基于猪丁型冠状病毒重组M蛋白间接ELISA抗体检测方法的建立与应用

猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)是近年新发现的一种猪肠道病原体,主要感染仔猪并引起仔猪腹泻甚至死亡,针对该病建立快速、准确的血清学检测方法尤为重要.本研究以PDCoVHNZK-04毒株为模板扩增M基因并将其克隆至原核表达载体pET-32a中,构建了重组质粒pET-32a-P...     

新疆地区规模化奶牛场牛支原体流行病学调查

为了调查新疆地区规模化奶牛场牛支原体的感染情况,采用牛支原体间接ELISA和特异性PCR检测牛血清中的牛支原体抗体及病料中牛支原体核酸,共检测9个地区15个规模化奶牛场437份血清,肺脏、关节液及鼻腔黏液44份。结果显示,血清抗体阳性率为76.43%（334/437）,其中脐带血抗体阳性率为40.00%（4/10）;病料阳性率为40.91%（18/44）。检测结果表明,新疆地区大部分奶牛场存在牛支原体感染,部分奶牛场发生牛支原体肺炎及关节炎病例,并存在垂直传播的风险。牛支原体感染可能成为危害新疆规模化奶牛场犊牛健康的主要疫病之一。     

猪萨佩罗病毒间接免疫荧光方法的建立与初步应用

以分离的猪萨佩罗病毒PSV Hu N1/2016株感染PK15细胞,制备抗原板,建立检测猪血清中PSV抗体的间接免疫荧光(IFA)方法。结果表明:一抗最佳稀释浓度为1∶300;FITC标记的羊抗猪荧光二抗最佳稀释浓度为1∶300。该方法特异性强,敏感度高,既能将PSV抗体与PRRSV、FMDV、PCV、CSFV抗体区分开,也可以检测到中和效价0.128的阳性血清。用该方法检测湖南省部分规模化猪场208份临床血清样品的总阳性率为68%,表明PSV在湖南省流行普遍,且其感染主要发生在保育阶段。     

对虾白斑综合征病毒单抗介导间接ELISA的建立

从白斑综合征病毒（WSSV）青岛株感染的克氏原螯虾（Canbarus proclarkii）中提纯病毒，用纯化病毒免疫BALB／c小鼠，采用细胞融合法获得4株阳性杂交瘤细胞，分别命名为1B1、1E4、4E6和4E5。4株单抗均为IgM。4E5株单抗在免疫转印中与37500左右的病毒蛋白条带呈阳性反应。用此株单抗作一抗，建立检测病毒蛋白的间接ELISA。该方法用于人工感染WSSV的螯虾组织样品中病毒的检测，48h后即有阳性检出，而正常螯虾组织均呈阴性。     

不同咸蛋腌制温度对禽流感毒灭活的影响

经人工接种H9亚型禽流感病毒(AIV)的鸭蛋,分别在12℃、22℃和32℃条件下按常规方法腌制于饱和盐水中,同时以置饱和盐水中的禽流感病毒和未经处理禽流感病毒样品作为对照,通过MDCK细胞培养、间接免疫荧光方法定期进行禽流感病毒毒力测定,结果在12℃、22℃和32℃条件下腌制鸭蛋中的禽流感病毒分别于49天、27天和4天失去毒力,置饱和盐水中的禽流感病毒分别在27天、9天和2天失去毒力,而未经处理的禽流感病毒分别在92天、29天和17天失去毒力;不同试验温度条件下,以荧光RT-PCR检测腌制鸭蛋和对照样品中的禽流感病毒,在100天后仍可检出病毒核酸(Ct<30)。以上检测结果表明,在腌制鸭蛋时于常温下置饱和盐水腌制40天以上的传统咸蛋生产工艺,可使禽流感病毒完全失去毒力,腌制的鲜咸蛋携带或传播禽流感病毒的风险极低或不存在风险。     

犬瘟热病毒抗体检测方法的比较研究

用犬瘟热（ＣＤ）弱毒和自制的高免犬血清，建立了检测犬瘟热病毒（ＣＤＶ）抗体的中和（ＳＮ）试验、间接荧光抗体技术（ＩＦＡＴ）和间接免疫酶染色法。这３种方法对３８份ＣＤ弱毒疫苗免疫犬血清抗体效价的测定结果表明，当被检血清稀释度为１∶１００时，ＳＮ试验、ＩＦＡＴ和间接免疫酶染色法测定时的阳性血清数分别为２９，７和３１份，ＩＦＡＴ和间接免疫酶染色法测定结果相对于ＳＮ试验结果的符合率分别为４２．１％和９４．７％。同时发现，当ＳＮ试验测定的血清效价在１∶１００以上时，ＩＦＡＴ测定结果总是在１∶２５以上，间接免疫酶染色法测定结果总是在１∶１００以上。３种方法以各自初步确定的ＣＤＶ血清抗体效价完全保护值指标计算所测血清的完全保护率，ＳＮ试验为７６．３％，ＩＦＡＴ为７８．９％，间接免疫酶染色法为８１．６％。据此认为，ＩＦＡＴ和间接免疫酶染色法均可部分代替ＳＮ试验用于ＣＤ疫苗免疫效果的评价、免疫犬抗体水平的监测以及ＣＤ的流行病学调查     

猪轮状病毒重组VP7蛋白抗原间接ELISA诊断方法的建立

本研究以纯化的原核表达的猪轮状病毒VP7抗原表位区域为抗原,建立了检测猪轮状病毒抗体的间接ELISA诊断方法。特异性试验表明,该抗原与其他7种常见猪病病毒（TGEV、PEDV、CSFV、PCV2、PRRSV、PPV、PrV）的阳性血清不发生交叉反应,批内和批间重复性试验的变异系数均小于10％;对来自不同猪场的血清的检测结果表明,该ELISA方法与中和试验检测结果符合率达94．8％。本试验建立的ELISA诊断方法具有良好的重复性、敏感性和特异性,为PRV的快速诊断、免疫猪群抗体监测和轮状病毒流行病学调查提供了一种快速、简便的血清学诊断方法。     

Longevity of Mycobacterium bovis in brushtail possum (Trichosurus vulpecula) carcasses,and contact rates between possums and carcasses

Abstract

AIM: To determine, for a variety of environmental conditions, how long Mycobacterium bovis might remain viable inside the carcass of a brushtail possum (Trichosurus vulpecula) that died of bovine tuberculosis (Tb), and to measure the rate of contact between free-ranging possums and possum carcasses.

METHODS: Lesions of M. bovis were simulated by inoculating excised spleens weighing 0.5–1 g with 0.2 mL liquid culture containing approximately 5 x 10⁷ cfu M. bovis/mL. Simulated lesions were inserted into possum carcasses (n=48) at the peripheral lymph nodes. Carcasses were placed in the field at two sites (a tussock grassland and a podocarp-broadleaved forest site) and in two seasons (summer and winter) for up to 62 days. Survival rates of M. bovis were estimated by sampling the simulated lesions over time, and culturing the recovered lesion to determine if any viable M. bovis bacteria were present.

The time taken for a free-ranging possum to first encounter a dead possum in its home range was estimated by live-trapping possums and fitting them with proximity loggers (n=13). A ‘contact’ was recorded if these possums came within 40–50 cm of proximity loggers fitted to possum carcasses.

RESULTS: There were strong seasonal and site effects in the survival rate of M. bovis in possum carcasses. In the grassland habitat, no viable bacilli were cultured from any carcass after 3 days in summer, whereas in winter all samples were culture-positive for the first 20 days, and some were still positive after 27 days. The survival rates for forest habitat were intermediate between the results for grassland, and there were no culture-positive carcasses after 9 days in summer or 27 days in winter.

In summer, infected carcasses (n=6) were first encountered by possums a mean 1.9 (range 0.4–6.7) days after placement.

CONCLUSIONS: Possum carcasses were contacted by free-ranging possums within the period that viable M. bovis were shown to survive in a carcass. The risk of such infection is likely to be most significant in winter or in areas with microhabitats where the survival of M. bovis is high. However, the generally low survival rate of M. bovis in possum carcasses and the low frequency of possum-to-carcass contacts indicate this route of transmission alone could not maintain Tb in a possum population.