Trypanosoma evansi and "Candidatus Mycoplasma haemolamae" Co-Infection in One-Humped Camel (Camelus dromedarius) from the Northwest of Iran: A Case Report.

A 4-year-old male one-humped camel (Camelus dromedarius) was referred to Veterinary Teaching Hospital of Urmia University, Iran in 2017 with anorexia, weakness, depression and pale mucosa. Decreased red blood cell count, packed cell volume and hemoglobin concentration were detected by complete blood cell count. In Giemsa-stained peripheral blood smears Trypanosoma spp. trypomastigotes scattered between erythrocytic spaces and Mycoplasma-like organisms were observed attached to the surface of erythrocytes. Species-specific PCR assay confirmed T. evansi and Candidatus Mycoplasma haemolamae (CMhl) co-infection. Administration of diminazene aceturate, oxytetracycline 20%, flunixin meglumine and phosphorus-vitamin B12 were not effective in treatment. Hemoplasmosis should be considered as an important differential diagnosis of conditions associated with hemolytic anemia in camel.

Introduction rypanosoma evansi is a widely-distributed haemoflagellate of veterinary importance that infects a variety of larger mammals including horses, mules, camels, buffalo, cattle and deer and mainly transmitted by hematophagous flies including, Tabanus sp., Stomoxyssp., Lyperosia sp. and Haematobia sp. (1,2). T. evansi infection incurs enormous economic losses including mortality, production losses costs of veterinary diagnosis/treatment and growth retardation in camel-raising flocks, throughout the tropical and subtropical regions; including Iran, with prevalence rates between 0% and 19.47% (3)(4)(5). It also is implicated as a zoonotic agent, causes of public health problem (2,6).
Hemotropic mycoplasmas (generally less than 1.0 μm in diameter), formerly known as Eperythrozoon or Haemobartonella, are small epierythrocytic bacterial agents lacking a cell wall. They are found attached to erythrocytes and may be observed free in the plasma of different species of vertebrates, including rodents, ruminants, pigs, alpaca (Lama pacos) and llamas. They are regarded as the most pathogenic species in livestock with clinical signs, especially in those with hemolytic anemia (7). Candidatus Mycoplasma haemolama (CMhl) was first described in 1990 in the USA in llamas and reported that CMhl infection alone, contributes to severe hemolytic anemia, fever, jaundice, hemoglobinuria, and weight loss in camelids (8). The present report describes the first case of a hemolytic disorder in an Iranian one-humped camel (Camelus dromedarius) coinfected with T. evansi and CMhl from northwest of Iran.

Case Presentation
In Jul 2017, a 4-year-old male one-humped camel (Camelus dromedarius) from Salmas county, Urmia, Iran belonging to a flock composed of 70 animals was referred to the Veterinary Teaching Hospital of Urmia University, Iran with poor body condition and signs of anorexia, weakness, depression and pale mucosa. According to the farmer, the symptoms started 5 d ago. The camels were reared in the pasture and only brought into the paddock during the night. The flock had no history of vaccination, anti-parasitic treatment, insecticides application or any other medication was recorded. Rectal temperature of the camel was 39.6 °C, heart rate was 50 beats per min and respiratory rate was 15 breaths per min. The body of camel was inspected carefully for the presence of ectoparasites. Jugular blood samples were taken into vacutainers (Kendall Company, Covidien, USA) containing EDTA-K2 as anticoagulant for determination of hematological and molecular analyses. During examination, the whole body of camel was examined for the presence of ticks by palpation, mainly on their ears, along their nape of neck, perineum, and udder/orchid, between thigh, shoulder region and tail base. The ticks were manually removed and transferred to the parasitology laboratory in tubes containing 70% ethanol solution.
Light microscopic examination of Giemsa stained peripheral blood films revealed the presence Trypanosoma spp. trypomastigotes (20×2.2 μm) and Mycoplasma-like organisms (up to 0.5 μm) (Fig. 1A). Mycoplasma spp. appeared as small, coccoid, basophilic epicellular bodies freely available in plasma or epicellular attached to the surface and periphery of erythrocytes.
Twelve adult ticks were collected that the most frequent and abundant tick species found on camel were Rhipicephalus sanguineus and Hyalomma annatolicum annatolicum.

Discussion
Previous studies from various southern regions of Iran employing PCR reported positive cases of T. evansi camels varying from 1.6% to 25.75% (2,(12)(13)(14)(15). However; this study is the first molecular identification of causative agent of surra in north-west of Iran. The disease is usually chronic and characterized by fever, anorexia, listlessness, pale mucous membranes, dullness, a very thin hump and drop to one side, abortions in pregnant females, and death in untreated camels (16)(17)(18).
Although, infection of camelids by hemomycoplasma has been described previously in camels (Camelus dromedarius) from central and south regions of Iran (Yazd and Kerman Provinces) using cytologic and molecular examination (19,20); however, to the best of the authors' knowledge the present report is the first documented and confirmed case of CMhl infection in Iranian one-humped camels from northern half of Iran. Anemia is a pathognomonic finding of surra and CMhl infections in the camelids. Decreased number of RBCs, hemoglobin concentration and PCV confirmed the occurrences of anemia in this case. These results are in agreement with earlier findings (19,21). This report was remarkable in several aspects I) molecular identification of T. evansi and CMhl co-infection in camel from Iran, II) association of hemoplasma infection with pyrexia and intravascular hemolysis in an in one-humped camel. Since ticks and hematophagous insects are important sources of Trypanosomes and CMhl infections in this area, it appears that such infections may be common in camels. The common clinical signs of both infections are fever, pale mucosa, and extremities edema. Since the hemomycoplasmiosis is a secondary infection, any kind of stressful, immune suppressing and retarding condition could aggravate the disease.
Rational treatment would appear to consist of correcting the anemia and eliminating of the infection. However, recovery was not achieved by administration of diminazene aceturate, oxytetracycline, and phosphorusvitamin B12.

Conclusion
Hemoplasmosis should be considered in the differential diagnosis of diseases characterized by hemolytic anemia and pyrexia. The PCR evaluation for hemoplasma DNA should be included in the investigation of such cases to enable the rapid definitive detection of this infection, which may be more common than previously estimated. Conducting further case studies are necessary to recommend successful treatment.