Chapter one Introduction & literature review 1.1. General introduction The reptiles and amphibians can be infected by different type of parasites. These parasites can be divided into enteric parasites and ectoparasites. Enteric parasites include protozoa, flagellates, , opalinids, amoebae, and coccidea. Also they can be infected by bacteria from ectoparasites like mites and ticks (De la Navarre, 2009). 1.2. Blood parasites of reptiles and amphibians

The blood parasites of the reptiles and amphibian may be divided into two classes depending on whether they infect the blood without attacking the corpuscles or whether they become established within the corpuscles; extracorpusular parasites such as the flagellates of the genera Trypanosoma, Leptomonas, and Leishmania, and nematodes, and intercorpuscular parasites such as many telosporidian of the suborder Adeleiing belonging to the genera , and species (Reichenow, 1953).

1.3. Kinetoplastid protozoa

This class contains species that parasitize a wide diversity of hosts ranging from humans to plants. Member of this group are characterized by a single large mitochondrion containing a body- the kinetoplast- that stain darkly in histological preparations. The Kinetoplastids differ considerably in their host distribution, life cycles, and medical and veterinary importance. (Battaglia et al. 1983)

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1.3.1. Sauroleishmania spp

Currently, the Leishmania parasites are classified into three subgenera, The subgenera Leishmania (Leishmania) which is transmitted by female sandfly of the Phlebotomus species in the Old World, and Lutzomyia species in the New World, the subgenera Leishmania (Viannia) which is only found in the New world and transmitted by Lutzomyia spp sanflies, and the subgenera Leishmania (Sauroleishmania) or ’ leishmania, which is transmitted by the sandflies of Sergntomyia spp (Chappuis, et al., 2007).

The Leishmania parasites have two forms in their life cycle, the promastigotes form in the sandfly, and the amastigotes form in the vertebrate host. In mammals, the organism is found in the macrophages and has a characteristic, distinctive, bar-shaped extra chromosomal DAN fragment called a kinetoplast. The motile promastigote form is found extracellularly and possesses a flagellum (Frye, 1991).

The Sauroleishmania has been reported in the peripheral blood of reptiles, primarily in (Keymer. 1981). The amastigote form in reptiles appears in the cytoplasm of blood cells, particularly the erythrocytes. It appears singly or may be numerous, and may condense to round basophilic inch with a central hollow (Paperna et al., 2001).

Microscopic diagnosis is the standard and routine diagnosis for leishmaniasis, which depends on detection of Leishmanai amastigotes in Geimsa stained aspirate materials from lymph nodes, bone marrow, spleen or liver, and in slit skin smears or in peripheral blood, and/ or promastigotes in dissected materials of infected sandfly (Zijlstra, et al., 1992).

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Hoare (1948) postulated that the lizard leishmania provide convincing evidence of evolution in which an invertebrate parasite has become adapted to life in a vertebrate host.

1.3.2. Trypanosoma spp These are large flagellate protozoa that possess a kinetoplast with a distinct or in distinct, undulating membrane (trypomastigote). These parasites may be found extracellularly in the peripheral blood of many reptiles’ species. They are transmitted by blood –sucking arthropods (biting dipteran flies such as Phlebtomine sandflies) in terrestrial reptiles, and by leeches in aquatic reptiles. Although trypanosomiasis can cause severe parasitemia, it is commonly associated with lifelong subclinical infection (Lane and Mader, 1996).

All species of trypanosomatidae have a single nucleus and are either elongated with a single flagellum or rounded with a very short, no protruding flagellum. Many members of the family are heteroxenous: During one stage of their lives they live in the blood and/or fixed tissues of all vertebrate classes, and during other stages they live in the intestine of blood sucking invertebrates (Tibayrenc & Ayala. 1999).

1.4. spp Apicomplexa species is a large group of protozoa, most of which possess a unique organelle called apicoplast and an apical complex structure involved in penetrating a host's cell. They are unicellular, spore-forming, and exclusively (Jadwiga, 1991) parasites of animals. Motile structures such as flagella or pseudopods are absent except in certain gamete stages. This is a diverse group including organisms such as , gregarines, piroplasms, haemogregarines, and plasmodia.

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1.4.1. Haemogregarina spp

Four genera of intracellular parasites are included among the Haemogregarines; Haemogregarina, Hepatozoon, Karyolysus, and Hemolivie.

These genera cannot be accurately classified based on their appearance in blood cells alone (Al-Farraj, 2008).

On blood films, the gamonts of Haemogregarines appear as sasusage-shaped inclusion with pale to purple cytoplasm and one centrally to slightly eccentrically placed, darker purple staining nucleus except in Haemogregarines infection where erythrocytic meronts may be present. These are unpigmented and are typically found of the cytoplasm of red cells and sometimes in white blood cells (Jacobson, 1983). The gamonts may push the nucleus of the host cells to inside or surround it. The hosts cells may appear irregular in shaped and size (Shazly, 2000). Rarely, two or more organisms may be found in one erythrocyte, or the gamonts may be found extracellularly. Because gamonts of different Haemogregarines are morphologically indistinguishable in the peripheral blood, the general terms Haemogregarines is used to report their presence. Haemogregarines belonging to the genus Hepatozoon are commonly found in terrestrial and aquatic snakes. Haemogregarines sporozoites are often transmitted by infected arthropods and leeches. Haemogregarines protozoa are well adapted to their natural host, but can cause significant clinical inflammatory disease in unnatural host species (Keymer, 1981).

1.4.2. spp

Five genera of the family are reported: plasmodium, Fallisia, Saurocytozoon, Haemocytstidium, and . Snakes and turtles may become infected with Hemoprotus. Whereas plasmodium, Fallisa,

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Saurocytozoon, and Haemocytstidium infections have been reported in lizards (Lane and Mader, 1996).

1.5. Microfilaria spp

Various genera of filaridae worms can be found in reptiles. Some of which are specific, such as Macdoualdius spp which have been detected in some species of snake and lizards, Saurositus spp which have been detected in lacertid lizards, Foleyella spp which have been detected in some species of chameleons lizards, and cardinema spp which have been detected in chelonians lizards (Lane & Mader, 1996).

Microfilariae are transmitted by blood sucking mosquitoes or ticks or some sandfly species such as Phlebotomus duboscqi.

Filariasis in reptiles is mostly subclinical, but with heavy infestation, thrombosis and blockage of blood vessels may occur resulting in edema, fibrosis and/or necrosis in the affective area (Lane & Mader, 1996).

1.6. Zoonotic diseases of reptiles and amphibian

There are several diseases which can be transmitted from reptiles and amphibain to humans such as Salmonellosis, Tuberculosis, also many reptiles can harbour some protozoan organisms capable of causing diseases such as Cryptosporidiosis which is caused by a coccidian protozoan (Kolle &Hoffmann, 1998).

Rationale:

Due to the lack of knowledge regarding the zoonoses diseases of reptiles and amphibian in the Sudan, there is a great need to identify those parasites firstly in order to know their zoonoses importance.

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Objectives:

Thus the present study was conducted to carry out the following:

 Survey of different blood parasites in reptiles and amphibians

 Identify types of blood parasites in two lizards’ species (Mabuya striata, Mobuya quinquetaeniatus) and toad (Bufo regularis).

 Comparison of blood parasites in reptiles and amphibians according to microscopic diagnosis.

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Chapter two Material and methods 2.1. Study area

The samples were collected from two locations in Khartoum state; Tuti Island which lies in the union of the Blue and the White Nile and Jebel Awlyia area near the White Nile in Khartoum south (Fig.1).

2.2. Sample collection and preparation

Thirteen lizards’ specimens from species Mabuya striata and two specimens from species Mobuya quinquetaeniatus from Tuti Island (Blue Nile), and 15 toads’ specimens from species Bufo regularis from Jebel Awlyia area (White Nile) were captured during this study.

The animals were dissected and tissue samples were obtained from spleen and liver and preserved in eppendorf tube in Isopropanol alcohol 95%. Blood samples were taken, blotted on filter paper (Whattman No.3) and air dried, then preserved in separated plastic bags.

2.3. Microscopic examination for tissue samples

Tissue from spleen and liver were spotted on slides, fixed with absolute methanol, air dried, stained with Giemsa, and examined under light microscope with oil emersion lens to search for parasites. Then the positive slides were photographed using a microscope digital camera (DCE-2).

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Fig.1: Satellite image showing the location of the two study areas in Khartoum.

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2.4. Identification of the parasites

The identification for microfilaria nematodes were done according to the characteristics described by Bain, et al., (1992), identification for Sauro- Leishmania amastigotes was performed according to Paperna et al., 2001, and the identification for Haemogregarina spp was documented based on the work described by Elwasila (1989).

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Chapter Three Results 3.1. Lizards and toads’ species captured:

A total number of 15 lizards belong to species Mobuya striatus (13 specimens) (plate.1) and Mobuya quinquetaeniatus (two specimens; plate. 2) were captured from Tutti Island, and a total number of 15 toads belong to species Bufo regularis were captured from Jebel Awlyia area near the White Nile (plate.3).

3.2. Microscopic detection of Sauroleishmania parasites in lizards and toads’ tissue samples

3.2.1. Sauroleishmania sp in lizards

Sauroleishmania sp amastigotes were only detected in eight specimens out of 13 of Mobuya striatus (plate. 4).

3.2.2 Sauroleishmania sp in toads

Two specimens out of 15 of the toad Bufo regularis showed the presence of amastigotes (plate. 5).

3.3 Microscopic detection of Haemogregarina sp in lizards and toads’ tissue samples

3.3.1 Haemogregarina species in lizards

Haemogregarina sp was detected in only one lizard specimen out of 13 belongs to the species Mobuya striatus (Plate. 6).

3.3.2. Haemogregarina species in toad (Bufo regularis)

Haemogregarina sp was detected in five specimens out of 15 (plate 7).

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3.4. Microscopic detection of Microfilaria species in lizard and toads

The Microfilaria spp were only detected in two specimens belonged to lizard Mobuya quinquetaeniaust (plate.8).

3.5. Microscopic detection of Trypanosoma species in lizards and toads

Only one specimen of the toad Bufo regularis showed the presence of Trypanosoma sp (plate.9).

3.6. The intensity of different parasites in lizards and toad species

Table (1) shows the intensity of different species of the haemoparasites identified in the lizards and toad species studied. Figure (2) shows the percentage of the haemoparasites in compare with hosts.

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9 cm

Plate .1: lizard species Mobuya striatus

14cm

Plate .2: lizard species Mobuya quinquetaeniatus

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Plate .3: toad species Bufo regularis

13

A

B

C

Plate .4 (A, B & C): WBCs rupture in lizard species Mobuya striatus

Showing amastigotes stage of Sauroleishmania spp 14

A

B

C

Plate .5 (A, B &C): the amastigotes in toad species of Bufo regularis

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A

B

Plate .6(A & B): the Haemogregarina species in lizards species in Mobuya striatus

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a A B

C D

Plate .7(A, B, C & D): the gamonts stages in Haemogregarina species in toad Bufo regularis

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A B

C D

Plate .8(A, B, C & D): the microfilaria species in lizard species of Mobuya quinquetaeniatus

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Plate .9: the Trypanosoma species in toad Bufo regularis

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Table (1): The intensity of different parasites in lizards and toad species

Parasites Toad Lizard

Bufo regularis Mobuya striatus Mobuya quinquetaeniatus

(n=15) (n=13) (n=2)

Sauroleishmania spp 13.33% (2) 61.53% (8) 0.00

Haemogregarina spp 33.33% (5) 7.69% (1) 0.00

Microfilaria spp 0.00 0.00 100% (2)

Trypanosome spp 6.66% (1) 0.00 0.00

Fig.2: the percentage of the haemoparasites in compare with hosts

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Chapter four Discussion

Most reptiles and amphibians may harbour a variety of different parasites, which could be causative agents of some zoonotic diseases. Historically, limited numbers of studies have focused on two major areas: parasite life histories and , and more of these studies were performed on amphibian than reptiles (Rose, 2005). The present study aimed at visually identifying of the blood parasites in samples obtained from common species in Khartoum state of two lizards’ species Mabuya striata, and Mobuya quinquetaeniatus, and common toad species Bufo regularis.

This study reports the detection of variety of blood parasites of kinitoplastids protozoa, Haemogregarina spp, and microfilaria spp. The kinetoplasitds protozoa which were detected in this study were sauroleishmania spp and only one Trypanosoma spp. Previously, Elwasila (1988) detected sauroleishmania promastigotes in the gecko Tarentola annularis. The present study reported the presence of the amastigotes of sauroleishmania in both lizard Mabuya striata and toad Bufo regularis. The molecular data indicates that the lizard Leishmania or Sauroleishmania are phylogenetically more closely related to the Old World L. (Leishmania) species than they are to the New World L. (Leishmania) species (Noyes, et al., 1997). Some lizard leishmania occurs in areas of human leishmaniasis, so there are sometimes needs to determine if promastigotes in the gut of sandflies are of mammalian or reptilian origin. Some species of lizard leishmania share antigens with species infective to mammals and can produce cryptic and temporary infections in mammals (Belova, 1971). Different species within the genus Trypanosma can infect various animals, including mammals, , reptiles and amphibians, and fish.

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Many investigators reported the detection of Trypanosma spp in amphibian before (Werner, et al., 1988) and recently, the natural infection of sandfly species with amphibian Trypanosma was reported in Phlebotomus kazeruni (Kato, et al., 2010). Among the kinetoplastid protozoa, the detection of Sauroleishmania promastigotes was highly recorded in comparison with Trypanosma spp , which may be attributed to seasonality of the presence of toad specimens under study, or to the parasite itself.

This study reports the identification of two Haemogregarina species, which were morphologically distinguishable, and showed a typical signs of Haemogregarine infection of un-pigmentation of host’ erythrocytes, their irregular shape and size, and the displacing of erythrocyte nucleus (Shazly, 2000). The detection of Haemogregarina spp was reported before in Sudan by Elwasila (1989) in the gecko Tarentola annularis and by Ibrahim (2001) in Bufo regularis. Mosquitoes and mites are the arthropod hosts most likely to transmit haemogregarines (Rose, 2005). The present study reports for the first time in Sudan the detection of Haemogregarina spp in lizard Mobuya striatus.

Detection of Microfilaria spp that infects other vertebrates hosts such as fish and amphibian was reported earlier in the Sudan by kirk (1957). However, the present study reports the detection of Microfilaria spp in lizard species Mobuya quinquetaeniat for the first time in Sudan.

This study showed that the toad Bufo regularis harbour more diversified parasites funa compared to the lizard. This may attributed to the nature of the aquatic behavior of this organism that lives in both aquatic as well as terrestrial environments.

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The unavailability of certain technique for capturing the lizards and the seasonality of toads, were the main problems that encountered the present study. Identifying of the parasites that invade non human hosts should be of great interest, as there is steadily increasing threat of recent zoonotic diseases. Without doubt, those unknown zoonotic diseases will emerge in the future, thus using of direct and indirect methods for detecting of the causative agents will contribute in the detection and thus the control of the newly emerging zoonotic diseases.

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