Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces

Avian intestinal spirochaetosis (AIS) is an infection of the caeca and/or colo-rectum of laying and meat breeder hens caused by anaerobic intestinal spirochaetes of the genus Brachyspira. AIS can result in a variety of symptoms, including delayed and/or reduced egg production, and increased faecal water content. The two most commonly reported Brachyspira species involved in AIS are Brachyspira pilosicoli and Brachyspira intermedia, and their detection and identification can be difficult and time consuming. In the current study a two-step nested duplex PCR (2S-N-D-PCR) was developed for the detection of these two species, using DNA extracted from washed chicken faeces. In the first step, a duplex PCR (D-PCR) amplifying Brachyspira genus-specific portions of the 16S rRNA and NADH oxidase (nox) genes was undertaken on the washed faeces. In the second step, a nested D-PCR was used that amplified species-specific portions of the 16S rRNA gene of B. pilosicoli and the nox gene of B. intermedia from the amplicons produced in the first step. The 2S-N-D-PCR was rapid and specific, and could be used to detect approximately 10(3) cells of each spirochaete species per gram of washed faeces. When tested on 882 chicken faecal samples from infected flocks, it detected 4-5% more positive faecal samples than did the standard method of selective anaerobic culture followed by individual species-specific PCR assays conducted on the growth on the primary plate. The application of this new technique should improve diagnostic capacity, and facilitate further studies on AIS.     

Detection of the astA (EAST1) gene in attaching and effacing Escherichia coli from ruminants

A total of 206 attaching and effacing Escherichia coli strains from ruminants were analysed for the presence of the astA (EAST1) and bfpA genes. None of these strains was bfpA-positive. The percentage of enteropathogenic E. coli (EPEC) strains astA-positive found in healthy cattle (15.6%) suggests that this animal species may be a significant reservoir of atypical EPEC potentially pathogenic for humans.     

Escherichia coli O115 forms fewer attaching and effacing lesions in the ovine colon in the presence of E. coli O157:H7

Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching-effacing (AE) lesions in the large intestine. Following previous observations of interactions between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.     

Spatial and temporal patterns at small scale in Austrocedrus chilensis diseased forests and their effect on disease progression

Austrocedrus chilensis is an endemic conifer of Patagonia that suffers a widespread mortality whose causes are a topic of discussion. Since Phytophthora austrocedrae is the most probable cause, we proposed that the spatial and temporal patterns of disease at small scale should reflect pathogen behavior. We aimed at characterizing the spatial and temporal patterns of diseased trees in different soil types and the effect of microsite variability on diseased trees spatial pattern. The spatial pattern of disease was influenced by soil type and tree density. In clay soils with low disease incidence (ca. 25%), the spatial pattern was random and not influenced by abiotic microsite conditions. When disease incidence increased (ca. 70%), concurring with denser plots, the spatial pattern was clustered, as a result of an infection process, and it was independent of microsite variability. In soils with better drainage conditions, that is, alluvial soils with volcanic ash input and coarse textured volcanic soils, the disease was clustered and associated with flat microtopographies. The progression of the disease at small scale was influenced by soil, precipitation and tree density. The spatial and temporal patterns of disease progression were associated with a contagion process and with environmental variables that affect drainage, coinciding with Phytophthora biology and requirements. Our results concur in pointing at Phytophthora as the cause of A. chilensis disease in the study area. Management practices should be urgently applied in order to minimize the spread of the inoculum.     

Lead Contamination in Feed Affects Performance, Egg Quality and Antioxidant Capacity of Laying Hens

本试验旨在研究不同剂量铅对蛋鸡生产性能、蛋品质以及血清、肝脏和肾脏中与抗氧化性能相关指标的影响.选用40周龄生产性能相近的海兰褐蛋鸡576羽,随机分为4组:对照组、试验1组(T1组)、试验2组(T2组)、试验3组(T3组),每组4个重复,每个重复36羽.对照组饲喂基础饲粮,T1、T2、T3组分别饲喂在基础饲粮中添加15、30、60 mg/kg铅的试验饲粮.试验期8周.结果表明:1)各组间产蛋率和料蛋比均无显著差异(P＞0.05),1～8周T3组平均蛋重显著低于对照组(P＜0.05),1～8周T1、T2、T3组平均日采食量分别较对照组降低了3.72％ (P ＜0.05)、1.93％ (P ＞0.05)、3.63％ (P ＜0.05).2)与对照组相比,试验组蛋白高度和哈夫单位均有降低趋势,除T3组蛋白高度(P＜0.05)外均差异不显著(P＞0.05);T3组蛋壳强度和蛋壳厚度最低,第4周和第8周时T3组蛋壳强度分别比对照组降低了21.89％、16.84％(P＜0.05);蛋壳厚度的变化趋势与蛋壳强度一致,均随着铅添加量的增加而降低,呈现一定的剂量-效应关系.3)与对照组相比,各试验组血清、肝脏和肾脏中谷胱甘肽过氧化物酶活性均显著降低(P＜0.05);丙二醛(MDA)含量均在一定程度上升高,T3组血清和肝脏中MDA含量均较对照组显著上升(P＜0.05),各试验组肾脏中MDA含量均显著高于对照组(P＜0.05);血清、肝脏和肾脏中还原型谷胱甘肽含量和总抗氧化能力以及总超氧化物歧化酶活性均呈现降低趋势.综上所述,饲料中铅污染可以导致蛋品质降低,诱导脂质过氧化作用,降低蛋鸡抗氧化能力.     

Microarray based comparative genotyping of gentamicin resistant Escherichia coli strains from food animals and humans

Recent data from the European and Hungarian Antimicrobial Resistance Monitoring Systems have indicated that the routine use of gentamicin in human and veterinary medicine frequently leads to the selection of gentamicin resistance in Escherichia coli. The aim of this study was to provide molecular characterization of gentamicin resistance in clinical and commensal E. coli strains representing humans and food producing animals by genotyping for antimicrobial resistance and virulence using a miniaturized microarray. All 50 strains tested proved to be multidrug resistant defined as resistance to three or more antimicrobial classes. Antimicrobial resistances genes such as aadA1-like, strB, bla(TEM), sul1 and tet(A) or tet(B), and corresponding phenotypes (streptomycin-, ampicillin-, sulfamethoxazole- and tetracycline resistance) were detected in >50% of isolates regardless of the host or clinical background. However, certain genes encoding gentamicin resistance such as aac(6')-Ib and ant(2″)-Ia as well as catB3-like genes for phenicol resistance were only detected in human isolates. Among virulence genes, the increased serum survival gene iss was predominant in all host groups. Although the majority of gentamicin resistant E. coli strains were characterized by diverse antimicrobial resistance, and virulence gene patterns, accentuated links between catB3-like, aac(6')-Ib, bla(CTX-M-1) and sat genes could be detected in human strains. Further resistance/virulence gene associations (tet(A) with iroN and iss) were detected in poultry strains. In conclusion, the simultaneous characterization of antimicrobial resistance and virulence genotypes of representative clinical and commensal strains of E. coli should be useful for the identification of emerging genotypes with human and or animal health implications.     

Modelling <Emphasis Type="Italic">Phytophthora</Emphasis> disease risk in <Emphasis Type="Italic">Austrocedrus chilensis</Emphasis> forests of Patagonia

Austrocedrus chilensis forests suffer from a disease caused by Phytophthora austrocedrae, which is found often in wet soils. We applied three widely used modelling techniques, with different data requirements, to model disease potential distribution under current environmental conditions: Mahalanobis distance, Maxent and Logistic regression. Each model was built using field data of health condition and landscape layers of environmental conditions (distance to streams, slope, aspect, elevation, mean annual precipitation and soil pH NaF). We compared model predictions by area under the receiver operating characteristic curve and Kappa statistics. A reasonable ability to predict observed disease distribution was found for each of the three modelling techniques. However, Maxent and Logistic regression presented the best predictive performance, with significant differences with respect to the Mahalanobis distance model. Our results suggested that if good absence data are available, Logistic regression should be used in order to better discriminate sites with high risk of disease. On the other hand, if absence data are not available or doubtful, Maxent could be a very good option. The three models predicted that around 50% (49–56%) of the currently asymptomatic forests are located on sites at risk of disease according to abiotic factors. Most of these asymptomatic forests surround the current diseased patches, at distances lower than 100 m from diseased patches. Management considerations and the scope of future studies were discussed in this article.     

西藏降水化学分析

1987-1988年及1997-2000年间,在西藏拉萨、定日、当雄、安多采集降雨样品及部分大气浮尘样品,分析测定了pH、电导率、CO2分压、总悬浮颗粒含量及10多种离子浓度。共测量了300场次降雨的pH值、电导率和其中60场次降雨的化学成分含量。结果显示：青藏高原降雨常呈弱碱性,1987-1988年间加权平均pH值为8．36,1997-1999年间为7．5。造成高原降雨呈弱碱性的主要原因是来自地面富含钙的粉尘。西藏是世界上空气污染最轻的地方,但是从20世纪80-90年代的降雨中,NO3^-与SO4^2-含量增加,pH值降低,表明保护这片脆弱环境的紧迫性。     

青海省2017—2019年州县级兽医系统实验室检测能力比对

为全面了解和提升兽医系统实验室检测能力,2017—2019年青海省动物疫病预防控制中心连续3年组织州县级兽医实验室开展检测能力比对工作。根据每年疫病的流行态势,设置PCR、ELISA以及传统类检测等比对项目。结果显示:2017—2019年实验室总体准确率分别为50.00%、56.82%、74.47%,样品总正确率分别为90.75%、93.56%、93.48%,整体呈上升趋势;各实验室PCR、ELISA、传统类检测项目的正确率差异不显著(P>0.05);州级实验室的检测正确率明显好于县级(P<0.05)。结果表明,州县级实验室检测能力逐年提升,绝大多数实验室具备PCR、ELASA、传统类检测等检测能力,但部分县级实验室的个别项目检测能力有待提高。建议进一步加大经费投入,加强基层实验室管理、业务培训等,整体提升县级实验室的检测能力。     

病原微生物药敏检测方法的研究进展

抗生素敏感试验在当前病原微生物耐药性持续增长的形势下,已成为指导临床科学合理用药不可或缺的重要环节.因此,药敏检测方法的完善与发展是快速获取科学、准确微生物耐药谱的前提,也是实现有效临床治疗的重要保障.本研究介绍了当前应用于微生物药物敏感检测的常规方法,如纸片扩散法、稀释法、Etest法等,简要分析了它们之间的优缺特性,并对近年来一些新兴发展的快速药敏检测方法进行了总结以及展望了不同类型药敏检测方法的发展和应用趋势.