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1.
Skin test negative cattle from a herd containing an unusually high proportion (194/382) of tuberculin skin test positive cattle were investigated for remaining Mycobacterium bovis infected animals. Blood samples from the skin test negative cattle, analysed by an antibody ELISA and an interferon-gamma assay, were mostly test negative for M. bovis. Radiometric culture of nasal mucus samples from 48 of the cattle yielded 22 culture positives with acid-fast bacilli and cording in 6 of these. Subculture on solid media was successful for 7, including 2 with cording of the 22 radiometric culture positives. Mycobacterium tuberculosis complex DNA probe testing using the Accuprobe (Gen-Probe, Inc.) and M. tuberculosis complex-specific PCR amplification, performed on the solid media subcultures, were negative. 16S rRNA PCR and sequence analysis were successful for 6 of the 7 solid media subcultures obtained and revealed the presence of Mycobacterium nonchromogenicum in all 6 subcultures. This is the first report of M. nonchromogenicum in nasal mucus of cattle. The observation highlights the importance of integrating definitive tests such as the PCR for diagnosis of bovine tuberculosis and indicates a possible zoonotic risk.  相似文献   

2.
A fluorescence polarization assay (FPA) utilizing fluorescein-labelled MPB70 protein as the antigen was developed and evaluated for its ability to detect antibodies to Mycobacterium bovis in cattle sera. Three panels of sera were examined in this study. These included: (A) sera (n=28) obtained from cattle from which M. bovis was cultured; (B) sera (n=5666) from Canadian field cattle which were presumed to be free from M. bovis; (C) sera (n=10) from cattle infected with Mycobacterium paratuberculosis and known to contain antibodies to this organism. Receiver operating characteristic (ROC) curve analysis of the results of panels A and B yielded an area under the curve value of 0.975 (95% confidence interval=0.971-0.979), which indicated that this FPA is an accurate indicator of M. bovis infection. At the cut-off point recommended by the ROC curve analysis, the FPA sensitivity and specificity estimates were 92.9% (95% confidence interval=76.5-98.9%) and 98.3% (95% confidence interval=97.9-98.6%) respectively. The FPA results were compared to the results of the single intradermal (SID) test for the 28 infected cattle. Fifteen of these animals were scored positive with the SID test (sensitivity=53.6%). The FPA detected 15/15 (100%) of the SID test-positive animals and 11/13 (84.6%) of the SID test-negative animals. Two of the culture-positive cattle were not detected by either test. None of the sera that were obtained from the M. paratuberculosis-infected animals cross-reacted in this assay.  相似文献   

3.
We evaluated by nested PCR reaction, different cow secretions from a herd with 48% of prevalence of bovine tuberculosis (BTB), seeking to determine niches where Mycobacterium bovis could be found. Postmortem examination of 18 (75%) tuberculin reacting cows allowed demonstrates BTB-compatible lesions in six, all of them PCR positives in milk and four in colostra samples. Our results showed that up to 62% of the colostra analysed contained M. bovis DNA, whereas only 18% of milk gave a positive reaction. Moreover, in bronchoalveolar lavages from cattle with compatible lesions in lungs or lymph nodes, where macrophages account up to 90% of cells, we did not find evidences of M. bovis. Altogether, these results suggest that differences in the anti-bacterial capacity of bovine macrophages, dependent upon microenvironment and organ-specific factors, exist. Alternatively, we hypothesize that hypoxic conditions that are encountered in mammary glands macrophages could induce M. bovis entrance into a 'dormancy-like' state, and that the high number of colostra samples were M. bovis was detected, could be an indicator of reactivation during 'peripartum'.  相似文献   

4.
Mycoplasma bovis was detected in 18/219 (8.2%) quarter milk samples collected from cases of bovine clinical mastitis in Northern Greece between November 1997 and March 1999. The cases occurred in 2/37 (5.4%) of the herds examined. The micro-organism was isolated from bulk milk samples (BTS) from the two positive herds but was not isolated from 111 composite milk samples collected from clinically healthy cows from all 37 herds. Isolates were identified as M. bovis by polymerase chain reaction (PCR) assay. Other micro-organisms were also isolated from the M. bovis positive samples. The M. bovis-positive cows had all been imported into Greece from other European countries.  相似文献   

5.
应用牛结核菌素对奶水牛进行牛结核病皮内变态反应的检测,然后对皮内变态反应阳性牛采样进行分离培养、γ-干扰素ELISA和聚合酶链反应检测,比较这四种检测方法的符合率。结果共对1850头奶水牛进行皮内变态反应检测,皮内变态反应阳性的奶水牛有78头,从78头反应阳性的奶水牛中分离鉴定为牛分枝杆菌的有2份,γ-干扰素ELISA方法检测为牛分枝杆菌阳性的有5份,PCR方法鉴定阳性的有4份;阳性检出率以变态反应为最高78/1850(4.21%),γ-干扰素ELISA方法为5/78(0.27%),PCR检测方法为4/78(0.21%),而分离培养为最低2/78(0.105%)。变态反应与分离鉴定的符合率最低,为0.105%;γ-干扰素ELISA方法、PCR方法与分离鉴定的符合率较接近,分别为40%和50%。γ-干扰素ELISA检测方法和PCR检测方法具有较好的特异性,可以作为变态反应检测的补充。  相似文献   

6.
牛分枝杆菌特异性PCR检测方法的建立及初步应用   总被引：12，自引：0，他引：12  
根据已发表的牛分枝杆菌的pncA的基因序列，设计和合成了一对可扩增294bp目的片段的引物，建立了特异性检测牛分枝杆菌的PCR方法。对牛分枝杆菌国际参考株和国内分离株成功扩增出294bp的特异性基因片段；对人结核分枝杆菌、副结核分枝杆菌、鸟胞内分枝杆菌和草分枝杆菌DNA的PCR扩增结果均为阴性。本PCR方法检测的敏感度可达到50pg。对10份牛分枝杆菌培养阳性和10份阴性样品的DNA分别进行了PCR检测，结果10份阳性样品中有9份样品为PCR扩增阳性，阳性符合率为90％（9／10）；而10份阴性样品则PCR扩增全部为阴性，阴性符合率为100％（10／10）。本方法可做为牛分枝杆菌的快速检测和流行病学调查的工具。  相似文献   

7.
The State of Texas had the most (cumulative) tuberculous cattle herds of any state in the United States during the decade ending in 1997. Of the cumulative 18 infected herds in Texas, 12 herds were concentrated in El Paso County (designated the 'El Paso milkshed'). To identify whether non-bovine reservoirs were a source of Mycobacterium bovis infection of cattle in this region, an investigation was conducted on the premises of 14 dairy herds (12 tuberculous and 2 non-affected herds) between May 1995 and June 1997. None of the 670 mammalian, avian and environmental (soil, water and air) samples collected and cultured from the premises of these herds was positive for the presence of M. bovis. None of the 119 human urine samples obtained from employees of these dairies was culture positive for M. bovis. Of 124 dairy-farm workers with tuberculin skin-test results, 48 showed positive test results. There was, however, no difference in percentages of positive skin-test results between farms without, and farms having, bovine tuberculosis within the last two years or longer. The percentage of positive reactions did not increase with length of time employed at a dairy with a history of confirmed tuberculosis. These findings suggest that non-bovine reservoirs appear not to be a factor responsible for tuberculosis of cattle in the El Paso milkshed.  相似文献   

8.
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.  相似文献   

9.
The intradermal tuberculin (IDTB) test and the interferon-gamma (IFN-gamma) assay are used worldwide for detection of bovine tuberculosis in cattle, but little is known about the effect of co-infecting agents on the performance of these diagnostic tests. This report describes a field trial conducted in a cattle herd with dual infection (bovine tuberculosis and paratuberculosis) during 3.5 years. It has been based on a strategic approach encompassing serial parallel testing (comparative IDTB test, the IFN-gamma assay and serology of paratuberculosis) that was repeated 8 times over the period, and segregation of animals into two herds. The IDTB test detected 65.2% and the IFN-gamma test detected 69.6% of the Mycobacterium bovis culture-positive cattle. However, the IDTB test performed better during the first part of the trial, while the IFN-gamma test was the only method that detected infected animals during the following three samplings. The number of false positive reactors with the IDTB and/or the IFN-gamma tests was remarkably high compared to other reports, and could be caused by cross-reactivity with M. avium subsp. paratuberculosis. Also, the M. bovis isolates from cattle and wildlife from the same property were characterised using molecular techniques to disclose an epidemiological link. The IDTB test may not be appropriate to eradicate bovine tuberculosis in herds with dual mycobacterial infections. This report highlights the need to use several diagnostic techniques for the accurate detection of M. bovis infected animals in these herds.  相似文献   

10.
Because of the frequent exposure of cattle to mycobacteria of the avium/intracellulare group, an investigation was carried out into the possible repercussions thereof on the diagnosis of bovine tuberculosis. Three calves from a bovine tuberculosis-free herd, scored avian reactors in the gamma-interferon assay for bovine tuberculosis, were sedated and inoculated endotracheally with a virulent Mycobacterium bovis strain. Then, three other avian reactors were housed with the above donor calves. Mycobacterium bovis was isolated from the nasal swabs of the three endotracheally infected, donor calves. On these samples, TB complex-specific polymerase chain reaction (PCR) tests for IS6110 were also positive, albeit with a different time kinetics. The three contact-infected calves showed clear immunological signs of infection; however, their nasal swabs were always PCR-negative and only Mycobacterium avium was isolated. In the endotracheally infected donor calves there was a rise of the gamma-interferon responses to avian and bovine purified protein derivative (PPD) tuberculins, which reached the same stable plateau levels over the whole experiment. The above effect was also observed in the contact-infected calves, even though the response to avian PPD tuberculin always remained at a higher level. By using conventional bovine and avian PPD tuberculins, the comparative intradermal test was generally positive in endotracheally infected, as opposed to contact-infected calves; a positive intradermal test for M. bovis was obtained in two contact-infected calves by different bovine PPD tuberculins based on M. bovis bacillus Calmette-Guerin (BCG) secreted or somatic antigens. It was concluded that M. bovis infection may be concealed for some time in cattle sensitized by mycobacteria of the avium/intracellulare group and that different diagnostic procedures should be adopted for such animals.  相似文献   

11.
OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.  相似文献   

12.
The presence of several Mycobacterium species was determined in 68 New World monkeys kept captive in the Cali Zoo. One hundred and thirty-three gastric lavage and blood samples were evaluated for mycobacterial presence by Ziehl-Neelsen (ZN) staining, culture and PCR amplification of the Mycobacterium tuberculosis Mtp40 species-specific gene. Mycobacteria other than tuberculosis (MOTT) were identified by PCR restriction fragment length polymorphism (RFLP). Different species of mycobacteria were detected in 65% of the primate population studied by Alpha Antigen PCR. Eleven percent were positive for Mtp40 PCR amplification, being diagnosed as having M. tuberculosis, and acid-fast bacilli were observed in 23% by ZN staining. MOTT were isolated from samples taken from 37 primates by culturing; according to the RFLP analysis, three strains were classified as belonging to the MAISS complex (Mycobacterium avium-intracellulare-scrofulaceum-simiae) and eight more, isolated from soil inside the cages, were categorized as environmental contaminants. Mycobacterium spp. were detected in 13 different New World primate species showing that PCR amplification of the Mtp40 gene is a better tool than culture for M. tuberculosis detection in captive animals and that RFLP is a useful technique for MOTT identification.  相似文献   

13.
A field comparison of the interferon-gamma (IFN-gamma) assay and the single intradermal cervical tuberculin (SICT) test for the diagnosis of bovine tuberculosis was conducted. A total of 1136 cattle belonging to 85 herds placed in 'Castilla y León' (northwestern Spain) were chosen, and 21 of these herds were subjected to the diagnostic assays two or three times at intervals of at least 4 months. All the animals positive to any of the tests were slaughtered and tuberculosis was confirmed by culture isolation method (CIM) and further identification by means of PCR. Only 10.6% of cattle reacted with the bovine PPD in the SICT test, a percentage that increased to 12.8% in the IFN-gamma assay. The sensitivity of the IFN-gamma assay compared to CIM was shown to be higher (84.9%) than that of the SICT test (80.2%), but the combination of both tests offered the highest sensitivity (92.9%). The number of false positive reactors (those animals in which CIM was negative) was considerably higher for the IFN-gamma assay than for the SICT test and, conversely, the number of false negative animals (M. bovis isolation but negative immunological result) was higher for the skin test than for the interferon assay. In the herds tested twice, tuberculosis was eradicated after the second cycle of testing in 50%, and in 75% after the third cycle in herds tested three times. The combination of these two techniques instead of separately seems, therefore, to be useful in eradication programmes against bovine tuberculosis.  相似文献   

14.
Fifty dairy herds in Alberta were tested for the presence of Mycobacterium paratuberculosis by fecal culture and serum enzyme linked immunosorbent assay (ELISA). Individual sera (1500) were tested for antibodies to M. paratuberculosis by ELISA. Fecal samples were combined in pools of 3 (10 pools/herd) for a total of 500 pools that were cultured for M. paratuberculosis. Thirty cultures, including all 10 pools from 1 herd, were not readable due to fungal contamination. The remaining 470 cultures, representing 49 herds, yielded 16 positive pools (3.4% +/- 2.1%) from 10 herds (20.4% +/- 11.3%). The ELISA of each of the 1500 sera detected 105 (7.0% +/- 2.4%) positive sera and 20 (40.0% +/- 13.6%) positive herds, based on 2 or more individual positive sera in the herd. The true herd-level prevalence, as determined by ELISA, was 26.8% +/- 9.6%. The true herd-level prevalence, as determined by M. paratuberculosis fecal culture, ranged from 27.6% +/- 6.5% to 57.1% +/- 8.3%, depending on whether 1, 2, or all 3 individual fecal samples in the positive fecal pool were culture positive.  相似文献   

15.
Tuberculosis, caused by Mycobacterium bovis is emerging as the most important disease affecting cattle. Furthermore, it results in a major public health problem when transmitted to humans. Due to its difficult and non-specific diagnosis, M. bovis has been declared to be one of the etiologic agents causing significant economic loss in the cattle industry. Our group evaluated a more rapid and specific method, based on a new polymerase chain reaction species-specific primers, which amplifies a 470-base pair fragment of the M. bovis genome. A total of 275 milk-producing cows were studied by intradermal tuberculin test (ITT) which gave 184 positive and 91 negative cases. From them, 50 animals were taken from a cattle ranch free of tuberculosis. Three different samples were collected from each animal (blood, nasal mucus, and milk). Positive results were obtained from 26 animals by PCR (11.4%), 1 by bacteriological culturing (0.4%) and 1 by bacilloscopy (0.4%). This finding suggests, as in previous reports, that ITT, normally used for bovine tuberculosis detection, has the inconvenience of having a broad range of specificity and sensitivity, and the PCR technique is a more specific and sensitive test to detect infection associated with M. bovis. Therefore, we propose this PCR assay as a useful tool in the epidemiological characterization of infected animals in areas considered to be at high risk of transmission.  相似文献   

16.
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.  相似文献   

17.
Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis   总被引：2，自引：0，他引：2  
An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.  相似文献   

18.
Sera from 100 herds of cattle located in the state of Washington were examined with an enzyme-linked immunosorbent assay for antibody to Fasciola hepatica in a screening procedure that included 5 to 10 samples/herd. Twenty-eight herds contained infected cattle and F hepatica was most prevalent in 3 distinct geographic areas. Subsequent retesting of all sera available from 14 herds (mean of 109 samples/herd) revealed that the screening procedure correctly detected 7 of 7 operations in which greater than 40% of samples were positive or suspect and 3 of 3 operations in which 12% to 13% of the samples were positive or suspect. One of 3 herds considered negative after screening was found to contain a few (7%) positive samples and 1 herd considered possibly infected was negative on retest. These results were compared with those obtained by fecal examination for F hepatica eggs in 9 of the 14 herds. A good correlation (5 of 5) was found in which a high percentage (48% to 85%) of sera were positive or suspect. Fasciola eggs were not found in samples from 2 herds with few (7% to 12%) positive or suspect sera or in 2 herds that were negative by an enzyme-linked immunosorbent assay.  相似文献   

19.
In an outbreak of Mycobacterium bovis infection in fallow deer in South Australia, 3 herds related by recent movement of deer were infected. From these 3 infected herds, 47 of 51 animals were tuberculosis at necropsy. A range of lesions was seen most of which differed from classical bovine tuberculosis in that pus was a white liquid, fibrous encapsulation was not marked and calcification was rare. Histopathology was of classical tuberculosis. M. bovis was cultured from lesions and M. avium-intracellulare was cultured from one deer with no visible lesions. The source of M. bovis infection has not been determined.  相似文献   

20.
In the US eradication program for bovine tuberculosis, a definitive diagnosis depends on the isolation of Mycobacterium bovis. However, in some cases bacterial culture is unsuccessful, even though the tissue is considered suspicious by histopathology because granulomatous lesions and acid-fast organisms are present. The purpose of this study was to determine if polymerase chain reaction (PCR) tests on formalin-fixed tissue would successfully identify the organisms observed in suspect lesions from culture-negative animals. Diagnostic laboratory records were used to select paraffin blocks of tissue from 102 ruminants that had suspect microscopic lesions but no bacterial isolation. Sections from these blocks were examined with PCR primers for IS6110 to detect Mycobacterium tuberculosis complex infection, or with 16S ribosomal RNA and IS900 primers for detection of Mycobacterium avium. The PCR tests successfully identified a mycobacterial infection in 58 of 102 tissues, including 41 M. tuberculosis complex and 17 M. avium (11 subspecies paratuberculosis). These results demonstrate that PCR testing of formalin-fixed tissue, in combination with bacterial culture, may increase the effectiveness of laboratory diagnostic efforts to detect and identify the most common mycobacterial diseases of ruminants.  相似文献   

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