Description of Trypanosoma dromedarius (n.sp.) Infecting Camels by Light and Electron Microscopy with Refer to its Life Cycle

Out of 195 Camelus dromedarius examined only 19 were infected (9.7 %) with this parasite. The life cycle of T. sp. involves many morphologically distinct stages-more than described for any other genus in the Trypanosomatidae. This parasite was appeared for the first time in Camelus dromedarius at Assiut, Egypt. Most of stages of T. dromedarius (n.sp.) which were appeared in the blood of Camelus dromedarius are amastigotes stages. At the same time spheromastigots, epimastigote stages and trypomastigote stages with two shapes slender and broad. In experimental infection, the trypanosome was found to be transmissible to laboratory white mice, also metacyclic and amstigote formes were seen.


Introduction
The majority of trypanosomes reported in bats have not been cultivated, and their classification has been based exclusively on the morphology of blood trypomastigotes. Large blood trypanosomes of the subgenus Megatrypanum, followed by small blood forms of the sub-genus Schizotrypanum, comprise the majority of the trypanosomes reported in bats throughout South America, Asia, Europe and, especially, Africa [1][2][3][4].
The subgenus Megatrypanum, originally comprising large blood trypanosomes from artiodactyls [5], was amended exclusively on a morphological basis to include any large tryp-anosome found in bats, monkeys and rodents [6].
Molecular phylogenetic analysis has demonstrated the polyphyly of the traditional subgenus Megatrypanum, which was revised as a clade comprising trypanosomes from ruminants headed by the type species T. theileri, a cosmopolitan parasite of cattle [7][8][9][10]. However, in the reappraisal of this subgenus, other species from non-ruminant hosts that putatively belong to this subgenus need to be phylogenetically positioned, especially those from bats.
Trypanosoma cruzi is the etiologic agent of Chagas disease, an endemic parasitosis in Latin America with 12 to 14 million people infected [11] The parasite's biological cycle includes three fundamental forms characterized by the relative positions of the flagellum, kinetoplast, and nucleus [12]. (a) Trypomastigotes: 20 μm long, fusiform, subterminal kinetoplast, constitute the infecting form, and are found in mammalian blood and the hindgut of triatomine bugs; they do not multiply.
In mammals they are the disseminators of blood-borne infection [12]. (b) Epimastigotes: Also 20 μm long, kinetoplast anterior to the nucleus, fusiform. They represent the parasite's multiplicative form in the triatomid's intestine, and are the predominant form in culture. For this reason it is the form most commonly used in biochemical studies [12]. (c) Amastigotes: Approximately 2 μm in diameter, round, without an emergent flagellum. They multiply by means of binary fission inside mammalian host cells, producing their rupture, and liberating trypomastigotes into the bloodstream that can once again invade any nucleated cell [12]. They can be grown in culture in muscle cells, fibroblasts, and macrophages among others [4,13].
Trypanosoma dromedarius (n.sp.) was infected Camelus dromedarius for the first time in Egypt through the present work so that; the present work aims to describe the different stages by both light and electron microscopy and to examine the zoonotic importance of the new parasite on the experimental animals (white rats).

Material and Methods
Out of 195 blood samples of camels (Camelus dromedarius) examined for blood protozoan parasites collected from different localities of Slaughter houses at Assiut city, Egypt (Dairout, Beni Ady, Elethamna). These freshly collected blood samples were divided in two groups one in a tube coated with EDTA, and the other in a test tube for Centrifugation to obtain sera. Thick and thin blood smears were made for morphological examination of some protozoan blood parasites. Electron microscopic studies.

TEM
Few drops from blood which is highly infected with Trypanosoma, Babesia and Theileria immediately fixed in 3 ml. of 3% glutaraldehyde solution in phosphate buffer (PH 7.2), for 24 hours and kept at 4°C in refrigerator. The samples were post fixed in 1% Osmium tetroxide in phosphate buffer (PH 7.2, 300 mom), for 30 minutes. They were washed several times with phosphate buffer solution. The samples

Journal of Bacteriology and Parasitology
were then embedded in Epon which can preserve in structure from distortion during processing then ultra-thin sections were cut by an Ultra microtome and examined by JEOL, 100 CXII operating at 80 KV (TEM).

SEM
For scanning electron microscope of blood; few drops were fixed in 3% Glutaraldehyde in buffer for 24 hours. Specimens were washed three times in Phosphate buffer and post fixed in 1% Osmium tetroxide for 2 hours and then washed in the same buffer. They were Dehydrated in different grades of ethyl alcohol and then mounted on special holders and coated with gold. Then they were examined in a JSM-T 200 L.V. 5400 Scanning Electron Microscopy (SEM).

Experimental infection
One group of laboratory animals representing in five white rates were injected with freshly infected blood camels by doses 3 ml blood which was infected with the new parasite of trypanosomes to examine the zoonotic importance for this parasites. Blood examination was performed daily for determine the infection of these laboratory animals.

Results
Out of 195 Camelus dromedarius examined only 19 were infected (9.7%) with this parasite. Most of stages of T. dromedarius (n.sp.) which were appeared in the blood of Camelus dromedarius are amastigotes stages and were measured (5-7.14 × 4.8-7 µm) in diameter (Figure 1). At the same time spheromastigots also were appeared ( Figure 2). This parasite was seen for the first time in Camelus dromedarius. The body was slightly slender, small in size and with appearing for the amastigote stages in a heavy in the blood cells. The cytoplasm was granular and the kinetoplast nearly was a half size of the nucleus. The nucleus was oval or rounded in shape and measured (1.68-2.76 µm) in length and (1.23-2 µm) in width.    Figure 6). Also trypomastigote stages were measured (16-18 µm) in length and (1.28-3.15 µm) in width (Figure 7). In addition that, the unequal division in the amastigote and trypomastigote stages was seen in Figures 8 and 9 respectively.      The ultrastructural organization of Tr. dromedarius revealed also all common organelles of trypanosomatids. However, some features as, the cytostome which forms together with the flagellar pocket the main structure involved in the endocytic process ( Figure 10), large number of reservosomes (Figure 11), which are compartments that accumulate endocytosed macromolecules found at the posterior region of epimastigotes and the compacted disk shaped kinetoplast structure and the division in stage as in Figure 12. In wet preparation, living trypanosomes were moving actively among the red blood cells. In experimental infection, the trypanosome was found to be transmissible to laboratory white rats, metacyclic trypomastigote and amastigote stages were appeared in the tissue and blood of the rat respectively as in Figures 13 and 14. The life cycle stages for this parasite was showing in Figure 15.

Discussion
By comparison this species with other trypanosomes in mammals. It was found that T. cruzi like trypanosomes [2], which the name trypanosome (schizotrypanum) assiutis sp. nov. was measured 19.4 µm in maximum body length, one nucleus was measured 1.16 µm and was encountered of local Egyptian bat Vesperugo kuhli and it was non-transmissible for white rat but in the present study this T. sp. was measured 18 µm in maximum body length, it was encountered Camelus dromedarius and the nucleus of the new trypanosoma was measured 1.68-2.7 µm and it was transmssible for white rat.
All trypanosoma evansi isolates described by [10], the maximum of body length was 34 µm. Also the mean length for T. vivax ranged between 18.73-25.4 µm [7,10]. But the present parasite was measured 18 µm in maximum length which differs from the mentioned species.
Another tropical trypanosome that can affect domestic animals is Trypanosoma congolens (Subgenus: Nanomonas) but morphologically it is distinguishable because of its small size mean (12.2-17.6 µm) and the absence of a free flagellum [10] but in the presence species there was a free flagellum and undulating membrane with 2-3 undulation.
From the previous comparison, the present species differs from the previous trypanosomes in some respects, so that the present trypanosome could be identified as a new species called T. dormedarius according to its host Camelus dromedarius.

Conclusion
Trypanosoma dromedarius (n.sp.) is new species trypanosomes and was appeared for the first time in Camelus dromedarius. This parasite was had a zoonotic importance for its transmissible to the experimental animals (white rats).