Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).     

Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 x 10(1) to 2 x10(6) copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.     

A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.     

New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan(mgb) and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10(2) CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.     

Peptide aMptD-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 10(2) CFU ml(-1) for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 10(2) CFU ml(-1)) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 10(3) and an association constant of 1.33 x 10(9). The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

Detection of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic

We investigated the presence of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure to M. avium subsp. paratuberculosis, albeit at a low level for viable cells.     

Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg(2+) concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 x 10(6) to 3 x 10(1) copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.     

建立液相芯片方法检测鉴别结核分枝杆菌复合群、鸟分枝杆菌与副结核分枝杆菌

运用液相芯片技术原理,以分枝杆菌菌种(群)特异基因序列IS6110、IS1081、IS1245和F57为目标基因,设计筛选4套扩增引物和杂交探针,建立同时检测鉴别结核分枝杆菌复合群、鸟分枝杆菌和副结核分枝杆菌的四重液相基因芯片检测方法。对13种共54株分枝杆菌菌株以及23种常见微生物样品的检测结果显示,四重液相芯片方法可特异检测鉴别目标菌种(群),与其它分枝杆菌菌种或微生物无非特异交叉反应;检测敏感性达2.1×101-2.5×102基因拷贝或0.06-0.74 fg DNA;组内检测变异系数和组间检测变异系数均<10%。采用四重液相芯片方法从临床结核疑似人痰样和牛组织样品中检出结核致病菌,检出率分别达75.6%(99/131)和94.9%(37/39),显著高于培养法(38.9%和53.8%)。对副结核疑似临床样品的检测试验结果显示,四重液相芯片方法与荧光PCR方法的阳性符合率为83%(24/29)。对四重混合模板的检测试验结果显示该液相芯片方法可鉴别不同菌种混合感染。四重液相芯片方法的检测周期<1 d,其中对纯化DNA模板的检测时间可在2-3 h内完成。     

Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn's disease cases in the city of Cardiff

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.     

Culture- and Quantitative IS900 Real-Time PCR-Based Analysis of the Persistence of Mycobacterium avium subsp. paratuberculosis in a Controlled Dairy Cow Farm Environment

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.     

Persistence of Mycobacterium avium subsp. paratuberculosis at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle

In this study, products from all steps of anaerobic digestion at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle were examined and quantified for the presence of the causal agent of paratuberculosis, Mycobacterium avium subsp. paratuberculosis, using culture and quantitative real-time PCR (qPCR). Viable M. avium subsp. paratuberculosis cells were detected using culture in fermentors for up to 2 months; the presence of M. avium subsp. paratuberculosis DNA (10(1) cells/g) was demonstrated in all anaerobic fermentors and digestate 16 months after initiation of work at a biogas plant, using IS900 qPCR. F57 qPCR was able to detect M. avium subsp. paratuberculosis DNA (10(2) cells/g) at up to 12 months. According to these results, a fermentation process that extended beyond 2 months removed all viable M. avium subsp. paratuberculosis cells and therefore rendered its product M. avium subsp. paratuberculosis free. However, M. avium subsp. paratuberculosis DNA was found during all the examined periods (more than 1 year), which could be explained by either residual DNA being released from dead cells or by the presence of viable cells whose amount was under the limit of cultivability. As the latter hypothesis cannot be excluded, the safety of the final products of digestion used for fertilization or animal bedding cannot be defined, and further investigation is necessary to confirm or refute this risk.     

Estimation of Mycobacterium avium subsp. paratuberculosis growth parameters: strain characterization and comparison of methods

The growth rate of Mycobacterium avium subsp. paratuberculosis was assessed by different methods in 7H9 medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), Tween 80, and mycobactin J. Generation times and maximum specific growth rates were determined by wet weight, turbidometric measurement, viable count, and quantitative PCR (ParaTB-Kuanti; F57 gene) for 8 M. avium subsp. paratuberculosis strains (K10, 2E, 316F, 81, 445, 764, 22G, and OVICAP 49). Strain-to-strain differences were observed in growth curves and calculated parameters. The quantification methods gave different results for each strain at specific time points. Generation times ranged from an average of 1.4 days for viable count and qPCR to approximately 10 days for wet weight and turbidometry. The wet-weight, turbidometry, and ParaTB-Kuanti qPCR methods correlated best with each other. Generally, viability has been assessed by viable count as a reference method; however, due to M. avium subsp. paratuberculosis clumping problems and the presence of noncultivable M. avium subsp. paratuberculosis cells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation of M. avium subsp. paratuberculosis bacterial numbers in a sample.     

Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies paratuberculosis

By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas of the genome that are expressed in vitro.     

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.     

Defining the stressome of Mycobacterium avium subsp. paratuberculosis in vitro and in naturally infected cows

Mycobacterium avium subsp. paratuberculosis causes an enteric infection in cattle, with a great impact on the dairy industry in the United States and worldwide. Characterizing the gene expression profile of M. avium subsp. paratuberculosis exposed to different stress conditions, or shed in cow feces, could improve our understanding of the pathogenesis of M. avium subsp. paratuberculosis. In this report, the stress response of M. avium subsp. paratuberculosis on a genome-wide level (stressome) was defined for the first time using DNA microarrays. Expression data analysis revealed unique gene groups of M. avium subsp. paratuberculosis that were regulated under in vitro stressors while additional groups were regulated in the cow samples. Interestingly, acidic pH induced the regulation of a large number of genes (n=597), suggesting the high sensitivity of M. avium subsp. paratuberculosis to acidic environments. Generally, responses to heat shock, acidity, and oxidative stress were similar in M. avium subsp. paratuberculosis and Mycobacterium tuberculosis, suggesting common pathways for mycobacterial defense against stressors. Several sigma factors (e.g., sigH and sigE) were differentially coregulated with a large number of genes depending on the type of each stressor. Subsequently, we analyzed the virulence of six M. avium subsp. paratuberculosis mutants with inactivation of differentially regulated genes using a murine model of paratuberculosis. Both bacterial and histopathological examinations indicated the attenuation of all gene mutants, especially those selected based on their expression in the cow samples (e.g., lipN). Overall, the employed approach profiled mycobacterial genetic networks triggered by variable stressors and identified a novel set of putative virulence genes. A similar approach could be applied to analyze other intracellular pathogens.     

副结核荧光PCR试剂盒研制与应用

采用TaqMan荧光标记探针技术原理, 建立副结核分枝杆菌特异的实时荧光PCR快速检测鉴定方法并组装形成临床诊断试剂盒。试剂盒提供荧光PCR与样品核酸提取试剂, 检测全程包括样品处理可在1 d内完成。特异性试验结果表明, 试剂盒对8株副结核分枝杆菌标准菌株的检测均呈典型阳性反应, 对牛分枝杆菌、结核分枝杆菌等其它12种分枝杆菌标准菌株以及大肠杆菌、肺炎链球菌等多种常见微生物均呈阴性反应。试剂盒检测灵敏度可达单个菌细胞、15个基因拷贝, 比常规PCR检测灵敏度提高100倍。对每份添加50~100个菌细胞的20份阴性牛奶样品进行检测, 均呈阳性反应。重复性试验结果显示, 试剂盒组内变异系数为1.41%, 组间变异系数为2.42%。采用所研制的副结核分枝杆菌荧光PCR试剂盒对来自广东地区7个奶牛场的250份牛奶样品和粪便样品、来自10个养猪场的143份猪血清样品, 以及3批次进口食蟹猴共100份血清样品进行检测, 检出牛奶样品中副结核分枝杆菌阳性率为7.7%, 牛粪样品中阳性率为3.7%, 猪血清样品中阳性率为8.2%, 进口猴血清样品中阳性率为3.0%。     

Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.     

Fate of Mycobacterium avium subsp. paratuberculosis in Swiss hard and semihard cheese manufactured from raw milk

Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 10(4) to 10(5) CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 10(3) to 10(4) cells of M. avium subsp. paratuberculosis per g will be inactivated.