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.     

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.     

Isolation of Mycobacterium avium subsp. paratuberculosis from free-ranging birds and mammals on livestock premises

Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants ("7,5") appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock.     

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.     

Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.     

Mycobacterium avium subsp. paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: diverse opportunities for environmental cycling and human exposure

Mycobacterium avium subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km2 containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium subsp. paratuberculosis in the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive for M. avium subsp. paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.     

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.     

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.     

Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7 degrees C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Etiology of Crohn's disease: the role of Mycobacterium avium paratuberculosis

Crohn's disease is a chronic inflammatory bowel disease characterized by transmural inflammation and granuloma formation. Several theories regarding the etiology of Crohn's disease have been proposed, one of which is infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), which causes a similar disease in animals, and is present in the human food chain. Considerable evidence supports the presence of M. paratuberculosis in the intestinal tissues of many patients with Crohn's disease including culture, detection of homologous mycobacterial DNA, detection of the mycobacterial insertion sequence IS900 by both PCR and in situ hybridization in tissues, and a serologic immune response to recombinant M. paratuberculosis antigens. Despite this evidence, and our personal belief that M. paratuberculosis is a cause of Crohn's disease, widespread acceptance of this hypothesis will require evidence that specific anti-mycobacterial chemotherapy will cure the disease.     

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.     

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.     

Comparative evaluation of PCR in Ziehl-Neelsen stained smears and PCR in tissues for diagnosis of Mycobacterium avium subsp. paratuberculosis

Thirty six tissues from sheep, previously diagnosed with paratuberculosis, were tested by PCR in positive Ziehl-Neelsen staining smears of tissues, and PCR in tissues targeting IS900 specific for Mycobacterium avium subsp. paratuberculosis. DNA amplification was achieved in 33.3% Ziehl-Neelsen smears, and in 61.1% tissue samples. Combination of both techniques found 66.7% samples as positive. Combination of techniques would, therefore, increase the sensitivity of diagnosis.     

Blowflies Calliphora vicina and Lucilia sericata as passive vectors of Mycobacterium avium subsp. avium, M. a. paratuberculosis and M. a. hominissuis

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.     

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.     

Evaluation of an automated system for non-radiometric detection of Mycobacterium avium paratuberculosis in bovine feces

Cultivation of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from feces remains the most reliable method to detect infected animals. The purpose of this study was to evaluate a broth-based automated system used for cultivation of mycobacteria such as M. tuberculosis from human hosts, for the detection of M. paratuberculosis in bovine feces. Bovine feces was spiked with tenfold serial dilutions of M. paratuberculosis (5x10(5) to 5x10(-1) organisms), then processed with a double-centrifugation technique that included disinfection prior to inoculation into broth tubes. The same pathogen dilution series was also inoculated directly into broth and broth with uninfected processed feces. All of the system signal-positive bottles were identified within 30 days, with the highest concentration of M. paratuberculosis detected by the system in as few as 8 days. The presence of the pathogen was confirmed with acid-fast staining and an IS900-based PCR assay when growth of M. paratuberculosis was indicated by the system. However, some of the signal-negative cultures inoculated with the equivalent of 0.5 organisms tested PCR-positive 56 days post-inoculation, indicating that longer culture periods may lead to detection of small quantities of the organisms. Additionally, it was indicated that the processing step had a detrimental effect on detection of the organism. Comparison of the broth- and Herrold's egg yolk medium (HEYM) solid media-based culture methods with defined check test specimens corroborated the experimental evaluation of this system, indicating that broth-based detection could provide a more rapid assay for M. paratuberculosis. These results suggest that this automated system could be used to detect this organism in bovine feces, but that new approaches to processing the feces for culture should be explored.     

Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards

From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates.     

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.     

Occurrence of Mycobacterium avium subsp. paratuberculosis in untreated water in Northern Ireland

Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.     

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.