Elsevier

Acta Tropica

Volume 185, September 2018, Pages 18-26
Acta Tropica

Unraveling cryptic epizootiology of equid trypanosomosis in Punjab state of India by parasitological and sero-molecular techniques

https://doi.org/10.1016/j.actatropica.2018.04.018Get rights and content

Abstract

To unravel equid trypanosomosis caused by Trypanosoma evansi in Punjab state of India, a cross sectional study was designed by utilizing parasitological and sero-molecular tools with objective to assess the prevalence of T. evansi in association with various risk factors in all agroclimatic zones of Punjab state of India. Parasitological Romanowksy stained thin blood smears (RSTBS) to detect patent infection, molecular techniques polymerase chain reaction I (PCR I; TBR 1/2 primers; targeting minichromosomal satellite DNA of T. evansi), polymerase chain reaction II (PCR II; TR 3/4 primers; targeting variable surface glycoprotein region DNA of T. evansi) & LAMP (Loop mediated isothermal amplification) assay to detect latent infection and serological assays card agglutination test (CATT/T. evansi) & ELISA (Enzyme linked immunosorbent assay) to detect exposure status of trypanosomosis were utilized in the present study. A total 429 equid blood and serum samples from all the five agroclimatic zones of Punjab state tested by these techniques showed a prevalence of 1.39% (CL: 0–15.28) by RSTBS, 6.52% (10.94–45.09) by both TBR 1/2 PCR and LAMP assay, 5.82% (11.57–38.42) by TR 3/4 PCR, 15.15% (36.57–135.42) with CATT/T. evansi and 22.84% (17.77–840.22) with ELISA. Interpretation of various risk factors revealed that the donkey/mules population (RR = 5.46, 95% [CI] = 0.15–15.56) was found to be at higher risk of T. evansi infection predominantly at ‘unorganized’ farms (RR = 4.06, 95% [CI] = 0.12–4.51). Animal used for commercial purposes (RR = 3.25, 95% [CI] = 0.06–7.42), rearing of equids with other domestic animals (RR = 2.36, 95% [CI] = 0.10–17.11) and farms without application of fly repellant/insecticides/net (RR = 3.68, 95% [CI] = 0.08-5.94) made them more prone to the disease. This comprehensive report utilizing the classical, serological and molecular diagnostic tools for epidemiology of T. evansi establishes the endemic stability of this infection in all agro climatic zones of Punjab with LAMP assay to be a promisingly sensitive and specific technique for the diagnosis of T. evansi under isothermal conditions in field situations.

Graphical abstract

A first report to utilize classical, serological and molecular diagnostic tools for unraveling cryptic epizootiology of Trypanosoma evansi infection from all agro climatic zones of Punjab. LAMP assay proved to be a field oriented easy diagnostic test for time and cost effectiveness and also indicate that equine population of unorganized farms with poor managemental practices were at higher risk of T. evansi infection.

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Introduction

Trypanosoma evansi, the first pathogenic mammalian trypanosome, responsible for the most widely distributed economically important, mechanically transmitted vector borne disease known as “Surra” in domestic livestock and wild animals in India (Singla et al., 2000; Kaur and Juyal, 2003). T. evansi was identified by Evans, (1880) a British Veterinarian while investigating camels and equids from Dera Ismail Khan Village of erstwhile Punjab (now in Pakistan). The lack of maxi circle DNA facilitate the utilization of a wide range of mechanical vectors by T. evansi (Masiga and Gibson, 1990) and thus making it widespread in Africa, Asia & South America in horses, camels, dogs, cattle, buffaloes and several wild animals (Hoare, 1972; Gill 1991; Singla et al., 2003; Sumbria et al., 2014).

The disease is mainly characterized by chronic weight loss, limb and ventral edema, urticaria, anemia, icterus, occasional abortions and neurological signs followed by death in the final stages (Losos, 1980; Brun et al., 1998). Parasite demonstration in blood smear is a gold standard microscopical examination technique (Muieed et al., 2010) but it suffers from limitations of sensitivity mainly due to scanty parasitaemia in peripheral blood during the chronic, intermission and prepatent phase (Gill, 1991; Juyal et al., 1994; Singla et al., 2013). Thus diagnosis of infection in blood is often challenging, especially in latent stage of infection, due to cryptic nature of parasitaemia and the sequestration of the parasite into less accessible tissues such as the nervous system (Desquesnes et al., 2009).

Serological antibody based tests such as enzyme linked immunosorbent assay (ELISA) and card agglutination test for trypanosomosis (CATT) can be helpful in mass field sero-surveillance, however may not be able to distinguish between current infection and residual antibodies from previous infection (Weiss, 1995). PCR tools improved the parasitological techniques from the 1990s, when primers were described for amplification of satellite DNA of trypanosomes (Masiga et al., 1992). Pruvot et al. (2010) have documented that highly repeated sequence of mini-chromosome satellite DNA (Masiga et al., 1992) as the gold standard for PCR based detection of Trypanozoon DNA.

The loop-mediated isothermal amplification (LAMP) technique is a novel DNA amplification method with high specificity, efficiency, rapidity and capability to amplify from a few copies of DNA to 109 copies in less than an hour under isothermal conditions. The test is simple and easy to perform, as it requires specific primers, Bst-DNA polymerase, and a regular laboratory heat block or a water-bath for the reaction (Notomi et al., 2000).

There is lack of comprehensive data on equine trypanosomosis covering all equid species from agroclimatic zones of Punjab, with the need of field oriented easy diagnostic test for time and cost effectiveness. Therefore, a cross-sectional study was designed to unravel the exact position of epidemiological status of equine trypanosomosis using a combination of parasitological and sero-molecular diagnostic test together with analysis of risk factor associated with them.

Section snippets

Ethical aspects

The study has the approval (IAEC/2016/310-35) of the ethics committee for animal experiments duly constituted by the Guru Angad Dev Veterinary and Animal Sciences University Ludhiana, Punjab-141004. Blood samples were collected in a humanly manner with prior consent of the equid keepers, so as to avoid any accidental injury to the equids.

Study areas

Punjab has 34 thousand equids which include 29 thousand horses/ponies and 5 thousand mules/donkeys (Fazili and Kirmani, 2011). The state covers a total area of

Romanowsky stained thin blood examination

Out of 429 animals screened, only six animals were found positive for T. evansi through classical Romanowsky stained thin blood smear examination. The RSTBS revealed several monomorphic trypanosomes, represented almost exclusively by thin (slender and intermediate) forms (Fig. 1). The slender forms are characterized by a long free flagellum with a narrow and drawn out posterior extremity which may appear as truncated or rounded. The kinetoplast is terminal or subterminal, i.e. placed at some

Discussion

The size and morphological structure of the T. evansi parasites were as per literature (Soulsby, 2005). No doubt, the conventional parasitological techniques will always remain important for understanding the biology, ecology and epidemiology of different strains of the parasites yet there is need of a highly sensitive test that can detect the lowest levels of parasitemia (Singh and Singla, 2012). Blood smear examination proved to be of limited value in diagnosis of subacute or chronic cases.

Conclusion

The present study is the first report to utilize classical, serological and molecular diagnostic tools for epidemiology of T. evansi which establishes the endemic stability of this infection in all agro climatic zones of Punjab. The entire samples positive by slide and PCR (TBR primers) were also positive by LAMP assay. This indicates LAMP assay to be a promisingly sensitive and specific technique for the diagnosis of T. evansi under isothermal conditions in field situations. The risk factors

Conflict of interest

None of the authors of this paper has a financial or personal relationship with other people or organizations that could in appropriately influence or bias the content of the paper.

Acknowledgements

Thanks are due to the authorities of Guru Angad Dev Veterinary and Animal Sciences University for providing financial support to carry out the research work.

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