This study aimed to develop an indirect ELISA detection method for antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in sheep, for providing an efficient and reliable technique for the epidemiological surveillance and serological testing of Johne's disease (JD) in sheep.

In this study, the culture supernatant of the strain (MAP-XJB13) was isolated in the laboratory during earlier stage, which was selected as the MAP-coated antigen. The optimal reaction conditions and critical values for indirect ELISA were determined through the systematic screening and optimization of various parameters, including the coating solution and conditions, blocking solution and conditions, antigen coating concentration, serum dilution ratio, antibody incubation and color development time, brand of color development solution, sample dilution solution and enzyme-labeled secondary antibody protective solution. The efficacy of the developed indirect ELISA antibody detection method for sheep MAP was assessed in terms of sensitivity, specificity, repeatability, preservation period, and coincidence rate. Finally, the initially assembled reagent kits were utilized for the clinical detection of samples from in Heilongjiang and Inner Mongolia.

The optimal conditions for the ELISA were determined as follows: the coating solution utilized as CBS buffer, with the coating condition process conducted at 37 ℃ for 4 hours. The blocking solution comprised 5% fish gelatin, 5% trehalose, and 12% PEG4000, with the blocking procedure also performed at 37 ℃ for 2 hours. The antigen coating concentration was set at 80 μg·mL^-1, the serum dilution ratio was 1:40, and the dilution ratio for the enzyme-labeled secondary antibody was 1:30000. The incubation parameters included primary antibody incubation at 25 ℃ for 30 minutes, followed by a 30-minute incubation of the enzyme-labeled secondary antibody, and a 15-minute color development phase. The color development solution employed was Biodragon, while the sample dilution solution consisted of 1% ovalbumin and 0.5% trehalose. Additionally, the protective solution for the enzyme-labeled secondary antibody contained 0.1% ovalbumin. The critical threshold for the developed indirect ELISA method for detecting antibodies against sheep MAP was determined to be 0.460, with a sensitivity of 95.89% and a specificity of 96.12%. The cross-reactivity analysis demonstrated that, based on the premise that the positive and negative results were valid, there was no cross-reactivity with the following: positive serum for Brucella in sheep, positive serum for Mycoplasma mycoides in sheep, positive serum for Corynebacterium pseudomycosis in goats, positive serum for tuberculosis in sheep, positive serum for peste des petits ruminants virus in goats, positive serum for peste des petits ruminants in sheep, and positive serum for poxvirus in sheep. The intra-batch and inter-batch coefficients of variation ranged from 0.754% to 7.812% and 1.252% to 7.277%, respectively, and the stability of the results was maintained for up to 8 months. The sheep MAP indirect ELISA antibody detection kit exhibited a positive concordance rate of 95.89% and a negative concordance rate of 95.55% when compared to the ID.vet MAP ELISA antibody detection kit, resulting in an overall concordance rate of 98.56%. The prevalence of MAP antibodies in sheep from Heilongjiang and Inner Mongolia was found to be 10.81%.

This study successfully developed an indirect ELISA method for the detection of antibodies MAP in sheep. The method exhibited exceptional specificity, high sensitivity and a long shelf life, thereby offering robust technical support for the prevention and management of JD.

Paratuberculosis (PTB) is a chronic, wasting infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in ruminants. PTB causes huge economic losses to the livestock industry and poses a serious threat to public health safety. Since the current clinical methods for the detection and control of PTB are inadequate, and the PTB vaccine used is ineffective and interferes with the diagnosis of bovine tuberculosis, there is a need for developing a vaccine with strong immunogenicity, good safety, and excellent protection for the prevention and control of PTB.

The immunogenic protein of MAP was screened and its immunoprotective effect was evaluated, so as to provide the data support for the prevention and control of PTB.

Five recombinant plasmids were constructed based on six genes of MAP: p22, map1272c, map3531c, map3783, map3701c, and map3527. The five recombinant proteins were combined with MONTANIDE ISA 61 VG adjuvant to immunize mouse by subcutaneous injection, and the best immunogen was screened by IFN-γ ELISPOT assay. The best immunogen was then mixed with the reported 66NC fusion protein. Mouse were immunized by subcutaneous multi-point injection. At 3 weeks after the second immunization, mice were immunized with 1×10⁸ CFU of the MAP K-10 strain intraperitoneally. The immunogenicity and immunoprotective effect of the candidate subunit vaccine were comprehensively evaluated by IFN-γ ELISPOT assay, monitoring antibody titers and serum cytokines, as well as detecting weight changes, liver pathological and histopathological observations and charge count differences of infected mouse.

Five recombinant proteins, such as 58F, 62F, 69F, 46F, and 52F, were expressed based on the genes p22, map1272c, map3531c, map3783 and map3701c. 58F produced the highest level of IFN-γ after immunization and was the most promising candidate immunogen. The fusion protein combination 66NC+58F induced persistent high titers of IgG, IgM, IgG₁ and IgG_2a, and also induced specific release of IFN-γ, TNF-α, and IL-17A. In the evaluation of protective effects, the fusion protein combination 66NC+58F resisted the weight loss caused by MAP infection, significantly reduced pathological damage in the liver, and decreased MAP colonization in the liver.

The fusion protein combination 66NC+58F induced Th1 and Th17-type immune responses in mouse, provided immune protection against MAP infection and was an important candidate subunit vaccine for PTB.