Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle.
Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398bp) and SapA-C (1422bp) fragments were amplified from the C. fetusSapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (P<0.05). Therefore, rSapA-N was selected to establish an indirect ELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P0.45: positive; S/P<0.4: negative; 0.45>S/P0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications.
Zhao
Hailing
H
Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 15001, PR China.
Liu
Huifang
H
Du
Yanfen
Y
Liu
Siguo
S
Ni
Hongbo
H
Wang
Yong
Y
Wang
Chunlai
C
Si
Wei
W
Yang
Jinguo
J
Ling
Jingkai
J
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis*; D000942:Antigens, Bacterial / Q000032:analysis / Q000235:genetics; D002169:Campylobacter Infections / Q000097:blood / Q000276:immunology / Q000662:veterinary*; D002168:Campylobacter fetus / Q000235:genetics / Q000276:immunology*; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood / Q000276:immunology / Q000382:microbiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D003612:Dairying; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000662:veterinary; D005260:Female; D005784:Gene Amplification; D008297:Male; D016133:Polymerase Chain Reaction; D012661:Semen / Q000382:microbiology; D014621:Vagina / Q000382:microbiology
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial