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PubMed2000

Paratuberculosis in red deer (Cervus elaphus hippelaphus) in the western Alps.

During the hunting seasons 1995-96 to 1997-98, 19 red deer from the Upper Susa Valley (Cottian Alps) were examined for paratuberculosis (Johne's disease). Specific DNA amplification on mesenteric lymph nodes detected Mycobacterium avium paratuberculosis in 17 animals. Ten of these red deer were tested for serum antibodies by the AGID and ELISA tests, nine being negative. Three isolates from infected deer were genetically characterized by an arbitrarily primed polymerase chain reaction, and showed similar genetic polymorphism to that of bovine strains isolated in different Italian areas. The study showed that paratuberculosis is present in red deer of the Upper Susa Valley and that serological tests are not an efficient means for monitoring this infection.

Animals↗
PubMed2001

An enzyme-linked immunosorbent assay using immonoaffinity-purified antigen in the diagnosis of caprine paratuberculosis and its comparison with conventional ELISAs.

An enzyme-linked immunosorbent assay (APA-ELISA) using an immunoaffinity-purified antigen was developed and compared with the unabsorbed and absorbed ELISA procedures, using a crude antigenic preparation, for its efficacy in detecting antibodies in goat sera against Mycobacterium ovium paratuberculosis. Serum samples from 89 goats belonging to three different flocks, two with a history and evidence of paratuberculosis and one without it, were subjected to each ELISA, which had been standardized on known positive sera from goats experimentally infected with paratuberculosis. Faecal culture, faecal examination and histopathology were used as indicators of infection. The diagnostic sensitivities of the unabsorbed, absorbed and APA-ELISA were 81.8%, 77.3% and 77.3% and the specificities were 90.6%, 93.7% and 96.8%, respectively. The positive predictive values of APA-ELISA (94.4%) was the highest, followed by absorbed ELISA (80.9%) and unabsorbed ELISA (72.0%). The negative predictive values for APA-ELISA, absorbed ELISA and unabsorbed ELISA were 93.0%, 92.7% and 93.8%, respectively. The results indicated the value of APA-ELISA in avoiding the need to absorb individual test sera with Mycobacterium phlei and giving more consistent results than the absorbed ELISA. The APA-ELISA was also better than the other two procedures in terms of specificity and positive predictive values.

Animals↗
PubMed2004

Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by culture and serology.

Faecal samples from 186 dairy cows representing ten commercial dairy herds with sporadic clinical paratuberculosis (group A), and from 100 dairy cows from herds without a history of paratuberculosis (group B) were cultured for Mycobacterium avium subspecies paratuberculosis (MAP). Two different decontamination methods, a NaOH/oxalic acid method and treatment with 0.75% hexadecylpyridinium chloride (HPC) were performed prior to inoculation of Loewenstein-Jensen agar slants with and without mycobactin. Cultures were incubated for 16 weeks. Acid-fast staining bacteria were identified as MAP on the basis of mycobactin dependency and by PCR-RFLP analysis of the IS 1311-insertion element of M. avium. MAP was grown from 15 out of 186 group A animals (8.1%) whereas faecal culture for MAP was consistently negative in group B. The growth rate of MAP was significantly higher (8.1% vs. 1.6%) and the contamination rate of cultures was significantly lower (17.6% vs. 21.5%) in faecal samples decontaminated with NaOH/oxalic acid than with HPC-treated faecal samples (p<0.01, McNemar's test). Atypical mycobacteria which were grown from 46.8% of NaOH/oxalic acid treated specimens were not obtained from any of the HPC-treated samples. A commercial ELISA with MAP-lipoarabinomannan as the antigen was used to detect MAP-antibodies in unabsorbed sera from all animals. The percentage of ELISA-positive cows was 16.8%. Overall agreement between antibody detection and MAP-positive faecal culture was 15.4%.

Animals↗
PubMed2003

Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis.

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.

Animals↗
PubMed2003

The results of using faecal culture as confirmation test of paratuberculosis-seropositive dairy cattle.

A total of 15,822 cattle aged 3 years and older, belonging to 378 randomly selected herds, were tested for paratuberculosis using an absorbed enzyme-linked immunosorbent assay (ELISA); 3.3% tested positive. This percentage was lowest for the group of cattle aged 3-4 years (2.3%) and highest for cattle with the age of 5-6 years (4.5%). The mean Sample to Positive (S/P) ratio of seropositive cattle vaccinated against paratuberculosis was higher (0.75 +/- 0.33) than that of seropositive, non-vaccinated cattle (0.58 +/- 0.26). Faecal samples of 422 ELISA-positive cattle were cultured for the presence of Mycobacterium avium subsp. paratuberculosis, 12% of these were contaminated. The percentage of non-contaminated samples with positive culture results was 17.3%, with a substantial difference between vaccinated (1.7%) and non-vaccinated cattle (20.2%). Of the positive cultures, the number of colonies varied from 1-10 (22% of cultures), 11-100 (22%), to more than 100 (55%). The percentage of ELISA-positive, non-vaccinated cattle tested culture-positive was positively correlated with the magnitude of the S/P ratio. This percentage varied from 12% (S/P ratio 0.3-0.5) to 58% (S/P ratio > 1.1), a result that might have implications for interpretation of the test. In this study, the percentage of ELISA-positive cattle with positive faecal culture results was limited and these individuals were mostly moderate to heavy shedders.

Animals↗
PubMed2002

Agar gel immunodiffusion test (AGID) evaluation for detection of bovine paratuberculosis in Rio de Janeiro, Brazil.

AIMS: The aim of this study is to evaluate the AGID serological test for detection of antibodies anti-Mycobacterium paratuberculosis and its possible adoption as diagnostic method in our field conditions. METHODS: Bovine serum samples from dairy herds in Rio de Janeiro State, Brazil, were screened for the presence of antibodies against Myco. paratuberculosis using three different ELISA tests. A panel of 48 randomly selected sera were evaluated by an Agarose Gel Immunodiffusion (AGID) test using Protoplasmatic Paratuberculosis Antigen (PPA). AGID results were compared to the standards--the results of the three ELISA tests, and the specificity and sensitivity were calculated. RESULTS: From 48 sera tested for AGID, 14 (29.17%) were positive and 34 (70.83%) were negative. AGID sensitivity was 57% with two false-positive reactions, and specificity was 92.5% with nine false-negative results. The positive predictive value was calculated in 85.7% for a confidence interval of 95%. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to its low sensitivity and specificity rates, AGID test has shown to be unsatisfactory as a screening diagnostic method for subclinical herd infection, but it can be useful as a confirmatory test for clinical suspect animals.

Agar↗
PubMed2001

How does Mycobacterium avium subsp. paratuberculosis resist intracellular degradation?

Paratuberculosis is a chronic, progressive disease of mainly ruminants caused by the facultative intracellular bacterium, Mycobacterium avium subsp. paratuberculosis. Infection usually occurs in young animals through oral uptake of food contaminated with the organisms. The ingested bacteria are transcytosed through M-cells overlying the Peyer's patches and are released in the stroma, where they are taken up by macrophages. Inside the macrophage, the mycobacteria resist enzymatic and toxic degradation and multiply until the infected macrophage ruptures. The thick, lipid-rich cell envelope is mainly responsible for micobacterial resistance. In addition to its barrier effect, which provides protections, the mycobacterial cell wall also contains several biologically active components that down-regulate the bactericidal function of macrophages. The basic survival strategy of pathogenic mycobacteria can be viewed at three levels: selective use of relatively safe entry pathways that do not trigger oxidative attack, modification of the intracellular trafficking of mycobacteria-containing phagosomes, and modulation of the cooperation between the innate and specific immunity. In doing so, pathogenic mycobacteria are successful intracellular organisms that survive and multiply inside macrophages. Current understanding about the survival strategies of M. a. paratuberculosis and its implications in the epidemiology, diagnosis, and control of the disease are discussed.

Age Factors↗
PubMed1994

Detection of Mycobacterium paratuberculosis by polymerase chain reaction in children with Crohn's disease.

Mycobacterium paratuberculosis, the causative agent of Johne's disease (a chronic enteritis in ruminants), has been suspected to be involved in Crohn's disease. In this study, polymerase chain reaction was used to detect the presence of IS900 DNA sequences specific to M. paratuberculosis genomes in biopsies and surgical resections from 53 children with various gastrointestinal diseases and disorders. IS900 sequences were found in 13 of 18 samples from patients with Crohn's disease (72%; P < .01 vs. samples from patients without Crohn's disease), in 1 of 5 with ulcerative colitis, in 2 of 6 with severe unclassified colitis, and in 7 of 24 with other gastrointestinal illnesses. These results appear to support the hypothesis that M. paratuberculosis is involved in the pathogenesis of Crohn's disease.

Child, Preschool↗
PubMed1992

Amplification of 16S rRNA sequences to detect Mycobacterium paratuberculosis.

A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne's disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.

Animals↗
PubMed1995

First report of paratuberculosis (Johne's disease) in sheep in Morocco.

Paratuberculosis (Johne's disease) was suspected in a flock of approximately 1000 sheep after weight losses and scouring had increased in adult animals despite repeated anthelmintic treatment. A total of 10 ewes showing clinical symptoms were submitted to laboratory examination. Gross pathological and histological examination of tissue samples rendered results compatible with Johne's disease in all 10 ewes. The diagnosis could only be confirmed by cultural isolation and identification of Mycobacterium paratuberculosis in the faeces of two ewes. This is considered the first documented isolation of Mycobacterium paratuberculosis in sheep in Morocco.

Animals↗
PubMed1996

Paratuberculosis in sheep flocks in Morocco: a serological, microscopical and cultural survey.

In an attempt to estimate the importance of Johne's disease in sheep in Morocco, a diagnostic survey was made in three flocks suspected of being infected with Mycobacterium paratuberculosis. Blood and faeces samples collected randomly from 188 adult sheep were investigated using direct microscopy of fecal smears, fecal culture and the serum complement fixation test (CFT). Microscopical examination of faeces revealed acid-fast bacteria in 67 (35.6%) samples. However, Ziehl-Neelsen staining lacks specificity and furthermore, acid-fast bacteria could only be demonstrated in 34 (60%) animals found to be culturally positive. Fecal culture succeeded in the isolation of mycobactin dependent strains of M. paratuberculosis in 56 (29.8%) of the sheep examined, with three strains growing in yellow-pigmented colonies. The CFT was regarded as positive (titres of 1:10 and higher) in 55 (29.2%) sera of animals, demonstrating a sensitivity of 48.2% and a specificity of 74.5% in relation to fecal culture From the results, it can be concluded that the combination of CFT and fecal culture might be a practical and useful procedure for detecting infected sheep within flocks and for controlling Johne's disease. This study supports the suspicion that paratuberculosis may constitute a serious problem in Morocco, particularly in sheep flocks involved in cross-breeding programmes.

Animals↗
PubMed2006

Serological, bacteriological and molecularbiological survey of paratuberculosis (Johne's disease) in Austrian cattle.

Paratuberculosis (Johne's disease) is a chronic infectious disease of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (MAP). Because of its long incubation period, high economic losses, difficulties in diagnosis and possible links to Morbus Crohn in humans, paratuberculosis is one of the most important diseases of ruminants today. An abattoir-based nationwide survey on the occurrence of MAP in the Austrian cattle population was performed using serology (SVANOVIR-ELISA) as well as culture, ZN-stain and IS900-PCR on faeces and lymph node samples. A total of 756 Austrian slaughter cattle were serologically, bacteriologically and molecularbiologically tested for the occurrence of MAP and specific antibodies respectively. Samples were collected following a statistical plan to obtain balanced specimens from the whole country. Nineteen per cent of the animals tested were serological positive, 10.1% gave an inconclusive result and 70.9% showed no specific antibodies against MAP. Only in four individuals MAP could be detected by stain, bacteriology or Polymerase Chain Reaction. The calculated prevalence of 19.0% positive cattle, each representing one farm, showing specific antibodies against MAP is rather high in terms of animal-level but low in herd level prevalence compared with other countries. When this study is compared with a similar study performed in Austria 1999, a significant increase of positive cattle and farms could be seen in Austria.

Abattoirs↗
PubMed2006

A comparison of ovine monocyte-derived macrophage function following infection with Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis causes Johne's disease in ruminants, whereas the antigenically and genetically similar subspecies Mycobacterium avium ssp. avium is less virulent. In this study, we compared one strain of each subspecies for its ability to survive, induce cytokines, suppress MHC class I and II expression and induce apoptosis or necrosis in ovine monocyte-derived macrophages. Both subspecies survived intracellularly and induced the secretion of IL-10. Low levels of TNF-alpha were detected after infection with both subspecies at 4 h. IL-12 was not upregulated after infection. Downregulation of MHC class I and II was evident in response to infection with both M. avium ssp. avium and M. avium ssp. paratuberculosis. No significant cytotoxicity was detectable in ovine macrophages after the addition of bacteria. M. avium ssp. paratuberculosis induced slightly more apoptosis than M. avium ssp. avium. Still the overall rate of apoptosis was very low and both subspecies suppressed LPS-induced macrophage apoptosis.

Animals↗
PubMed2002

Antigenicity of Mycobacterium paratuberculosis superoxide dismutase in mice.

Mycobacterium paratuberculosis (MPT) is the etiologic agent of paratuberculosis. The disease is prevalent in cattle worldwide, and exacts a heavy financial toll. Effective control requires the development of acellular vaccines offering a better protection than the current available vaccines without side effects and allowing the discrimination between infected and vaccinated animals. We studied the immune response of mice to the MPT superoxide dismutase (SOD) alone or adjuvanted by Ribi. We cloned, overexpressed and purified this antigen in Escherichia coli. Spleen cells from immunized mice, after exposure to recombinant MPT SOD (MPT rSOD), produced significant levels of IFNgamma, TNFalpha and IL-6. IFNgamma and TNFalpha production was increased by the addition of Ribi. In contrast, low levels of NO, IL-4 and IL-10 were secreted by spleen cells culture from immunized mice. The immunoglobulin isotype distribution analysis showed that Ribi adjuvant clearly induced a significantly higher anti-MPT rSOD antibody production of all classes tested and decreased the IgG1/IgG2a ratio thus improving the Th1 response. Delayed-type hypersensitivity responses in mice footpads were observed only in mice immunized with MPT rSOD emulsified in Ribi. Vaccination of MPT rSOD emulsified with Ribi induced both a Th2 and Th1 type of immune response with the later slightly more pronounced. The results presented here on the immunogenicity of MPT SOD suggest that this antigen should be further tested as a candidate antigen for a future acellular vaccine against paratuberculosis.

Adjuvants, Immunologic↗
PubMed1999

Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction.

OBJECTIVES: To evaluate and compare methods for DNA extraction from formalin-fixed, paraffin-embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's disease in ruminants. DESIGN: A laboratory study. PROCEDURE: Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS900 primers were compared. Sensitivity and specificity were assessed using samples from ruminants with and without histological evidence of Johne's disease. RESULTS: The simplest method of DNA extraction, which involved two cycles of boiling and freezing followed by centrifugation, gave more consistent results than two methods that required solvent extraction of paraffin, proteinase digestion and DNA purification. The sensitivity of detection of M avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 years from 34 cases of Johne's disease in sheep, cattle and goats was 88% for a 229 bp IS900 PCR assay and 71% for a 413 bp assay, using the detection of acid-fast bacilli by Ziehl Neelsen staining of histological sections from the same blocks as the gold standard test. PCR results correlated with the abundance of acid-fast organisms in the tissues. No false positive reactions were detected. CONCLUSION: PCR for identification of M avium subsp paratuberculosis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is effective. Amplification of short fragments of IS900 is more effective than amplification of longer fragments.

Animals↗
PubMed2000

DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP.

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.

Animals↗
PubMed2001

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.

Animals↗
PubMed2004

Field evaluation of tracer sheep for the detection of early natural infection with Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To determine whether tracer sheep could be used to detect S strain Mycobacterium avium subsp paratuberculosis on pasture, and to provide further insight into the early stages of infection. DESIGN: A field study on two farms in an endemic area for ovine Johne's disease in New South Wales. Procedure Lambs, weaners and adult ewes were introduced to pasture with varying amounts of M. a. paratuberculosis contamination and monitored using skin tests, gamma interferon assay, faecal culture and serial necropsy of small groups for up to 15 months after first exposure. RESULTS: Culture from tissues was the most sensitive method for detecting early infection in sheep after natural exposure to S strain M. a. paratuberculosis. The organism was detected in at least one introduced sheep from every exposed group, 6 to 12 months after first exposure. Histopathological lesions were detected in only 17% of culture-positive sheep, and only after at least 8 months of exposure. Similarly, antemortem diagnostic tests had low sensitivity during the early stages of naturally acquired infection. There was no evidence of any differences in infection rate between sheep first exposed as neonates, as weaners or as adults. A higher proportion of lambs born to ewes from an infected flock were infected than lambs suckling uninfected ewes introduced to the same infected environment, and infection was detected earlier in these 'resident' lambs. CONCLUSION: These findings indicate that groups of unexposed 'tracer' sheep, tested by culture of tissues at slaughter 6 to 12 months after first exposure, might be a useful way to assess pasture infectivity in control programs for ovine Johne's disease.

Animals↗