Detection of Mycobacterium avium subsp. paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture

One of the most relevant aspects in the diagnosis of paratuberculosis (Johne’s disease) in cattle is the availability of a method for the rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis (MAP) in order to facilitate the prompt removal of pathogen-shedding animals from a herd. To meet this requirement, methods for pre-treatment of bovine faecal samples and subsequent extraction of DNA for detection of MAP by real-time PCR were compared with MAP culture results. A total of 116 bovine faecal samples that showed weak (64.7%), moderate (18.1%) or strong (17.2%) growth of MAP on solid HEY medium were investigated.For PCR, supernatants, sediments or bacterial pellets were obtained from faecal samples by pre-treatment before extraction of MAP DNA based on silica membranes or magnetic particles. Samples then were tested by MAP IS900 and ISMav2 real-time PCR with an analytical sensitivity of 6 and 28 genome equivalents (GE) per mL, respectively.The best results were obtained by including a microfiltration step in the sample pre-treatment in combination with silica membrane-based mini-columns or magnetic particles for DNA extraction. This approach enhanced the detection rate of MAP in IS900 real-time PCR from 58.6% to 84.5% using silica membrane mini-columns and from 61.2% to 64.7% using magnetic particles.     

Comparison of BACTEC 460 and MGIT 960 systems for the culture of Mycobacterium avium subsp. paratuberculosis S strain and observations on the effect of inclusion of ampicillin in culture media to reduce contamination

To compare the utility and diagnostic accuracy of BACTEC and MGIT culture systems, a total of 41 pooled faecal samples, each containing faeces from one sheep infected with the S strain of Mycobacterium paratuberculosis and four uninfected sheep was cultured. The MGIT culture system did not support the growth of the S strain of M. paratuberculosis from faeces within the time frame of the experiments, although a laboratory adapted S strain grew slowly in MGIT provided that sufficient bacteria were inoculated. In contrast, C strain grew rapidly in MGIT. The sensitivity of culture was calculated relative to the infection status of the animals, none of which had clinical signs of ovine Johne's disease. The overall sensitivity of pooled faecal culture in the BACTEC culture system was 21.9% (95% confidence limits, 10.5-37.6), a figure dependant on the proportion of multibacillary cases. The sensitivities of the BACTEC culture system for pools containing animals with multibacillary and paucibacillary lesions were 100.0% (95% confidence limits, 47.2-100.0) and 17.8% (95% confidence limits 6.06-36.8), respectively. The contamination rate of BACTEC cultures was 9.7% compared to 14.3% for MGIT. The effect of 100 microg/ml ampicillin on the S strain of the M. paratuberculosis was examined and in both BACTEC and MGIT media it delayed growth by about 1 week. The composition of MGIT medium, particularly presence of vancomycin hydrochloride, slowed the growth of the S strain. The low content of egg yolk was considered to be another possible factor. The radiometric BACTEC culture system remains the best alternative for the culture of S strain and is recommended in circumstances where the genotype (s) of the strains present in a region/farm is either unknown or S strain.     

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I^®. The method, consisting of amplification of a 235 bp fragment of the 18S rRNA gene, is able to detect at least 0.1 fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1 ng–0.1 fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).     

Cost-effectiveness of diagnostic strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis cases in large dairy herds

Diagnostic strategies to detect contagious mastitis caused by Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae in dairy herds during an outbreak have been minimally studied with regard to cost and diagnostic sensitivity. The objective of this cross-sectional study was to compare the cost-effectiveness of diagnostic strategies for identification of infected cows in two California dairy herds during contagious mastitis outbreaks.     

A preliminary study of possible genetic influences on the susceptibility of sheep to Johne's disease

OBJECTIVE: To investigate possible genetic influences on susceptibility or resistance of sheep to Johne's disease. DESIGN: A field and laboratory study of two fine-wool Merino flocks with a high prevalence of disease due to Mycobacterium avium subsp paratuberculosis infection. PROCEDURE: Adult sheep were phenotypically classified as having severe, mild or no disease on the basis of clinical, pathological and cultural tests for paratuberculosis, and as positive or negative in tests for humoral immunity (agar gel immunodiffusion test) or cell mediated immunity (skin test for delayed type hypersensitivity). Correlations with phenotype were sought for polymorphisms at loci within selected immune function genes (NRAMP, MHC complex, IFN-gamma, lysozyme, leukaemia inhibiting factor). RESULTS: Possible associations of particular NRAMP and MHC alleles with susceptibility or resistance to Johne's disease were detected. CONCLUSION: If the results of this preliminary study are confirmed in further work, then the use of rams with "resistant" genotypes may assist in the control of Johne's disease in infected flocks.     

Longitudinal study of the spread of ovine Johne's disease in a sheep flock in southeastern New South Wales

OBJECTIVE: The aim of this study was to apply whole flock testing over time to determine the prevalence, distribution and spread of infection in a recently infected flock, with a view to planning intervention strategies for disease control. PROCEDURE: Serology, pooled faecal culture (PFC) and histology were used to determine the distribution and persistence of infection in a sheep flock in south east New South Wales between 1997 and 2002. Partial flock testing was done up to June 2000, after which annual whole flock testing, using PFC was performed. RESULTS: Faecal shedding of M a paratuberculosis was not detected in home-bred sheep until 7 years after the introduction of infected sheep in 1993. For at least 7 years there was clustering of infection and shedding within two age groups only. The infected groups appeared to have been exposed to infection (mycobacterial contamination) at an early age (<12 months) and commenced shedding at 5 years of age or older. Groups that were exposed to contamination as adults did not shed detectable amounts of M a paratuberculosis during the study period. CONCLUSION: Clustering of detectable infection in age groups of sheep that were exposed as lambs was a feature on this farm, providing indirect evidence of finite duration of survival of M a paratuberculosis on pasture and the influence of age on the susceptibility of sheep to develop detectable M a paratuberculosis infection. Spread of infection occurred very slowly and was probably related to the long incubation period (exposure to shedding interval) of 5 years observed on this farm. The findings suggest that partial flock culling, selective grazing management and vaccination could lead to a reduction in mycobacterial contamination on farm to a level at which patent infection no longer occurs. Better understanding of disease spread within flocks over time through flock profiling using PFC will help in devising surveillance strategies (including testing protocols for market assurance testing) to detect infected flocks where there has been clustering and slow spread of infection.     

Further potential limitations of the undifferentiated faecal egg count reduction test for the detection of anthelmintic resistance in sheep

Fifteen (36%) of 42 mixed gastro-intestinal nematode infections in sheep, identified as drench-susceptible by the undifferentiated faecal egg count reduction test, were found to harbour anthelmintic-resistant worms when analysed on the basis of changes in the egg counts of individual nematode genera. Most of these cases involved resistance in a single nematode genus, with Ostertagia and Trichostrongylus being implicated most frequently. The possible contributory role of larval culture results in helping to reduce the chances of the faecal egg count reduction test producing these and similar types of errors is discussed.     

羊鞭虫病实时荧光PCR检测方法的建立

为建立一种快速检测羊鞭虫病实时荧光PCR方法,根据GenBank已经公布的羊鞭虫(Trichuris ovis)ITS基因序列(登录号:JF680987.1),设计特异性引物和TaqMan探针,以重组质粒作为绝对定量模板,建立检测羊鞭虫病的TaqMan实时荧光PCR方法。对反应体系的特异性、敏感性和稳定性进行评价,并用该方法对临床样品进行检测。结果显示,该检测方法线性关系良好,标准曲线的相关系数R^2=0.994,扩增效率E=1.05。该方法特异性强,与其他8种常见家畜寄生性线虫病不发生交叉反应;灵敏度高,最低检出下限为31.7拷贝/μL;重复性好,组内和组间变异系数分别为0.43%~1.04%和1.20%~1.91%,均小于2.00%。用建立的实时荧光PCR方法和显微镜检查方法分别对20份临床样品进行检测,实时荧光PCR和显微镜检查方法检出的阳性样品分别为14和9份。本研究建立的TaqMan实时荧光PCR方法能在粪便中快速、准确、灵敏检测羊鞭虫卵,为羊鞭虫病的检测和防控提供新的方法。     

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10¹ to 10⁸ copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (C_t) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of C_t values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R² > 0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID₅₀/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.     

A model of the relationship between the epidemiology of Johne's disease and the environment in suckler-beef herds

A non-predictive, dynamic and stochastic herd-level simulation model of an outbreak of Johne's in a suckler-beef herd is reported. Importantly, the model incorporates, with a simple method, the environment as the primary source of infection, reflecting the consensual understanding of the disease. The model also takes into account the density of the infectious agent in the environment. A sensitivity analysis suggests that the model is highly and equally sensitive to certain parameters (probability of infection in the presence of one unit of bacterial density, infectious area and bacterial shedding rate). Mathematical reasons for this similarity in sensitivity are presented. Compared to many other diseases, data for Johne's are scarce. Therefore models of Johne's outbreaks including this one cannot be predictive or easily validated. The qualitative results: (a) demonstrate the modelled effect of inclusion of infection via the environment; (b) suggest management factors that could be tested by experimentation or observation. Estimates for the rate of transmission, arising from the model output, are similar to published empirical estimates. The results of future empirical research should aid scientific understanding of the disease, help validate this model and might bring economic benefits through improved management.     

藏系绵羊VEGF基因的克隆、同源性分析及其表达水平的实时荧光定量PCR检测

采用PCR技术从藏系绵羊皮肤组织细胞中扩增出了血管内皮细胞生长因子(VEGF)基因,长度为573 bp,共编码190个氨基酸;序列比对发现,VEGF基因在所选择的多个物种之间具有高度同源性,相似性在100%～74.5%之间,其中藏系绵羊与普通绵羊的VEGF基因序列完全相同,其次为牛和猪,相似性分别为99.1%和96.2%,而与鸡的同源性最低,相似性为74.5%。采用实时荧光定量PCR SYBR Green I荧光染料法,对藏系绵羊皮肤组织细胞中VEGF基因的表达水平进行了相对定量分析,建立了快速检测VEGF基因表达量的方法。检测结果表明:给妊娠后期藏系母羊补饲自制的营养舔砖和颗粒饲料后,所产羔羊体内VEGF基因的表达水平分别为对照组的11.5倍和0.85倍。为提高羔羊皮肤组织中VEGF基因mRNA的表达水平,对妊娠后期藏系母羊补饲自制的营养舔砖,效果优于补饲颗粒饲料。     

Development of a faecal occult blood test to determine the severity of Haemonchus contortus infections in sheep

Haemonchus contortus commences feeding on host blood by day 11 of infection, which leads to the presence of blood in the host's faeces. This study examined the capacity for a faecal occult blood (FOB) test to determine the severity of H. contortus infection in sheep at pasture, and to predict a rise in worm egg count (WEC) as infection matures. Diluted faeces were assayed with Bayer Hemastix and the change in colour of the reagent patch was scored on a 9-point scale from 1 (negative) to 5 in half unit increments. Performance of the test was compared with four benchmarks for severe infection: (1) WEC>2000 on test day; (2) WEC>2000 on test day or 3 days later; (3) WEC>2000 on test day or 3 or 7 days later; and (4) WEC>2000 on test day or 3, 7 or 10 days later. For a FOB score > or = 3, the frequency of false positive results was high (31.6%) for benchmark 1 but decreased to 3.6% as the definition of severe infection was extended to include WEC>2000 on the test day or 3 or 7 days later. Sensitivity (92.0%), specificity (94.2%) and predictive value of a negative test result (87.5%) were also high for benchmark 3. By detection of blood in faeces during heavy H. contortus infections prior to the emergence of high WECs, the test provided an early indication of imminent haemonchosis. Positive FOB test results are also likely to arise from other causes of blood in faeces such as fascioliasis, coccidiosis and some bacterial enteritides. Further field studies are needed to validate the method as a diagnostic test for determining the severity of H. contortus infections under diverse environmental and sheep husbandry conditions.     

瘤胃总甲烷菌实时荧光定量PCR方法的构建

试验构建了瘤胃总甲烷菌实时荧光绝对定量PCR的标准品及标准曲线用于总甲烷菌的定量测定,并提取瘤胃微生物总DNA, 以甲烷菌特异性引物进行PCR扩增, 回收纯化PCR产物,与pBS-T载体连接并转化到大肠杆菌,再用氨苄西林培养基筛选阳性重组质粒, 提取含目的片段质粒DNA, 通过PCR及测序鉴定重组质粒.根据OD值确定浓度, 将梯度稀释的质粒作为模板, 进行荧光定量PCR反应并作出标准曲线.结果表明: 所构建的标准曲线具有很高的相关性(R2=0.992), 并获得了高扩增效率产物.     

Financial modelling of the potential cost of ovine Johne's disease and the benefit of vaccinating sheep flocks in southern New South Wales

OBJECTIVE: To develop an enterprise gross margin (GM) model that predicts the on-farm financial impact of ovine Johne's disease (OJD) for various sheep enterprises in Australia. In addition, to estimate the benefits and costs of control through the use of the Gudair vaccination, including a breakeven point. DESIGN AND POPULATION: Data for the model was gained from an observational study conducted over a 3-year period from 2002 to 2004 using sheep from 12 OJD-infected flocks from southern New South Wales. Flocks ranged between 3500 and 20,000 sheep, with owner estimates of 5% or greater OJD mortality at the start of the study. PROCEDURE: A GM model was developed to predict the on-farm financial impact of OJD for various sheep enterprises in Australia, comparing non-infected, infected (status quo) and infected (vaccination) disease scenarios. RESULTS: Vaccination breakeven points are achieved within 2 to 3 years for breeding enterprises if OJD mortalities are high, rising towards 7 years for a Merino ewe enterprise if OJD mortalities are low. CONCLUSION: The GM model demonstrates the returns to investment of vaccination for Australian sheep producers with OJD-infected flocks.     

Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats

There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA - extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method - using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results.     

A PCR technique for the detection of Jaagsiekte sheep retrovirus in the blood suitable for the screening of ovine pulmonary adenocarcinoma in field conditions

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring contagious lung neoplasia caused by jaagsiekte sheep retrovirus (JSRV). Although no specific circulating antibodies against the virus can be detected in infected sheep, JSRV proviral DNA sequences can be found in peripheral blood leukocytes (PBLs) in clinically affected and in a proportion of in contact animals. In this study, existing hemi-nested PCR procedure is compared with a new one-step PCR technique that was developed to minimise potential DNA contamination and reduce sample and reagent handling. Different blood preparations were assessed and the best results were achieved on DNA prepared from buffy coat. The sensitivity of this PCR was lower in JSRV infected sheep without lesions of OPA than in clinically affected sheep, which indicate that this PCR may not be not fully appropriate for screening of individual sheep, but rather to provide results at flock level. This PCR is the only currently available blood test for detection of JSRV infected sheep and may be useful in epidemiological studies and in control programmes of OPA.     

检测猪戊型肝炎病毒的荧光定量PCR方法的建立

根据GenBank中猪戊型肝炎病毒的ORF2核苷酸序列的保守区域设计合成一对特异性引物,建立了一套SYBRGreen Ⅰ荧光定量PCR检测猪源戊型肝炎病毒(swHEV)的方法,并评价了该方法的灵敏度、稳定性和特异性,同时与常规的RT-nPCR进行对比分析.结果表明,建立标准曲线的相关系数为0.998,斜率为-3.039,Ct值变异系数(CV)在0.17％～1.41％之间,有良好的稳定性.同时在检测猪群常见病中显示出很好的特异性,并且比RT-nPCR更灵敏,适合于swHEV的检测.     

Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease.