甘肃某羊场免疫布鲁菌病疫苗后绵羊流产原因的调查及分析

Investigation and analysis of abortion in sheep after immunization against brucellosis in a Gansu sheep farm

应用疫苗免疫绵羊后,羊场依然出现大规模流产和不孕,为查明原因,收集该羊场31份流产羊和23份不孕羊的阴道分泌物拭子及其对应的羊血清进行检测。用竞争酶联免疫吸附试验(c ELISA)、布鲁菌抗体Ig G检测试验和试管凝集试验(SAT)检测血清;用荧光PCR做54份拭子流产相关的布鲁菌病、Q热和衣原体病的病原检测;对检测布鲁菌阳性的样本用AMOS-PCR进行布鲁菌种属鉴定,扩增利福平耐药基因(rpoB)并测序,将序列与其标准参考菌株比对后构建进化树。结果显示:用布鲁菌c ELISA检测54份血清,其中22份血清初筛阳性,阳性率为40.7%;SAT确诊其中16份样本为阳性,其中流产羊有6只阳性,阳性率为19.4%,不孕羊有10只阳性,阳性率为43.5%,总体阳性率为29.6%。用布鲁菌抗体Ig G检测试验检测感染的样本19份发现,流产羊有6只阳性,阳性率为19.4%,不孕羊有13只阳性,阳性率为56.5%,总体阳性率为35.2%。荧光PCR结果显示:拭子样本10份为布鲁菌阳性,阳性率为18.5%;12份为贝氏柯克斯体(Q热病原)阳性,阳性率为22.2%;衣原体阳性率为0。布鲁菌阳性样本AMOS-PCR扩增得到731 bp的条带,rpoB基因分析发现所有氨基酸序列都未发现利福平耐药突变,AMOS-PCR和rpoB基因系统进化树分析结果显示羊感染的为马耳他布鲁菌。结论:本场群发性流产的发生可能是由于布鲁菌病疫苗免疫失败后病原感染,并伴随着贝氏柯克斯体等病原的混合感染引起。

Massive abortion and infertility still occurred after the application of the vaccine to immunize the sheep.In order to find out the causes of abortion and infertility in those sheep,a total of 54 vaginal swab samples,including 31 samples of aborted and 23 samples of infertile sheep,and their corresponding sera were collected from this sheep farm for testing.Sera were subjected to competitive enzyme-linked immunosorbent assay(c ELISA),Brucella antibody Ig G detection test and tube agglutination test(SAT).Fifty-four swabs were subjected to fluorescent probe PCR for detection of miscarriage-associated pathogens such as Brucella,Coxiella burnetii,and Chlamydia.Identification of Brucella species were carried out on Brucella-positive samples by using AMOS-PCR.Rifampicin-resistant(rpoB)gene was amplified and sequenced.After the sequencing results were compared with its standard reference strains,an evolutionary tree was constructed.The results showed that 22 samples were anti-Brucella positive from 54 sera tested by c ELISA,with a positive rate of 40.7%,and SAT confirmed 16 samples of these to be anti-Brucella positive,of which 6 samples were positive in aborted sheep,with a positive rate of 19.4%,and 10 samples were positive in infertile sheep,with a positive rate of 43.5%,and an overall positive rate of 29.6%.Nineteen samples were tested for infection by Brucella antibody Ig G.Six aborted sheep were positive,with a positive rate of 19.4%,and 13 infertile sheep were positive,with a positive rate of 56.5%and an overall positive rate of 35.2%.There were 10 swab samples positive for Brucella with a positive rate of 18.5%,12 positive samples for C.burnetii with a positive rate of 22.2%,and 0 positive for Chlamydia,as detected by fluorescent PCR.After identification by AMOS-PCR,a band of 731 bp was amplified,and analysis of the rpoB gene revealed that all the amino acid sequences showed that no rifampicin-resistant mutation was detected.AMOS-PCR and the rpoB gene phylogenetic tree analysis showed that sheep were infected with B.melitensis.In conclusion,the occurrence of herd abortion in the farm could be caused by pathogenic infection after Brucella immunization failure,accompanied by mixed infection with C.burnetii and other pathogens.