Farm factors associated with the presence of Mycobacterium paratuberculosis infection in dairy herds on the New York State Paratuberculosis Control Program

An extensive questionnaire was developed and used to collect data from 33 herds that were on the New York State Paratuberculosis Control Program, to study farm factors associated with the presence of Mycobacterium paratuberculosis infection in dairy herds. The results of the last whole herd paratuberculosis fecal culture were used to indicate presence of infection in a herd, with herds having one or more animals positive classified as ‘infected’. The average prevalence within herds was 5.2%. Fourteen herds were uninfected and 19 herds had prevalences ranging from 0.7%–28.2%. Data on 31 continuous and 67 categorical risk factors were collected by questionnaire. Ten factors were significantly associated with prevalence risk of infection in the univariable logistic regression. These factors were: the type of farm operation (commercial/registered or both); earlier diagnosis of the disease before entering the control program; number of clinical cases in the previous year; whether clinical cases were raised or purchased animals; typical signs in clinical cases; exposure of calves 0–6 weeks of age to feces of adult cows; contact of young stock with adult animal feces from using the same equipment to clean the housing for both groups of animals; spreading feces on fields from which forage is later harvested and fed to animals of any age group; what is done with animals that are suspected of having paratuberculosis or test positive on culture; and frequency of cleaning the cow barn. Stepwise logistic regression was used to determine the significance of each risk factor while controlling simultaneously for the effect of other factors. The significant factors were the type of farm operation, clinical signs, and exposure of calves to feces of adult cows. Commercial herds, presence of clinical signs typical of paratuberculosis in animals, and exposure of calves 0–6 weeks old to feces of adult cows all indicate a higher likelihood that a herd is infected with M. paratuberculosis.     

奶牛副结核病流行牛场的处置方案分析

为协助新疆某副结核病严重流行的奶牛场抑制该病的流行,2014-2015年间采用副结核分枝杆菌(MAP)间接ELISA抗体检测试剂盒对该牛场的965头牛进行了4次血清抗体检测,并通过淘汰部分阳性牛控制该病的流行。结果显示,在两年内累计检测出297头阳性牛,检出率为30.78%(297/965)。首次检测发现104头阳性牛,检出率为13.51%(104/770)。淘汰处理后,间隔3个月再次检出2.91%(19/652)的阳性牛,再次淘汰阳性牛后进行了两次监测,其中间隔11个月时检测阳性率为7.55%(41/543),间隔17个月时检出率为33.33%(171/513)。同时,检测结果显示:抗体阳性牛的检出率随年龄的增加而呈现上升趋势,在4~5岁检出率最高,而0.5~1和7~8岁牛中检出率较低。统计分析发现,在副结核病严重流行的奶牛场开展该病的控制或净化时,检测和主动淘汰的间隔期设置为3个月时的控制效果优于11个月和17个月(P<0.001)。虽本次处置未达到预期目标,但根据检测数据和处置措施效果,结合现有的研究数据,初步制订了牛群副结核病流行程度的划分标准和防控、净化方案。本研究将为牛副结核病的防控提供案例借鉴和防控方案。     

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock

Paratuberculosis (pTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) in a wide variety of domestic and wild animals. Control of pTB is difficult due to the lack of sensitive, efficacious and cost-effective diagnostics and marker vaccines. Microscopy, culture, and PCR have been used for the screening of MAP infection in animals for quite a long time. Besides, giving variable sensitivity and specificity, these tests have not been considered ideal for large-scale screening of domestic livestock. Serological tests like ELISA easily detects anti-MAP antibodies. However, it cannot differentiate between the vaccinated and infected animals. Nanotechnology-based diagnostic tests are underway to improve the sensitivity and specificity. Newer generation diagnostic tests based on recombinant MAP secretory proteins would open new paradigm for the differentiation between infected and vaccinated animals and for early detection of the infection. Due to higher seroreactivity of secretory proteins vis-à-vis cellular proteins, the secretory proteins may be used as marker vaccine, which may aid in the control of pTB infection in animals. Secretory proteins can be potentially used to develop future diagnostics, surveillance and monitoring of the disease progression in animals and the marker vaccine for the control and eradication of pTB.     

DNA探针、粪检菌和ELISA三种方法在诊断副结核病中的比较研究

本研究在已构建的副结核分枝杆菌Ｃ２株ＤＮＡ基因文库的基础上,应用正、反向杂交试验从基因文库中筛选出特异性片段ＰＴＰ３１,以光敏生物素标记制成ＤＮＡ探针。用此ＤＮＡ探针以及粪检菌和ＥＬＩＳＡ三种方法分别对３２份副结核菌素（ＰＰＤ）变态反应阳性牛粪便及相应血清样品进行检测,其检出率分别为４７％（１５／３２）、５６％（１８／３２）和３４％（１１／３２）。对随机采集的２７６份牛粪便及血清样品,３种方法的检出率分别为１０％（２７／２７６）、１３％（３６／２７６）和７％（７９／２７６）。本研究结果表明,ＤＮＡ探针与粪检菌呈现正相关性,ＤＮＡ探针比ＥＬＩＳＡ方法能够检出更多的阳性数。     

副结核分枝杆菌SOD基因在BHK-21细胞中的表达

将重组质粒pGEM-T-SOD中的副结核分枝杆菌SOD基因亚克隆至真核表达载体pVAX1中,构建真核表达重组质粒pVAX1-SOD,以脂质体介导法转染至BHK-21细胞中,并采用RT-PCR和间接免疫荧光技术检测SOD基因在BHK-21细胞中的表达。结果显示,成功地构建了副结核分枝杆菌SOD基因的真核表达载体,且SOD基因在哺乳动物细胞中获得了表达。为研究副结核分枝杆菌SOD基因的免疫效果及作为牛副结核病DNA疫苗奠定基础。     

DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.     

新疆北疆某规模化牛场奶牛副结核病的检测与分析

为查明导致新疆北疆某规模化牛场奶牛腹泻、消瘦的主要病原菌,本研究采用微生物学与分子生物学方法对采集的患牛粪便进行检测与鉴定。结果显示:2份粪样抗酸染色阳性,IS900基因及亚型分型基因检测阳性,IS900基因与GenBank中多个副结核分枝杆菌序列同源性在99%以上,亚型分型基因与Ⅱ型同源性为99.81%,从而确定2份样本均被Ⅱ型(牛型)副结核分枝杆菌感染。     

羊副结核病的致病特点及综合防控措施

羊副结核病是由副结核分枝杆菌引起的一种以顽固性腹泻、肠粘膜显著增厚为主要临床特征的慢性传染病,多呈现隐形感染,该病潜伏期长,传播迅速,呈全球性分布,对畜牧业的危害巨大。本文立足羊副结核病研究进展的实际情况,梳理总结了目前学术界对羊副结核病病原学、流行病学、致病机制、临床诊断和免疫综合防控等方面的研究讨论,为羊副结核病的诊断与防控提供了理论基础。在此基础上提出防控羊副结核病政府需制定有效政策,养殖场需加强饲养管理,形成科学有效的防控系统,为我国畜牧业的可持续健康发展提供了理论支撑。     

Analysis of antigens in Mycobacterium paratubercuolsis.

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good.Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations.Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity.The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

Changes in the acid‐base parameters of venous porcine blood caused by the period of storage and the method of sampling

Summary

The experiments demonstrate that the acid‐base status of porcine blood does not change significantly during a six hours’ storage period at 0° C. They further show that the sampling of blood via a vena cava puncture in comparison with sampling via an indwelling ear vein catheter is associated with severe stress that affects the acid‐base statuts of the blood.     

牛副结核分枝杆菌LAMP快速检测方法的建立

为建立牛副结核分枝杆菌(MAP)的快速检测方法,本研究采用环介导等温扩增(LAMP)技术,以MAP的IS900基因序列为靶基因设计特异性引物,建立了MAP特异性的LAMP快速检测方法。经反应条件优化,检测结果显示,建立的LAMP检测方法具有良好的特异性,最低见测量为0.53 pg;与其他病原菌无任何交叉反应。临床样品检测与行业标准的符合率为100%。该LAMP检测方法可以用于MAP的常规检疫。