Veterinary Parasitology, 49 (1993) 319-323 319 0304-4017/93/$06.00 © 1993 - Elsevier Science Publishers B.V. All rights reserved

Short Communication

Camel trypanosomosis in Rajasthan, India

K.M.L. Pathak *'a, J.K. Arora b, M. Kapoor c aCollege of Veterinary and Animal Science, Bikaner 334001, Rajasthan, India

b Veterinary Hospital, Rainsinghnagar, Rajasthan. India CDepartment of Pharmacology, College of Veterinary and Animal Science, Bikaner 334001,

Rajasthan, India

(Accepted l August 1992)

Abstract

Blood samples from 240 camels ( Camelus drornedarius) were examined for trypanosome infection. Of these, 18 (7.50%) were found to be infected using the wet blood Giemsa stain technique, while 76 (31.66%) camels were found to be positive for Trypanosoma evansi antigen using the double antibody sandwich enzyme-linked immunosorbent assay (ELISA). The latter was found to be a more useful method for the detection of current infection.

Introduction

India possesses a large dromedary populat ion of 1.174 million, approxi- mately 7.8% of the world's populat ion of camels. Rajasthan alone has almost 70% of the entire camel populat ion of India, fostered by the nomads in the northwestern arid and semi-arid regions. Though the camel is a hardy animal, well-adapted anatomically and physiologically to the arid and semi-arid de- sert, it suffers from many parasitic diseases. Trypanosomosis , caused by Try- p a n o s o m a evansi, is one of the major parasitic diseases of camels, causing 30% morbidi ty and 3% mortal i ty (Rutter , 1967).

In India, T. evansi is associated with arid deserts and semi-arid steppes, but has been reported from other parts o f the country also. It is very common in Rajasthan, Haryana, Gujrat , Punjab and Ut tar Pradesh, comparat ively less in Madhya Pradesh, Tamil Nadu and Maharastra. Menon (1957) reported surra to be the most prevalent and serious infection of camels and horses in Rajasthan. It has been reported in 18 districts o f Rajasthan (Raisinghani and Lodha, 1989); apparently the disease is on the increase, as evidenced by re- ports received from the various veterinary hospitals in the affected area. This increase can be at tr ibuted to the Indra Gandhi Canal Project, which is allow-

*Corresponding author.

320 K.M.L, Pathak et aL /Veterinary Parasitology 49 (1993) 319-323

ing the establishment of ecological conditions favourable for the breeding of biting flies responsible for transmission of T. evansi (Menon, 1957).

In spite of the camel's importance, no systematic studies on camel trypa- nosomosis have been carried out and no up-to-date information is available on the prevalence of T. evansi in any part of India. The purpose of this study was to investigate the present status of camel trypanosomosis in the western part of Rajasthan, where the disease occurs frequently.

Materials and methods

Blood samples

Blood samples were collected from 240 apparently healthy camels (age range 4-5 years) from the western part of Rajasthan. Serum from these blood sam- ples was collected and used for sandwich assay.

Blood smears

Approximately 0.2 ml of blood from each sample was used for preparing wet and Giemsa-stained thin smears which were examined for trypanosomes; the parasite species was identified by morphology.

Antigen preparation

Trypanosome antigen was prepared from T. evansi maintained in dogs, originally isolated from a camel. Trypanosomes were separated from the blood of dogs, as described by Purohit and Jatkar (1979). The whole cell soluble antigen of the separated trypanosomes was prepared according to the method of Pathak et al. (1993). The protein content of the antigen was determined by the method of Lowry et al. ( 1951 ). A drop of merthiolate was added and the samples were stored at - 20 ° C until further use.

Preparation of hyperimmune serum

Rabbits were immunised subcutaneously against the whole cell soluble an- tigen of T. evansi using four injections, with doses gradually increasing from 1.0 to 3 ml, given at intervals of 6 days. The rabbits were bled by cardiac puncture 7 days after the last injection; the serum was separated under strict aseptic conditions.

Preparation of conjugate

The immunoglobulin fractions of the hyperimmune rabbit serum were pre- cipitated using 50% saturated ammonium sulphate. The precipitated globu-

K.M.L, Pathak et al. /Veterinary Parasitology 49 (1993) 319-323 321

lins were washed twice with 50% saturated a m m o n i u m sulphate, dissolved in phosphate-buffered saline (PBS) (pH 7.2) and then dialysed against PBS at 4 ° C. The protein content of the globulin preparations was determined pho- tometrically and the antisera were then stored at - 20 ° C. Each globulin prep- aration was conjugated to the enzyme horseradish peroxidase (Sigma, St. Louis, MO) using the periodate method described by Wilson and Nakane (1978).

Enzyme-linked immunosorbent assay (ELISA )

The assay system used was based on the double antibody sandwich assay described by Bidwell and Voller ( 1981 ). Immunoglobulin fractions of rabbit antiserum to T. evansi were diluted with 0.05 M carbonate buffer (pH 9.6) so that they contained approximately 200 mg protein ml-~. Two hundred microlitres of antisera were used to coat each well of 96-well polystyrene ELISA plates (Laxbro, India) by incubation for 24 h at 4°C. The plates were washed three times for 3 min each with PBS containing 0.05% Tween 20 (PBST) and were then incubated overnight at 4 °C with 1% bovine serum albumin (BSA) to reduce non-specific binding. After washing, 200/tl of undiluted test serum were added to the wells and incubated for 4 h at 37°C. The plates were then washed and 200/ t l of rabbit anti-T, evansi immunoglobulin conjugated to horseradish peroxidase and diluted to 1 : 1000 with PBST (pH 7.4) was added to each well. After incubation for 1.5 h at 37 ° C, the plates were washed three times with PBST (7.2 pH) and 200/tl of the freshly prepared substrate O- phenylenediamine was added and after incubation for 1 h at 37°C the reac- tion was stopped by the addition of 50/~1 of I N NaOH. The optical density (OD) of the contents of the wells was read at 450 nm and the results ex- pressed as ELISA values. Controls were included with each plate consisting of wells reacted with substrate only, negative control wells using serum from uninfected healthy camels, and positive control using serum from uninfected animals to which had been added antigen of T. evansi. An OD value of more than 0.209 was considered as the cut-offvalue for the positive sera.

Results

Out of 240 camels 18 (7.50%) were found positive for T. evansi when screened using the wet Giemsa-stained smear technique, whereas the number found positive for T. evansi antigen by sandwich ELISA was 76 (31.66%). Out of 18 camels found positive on screening of stained blood smears, 17 were found positive by sandwich ELISA.

The optical density of each well measured photometrically at 450 nm after 1 : 30 dilution varied from 0.087 to 0.209 (average 0.143 _+ 0.07 ) for negative control sera; for T. evansi positive sera the optical density varied from 0.349

322 K.M.L. Pathak et aL/Veterinary Parasitology 49 (1993) 319-323

to 0.644 (average 0.484 _ 0.34). The calculated sensitivity of the sandwich ELISA test was 92%.

Discussion

The present work has established that camel trypanosomosis, caused by T. evansi, occurs in the western part of Rajasthan, as circulating T. evansi anti- gens were demonstrated in the serum of camels using a double antibody en- zyme immunoassay. No other species of trypanosome was detected.

Parasitological diagnostic tests such as examination of wet blood films and stained smear examinations are at present the only methods available for the identification of current T. evansi infection in camels. In the present study, only 7.50% camels were found to be infected when the Giemsa-stained smear examination was used, while detection of circulating antigen of T. evansi was found to be a more sensitive means (92%) of practical diagnosis and was a more reliable method of detecting current infection in animals. Luckins et al. ( 1978, 1979) and Rae and Luckins (1984) reported that in both laboratory and naturally infected animals, antigen detection is the better method be- cause antibody levels decline slowly following trypanosomal drug therapy and remain high for many weeks after the parasite has been eliminated. The de- tection of antigenaemia indicates the current status of infection as well as the latent infection. As an epidemiological tool for use in surveys of trypanoso- mosis, this test could prove a useful immunodiagnostic test of active trypan- osome infection. In countries like India, where a single species of trypano- some infects animals, the test could be extremely effective, since it does not require access to freshly collected blood.

Trypanosomosis caused by T. evansi was found to be in the chronic form. The high incidence of surra as recorded in this study may be due to close proximity with Indira Gandhi Canal Command area which offers very con- genial environment for breeding of tabanid flies, the chief vector of surra in Rajasthan (Menon, 1957).

Acknowledgements

We are grateful to the Head of the Department of Parasitology, and the Dean of the College of Veterinary and Animal Science, Bikaner, for providing facilities for research.

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