Development of an epitope-based competitive ELISA for the detection of antibodies against Tibetan peste des petits ruminants virus.

AIMS: To develop an effective diagnostic kit, based on a competitive ELISA-based system (cELISA), for detecting serum antibody against peste des petits ruminants virus (PPRV). METHODS: Epitope peptides of the nucleocapsid (N) protein of Tibetan PPRV were synthesized chemically and injected into rabbits to prepare hyperimmune antisera. Test sera were incubated simultaneously with hyperimmune antisera and added to the wells of ELISA plates coated previously with recombinant N protein. Horseradish peroxidase-conjugated goat anti-rabbit antibody was employed to detect the quantity of hyperimmune antisera combined with recombinant N protein. RESULTS: A cELISA has been developed for monitoring PPRV infections with a cutoff value of 35. Relative sensitivity and specificity values of the epitope-based cELISA were 96.18 and 91.29%, respectively, when compared with a commercial cELISA kit in a test involving 1,039 serum samples. CONCLUSION: We report an efficient method for preparing antibody suitable for incorporation into a cELISA that can be used routinely for the detection of PPRV antibodies in serum samples. The method eliminated the requirement for virus culture and monoclonal antibody preparation, reduced the biorisk posed by virus-dependent manipulations, and the performance of the resultant cELISA compared favorably with a commercially available cELISA kit.

Development of an indirect ELISA with artificially synthesized N protein of PPR virus.

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 µg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.

Evaluation of the economic impact of brucellosis in domestic yaks of Tibet.

Brucellosis is considered as an endemic disease in yaks (Bos grunniens) in China, but few economic analyses describing the cost of the disease and potential benefits of control have been reported. The aim of the study was to estimate the economic cost of brucellosis in yaks and the economic value of three control strategies: (a) vaccination; (b) test-and-slaughter; and (c) a combination of vaccination and test-and-slaughter programs in Damxung and Maizhokunggar counties and Pali township of Yadong county in Tibet. Using data from a cross-sectional seroprevalence survey conducted in 2015, combined with financial data, the predicted costs and benefits of the different control strategies were simulated over a 6-year period. The annual estimated cost of brucellosis in yaks within the study area was US$ 521,043 (95% CI: US$ 334,441; US$ 759,862), with an annual average cost per yak estimated at US$ 1.42 (95% CI: US$ 0.91, US$ 2.07). The benefit-cost analysis predicted that vaccination was the most effective control method with a benefit-cost ratio (BCR) of 3.19 (95% CI: 2.17, 4.66) and a net present value (NPV) of US$ 313,355 (95% CI: US$ 157,679, US$ 541,062) over a 6-year period. A sensitivity analysis found the NPV was most sensitive to the loss from a female yak aborting in the vaccination control program. In contrast, the price of yaks that were slaughtered had the largest influence on the NPV for both the test-and-slaughter control program and the combination control program. These estimates provide valuable information and establish a foundation for formulating and implementing cost-effective measures for controlling the disease in yaks on the Tibetan plateau, and more broadly in China.