Neospora Caninum and Hammondia Heydorni Are Two Coccidian Parasites with Found N

66 Opinion TRENDS in Parasitology Vol.18 No.2 February 2002

from the infective larval stage of Toxocara canis 22 Hunter, S.J. et al. (1999) The isolation of extracellular CuZn superoxide dismutases in by an expressed sequence tag strategy. Infect. differentially expressed cDNA clones from the the human parasitic nematode Onchocerca Immun. 67, 4771–4779 filarial nematode Brugia pahangi. Parasitology. volvulus. Mol. Biochem. Parasitol. 88, 20 Gregory, W.F. et al. (2000) The abundant larval 119, 189–198 187–202 transcript-1 and 2 genes of Brugia malayi encode 23 Au, X. et al. (1995) Brugia malayi: Differential 25 Selkirk, M.E. et al. (2001) Acetylcholinesterase stage-specific candidate vaccine antigens for susceptibility to and metabolism of hydrogen secretion by nematodes. In Parasitic filariasis. Infect. Immun. 68, 4174–4179 peroxide in adults and microfilariae. Exp. Nematodes: Molecular Biology, Biochemistry 21 Blaxter, M.L. et al. (1996) Genes expressed in Parasitol. 80, 530–540 and Immunology (Kennedy, M.W. and Brugia malayi infective third stage larvae. Mol. 24 Henkle-Dührsen, K. et al. (1997) Localization Harnett, W., eds), pp. 211–228, CABI Biochem. Parasitol. 77, 77–93 and functional analysis of the cytosolic and Publishing

N. caninum and T.gondii Neospora caninum In 1984, Bjerkås et al. [5] first discovered a toxoplasmosis-like disease of Norwegian dogs that had no demonstrable antibodies to T. gondii. In 1988, and Hammondia Dubey et al. [6] described in detail a similar neurological disease of dogs in the USA, distinguished the parasite from T. gondii based on antigenic and heydorni are separate ultrastructural differences, and proposed the genus Neospora with N. caninum as the type species. Isolation and in vitro cultivation of N. caninum [7,8] species led to the development of serological and immunohistochemical tests [7,9], which confirmed that N. caninum is distinct from T. gondii. Today, J.P.Dubey, Dolores E. Hill, David S. Lindsay, N. caninum is recognized as a major cause of abortion Mark C. Jenkins, Arvid Uggla and in cattle worldwide [10,11]. In 1998, Marsh et al. [12] described another species Clarence A. Speer of Neospora, N. hughesi, as a cause of neurological disease in horses. The full life cycle of N. caninum was not discovered until 1998, when McAllister et al. [13] Neospora caninum and Hammondia heydorni are two coccidian parasites with found N. caninum oocysts in the feces of dogs fed morphologically similar oocysts in canine feces. It was recently proposed that tissues from experimentally infected mice. When they are one species. In this paper, we review the biology and morphology of these oocysts were inoculated into immunodeficient these parasites and present evidence that N. caninum and H. heydorni are mice [13] and normal gerbils [14], neosporosis was separate species. induced. Recently, Lindsay et al.[15] found that dogs fed tissues of mice infected with cloned N. caninum Toxoplasma gondii, Neospora caninum, Hammondia tachyzoites produced N. caninum oocysts, thus hammondi and Hammondia heydorni are closely confirming the dog as a definitive host of N. caninum. related coccidian parasites with similarly sized oocysts Since the discovery of N. caninum in 1988, >500 [1]. Cats (felids) are definitive hosts for T. gondii and papers have been published on this parasite in H. hammondi, and dogs (canids) are definitive hosts peer-reviewed scientific journals. The description of for N. caninum and H. heydorni. However, these numerous morphological, biological, antigenic and species have important biological and ultrastructural molecular differences [10,16–20] between N. caninum differences. Whereas T. gondii causes a disseminated and T. gondii leave no doubt that they are separate infection in humans and animals, and N. caninum is species, and this issue will not be discussed further in one of the most important causes of abortion in cattle, this communication. H. hammondi and H. heydorni are not known to be of clinical significance. Recently, Mehlhorn and Heydorn N. caninum and H. heydorni [2] and Heydorn and Mehlhorn [3] questioned the Much of the confusion regarding the identity of validity of the genera Hammondia and Neospora and N. caninum and H. heydorni results from incomplete argued that, in this group, only T. gondii and T. heydorni knowledge of the details of the life cycles of these are valid. In a rebuttal paper, Frenkel and Dubey [4] parasites. Mugridge et al. [21] and Mehlhorn and stressed the ultrastructural, biological and genetic Heydorn [2] tabulated differences and similarities differences between T. gondii and H. hammondi. between the two species. They gave reasons why Hammondia and Toxoplasma Heydorn [22] discovered that dogs fed naturally should be deemed separate genera, and why T. gondii infected beef shed sporulated oocysts (which should be regarded as a species distinct from were of a species of Sarcocystis) and small, H. hammondi. This discussion is not repeated here; unsporulated oocysts (which he then called instead, this article discusses the relationships Isospora bigemina). Isospora bigemina was first between N. caninum, T. gondii and H. heydorni. named Coccidium bigeminum in 1891, based on the

(a) (b) to dogs, coyotes and foxes have been identified as definitive hosts for H. heydorni [1].

Tissue cyst stage of H. heydorni The tissue cyst stage of H. heydorni has not been conclusively identified in naturally infected animals. The only report describing tissue cysts of H. heydorni is by Matsui [26], who examined tissues of guinea pigs inoculated with oocysts. He found two tissue cysts in the brain of an animal killed 77 days post-infection, and in a brain smear of a guinea pig killed 189 days post-infection. The tissue cysts were 10.7 × 10.6 µm in the histological section and 13.0 × 12.1 µm in the tissue smear [26]. They had thin walls and were indistinguishable from T. gondii tissue cysts. Guinea pigs are generally resistant to T. gondii, but latent infections have been reported. Furthermore, it cannot be completely ruled out that the cysts were young Fig. 1. A comparison of tachyzoites of (a) Neospora caninum and (b) Hammondia heydorni in cell N. caninum tissue cysts. Thus, the nature of the tissue culture. Both organisms are located in parasitophorous vacuoles (P). In both figures, tachyzoites have cysts described by Matsui remains in doubt. divided and remain attached to each other at one end. Tachyzoites of N. caninum have many more micronemes (M), few amylopectin granules (A) and no vacuole (V) compared with tachyzoites of H. heydorni. Abbreviations: C, conoid; D, dense granules; N, nucleus; R, rhoptry. Differences between tachyzoites of N. caninum and H. heydorni finding of oocysts with two sporocysts in the intestinal Neospora caninum has been cultivated in vitro in mucosa of a dog [1,23]. Oocysts morphologically different cell lines [8,16]. It can be maintained resembling those of I. bigemina were later reported continuously in cell culture by sub-inoculation of new

J.P.Dubey∗ from several other hosts including cats and humans, cultures. The NC-1 isolate of N. caninum isolated in Dolores E. Hill and dogs and cats were found to shed sporulated 1988 [7] has been cultivated in this way for >12 years. Mark C. Jenkins oocysts of several species of Sarcocystis [23]. Because Unlike N. caninum, there is only one report of cell Parasite Biology, the name I. bigemina was first used for the parasite culture of H. heydorni [27]induced with sporozoites. Epidemiology and Systematics Laboratory, developing in the lamina propria of the dog (which is The parasite grew in several cell lines, but died out Animal and Natural now Sarcocystis bigemina), a new name, after a few asexual cycles and could not be transferred Resources Institute, Isospora heydorni, was proposed by Tadros and to other cell lines by inoculation with merozoites [27]. Agriculture Research Service, United States Laarman [24] for the canine I. bigemina developing in Although H. heydorni and N. caninum both multiply Dept of Agriculture, the intestinal surface epithelium. Dubey and Fayer in cell culture by endodyogeny, the organisms are Building 1001, Beltsville, [25] described I. bigemina-like oocysts in the feces, ultrastructurally distinct [28] (Fig. 1). A noticeable MD 20705-2350, USA. and asexual and sexual stages in the intestine, of a difference is the presence of a large vacuole near the ∗e-mail: jdubey@ anri.barc.usda.gov naturally infected dog. Dubey [23] proposed that the conoidal end in H. heydorni (Fig. 1), a feature that is canine I. bigemina to be transferred to the genus absent in N. caninum. Furthermore, H. heydorni has David S. Lindsay Center for Molecular Hammondia because of its biology, including an several amylopectin granules that are absent or rare Medicine and Infectious obligatory two-host life cycle; Isospora species in N. caninum tachyzoites, and N. caninum has more Diseases, Dept of generally have a fecal–oral one-host life cycle. micronemes than H. heydorni (Fig. 1a,b). In addition, Biomedical Sciences and Dogs fed H. heydorni oocysts became infected, N. caninum can be transmitted to a variety of hosts by Pathobiology, Virginia-Maryland but did not shed oocysts, and the organism could subcutaneous inoculation of infected tissues, whereas Regional College of not be transmitted to mice by subcutaneous H. heydorni has an obligatory two-host cycle. Veterinary Medicine, inoculation of infected tissues. Thus, the name Following the single report of successful in vitro Virginia Tech, 1410 Prices Fork Road, Blacksburg, became Hammondia heydorni [23,24]. Because cultivation of H. heydorni [27], the oocysts used in this VA 24061-0342, USA. it is now impossible to determine the true culture were morphologically described by Blagburn identity of I. bigemina, this name is regarded et al. [29]. Oocysts from the same stock as used for Arvid Uggla Dept of Parasitology as nomen nudum [23]. induction of this cell culture had been stored at 4°C (SWEPAR), National Subsequently, several herbivores (cattle, water and, ten years later, they could still be used for Veterinary Institute and buffaloes, sheep, moose, deer, goats, reindeer, roe molecular studies [20,30]. At PCR analysis, only Swedish University of Agricultural Sciences, deer, moose, camels, horses and guinea-pigs) were H. heydorni and not N. caninum [2] was detected in SE-75189 Uppsala, identified as natural or experimental intermediate the same material, which provided evidence that Sweden. hosts for H. heydorni based on shedding of oocysts by the original cell culture inoculum of Speer et al. [27] Clarence A. Speer dogs fed tissues from these host species [1,23]. Thus was H. heydorni. College of Agricultural far, no parasite stages of H. heydorni have been The stages of H. heydorni and N. caninum shown Sciences and Natural identified in any of the natural intermediate hosts, in Fig. 1 are not strictly comparable because the Resources, University of Tennessee, Knoxville, and none of the hosts experimentally fed H. heydorni N. caninum culture was initiated by in vitro-adapted TN 37901-1071, USA. oocysts have developed clinical signs [1]. In addition tachyzoites, whereas the H. heydorni culture was

http://parasites.trends.com 68 Opinion TRENDS in Parasitology Vol.18 No.2 February 2002

initiated by sporozoites from oocysts. However, tachyzoites resulting from both processes are not 123456 likely to be ultrastructurally different. For example, in the case of the closely related coccidium T. gondii, 500 bp tachyzoites in cultures induced by sporozoites are 450 bp indistinguishable from tachyzoites in cultures induced by tachyzoites [31]. Although there is now uncertainty as to whether Heydorn [22] initially 220 bp found I. bigemina (H. heydorni) or N. caninum oocysts

[32], we believe in the existence of two canine TRENDS in Parasitology coccidian species with T. gondii-like oocysts. Fig. 2. Random primer F6 amplification by PCR of Neospora caninum Molecular differences between N. caninum and DNA isolated from gerbil brain (Lane 1), N. caninum tachyzoite control DNA (Lane 2), N. caninum oocyst DNA from dog (Lane 3) N. caninum H. heydorni oocyst control DNA (Lane 4), Hammondia heydorni oocyst DNA Recent phylogenetic studies based on ITS-1 and (Lane 5) and N. hughesi tachyzoite DNA (Lane 6). large-subunit ribosomal DNA sequences suggested that N. caninum and H. heydorni are closely related Validity of the genus Neospora to each other and to T. gondii and H. hammondi, The genus Neospora, as distinct from Toxoplasma, and also that N. caninum and H. heydorni do not was originally proposed to draw attention to the necessarily form a sister group relationship [33]as newly identified neurological disease of dogs. Dubey previously suggested [20]. It has been shown that et al. [6] recognized that N. caninum and T. gondii are N. caninum genomic DNA could be distinguished related but distinct. Naming the new parasite from H. heydorni using the Np6/21 primer set, (N. caninum) as a species of Toxoplasma would have which targets the Nc5 genomic sequence of perpetuated their likeness rather than their N. caninum [30]. Results of competitive PCR distinctiveness. In the early 1900s, many species of assays demonstrated that H. heydorni oocyst DNA Toxoplasma were named on the basis of the hosts was not amplified, and did not interfere with from which they were derived, including T. canis from amplification of N. caninum oocyst DNA in mixed a dog [39]. However, because the strains of T. gondii samples using the Nc5-specific primers [30]. These derived from different warm-blooded hosts cannot be results were recently confirmed by Dijkstra et al. [34], distinguished morphologically or serologically, they who showed that dogs fed placenta from naturally are considered to belong to one species, T. gondii. infected cows shed N. caninum oocysts. The identity In 1988, the genus Neospora was distinguished from of the oocysts as N. caninum was confirmed by Toxoplasma by the antigenic differences and bioassay in gerbils and by PCR using the N. caninum- ultrastructure of their tissue cysts and tachyzoites. specific Np6/21 primers, which did not amplify Neospora caninum tissue cysts are thick walled H. heydorni DNA [34]. (1–4 µm) and normally found in neural tissues, Neospora caninum oocysts were recently isolated whereas T. gondii tissue cysts are thin walled from the feces of a naturally infected dog [35]. (0.5 µm) and occur in many tissues. Irrespective These oocysts were determined to be N. caninum of the fixative used, rhoptries in N. caninum based on results from bioassay and molecular tachyzoites are numerous and contents are electron characteristics. Gerbils that had been fed feces from dense, whereas those in T. gondii tachyzoites are few the dog subsequently developed antibodies to and electron lucent. N. caninum, and N. caninum from the gerbil brains Although much has been written about clinical was isolated in cell culture. DNA obtained from the neosporosis in dogs and cattle in the past 13 years, brains of the infected gerbils, from the culture-derived many aspects of the biology of N. caninum are still parasites, and from the oocysts of the naturally unknown. As yet, there is no good animal model to infected dog was determined to be N. caninum and study the different phases of the life cycle of not H. heydorni. Figure 2 shows differences in N. caninum, especially the tissue cyst production. In amplified genomic DNA obtained from H. heydorni animals such as cats, dogs, mice, gerbils, rats, sheep and N. caninum oocysts using the F6 primer used by and goats experimentally infected with N. caninum, Guo and Johnson [36] and Spencer et al. [37]to tissue cysts were thick walled and found only in differentiate apicomplexan parasites and neural tissues. Recently, Peters et al. [40] reported Neospora species. More recently, Slapeta et al. [38] thin-walled (0.3–1.0 µm) tissue cysts in muscles of described an H. heydorni-specific PCR based cattle and dogs naturally infected with N. caninum. on amplification of the ITS-1 region that can be used These intramuscular tissue cysts were labeled with for identification of H. heydorni oocysts. In antibodies against N. caninum and were apparently conclusion, a variety of molecular studies and situated in myofibers, although the location could not evaluations of phylogenetic relationships have be confirmed because of poor fixation [40]. The indicated that N. caninum and H. heydorni are intramuscular cysts contained 14–50 bradyzoites distinct species[20,21,30,38]. 5.2 × 1.6 µm in size, and morphologically they most

http://parasites.trends.com Opinion TRENDS in Parasitology Vol.18 No.2 February 2002 69

closely resembled Neospora rather than Toxoplasma relationships and partly a matter of convenience. or Sarcocystis. However, the bradyzoites were smaller Neosporosis is now a well-defined, economically than those of N. caninum, which are 8.1 × 2 µm [17]. important disease of cattle. Before making a final Many other aspects of the life cycle of N. caninum determination about the taxonomic status of are also unknown, including the possiblility of dogs N. caninum, one should reflect upon whether the to produce oocysts after ingesting sporulated science will be advanced by calling the disease it oocysts. Dogs fed sporulated H. heydorni oocysts do produces hammondiosis, isosporosis or not shed oocysts, indicating that H. heydorni has an toxoplasmosis. In fact, the rules of zoological obligatory two-host life cycle. Furthermore, the nomenclature allow us the use of well-established, entero-epithelial stages of N. caninum in the clearly defined taxa. However, irrespective of the definitive host are unknown. genus status, in our opinion, morphological, biological, antigenic and molecular studies have Conclusion together provided compelling evidence that As stated by Mugridge et al. [21], biological N. caninum and H. heydorni are two separate classifications are partly a matter of phylogenetic coccidian parasite species of dogs. References 15 Lindsay, D.S. et al. (2001) Oocyst excretion in dogs 29 Blagburn, B.L. et al. (1988) Further 1 Dubey, J.P. (1993) Toxoplasma, Neospora, fed mouse grains containing tissue cysts of a characterization of the biology of Hammondia Sarcosystis, and other tissue cyst-forming coccidia cloned line of Neospora caniunum. J. Parasitol. heydorni. Vet. Parasitol. 27, 193–198 of humans and animals. In Parasitic Protozoa 87, 909–911 30 Hill, D.E. et al. (2001) Specific detection of (Vol. 6) (Kreier, J.P., ed.), pp. 1–158, New York 16 Hemphill, A. (1999) The host–parasite Neospora caninum oocysts in fecal samples from Academic Press relationship in neosporosis. Adv. Parasitol. 43, experimentally-infected dogs using the 2 Mehlhorn, H. and Heydorn, A.O. (2000) 47–104 polymerase chain reaction. J. Parasitol. 87, Neospora caninum: Is it really different from 17 Speer, C.A. et al. (1999) Comparative 395–398 Hammonida heydorni or is it a strain of ultrastructure of tachyzoites, bradyzoites, and 31 Dubey, J.P. et al. (1997) Oocyst induced murine Toxoplasma gondii? An opinion. Parasitol. Res. tissue cysts of Neospora caninum and toxoplasmosis: Life cycle, pathogenicity, and stage 86, 169–178 Toxoplasma gondii. Int. J. Parasitol. 29, conversion in mice fed Toxoplasma gondii oocysts. 3 Heydorn, A.O. and Mehlhorn, H. (2001) Further 1509–1519 J. Parasitol. 83, 870–882 remarks on Hammondia hammondi and the 18 Romand, S. et al. (1998) Direct agglutination test 32 Schares, G. et al. (2001) Hammondia taxonomic importance of obligate heteroxeny. for serologic diagnosis of Neospora caninum heydorni-like oocysts shed by a naturally Parasitol. Res. 87, 573–577 infection. Parasitol. Res. 84, 50–53 infected dog and Neospora caninum NC-1 4 Frenkel, J.K. and Dubey, J.P. (2000) The 19 Howe, D.K. and Sibley, L.D. (1999) Comparison of cannot be distinguished. Parasitol. Res. 87, taxonomic importance of obligate heteroxeny: the major antigens of Neospora caninum and 808–816 Distinction of Hammondia hammondi from Toxoplasma gondii. Int. J. Parasitol. 29, 33 Mugridge, N.B. et al. (2001) Effects of sequence Toxoplasma gondii – another opinion. Parasitol. 1489–1496 alignment and structural domains of ribosomal Res. 86, 783–786 20 Ellis, J.T. et al. (1999) The genus Hammondia is DNA on phylogeny reconstruction for the 5 Bjerkås, I. et al. (1984) Unidentified cyst-forming paraphyletic. Parasitology 118, 357–362 protozoan family Sarcocystidae. Mol. Biol. Evol. sporozoon causing encephalomyelitis and 21 Mugridge, N.B. et al. (1999) Phylogenetic analysis 17, 1842–1853 myositis in dogs. Z. Parasitenkd. 70, based on full-length large subunit ribosomal RNA 34 Dijkstra, T. et al. (2001) Dogs shed 271–274 gene sequence comparison reveals that Neospora caninum oocysts after ingestion of 6 Dubey, J.P. et al. (1988) Newly recognized fatal Neospora caninum is more closely related to naturally infected bovine placenta but not protozoan disease of dogs. J. Am. Vet. Med. Assoc. Hammondia heydorni than to Toxoplasma gondii. after ingestion of colostrum spiked with 193, 1259–1263 Int. J. Parasitol. 29, 1545–1556 Neospora caninum tachyzoites. J. Parasitol. 31, 7 Dubey, J.P. et al. (1988) Neonatal Neospora caninum 22 Heydorn, A.O. (1973) Zum Lebenszkylus der 747–752 infection in dogs: Isolation of the causative agent kleinen Form von Isospora bigemina des Hundes 35 Basso, W. et al. (2001) First isolation of Neospora and experimental transmission. J. Am. Vet. Med. I. Rind und Hund als mögliche Zwischenwirte. caninum from the feces of a naturally infected Assoc. 192, 1269–1285 Berl. Münch. Tierärztl. Wochensch. 86, 323–329 dog. J. Parasitol. 87, 612–618 8 Lindsay, D.S. and Dubey, J.P. (1989) In vitro 23 Dubey, J.P. (1977) Toxoplasma, Hammondia, 36 Guo, Z.G. and Johnson, A.M. (1995) Genetic development of Neospora caninum (Protozoa: Besnoitia, Sarcocystis, and other tissue cyst- comparison of Neospora caninum with Apicomplexa). J. Parasitol. 75, 163–165 forming coccidia of man and animals. In Parasitic Toxoplasma and Sarcocystis by random amplified 9 Lindsay, D.S. and Dubey, J.P. (1989) Protozoa (3rd edn) (Kreier, J.P., ed.), pp. 101–237, polymorphic DNA-polymerase chain reaction. Immunohistochemical diagnosis of Neospora New York Academic Press Parasitol. Res. 81, 365–370 caninum in tissue sections. Am. J. Vet. Res. 50, 24 Tadros, W. and Laarman, J.J. (1976) Sarcocystis 37 Spencer, J.A. et al. (2000) A random amplified 1981–1983 and related coccidian parasites: A brief general polymorphic DNA polymerase chain reaction 10 Dubey, J.P. and Lindsay, D.S. (1996) A review of review, together with a discussion on some technique that differentiates between Neospora caninum and neosporosis. Vet. biological aspects of their life cycles and a new Neospora species. J. Parasitol. 86, Parasitol. 67, 1–59 proposal for their classification. Acta Leiden. 44, 1366–1368 11 Dubey, J.P. (1999) Recent advances in 1–107 38 Slapeta, J.R. et al. Coprodiagnosis of Neospora and neosporosis. Vet. Parasitol. 84, 25 Dubey, J.P. and Fayer, R. (1976) Development of Hammondia heydorni in dogs by PCR based 349–367 Isospora bigemina in dogs and other mammals. amplification of ITS 1 rRNA: Differentiation from 12 Marsh, A.E. et al. (1998) Description of a new Parasitology 73, 371–380 morphologically indistinguishable oocysts of Neospora species (Protozoa: Apicomplexa: 26 Matsui, T. (1991) The tissue stages of Isospora Neospora caninum. Vet. J. (in press) Sarcocystidae). J. Parasitol. 84, 983–991 heydorni in the guinea pig as an intermediate 39 Mello, U. (1910) Un cas de toxoplasmose du chien 13 McAllister, M.M. et al. (1998) Dogs are definitive host. Jap. J. Parasitol. 40, 581–586 observé à Turin. Bull. Soc. Pathol. Exot. 3, hosts of Neospora caninum. Int. J. Parasitol. 28, 27 Speer, C.A. et al. (1988) Development of 359–363 1473–1478 Hammondia heydorni in cultured bovine and 40 Peters, M. et al. (2001) Immunohistochemical and 14 Dubey, J.P. and Lindsay, D.S. (2000) Gerbils ovine cells. J. Protozool. 35, 352–356 ultrastructural evidence for Neospora caninum (Meriones unguiculatus) are highly susceptible to 28 Speer, C.A. and Dubey, J.P. (1989) Ultrastructure tissue cysts in skeletal muscles of naturally oral infection with Neospora caninum oocysts. of sporozoites and zoites of Hammondia heydorni. infected dogs and cattle. Int. J. Parasitol. 31, Parasitol. Res. 86, 165–168 J. Protozool. 36, 488–493 1144–1148

http://parasites.trends.com