every 24 hours) for 10 days. screening of animals, mainly in the Control. The most efficient and widely international trade is indirect fluorescent used method employed for controlling antibody test. theileriosis is the control of ticks using Other serological methods (i.e. ELISA) are acaricides. Factors contributing to an also used. Molecular methods are highly unsuccessful control include acaricide sensitive but not implemented in the resistance of ticks, improper use of routine surveys. acaricide drugs (cheap, poor quality products, underdosing) or illegal animal Treatment. Most of the drugs are movements. Chemoprophylaxis with improving the clinical presentation but theilericidal drugs is an option for are not eliminating the infection. animals newly introduced to an endemic The treatment of infected cattle is area. recommended only during the very early Prevention by immune prophylaxis is phase of the clinical diseases. Two possible in cattle. Live vaccines have been molecules are widely used: parvaquone developed for T. parva and T. annulata. and its derivative buparvaquone.

Parvaquone is used as single dose (20 mg/kg, intramuscularly) in cattle infected 2.5 Ciliophora with T. annulata or at 10 mg/kg, intramuscularly, twice at 48 hours Phylum Ciliophora includes very diverse interval against the infection with T. groups of highly mobile protozoans parva and T. mutans. Buparvaquone (2.5 known generically as ciliates. Their mg/kg, single dose, intramuscularly) is defining feature is the presence of hair- effective against bovine theilerioses. like movement organelles, known as cilia In cattle but also other species, various (singular cilium). Most species are free- other drugs are used with variable living, and they usually inhabit aquatic efficacy. In equids infected with T. equi, environments, both marine and chlortetracycline hydrochloride and freshwater. The majority of them are oxytetracycline hydrochloride are predators of smaller microorganisms (i.e. effective when given intravenously (5.5 bacteria). Other species are symbiotic and they are associated with a huge

189 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | variety of hosts within most animal phyla, There are two types of reproduction in both invertebrates and vertebrates. ciliates. The asexual reproduction is by Certain groups inhabit the digestive binary fission. The sexual reproduction is system of their host where they are a very interesting process and it takes mutualists, commensals and few species place by conjugation. During conjugation, parasitic. Others are living on the two cells which are of complementary tegumentary surface of aquatic animals, mating type, are fertilizing each other. mostly fish as commensals or parasites. Two genera are of veterinary importance, Their medical importance is mainly both included in Class Litostomatea: known in aquaculture, as they produce Neobalantidium and Buxtonella. outbreaks in farmed fish (e.g. Ichthyo- phthirius, Trichodina, Chilodonella). In 2.5.1 Ciliates of domestic animals domestic animals very few species are known to be potentially pathogenic, and Ciliates are the least represented the most prominent example is protozoans in the veterinary Neobalantidium coli. parasitology. They are usually All species are unicellular and they bear opportunistic pathogens, and the clinical on their surface variable number of cilia. infections are sporadic. Two diseases will Their arrangement and patterns are be discussed: balantidiosis of swine and important in classification. The ingestion buxtonellosis of cattle. of nutrients is through a small opening in the cell, known as cytopharynx, surrounded by a group of cilia. Waste 2.5.1.1 Balantidiosis material is eliminated from the cell through another opening, known as Introduction. Balantidiosis is a cytoproct (or “cellular anus”). The worldwide distributed parasitic disease internal structure of ciliates is also very of the lower intestinal tract of pigs and characteristic. They have two nuclei, one humans, with usual subclinical course but larger (macronucleus) and one smaller problematic in immunocompromised (micronucleus). The macronucleus is the patients. true-nucleus and it is controlling the Historical notes. In 1857, Malmstein metabolic activity of the cell. The role of described a new species of ciliates from micronucleus is in the sexual the feces of dysenteric patients. He reproduction. Like all eukaryotic cell, named it coli. One year later, ciliates have all the typical cell organelles Claparède and Lachmann erected the (mitochondria, Golgi body etc.). genus for a newly described The life cycle of all parasitic ciliates is species, B. entozoon from frogs. In 1858, homoxenous, and transmission is directly Stein moved Paramecium coli into genus by contact (in species parasitic on the Balantidium. However, due to evident skin of fish) or via ingestion of cysts (in phylogenetic differences, in 2013, species parasitic in the digestive tube). Pomajbíková et al., erected the new genus

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Neobalantidium and placed within it The newly formed cysts are passed via Paramecium coli. the feces.

Etiology. The species responsible for the Epidemiology. The disease is virtually infection of pigs is Neobalantidium coli. It present wherever pigs are present. is not clear yet if all Balantidium-like Neobalantidium coli is found in more than ciliates described from pigs, primates and half of the adult pigs. In young pigs the camels are conspecific with N. coli. prevalence is lower. In some pig However, until new data is available, we populations the prevalence can be 100%. list N. coli as a zoonotic agent. In humans, the disease is sporadic and Morphology. According to the the geographic areas with high description by Pomajbíková et al. (2013), prevalences include Latin America, trophozoite is elongated, rounded at the Philippines, Indonesia, Papua New posterior end and narrow at the anterior Guinea and Middle East. Pigs-to-humans end. The size of the trophozoite is 30-300 and humans-to-humans cycle are x 30-100 µm. The surface is covered by described. The occurrence of the disease cilia, arranged in longitudinal rows. The in humans is influenced by several cytostome-cytopharyngeal complex is factors. The close contact between located at the apical end and surrounded humans and pigs together with improper by longer cilia. The cysts (40-60 µm) are waste management seem to be such spherical or ovoidal s one hypobiotic factors. trophozoite. Survival of cysts is longer in warm and Life cycle. Neobalantidium coli inhabits humid climate. This is why the the cecum and colon of its hosts. It is not prevalence of human disease is higher in clear if interspecific transmission is humid tropical areas. possible. The life cycle is direct Pathogenesis. Neobalantidium coli is (homoxenous) (figure 2.85) typically a commensal of the large The organisms are passed in feces as intestine. It typically feeds through the cysts. Cysts are ingested via cytopharynx with debris and other contaminated food or water by a new particles from the intestinal content host (figure 2.87 - 1). In the intestine of without any pathogenic effect. Under the new host, trophozoites excyst and certain condition (decrease of immunity, grow. They inhabit the surface of the intestinal dysmicrobism) it becomes mucosa where they feed through their pathogenic by producing proteolytic cytopharynx with various particles. They enzymes which destroy the intestinal multiply asexually by binary fission epithelium resulting in acute diarrhea. (figure 2.87 - 2) or sexually, by Persistent mucosal damage may result in conjugation (figure 2.87 - 3). When the humans in the production of ulcers. Deep intestinal content begins dehydration ulcers can be complicated by potential prior to fecal transmission, N. coli starts fatal intestinal perforations. the encystment process (figure 2.87 - 4).

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Figure 2.85 Life cycle of Neobalantidium coli. For the meaning of numbers, please refer to the text.

In pigs, they can invade lesions produced Immunology. The role of immunity is not by other pathogenic organisms. clear. Indirect evidences however suggest Production of hyaluronidase by N. coli the role of the immune system in aggravates the ulcers. maintaining the infection asymptomatic. In immunocompromised patients, Neobalantidium coli can produce a

192 | P r o t o z o a M I H A L C A | T e x t b o o k o f V e t e r i n a r y P a r a s i t o l o g y | systemic disease, infecting the lungs, sulcata. The signs may include mild lymph nodes. diarrhea.

Clinical signs. The vast majority of Identification of “Balantidium”-like cysts infected pigs have no clinical signs. Under in the feces of cows is indication of certain conditions, some pigs will show Buxtonella sulcata. moderate to severe acute diarrhea.

Pathology. In non-clinical infections, even large numbers of organisms are not Selected references and further associated with lesions. In clinical cases, reading the lesions consist in focal colitis.

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