Surveillance of wildlife for Mycobacterium bovis infection using culture of pooled tissue samples from ferrets (Mustela furo)

AIM: To compare culture results of homogenates of pooled lymph nodes from individual ferrets with and without macroscopic lesions of bovine tuberculosis for the presence of Mycobacterium bovis, and to determine whether homogenates from 10-30 ferrets could be combined and cultured without loss of sensitivity as a possible method for improving cost-effectiveness of surveillance for M. bovis infection in wildlife populations. METHODS: Numbers of colony forming units (cfu) of M. bovis present in cultures of homogenates of pooled lymph nodes from individual ferrets known to be infected and having no visible lesions (NVL) or macroscopic lesions consistent with bovine tuberculosis were determined. Prevalences of M. bovis infection in populations of ferrets in the Marlborough region of the South Island of New Zealand were determined by culturing homogenates of pooled lymph nodes from individual animals. Samples from homogenates from North Canterbury were combined to form pools representing 10, 20 and 30 animals and also cultured for M. bovis. RESULTS: Fewer M. bovis cfu were isolated from ferrets with NVL (mean=0.77 log10) compared with ferrets with macroscopic lesions (mean=3.22 log10; p<0.05). The mean prevalence of infection in eight different surveys involving 427 ferrets from the Marlborough region was 18% (range 8-44%), which included a small number of animals with macroscopic lesions of tuberculosis. Pooling of samples from up to 30 different ferrets with NVL did not reduce the sensitivity of detecting M. bovis infected populations. CONCLUSION: Culturing of pools of lymph node samples detected a significant proportion of M. bovis-infected ferrets that would otherwise have gone unnoticed based on samples that had only macroscopic lesions. Culturing of samples pooled from up to 30 different ferrets could provide significant cost savings in surveys of wildlife for the presence of M. bovis infection without any apparent loss of sensitivity.     

Comparison of the recovery of Mycobacterium bovis isolates using the BACTEC MGIT 960 system, BACTEC 460 system, and Middlebrook 7H10 and 7H11 solid media.

The BACTEC Microbacteria Growth Indicator Tube (MGIT) 960 system was evaluated to determine how it compares with the BACTEC 460 radiometric system and solid media for recovery of Mycobacterium bovis from tissue samples. A total of 506 bovine lymph node samples were collected from abattoirs in the United States and Mexico between November 2003 and September 2004. Processed samples were inoculated into an MGIT 960 tube, BACTEC 460 vial, and Middlebrook 7H10 and Middlebrook 7H11 solid media. Ziehl-Neelsen slides were prepared to check for contaminants and confirm the presence of acid-fast positive bacilli. Samples containing acid-fast bacilli were confirmed as members of the Mycobacterium tuberculosis complex by a nucleic acid assay. Niacin and nitrate biochemical tests were used to distinguish M. bovis from M. tuberculosis isolates. Statistical analyses were performed to compare recovery rate, mean time to detection, contamination rates, as well as pair-wise comparisons in each category. The results showed that the MGIT 960 system had a higher recovery rate of M. bovis (122/129) than did the BACTEC 460 (102/129) and solid media system (96/129). The average time to detection was 15.8 days for the MGIT 960 system, 28.2 days for the BACTEC 460 system, and 43.4 days for solid media. Contamination rates were 6.9% for the MGIT 960 system, 3.4% for the BACTEC 460 system, and 21.7% for solid media. These results indicate the MGIT 960 system can be used as an alternative to the BACTEC 460 system for recovering M. bovis from tissue samples.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

牛支原体、巴氏杆菌A型和化脓隐秘杆菌多重PCR快速检测方法的建立

本试验旨在建立一种可同时鉴别牛支原体、巴氏杆菌A型和化脓隐秘杆菌的多重PCR方法。分别针对多杀性巴氏杆菌A型特异的hyac-hvaD基因区段、化脓隐秘杆菌的16SrRNA基因上保守区段和牛支原体的UvrC基因设计特异性引物,多重PCR的最佳扩增条件确定为：95℃ 10min预变性;95℃ 1min,56℃ 50s,72℃ 1min,循环30次;72℃ 210min延伸。结果表明,该多重PCR方法可同时扩增出以上三种致病菌的特异性片段,不能扩增出其他病原菌的相关片段;对多杀性巴氏杆菌A型、化脓隐秘杆菌和牛支原体的最低检测浓度分别为8×10^5CFU／mL、8×10^5CFU／mL和4×10^6CFU／mL。同时用该方法检测了牛支原体肺炎患牛的鼻拭子与肺组织,发现12h预增菌后,肺组织检测与牛支原体培养的阳性符合率为92％。对临床样本进行牛支原体分离培养需要3-4d时间,而采用多重PCR方法检测12h预增菌则能在24h内出结果。该多重PCR方法显著加快了临床诊断速度,具有推广应用价值。     

Naturally occurring tuberculosis in white-tailed deer

OBJECTIVE: To determine the distribution of lesions and extent of tissues infected with Mycobacterium bovis in a captive population of white-tailed deer. DESIGN: Cross-sectional study. ANIMALS: 116 captive white-tailed deer. PROCEDURE: Deer were euthanatized, and postmortem examinations were performed. Tissues with gross lesions suggestive of tuberculosis were collected for microscopic analysis and bacteriologic culture. Tissues from the head, thorax, and abdomen of deer with no gross lesions were pooled for bacteriologic culture. Tonsillar, nasal, oral, and rectal swab specimens, fecal samples, and samples of hay and pelleted feed, soil around feeding sites, and water from 2 natural ponds were collected for bacteriologic culture. RESULTS: Mycobacterium bovis was isolated from 14 of 116 (12%) deer; however, only 9 of 14 had lesions consistent with tuberculosis. Most commonly affected tissues included the medial retropharyngeal lymph node and lung. Five of 14 tuberculous deer had no gross lesions; however, M bovis was isolated from pooled tissue specimens from the heads of each of these deer. Bacteriologic culture of tonsillar swab specimens from 2 of the infected deer yielded M bovis. Mean (+/- SEM) age of tuberculous deer was 2.5 +/- 0.3 years (range, 0.5 to 6 years). Mycobacterium bovis was not isolated from feed, soil, water, or fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Examination of hunter-killed white-tailed deer for tuberculosis commonly includes only the lymph nodes of the head. Results of such examinations may underestimate disease prevalence by as much as 57%. Such discrepancy should be considered when estimating disease prevalence.     

Detection of mycoplasma in mastitic milk by PCR analysis and culture method.

Mycoplasma alkalescens, M. bovigenitalium, M. bovirhinis and M. bovis were directly detected from milk specimens by a polymerase chain reaction (PCR) when milk specimens were centrifuged and treated with mycoplasmal lysis buffer. The sensitivity of this PCR method was 110 to 1,400 colony forming units (CFU). This method was useful for the detection of mycoplasmas in milk specimens from cows at an early stage of mycoplasmal mastitis since a small amount of mycoplasma could be detect in milk without culture. The results were available within 12 hr, which is faster than conventional culture techniques. M. bovirhinis was detected in more than 70% of mastitic milk specimens when mycoplasmas were detected in milk specimens from 30 cows with mastitis by this PCR method.     

The emergence of Mycobacterium paratuberculosis in farmed deer in New Zealand - a review of 619 cases

AIM: To review cases in which Mycobacterium paratuberculosis was identified in farmed deer in New Zealand. METHODS: Case histories were reviewed where M. paratuberculosis was identified in deer by either culture or a polymerase chain reaction (PCR) test using primers from IS900. RESULTS: Between 1986 and 2000, M. paratuberculosis was identified by bacterial culture and/or PCR in 619 farmed deer from 299 herds, representing approximately 6% of deer herds in New Zealand. Over 85% of cases were identified during the last 6 years. In 60% of the infected herds, only one infected animal was identified. The maximum number of cases identified in a single deer herd was 47, and these were identified over a period of 8 years. Only 36 (5.8%) cases came from clinically affected animals identified on farms by veterinarians. The majority (89.7%) of the 619 cases were identified from lesions in mesenteric lymph nodes, including the ileocaecal lymph nodes, identified at meat inspection as being macroscopically either typical or equivocal of bovine tuberculosis (M. bovis). While the overwhelming majority of lesions were identified in mesenteric lymph nodes, M. paratuberculosis was also identified in 27 lesions in lymph nodes of the head, especially the retropharyngeal lymph node. CONCLUSIONS: The figures presented underestimate the true prevalence of infection with M. paratuberculosis, especially since not all suspect cases were submitted for culture or PCR. However, they do show that M. paratuberculosis appears to be spreading in farmed deer in New Zealand and highlight the possibility that Johne's disease is emerging as a potential major problem affecting this species. Identification of the organism by bacterial culture or PCR is required in many cases to distinguish lesions in mesenteric lymph nodes and lymph nodes of the head caused by M. paratuberculosis from those caused by M. bovis and M. avium.     

Investigation of the viability of M. bovis under different environmental conditions in the Kruger National Park

The survival time of Mycobacterium bovis in the natural habitat of infected free ranging wildlife was investigated. Seven different experimental sites near Skukuza in the Kruger National Park, South Africa, were selected to expose macroscopically affected lung or lymph node tissue of African buffalo (Syncerus caffer) origin and spiked faecal specimens for various lengths of time over a 1-year period. Mycobacterium bovis could be isolated for a maximum period of 6 weeks from tissue specimens and 4 weeks from faeces. The longest survival of M. bovis in both specimen types was observed in winter and under moist conditions. Surprisingly, the survival time of M. bovis in buried specimens seemed greatly reduced to a maximum of 5 days.     

Use of BACTEC radiometric culture method and polymerase chain reaction for the rapid screening of faeces and tissues for Mycobacterium paratuberculosis

SUMMARY The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC - PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.     

A one-tube nested polymerase chain reaction for the detection of mycobacterium bovis in spiked milk samples: an evaluation of concentration and lytic techniques.

The objective of this study was to evaluate the use of a one-tube nested polymerase chain reaction (OTN PCR) with 5 concentration and lytic treatments for the detection of Mycobacterium bovis in experimentally inoculated milk samples (spiked samples). OTN PCR and the following treatments were tested in inoculated samples: 1) centrifugation; 2) C18-carboxypropylbetaine + capture resin 1 + Proteinase K (CB18-CH-PK); 3) centrifugation + capture resin 1 + Proteinase K; 4) centrifugation + capture resin 2 + Proteinase K; and 5) centrifugation + immunomagnetic separation (IMS). The OTN PCR and the 5 treatments were evaluated in 2 different sets of spiked milk samples. One set consisted of 10-fold serial dilutions of a phenol-killed M. bovis in milk to final concentrations ranging from 5 to 50,000 cells/ml of milk. The other set of samples consisted of 2.5 serial dilutions of milk spiked with M. bovis to final concentrations ranging from 20.5 to 5,000 cells/ml of milk. Each treatment was repeated 5 times at each cell concentration. CB18-CH-PK and IMS were significantly more sensitive than other treatments. The lowest detection limit for these techniques was 20-50 cells/ ml of spiked milk. The specificity of OTN PCR in this study was high as demonstrated by the lack of DNA amplification products when M. bovis cells were not present in the samples. [The OTN PCR used in conjunction with CB18-CH-PK or IMS could be effectively used as a diagnostic and/or screening test for the detection of M. bovis in milk from herds with bovine tuberculosis.]     

Use of a macrophage cell line for rapid detection of Mycobacterium bovis in diagnostic samples

Mycobacterium bovis isolation on bacteriological media from suspected cases of bovine tuberculosis (TB) demands laborious and time-consuming procedures. Even polymerase chain reaction (PCR) and radiometric analyses are secondary procedures and not alternatives to bacteriological procedures. Therefore, there is a need to develop new techniques aimed at rapid M. bovis detection in diagnostic samples. The human macrophage cell line THP-1 was thus investigated in experiments of M. bovis propagation and isolation from reference lymph node suspensions. THP-1 cells were shown to support a high-titered propagation within 48h of minute amounts of both M. bovis BCG and fully pathogenic M. bovis strain 503. A semi-nested PCR for TB-complex-specific insertion sequence IS6110 revealed M. bovis infection in THP-1 cells. The same was true of a flow cytometry (FC) assay for expression of M. bovis chaperonin 10 in infected cells. The reduced time for isolation and identification of M. bovis (48-72h) and the consistency of the test results make the use of macrophage cell cultures attractive and cost-effective for veterinary laboratories involved in TB surveillance.     

牛支原体间接免疫酶标组织化学检测方法的建立

为建立检测牛支原体的间接免疫组织化学(简称免疫组化)方法,分别以鸡抗牛支原体(Mycoplasma bovis)IgY、羊抗鸡IgG-HRP为一抗和二抗,对牛支原体菌体涂片和牛支原体阳性肺脏组织切片进行免疫组化染色,并对染色条件进行优化,确定组织触片及组织切片的最佳免疫组化条件。在最佳条件下对病死犊牛病料切片进行免疫组化检测,并以细菌分离培养和PCR方法进行验证。结果显示,免疫组化法阳性标本与分离培养和PCR结果相符,而阴性对照和替代试验均为阴性。结果表明本试验建立的间接免疫组化方法可用于检测临床病例组织样本及培养物中的牛支原体,具有特异性和可靠性,为探究该病原在机体内的定位、动态分布规律及致病机理提供了手段。     

Isolation and identification of mycobacteria from cattle slaughtered in Pakistan.

Specimens of lung, liver and mesenteric lymph node from cows and buffaloes slaughtered in the Lahore area were cultured to investigate the type of mycobacteria involved in bovine tuberculosis. Employing the concentration method, 56 out of 530 cattle were found to be culture positive for acid-fast bacteria, 48 being Mycobacterium bovis and eight atypical mycobacteria. No M tuberculosis or M avium was isolated. Most of the isolated M bovis strains were found to be highly virulent for rabbits.     

Immunohistochemical markers augment evaluation of vaccine efficacy and disease severity in bacillus Calmette-Guerin (BCG) vaccinated cattle challenged with Mycobacterium bovis

Development of necrotic granulomas in response to Mycobacterium bovis infection in cattle is pathognomonic for bovine tuberculosis. Previously our laboratory reported on M. bovis granuloma classification by stage of lesion advancement within bovine lymph nodes and developed immunohistochemical markers to further characterize these granulomas. In this study of bovine lymph node granulomas we applied this classification system to assess the dynamics of vaccination challenge. Lymph nodes collected from cattle vaccinated with M. bovis bacillus Calmette-Guerin (BCG) and subsequently challenged with virulent M. bovis were compared to lymph nodes from unvaccinated, challenged cattle. Expression of interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), type I procollagen and cell marker identification of T cells, B cells, macrophages and WC1(+)gammadelta TCR+ cells were assessed. Granulomas formed in vaccinated cattle were greatly reduced in number, area, degree of necrosis and peripheral fibrosis and contained fewer Langhans' giant cells, acid fast bacilli, WC1(+)gammadelta TCR+ cells and less TGF-beta expression in comparison to controls. B cells clustered intensely along the outer granuloma margins within vaccinated calves, with significantly more IFN-gamma producing cells identified in the medullary regions of lymph nodes from BCG-vaccinated animals compared to unvaccinated controls. This may be indicative of immune activation and surveillance in regions not directly associated with ongoing disease. Lymph node evaluation using light microscopy and immunohistochemical markers is useful to assess the immune response and discriminate granulomas to determine vaccine efficacy and disease severity.     

利用聚合酶链反应技术检测牛结核杆菌病的研究

应用聚合酶链反应（ Ｐ Ｃ Ｒ） 技术检测牛结核分枝杆菌纯化 Ｄ Ｎ Ａ, 其敏感性为２５０ｆｇ 。所用引物序列对９种抗酸分枝杆菌 Ｄ Ｎ Ａ 进行扩增, 经琼脂糖凝胶电泳证实, 只有人型结核分枝杆菌、牛型结核分枝杆菌产生了３１７ｂｐ 的特异性扩增带。将 Ｐ Ｃ Ｒ 法与皮内变态反应试验（ Ｐ Ｐ Ｄ） 检测方法比较; ５４ 份血样标本中 Ｐ Ｃ Ｒ 的阳性率为１ ％ , Ｐ Ｐ Ｄ 试验的阳性率为０ 。同时对奶样标本的检测与血样结果一致。结果表明, Ｐ Ｃ Ｒ 在直接检测患牛血样、奶样标本中显示出快速、敏感、特异的优点。为今后牛结核病的检测工作提供了一条新的途径。     

致犊牛肺炎牛支原体南疆株的分离鉴定及oppF基因序列分析

本研究旨在调查新疆喀什某规模化奶牛场的犊牛死亡原因,并确定病原体.无菌采集3份因肺炎死亡的犊牛肺脏病料样品.采用牛支原体专用液体培养基和1.0%牛支原体琼脂固体筛选培养基从3份病死犊牛肺脏病料中分离得到2株牛支原体(Mycoplasma bovis,M.bovis),分别命名为M.bovis-NJ-1和M.bovis-NJ-2.通过菌落形态学观察、特异性PCR和oppF测序比对对分离株进行鉴定.结果显示,2个分离株在固体培养基上的菌落呈现典型的"煎蛋状",且Dienes染色特点符合牛支原体菌落着色特征,中心呈深蓝色;PCR能扩增出牛支原体特异的448 bp目的片段;2个分离株的oppF基因序列与牛支原体国际标准株PG45的同源性分别为96.7%和95.3%.结果表明,引起犊牛发病死亡的病原是牛支原体,本研究为犊牛支原体肺炎的快速诊断和防制提供依据.     

Granuloma development in cattle after intratonsilar inoculation with Mycobacterium bovis

OBJECTIVE: To examine the temporal development of tuberculous lesions in cattle inoculated with Mycobacterium bovis. ANIMALS: 15 mature crossbred cows obtained from a herd with no history of M bovis infection. PROCEDURE: Inoculation of cattle was done by intratonsilar instillation of 1.48 X 10(5) to 5.4 X 10(7) colony-forming units of M bovis strain 2045T. At 3 to 4 hours, 4 weeks, 6 weeks, and 8 weeks after inoculation, tissues were examined for gross and microscopic lesions and processed for isolation of M bovis. RESULTS: Retropharyngeal lymph nodes from cattle examined 4 weeks after inoculation contained microgranulomas consisting of aggregates of macrophages with few neutrophils. Retropharyngeal lymph nodes from all cattle examined 6 and 8 weeks after inoculation contained multiple, large, coalescing granulomas consisting of central areas of necrosis with mild fibrosis, numerous macrophages, lymphocytes, plasma cells, multinucleated giant cells, and neutrophils. Three of 8 cattle examined 6 or 8 weeks after inoculation had lesions in nonretropharyngeal sites with morphologic characteristics similar to that seen in retropharyngeal lymph node granulomas from cattle examined 4 weeks after inoculation. CONCLUSION: Granulomas can develop in draining lymph nodes of cattle in as little as 4 weeks after inoculation via intratonsilar instillation of M bovis. Intralesional morphologic changes between 4 and 6 weeks after inoculation indicate an increase in cellular chemotaxis and differentiation. Dissemination of bacteria to distant sites most likely was by lymphatic and hematogenous routes after establishment of the primary infection in retropharyngeal lymph nodes.