ELISA based Sero- epidemiology exploration of ‘surra’ in bovines and camels in Karnataka and Rajasthan: A pilot survey 1National
Ligi Milesh1,2*, Sengupta. P. P1, Rudramurthy. G. R1 ,Twinkle Mathew2 Institute of Veterinary Epidemiology & Disease Informatics (NIVEDI), Ramagondonahalli, Yelahanka, Bangalore 560064 2 REVA University, Department of Biotechnology, Bangalore 560064, Karnataka *Presenting author and Corresponding author: Dr. Ligi Milesh, Assistant Professor, Department of Biotechnology, REVA University Email id: ligi.milesh@reva.edu.in ; ligimilesh81@gmail.com
ABSTRACT Trypanosomosis or ‘surra’ is caused by Trypanosoma evansi. It affects wide range of host including domestic and wild animals and is transmitted by the bites of tabanid fly. The disease is characterized by undulating fever, hypoglycemia, anemia, reduced animal production, milk yield and draughtability. At high parasitemia, it can be easily detected by conventional blood smear microscopic examination but become difficult when the infection is very low and in animals with carrier status. For successful control of the disease, it is always important to detect the carrier animals followed by its successive treatment. Thus, to overcome the issue, there was an urgent need to develop a reliable ELISA based serological assay for mass screening to understand the etiology, pathophysiology and behavior of the disease transmission for the successful treatment. In the present study, sero-epidemiology surveillance study was carried out using ELISA technique developed using cell membrane (CM) antigen of T. evansi (CM-I-ELISA) for the detection of antibody against surra. The assay was further compared with standard CATT/T. evansi test for its diagnostic potentiality and agreement correlation for the presence trypanosomosis or ‘surra’. The epidemiological survey was carried out with total of 220 field sera samples and showed overall 22.72% Sero-Positivity (SP) among bovines and camels in Karnataka and Rajasthan respectively and thus can be widely used as an effective tool for the sero prevalence study for the detection of carrier status of surra in livestock in India. Keywords: Trypanosoma evansi; trypanosomosis or ‘surra’; CM antigen; I- ELISA; CATT/T. evansi; sero- epidemiology analysis SUMMARY: C.M antigen based Indirect ELISA assay will help in the detection of carrier status of the infection and the prevalence of surra in India by CM-I-ELISA was found to be 22.72%
RESULTS INTRODUCTION: • Trypanosoma evansi is an important and highly pathogenic haemoflagellate parasite. • It causes trypanosomosis / surra. When parasitaemia is high, the clinical form of the disease can easily be detected by the conventional Giemsa’s stained blood smear examination which is very widely practiced in the field. • Many times, animals exhibit low levels of parasitaemia for years even after recovery and serves as parasite reservoirs. • Moreover, now T.evansi is not only restricted in animal hosts but also reported its adaptations in human host (Joshi et al., 2005), leading to its importance of zoonotic potentiality. • In this scenario, there is an urgent need of development of sensitive diagnostic tool for mass screening and treatment of the animals. • Some serological tests with improved technique, can detect both antibodies and the presence of the organisms, may be very much useful to screen out a large number of animal population. • Monoclonal antibody-based techniques are very much helpful to detect the parasitic antigen/antibody with high sensitivity and specificity.
Camel
Revival of Isolate
Propagatio n in lab animals
Purification of trypanosoma by DE-52 Cellulose Column
Whole Trypanoso ma evansi (100X)
INDIRECT ELISA Aim: To detect trypanosomal antibody
CONCLUSION: The present study using I-ELISA assay has a potentiality to be used in sero-diagnosis of surra for mass screening and the epidemiological data available can be used further for the treatment and control strategies.
anti-
COATING ▼ BLOCKING ▼ PRIMARY ANTIBODY ▼ SECONDARY ANTIBODY ▼ SUBSTRATE ▼ STOP REACTION ▼ OBSERVATION
ACKNOWLEDEGMENT: The entire work was carried out at ICAR-NIVEDI. My guide Dr. P.P. Sengupta of ICAR- NIVEDI, DBT for the financial support and REVA University. REFERENCES: 1.Bajyana Songa, E., Hamers, R., 1988. A card agglutination test (CATT) for veterinary use based on an early VAT Ro Tat 1.2 of Trypanosoma evansi. Ann. Soc. Belg. Med. Trop. 68, 233-240. 2.Laha, R. and Sasmal, N. K., 2009. Detection of Trypanosoma evansi infection in clinically ill cattle, buffaloes and horses using various diagnostic tests. Epidemiol. Infect,137: 1583-1585. 3.Ligi Jose., Sengupta P. P., Rudramurthy G. R and Rahman. H 2015. A pilot sero-survey for surra in livestock in Karnataka by ELISA using flagellar antigen of Trypanosoma evansi. International Journal of fundamental and applied sciences 4, 99-103 4.Ligi Jose.,Sengupta P. P., Rudramurthy G. R and Rahman. H., 2016 a. Seroprevalence of surra in camels using flagellar antigen in some areas of Rajasthan: A pilot survey International Journal of fundamental and applied sciences 5, 23-26 5. Ligi, M., Sengupta, P. P., Rudramurthy, G. R. and Rahman, H., 2016 b. Flagellar antigen-based CI-
ELISA for sero-surveillance of surra. Vet. Parasitol.219, 17–23
Species wise sero-prevalence and chi- square (χ²) analysis of CM-I-ELISA Comparision
Bovine Tabanid fly (host)
Trypanosoma evansi in blood
OBJECTIVES 1. To develop CM-I-ELISA based serological assay. 2. To screen field sera samples from Karnataka and Rajasthan for seroprevalence study for the presence of antibodies.
MATERIALS AND METHODS Preparation of cell membrane (CM) antigen of T. evansi from whole trypanosomes ▼ Characterization of CM antigen ▼ Development of CM-I-ELISA ▼ State wise sero- epidemiology study of trypanosomosis or surra ▼ Statistical analysis of the test (CM-I-ELISA)
and Immunoreact ivity of CM antigen with whole cell lysate (WCL) antigen by indirect ELISA
A total of 220 filed sera samples were collected randomly from different districts of Karnataka and Rajasthan and were subjected to CM-I-ELISA and observed around 22.72% sero-positivity.
CM-I-ELISA CM-I-ELISA State Specie wise s wise
Karnat Bovin aka e 130 Rajasth an Camel 90
CM-I-ELISA
Total
CATT/ T. evansi
22.30% T
220
P
29
21
50
N
%P
T
101 22.30 130
69
CATT/T.evansi 23.33% 23.33%
23.33
90
170 22.72 220
P
N
%P χ² analysis
28
χ² =0.022, df=1, 102 21.53 p>0.05
21
χ² =0.000, df=1, 69 23.33 p>0.05
49
χ² =0.013, df=1, 171 22.27 p>0.05
21.53%
Bovine (Karnataka)
Cattle (Rajasthan)