The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy

The detection of Mycobacterium paratuberculosis organisms in bovine faeces by isolation was compared with that by the microscopical examination of Ziehl-Neelsen stained faecal smears for the presence of clumps of acid-fast M. paratuberculosis organisms. Faeces were obtained from cattle naturally or experimentally infected with M. paratuberculosis as well as from uninfected cattle. Microscopical examination was an unreliable method for the detection of M. paratuberculosis organisms since the organisms were only detected in 99 (=55.9%) of 177 culturally positive faecal samples. In addition, clumps of acid-fast organisms indistinguishable from M. paratuberculosis were also observed in three of 18 samples from cattle free from Johne's disease and in 18 of 37 culturally negative samples from paratuberculous cattle. When M. paratuberculosis organisms were added to faeces from an uninfected cow, results showed that isolation attempts should be positive when 15 or more M. paratuberculosis organisms per gram of faeces are present.     

Mycobacterium paratuberculosis and the bovine immune system

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a deadly intestinal ailment of ruminants. Johne's disease is of tremendous economic importance to the worldwide dairy industry, causing major losses due to reduced production and early culling of animals. A highly controversial but developing link between exposure to M. paratuberculosis and human Crohn's disease in some individuals has led to the suggestion that M. paratuberculosis is also a potential food safety concern. As with many other mycobacteria, M. paratuberculosis is exquisitely adapted to survival in the host, despite aggressive immune reactions to these organisms. One hallmark of mycobacteria, including M. paratuberculosis, is their propensity to infect macrophages. Inside the macrophage, M. paratuberculosis interferes with the maturation of the phagosome by an unknown mechanism, thereby evading the host's normal first line of defense against bacterial pathogens. The host immune system begins a series of attacks against M. paratuberculosis-infected macrophages, including the rapid deployment of activated gammadelta T cells, CD4+ T cells and cytolytic CD8+ T cells. These cells interact with the persistently infected macrophage and with each other through a complex network of cytokines and receptors. Despite these aggressive efforts to clear the infection, M. paratuberculosis persists and the constant struggle of the immune system leads to pronounced damage to the intestinal epithelial cells. Enhancing our ability to control this important and tenacious pathogen will require a deeper understanding of how M. paratuberculosis interferes with macrophage action, the cell types involved in the immune response, the cytokines these cells use to communicate, and the host genetic factors that control the response to infection.     

Comparison of two methods for isolation of Mycobacterium paratuberculosis from bovine fecal samples

Fecal samples from 131 cattle clinically suspect for paratuberculosis were cultured bacteriologically, using the traditional sedimentation processing method and a processing method that included a centrifugation step. Of 16 samples that were contaminated, 6 were culture-positive on at least 1 medium and by 1 processing method. Ten of 131 (7.6%) fecal samples processed by both methods were lost because of contamination. The number of culture-positive samples (using both processing methods) were 65 of 121 (53.7%) on media without miconazole and 60 of 121 (49.6%) on media with miconazole. Seven of the 121 (5.8%) samples were culture-positive, using centrifugation, after 16 weeks' incubation at 37 C. Thirteen of 60 (21.7%) isolates were obtained only with centrifugation, and 10 of these had low colony counts, suggesting that a centrifugation step may have concentrated microorganisms that would have gone undetected without centrifugation. Six of 60 (10%) isolates positive for M paratuberculosis on the sedimentation method were negative on the centrifugation method. Contamination rates were significantly (P less than 0.001) increased when centrifugation was used. The miconazole significantly (P less than 0.001) decreased contamination rates when centrifugation was used.     

Factors influencing the isolation of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples.

A modified procedure was used for culture of Mycobacterium paratuberculosis (Mptb) from bovine feces. Bovine fecal samples were decontaminated with NaOH, exposed to a mixture of oxalic acid and malachite green, incubated in a mixture of neomycin and amphotericin B. Decontaminated specimens were inoculated onto modified L?wenstein-Jensen medium. Specimens processed by high-speed centrifugation showed growth earlier than specimens prepared by low-speed centrifugation. However, the overall number of positive cultures at 16 weeks was not different for the 2 methods. When infected dairy herds were sampled 4 times at 6-month intervals and culture-positive cows were culled, the prevalence of infected cattle declined over time. After selective culling, the cattle left in the herds shed low numbers of Mptb, which explains why it took longer for cultures to become positive. No heifers younger than 11 months were culture positive, but heifers 13-14 months of age were more frequently culture positive than were heifers of any other age. The 16-week culture period is needed with this method to detect cattle shedding low numbers of Mptb. High-speed centrifugation of samples does not increase the efficiency of identification of animals shedding Mptb.     

Evaluation of ELISA and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces

Enzyme-linked immunosorbent assay (ELISA) was compared with electron microscopy in the examination of faeces from experimental calves and showed 100 per cent agreement in the detection of 19 bovine coronavirus and 15 bovine rotavirus electron microscope positive samples. In a limited field survey of calf diarrhoea 75 selected faeces were examined independently by ELISA and electron microscopy and the agreement between the two tests was 95 per cent for coronavirus and 84 per cent for rotavirus. A further comparison was made with 74 samples submitted for routine diagnosis and this yielded agreements of 82 per cent (coronavirus) and 89 per cent (rotavirus). Factors contributing to discrepant results were examined and the relative advantages and disadvantages of the two tests for routine detection of these enteric viruses are discussed.     

Nested PCR on blood and milk for the detection of Mycobacterium avium subsp paratuberculosis DNA in clinical and subclinical bovine paratuberculosis

OBJECTIVE: To determine the potential of PCR on blood and milk to detect cattle infected with Mycobacterium avium subsp paratuberculosis. PROCEDURE: A nested PCR method probing for IS900 was developed and compared to ELISA serology in 11 clinically infected and 46 subclinically infected, lactating Holstein cows from a herd with confirmed paratuberculosis (Johne's disease). RESULTS: When compared to serum ELISA the nested blood- and milk PCRs were equal in identifying DNA from clinically infected animals. The PCR procedures also gave positive DNA results with some subclinically infected animals when these only gave suspicious or negative results in the ELISA test. Most clinically and subclinically infected animals were detected with milk PCR. CONCLUSION: Since there may well be a haematological phase in paratuberculosis, nested PCR testing of blood and milk samples shows potential to detect animals subclinically infected with M a paratuberculosis. More subclinically infected animals need to be tested and confirmed infected before estimates of sensitivity and specificity can be made.     

Comparison of DNA probe test and cultivation methods for detection of Mycobacterium avium subsp paratuberculosis in caprine and ovine faeces

OBJECTIVE: To compare a DNA probe test with two cultivation methods for the detection of Mycobacterium avium subsp paratuberculosis in goat and sheep faeces. DESIGN: Comparison of the results of the three methods with histological examination as the reference standard. PROCEDURE: Faecal specimens were obtained from goats and sheep originating from flocks known to be affected with paratuberculosis and tested for Mycobacterium avium subsp paratuberculosis with a DNA probe test and two cultivation methods (old conventional culture and new double incubation method in Herrold's and Lowenstein-Jensen medium). RESULTS: In goats, the sensitivity of the various tests were for the DNA probe test 17.2%, for the double incubation culture method 25.4% and for the old conventional culture method 22.8% using the histopathological results as reference. In sheep the sensitivity of the various tests were for the DNA probe test 13.2%, for the double incubation culture method 8.8% and for the old conventional culture method 5.9% using the histopathological results as reference. The specificity of the above tests was 100% in goats and sheep and the specificity of the double incubation culture method in goats was 91%. CONCLUSIONS: The DNA probe test is a rapid and specific test that could be used in a control program if the sensitivity of the test were improved and possibly in combination with another test.     

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture

In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.     

Immunohistochemical methods for the visualization of Mycobacterium paratuberculosis in bovine tissues

The peroxidase-antiperoxidase (PAP), streptavidin-biotin (SB), and avidin-biotin-complex (ABC) techniques have been evaluated for the visualization of Mycobacterium paratuberculosis (Mp) in formalin-fixed, paraffin-embedded bovine tissues. The used immunoperoxidase techniques were comparatively better than the Ziehl-Neelsen stain, specially for the demonstration of small number of mycobacteria in tissue sections.     

Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.     

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.     

A DNA probe for the detection of Mycobacterium paratuberculosis

A genomic library of DNA extracted from Mycobacterium paratuberculosis was constructed in the expression vector lambda gt 11. The library was screened by plaque hybridization with labelled M. paratuberculosis genomic DNA as probe. Strongly hybridizing plaques were isolated and their DNA extracted and characterised for M. paratuberculosis specificity by hybridization to DNA from other Mycobacteriaceae. A clone was obtained which was specific for M. paratuberculosis. DNA from this clone could detect 7 ng M. paratuberculosis DNA.     

Effectiveness of Mycobacterium paratuberculosis isolation from raw milk by means of direct isolation of DNA and classic culture

Detection of Mycobacterium paratuberculosis (MAP) in tissues of patients suffering from Crohn's disease has given rise to speculation that this mycobacterium may play some role in the development of this disease in humans. Food products, especially milk obtained from animals infected with paratuberculosis, may be a potential vector of MAP to humans, yet the detection of this pathogen poses a number of difficulties. This study was aimed at comparing the effectiveness of MAP isolation from milk samples. Mycobacteria were detected by means of two methods: direct isolation of DNA using a QIAamp DNA Mini Kit by Qiagen, and a culture method with the use of HEYM culture medium. Analyses were carried out on 87 samples of udder cow milk originating from a herd that exhibited seropositive and serodoubtful reactions against paratuberculosis. The presence of an insertion sequence IS-900 was detected in 18 samples of udder milk analyzed with the method of direct DNA isolation and in two samples analyzed by means of the culture method.     

牛乳头状瘤病毒临床病理学检测及感染C127细胞研究

为建立犊牛乳头状瘤病毒(BPV)的实验室检测和体外感染C127细胞的方法,本研究通过光镜和电镜对牛乳头状瘤组织进行观察,采用PCR方法对瘤组织BPV进行检测,测序后确定其基因型。并将分离的BPV感染C127细胞,利用光镜观察细胞形态学变化,透射电镜观察C127细胞的病毒粒子分布。结果显示,光镜可见瘤体组织呈多个指状突起,每个突起的中心为纤维组织和血管,突起外为多层鳞状上皮细胞;异型性低,未见明显角化且无异常核分裂象。透射电镜观察可见基底细胞、棘细胞及颗粒细胞均出现细胞形状不规则、线粒体空泡状等病理变化。采用PCR方法检测到在约450 bp处出现特异片段,测序结果显示该基因序列与分离自中国的BPV-2SW株(KC256805.1)L1基因的相似性高达99%,表明该病毒株为BPV-2型。利用BPV感染C127细胞经培养后可见细胞变小、细长、生长密集、排列紊乱等明显的形态变化。本研究结果表明牛乳头状瘤是由BPV-2感染引起的,并且BPV-2能够感染C127细胞,呈现出明显的形态学病理变化。     

A cloned DNA probe for the detection of Mycobacterium paratuberculosis

DNA extracted from Mycobacterium paratuberculosis, which had been isolated from a cow with clinical Johne's disease, was used to make a gene library in the Escherichia Coli expression vector phage lambda gt11. Plaque-lifts were made from the library onto nitrocellulose membranes. These were screened by differential hybridization using radiolabelled chromosomal DNA from M. paratuberculosis and Mycobacterium phlei. By this method six recombinants that hybridized to M. paratuberculosis but not to M. phlei were identified. Three of these, designated lambda gt-R3, lambda gt-R4 and lambda gt-RS, containing DNA inserts of 2.5,1.5 and 3.7 kilobases (kb), respectively, were chosen for further analysis of their insert specificities. Following restriction with the endonucleases EcoRI and BamHI, the digestion fragments from the three recombinants were transferred to nitrocellulose membranes and probed with radiolabelled DNA from M. paratuberculosis and M. phlei. As expected, M. paratuberculosis DNA hybridized to all the fragments. M. phlei DNA hybridized to both the fragments that were generated from lambda gt-R3, to the single fragment from lambda gt-R4 and to two of the three fragments generated from lambda gt-RS. The fragment with which M. phlei DNA failed to hybridize was 0.45 kb in length. Multiple copies of this fragment were made in the plasmid pGEM-2; the plasmid DNA was then harvested and radiolabelled. Designated PAM-1, the radiolabelled material hybridized to a 3.7 kb fragment of EcoRI-digested M. paratuberculosis and to 2.2 kb fragments of similarly digested M. avium serovars 2 and 3. PAM-1 did not hybridize to DNA from the other four mycobacterial species examined or from Nocardia asteroides. The restriction fragment length polymorphism thus demonstrated distinguishes M. paratuberculosis from M. avium serovars 2 and 3.     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.