Rawool D B, Malik S V S, Shakuntala I, Sahare A M, Barbuddhe S B
Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar 243 122, India.
Int J Food Microbiol. 2007 Jan 25;113(2):201-7. doi: 10.1016/j.ijfoodmicro.2006.06.029. Epub 2006 Sep 18.
Clinical samples (n=725) were collected from bovines (n=243) which were positive for mastitis using the California mastitis test (CMT) and somatic cell count (SCC). The clinical samples comprising blood (n=239), milk (n=243), and faecal swabs (n=243) were examined for the presence of pathogenic Listeria spp. Isolation of the pathogen was done using selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Confirmation of the isolates was based on biochemical tests and Christie, Atkins, Munch-Petersen (CAMP) test followed by pathogenicity testing. Pathogenicity of the isolates was tested by phosphatidylinositol-specific phospholipase C (PI-PLC) assay as well as in vivo tests namely, chick embryo and mice inoculation tests. The isolates were subjected to PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. Listeria spp. were isolated from 12 (1.66%) samples. Of these 4 (0.55%) and 1 (0.14%) were confirmed as Listeria monocytogenes and Listeria ivanovii, respectively. L. monocytogenes and L. ivanovii were recovered from milk samples (2) and faecal (3) of mastitic cattle (3) and buffaloes (2). L. monocytogenes recovered from the milk of mastitic cattle and L. ivanovii from the faecal swab of buffalo turned out to be pathogenic. However, the remaining three hemolytic isolates exhibiting positive CAMP test turned out to be negative in PI-PLC assay, chick embryo and mice inoculation. L. monocytogenes and L. ivanovii isolates characterized as pathogenic by PI-PLC assay and in vivo pathogenicity tests were found to possess all the five virulence-associated genes and three genes, plcA, prfA and actA respectively. The remaining three hemolytic but non-pathogenic L. monocytogenes isolates were negative for plcA by PCR. It seems that the plcA gene and its expression (in the PI-PLC assay) have an important role as virulence determinants in pathogenic Listeria spp. In conclusion, the PI-PLC assay and virulence genes targeted PCR (plcA, prfA and hlyA genes for L. monocytogenes and plcA, prfA and actA genes for L. ivanovii) hold a good promise as rapid and reliable in vitro alternatives to in vivo pathogenicity tests.
从使用加利福尼亚乳房炎检测法（CMT）和体细胞计数（SCC）检测为乳房炎阳性的牛（n = 243）中采集了临床样本（n = 725）。对包括血液（n = 239）、牛奶（n = 243）和粪便拭子（n = 243）在内的临床样本进行检测，以确定是否存在致病性李斯特菌属。使用佛蒙特大学培养基进行选择性富集，并接种到多明格斯 - 罗德里格斯分离琼脂上，以分离病原体。基于生化试验、克里斯蒂、阿特金斯、蒙克 - 彼得森（CAMP）试验以及致病性测试对分离株进行确认。通过磷脂酰肌醇特异性磷脂酶C（PI - PLC）测定以及体内试验（即鸡胚和小鼠接种试验）来测试分离株的致病性。对分离株进行五个毒力相关基因plcA、prfA、hlyA、actA和iap的PCR检测。从12份（1.66%）样本中分离出了李斯特菌属。其中，分别有4份（0.55%）和1份（0.14%）被确认为单核细胞增生李斯特菌和伊氏李斯特菌。单核细胞增生李斯特菌和伊氏李斯特菌分别从患乳房炎的牛（3头）和水牛（2头）的牛奶样本（2份）和粪便样本（3份）中分离得到。从患乳房炎的牛的牛奶中分离出的单核细胞增生李斯特菌和从水牛粪便拭子中分离出的伊氏李斯特菌被证明具有致病性。然而，其余三个CAMP试验呈阳性的溶血分离株在PI - PLC测定、鸡胚和小鼠接种试验中结果为阴性。通过PI - PLC测定和体内致病性测试被鉴定为致病性的单核细胞增生李斯特菌和伊氏李斯特菌分离株被发现分别拥有所有五个毒力相关基因以及三个基因，即plcA、prfA和actA。其余三个溶血但无致病性的单核细胞增生李斯特菌分离株通过PCR检测plcA为阴性。似乎plcA基因及其表达（在PI - PLC测定中）在致病性李斯特菌属中作为毒力决定因素起着重要作用。总之，PI - PLC测定以及针对毒力基因的PCR（针对单核细胞增生李斯特菌的plcA、prfA和hlyA基因以及针对伊氏李斯特菌的plcA、prfA和actA基因）有望成为快速可靠的体内致病性测试的体外替代方法。
