摘要
多杀性巴氏杆菌(Pasteurella mutocida, Pm)是牛出血性败血症的主要病原菌,由该菌引起的疾病呈散发或地方流行性,主要表现为败血症和出血性炎症,以高热、肺炎或急性胃肠炎并内脏广泛出血为主要特征。国内外有关该菌引起犊牛肺炎的研究报道甚少。2007年,新疆石河子先后有6个规模化奶牛场1-9周龄犊牛突然发病,共有90余头犊牛死亡,主要临床症状以体温升高、咳嗽、腹泻为特征。
为确定引起犊牛发病死亡原因,避免疫情扩大并有效地控制该病的传播。本文采集发病犊牛病料进行病原菌的分离、培养特性鉴定、生理生化以及分子生物学鉴定,对分离菌进行动物感染试验、毒力因子的检测以及药敏试验等研究。结果如下：
1、4株分离株均为革兰氏阴性短杆菌,两端钝圆。分离菌形态特征及培养特性与以往引起成年牛败血症状的巴氏杆菌相符,分离菌株生化鉴定结果与多杀性巴氏杆菌的生化特性一致。
2、参考多杀性巴氏杆菌种特异性基因kmtl及荚膜血清型特异性基因hyaD-hyaC、bcbD、dcbF、ecbJ、fcbD序列,设计引物对4株分离菌用多重PCR方法扩增,结果分离株均可扩增出小于500bp和1000bp左右的条带,用胸膜肺炎放线杆菌、肠球菌及大肠杆菌做模板都不能扩增出特异性条带,送测序并与GenBank中核苷酸进行比对,表明分离菌株为荚膜血清A型多杀性巴氏杆菌。且凡是引物Kmtl扩增为阳性的菌株生化结果都符合Pm标准。
3、采用分离株的纯培养物分别接种小白鼠、家兔,进行动物致病性实验,分离株表现出较强的致病性,所有试验组小白鼠在48h内全部死亡,家兔均在72h后死亡,从死亡家兔和小鼠体内均可回收到接种细菌。
4、药敏试验结果表明,所分离的4株Pm对氧氟沙星、庆大霉素表现较高的敏感性。对环丙沙星、新霉素敏感性不高,但对于氨苄青霉素钠无敏感性。
5、本实验选取多杀性巴氏杆菌与铁摄取有关的tbpA基因和编码4型菌毛ptfA基因,对分离株进行毒力因子的检测,结果经测序比对,分析后仅能检测到ptfA基因。
6、从犊牛、小鼠、家兔病理组织学变化可见主要病变发生在肺部,可见肺泡壁毛细血管扩张充血,肺泡浆液性水肿,肺泡腔内有纤维素渗出物,其中可见少量中性粒细胞和红细胞。其它脏器均出现不同程度的出血现象。
研究结果证实,该分离菌为多杀性巴氏杆菌。本研究首次证实新疆地区多杀性巴氏杆菌荚膜血清A型引起犊牛肺炎,并系统地研究了新疆多杀性巴氏杆菌生物学特性,同时建立一个从普通生化鉴定到分子生物学快速检测的诊断模式。
Pasteurella mutocida was an important pathogen of hemorraghic septicemia in cattle.It was endemic or diverging. HS charactered by significant form of hemorrhage, septicaemic,,hyperthermy,pneumonia or acute gastroenteritis predominantly in cattle. In 2007,The sick calf with age between one to nine weeks was characterized by fever, dyspnea and pathological changes in lung,part of which have diarrhea and about ninety calf died of pneumonia in six pastures of dairy cow in Xinjiang shihezi.
In order to determine the etiology of sick calves, preventing and to control the spread of the disease effectively. We made a description of isolated bacteria on the basis of morphological features, physiologeical,biochemical characteristic,molecule biology, animal infection assay,drug sensitivity test and detection of virulence factor.Result showed that:
The four isolates were Gram-negative rod bacteria.Biochemical characteristics and morphological features of this isolated bacteria were identified uniformly with Pasteurella mutocida which was reported, causing of HS.
The designed of primer refer to species—specific kmtl gene and serogroup—specific hyaD-hyaC, bcbD, dcbF, ecbJ and fcbD gene and amplificated by PCR.500bp kmtl gene fragement and 1000bp hyaD-hyaC gene fragemen were amplified respectively with the genome of each isolate as template.PCR products was contrasted by BLAST. Results indicated that the four bacterial isolates belong to capsular-serotype A of Pasteurella mutocida.
By injecting isolates pure culture to Mus musculus albus and rabbit,the experiment animals were all died after 72h.The pathogenicity test showed the isolate had fort pathogenicity to experiment animal and aim bacteria was obtained from the infected Mus musculus albus and rabbit.
The results of drug sensitivity test showed that isolated bacteria was sensitive to GEN, OFX,but resistantedto AMP.
Primers were designed and amplificated by PCR to detected the virulence factor of four isolates. Comparison of sequencing results of iron acquisition-related factors gene tbpA sequence and type four fimbriae gene ptfA sequence of Pasteurella mutocida by GenBank showed that only ptfA gene can be detection.
Pathologic histology observation showed that the affection mainly occurred on pulmones Telangiectasis on alveolar wall, serous edema of pulmonary alveoli, fibrin exudation on alveolar space,small amounts neutrophilic granulocyte and akaryocyte were observed.But other organ only had light hemorrhage.
This was the first reported capsular-serotype A of Pasteurella mutocida Inducing Pneumonia of Calf in Xingjiang. This paper systematacially studied some of biological chatacteristics of this isolated Pasteurella mutocida.
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