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PubMed1998

Factors associated with seroprevalence to Mycobacterium paratuberculosis in small-ruminant farms in the Madrid region (Spain).

A cross-sectional study was conducted in a population of small ruminants in the Madrid region (Spain) to determine the Mycobacterium paratuberculosis seroprevalence and to identify farm factors possibly associated with paratuberculosis (PTB). Farming-management information and sera were collected from 60 sheep or goat flocks. The relationship between seropositivity and the variables in the questionnaire was assessed by unconditional logistic regression, followed by random-effects logistic regression analysis to adjust for overdispersion between herds. The seroprevalence to M. paratuberculosis was 11.7% (64 out of 546) using agar-gel immunodiffusion assay (AGID). According to the sensitivity and specificity of the AGID test the true prevalence could be as high as 44%. A herd size of between 200-400 head and the presence of foreign breeds and their crosses were significantly associated with seropositivity (OR = 4.05 and OR = 4.32, respectively). A higher replacement rate was also associated with seroprevalence to M. paratuberculosis (24.2% in positive herds vs. 18.1% in negative). All these three factors were related to more intensive management in the surveyed area. In contrast, membership of a professional livestock association appeared to be a protective factor against PTB (OR = 0.28). No variables related to veterinary assistance were associated with seroprevalence, probably reflecting the current lack of interest in PTB on the part of the animal-health administration and veterinary services in Spain.

Animal Husbandry↗
PubMed2003

Efficacy of commercial and field-strain Mycobacterium paratuberculosis vaccinations with recombinant IL-12 in a bovine experimental infection model.

The efficacy of commercial (Strain 18) and field-isolate paratuberculosis vaccine preparations was investigated. The effect of prior exposure to Mycobacterium paratuberculosis and the adjuvant effect of rIL-12 on vaccine efficacy were also tested. Both Strain 18 and field-isolate vaccines induced strong local, systemic and enteric IFN-gamma responses. A significant reduction in mycobacterial colonization was observed when calves were vaccinated with the field-isolate prior to challenge, but not following vaccination with Strain 18 vaccine. Vaccination with rIL-12 prevented infection in some calves but its overall effect on IFN-gamma response and total mycobacterial load was not statistically significant. Efficacy of paratuberculosis vaccines may be enhanced if calves are vaccinated prior to M. paratuberculosis exposure with field-isolate vaccine instead of Strain 18 vaccine currently in use.

Adjuvants, Immunologic↗
PubMed2000

HspX is present within Mycobacterium paratuberculosis-infected macrophages and is recognized by sera from some infected cattle.

A portion of the gene encoding HspX has been previously identified as a sequence specific to Mycobacterium avium subspecies paratuberculosis (hereafter referred to as M. paratuberculosis) based on DNA hybridization experiments. In this study, rabbit antisera were raised against a recombinant protein of HspX fused to the Escherichia coli maltose binding protein (MBP/HspX). Immunoblots of lysates of M. paratuberculosis-infected macrophages probed with the rabbit antisera showed that HspX was present within infected macrophages of bovine and murine origin. This observation was confirmed by immunofluorescence microscopy of infected macrophages. Lysates of E. coli expressing HspX without the MBP fusion partner were loaded onto preparative SDS-PAGE gels and used to determine whether infected cattle generated a humoral immune response to the antigen. Sera from four of 24 paratuberculous cows (17%) detected HspX. No reactivity was present in sera from control cows. While HspX may be immunogenic during infection in some cows, the protein is not secreted and it does not stimulate cell-mediated immunity. Collectively, these data give a preliminary characterization of the first described M. paratuberculosis protein identified within infected macrophages.

Animals↗
PubMed2000

Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis.

Crohn's disease (CD) is a chronic inflammatory bowel disease that is similar to Johne's disease in ruminants. Recent data have strengthened the association of M. avium subsp. paratuberculosis (M. paratuberculosis) with CD. To provide more evidence of an etiological association, antibody reactivities from CD patients were tested by immunoblotting against recombinant antigens that were identified previously from our M. paratuberculosis genomic library. Two clones (designated pMptb#40 (3.2-kb insert) and #48 (1.4-kb insert) expressing a 35K (p35)- and 36K(p36)-antigens showed specific reactivities with serum samples from CD patients. Serum samples from 75% of 53 CD patients, 14% of 35 normal individuals and 10% of 10 ulcerative colitis patients reacted to p35 antigen. Reactivities were also observed with serum samples from 89% of 89 CD patients, 14% of 50 normal controls and 15% of 29 ulcerative colitis patients reacted with p36 antigen. When the reactivity results from p35 and p36 were combined, the background from the controls was eliminated, i.e. only the CD patients reacted to both p35 and p36. The positive predictive value was 98% with specificity of 98% and the negative predictive value was 76% with sensitivity of 74% (39 positive out of 53). A statistical significance (p<0.0001) was observed when the results from CD serum samples reacting with either or both antigens were compared to the controls. The reactivity of anti-M. paratuberculosis (specifically against p35 and p36 antigens) antibodies in a significant proportion of CD patients would suggest a causal role for the organism in CD.

Adolescent↗
PubMed2001

Recovery of Mycobacterium avium subsp. paratuberculosis from nematode larvae cultured from the faeces of sheep with Johne's disease.

A study was conducted to determine whether trichostrongylid nematode larvae become contaminated with Mycobacterium avium subsp. paratuberculosis when they develop in the faeces of sheep with Johne's disease. Nematode larvae were hatched from ova in the faecal samples of affected sheep. Larval sheaths were removed and these as well as exsheathed larvae were subjected to radiometric culture for M. paratuberculosis. The organism was recovered from washing water used to prepare the larvae, third stage larvae and larval sheaths, but not from exsheathed larvae. The recovery of M. paratuberculosis from larvae was associated with the severity of the histological lesions in affected sheep and with the results of culture of the organism from intestinal tissues and faeces. Nematode parasites of sheep might be able to act as mechanical vectors for M. paratuberculosis as the organism associates with infective third stage larvae when these develop in the faeces of sheep with Johne's disease.

Animals↗
PubMed1997

Differentiation of Mycobacterium paratuberculosis isolates by rDNA-spacer analysis and random amplified polymorphic DNA patterns.

The ability to distinguish between isolates of Mycobacterium paratuberculosis isolates was studied using two molecular techniques. Nucleotide sequence analysis of the rDNA-spacer and random amplified polymorphic DNA (RAPD) analysis using decamer primers with GC contents of 60 to 70% were evaluated on 16 isolates of M. paratuberculosis from cattle. The rDNA spacer analysis did not discriminate between isolates as it revealed an identical sequence for all 16 strains tested but it showed one common difference to the sequence previously described for M. paratuberculosis J2A. In the RAPD analysis, 14 of the 60 decamer primers used resulted in distinct amplification products for most of the isolates. For seven of the primers the size of the amplification products varied among strains thus allowing the specific identification of eight of the 16 isolates; of the remaining eight isolates five could each be differentiated from 14 other isolates, two from 13, and one from 12 isolates. Therefore, these data illustrate the possibility of using RAPD-analysis with certain primers for the differentiation of individual M. paratuberculosis isolates.

Animals↗
PubMed1999

Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in ruminants. The current methods for detection of M. avium subsp. paratuberculosis are slow and insensitive. We report the use of a polymerase chain reaction (PCR) based on IS900 to confirm growth of M. avium subsp. paratuberculosis in primary bacterial cultures from bovine tissue and fecal samples. The use of PCR on single colonies reduced the time for analysis by 2 months compared with conventional methods. We also report the development of a nested PCR based on IS900 and the development of a positive internal control molecule, a so-called mimic. The system was tested with spiked tissue samples, and the sensitivity was estimated to 10 CFU per sample. Seventeen tissue samples, previously found M. avium subsp. paratuberculosis positive by microbiological culture, were analyzed by nested PCR and the efficiency of the PCR was checked by co-amplification of the mimic. Absence of the mimic amplicon indicated inhibition of the amplification. Ten of the samples were positive and five were negative, as judged from the presence or absence of the IS900 PCR product. Two negative samples could not be judged because of inhibition revealed by mimic molecules. It was concluded that the nested PCR, together with the mimic, could be a useful tool in screening tissue materials.

Animals↗
PubMed2003

Immunogenicity of a 16.7 kDa Mycobacterium paratuberculosis antigen.

Mycobacterium paratuberculosis (MPT), the agent of paratuberculosis is a slow growing mycobacteria that causes important economic losses mainly due to lower weight gains and drastic decrease in milk production. Existing paratuberculosis vaccines are not completely protective and induce antibodies/delayed type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are needed to improve the control of this disease. We have identified, expressed and purified a hypothetical thiol peroxidase of MPT (MPT-TP) in mice. We also characterized the immunogenicity of this antigen in mice. The recombinant MPT-TP (rMPT-TP) antigen induced a high production of IFNgamma, IL-6, and NO and a low production of IL-10 by spleen cells of immunized mice. Addition of Ribi adjuvant to rMPT-TP resulted in lower IFNgamma secretion and higher NO production in spleen cells. A similar level of proliferation of spleen cells exposed to rMPT-TP was found in immunized groups (rMPT-TP and rMPT-TP emulsified in Ribi). DTH responses in mice footpads were observed only in mice immunized with rMPT-TP emulsified in Ribi. Addition of Ribi adjuvant clearly induced a significantly higher anti-rMPT-TP antibody production of all classes tested and decreased the IgG1/IgG2a ratio. MPT-TP demonstrated antigenic characteristics that make this antigen a potential component in the development of a future subunit vaccine against paratuberculosis.

Amino Acid Sequence↗
PubMed1999

Serological cross-sectional study of paratuberculosis in cattle in Austria.

From 1995 to 1997, the prevalence of serum antibodies against Mycobacterium avium subspecies paratuberculosis (M. av. ssp. ptbc.)--the causal agent of paratuberculosis (Johne's Disease)--was examined in 11,028 Austrian cattle. Samples from the four oldest cattle on 2757 farms were collected according to a specific sampling schedule for this epidemiological study. District, age and breed of animals were included as variables in this study. For antibody screening against M. avium subspecies paratuberculosis, a modified, commercially available ELISA (ALLIED Monitors, Fayette, USA) was employed. A total of 2253 samples that were found to be positive or questionable were subjected to further testing with a more specific ELISA (Institute of Microbiology and Infectious Animal Diseases). Results of this study were used for statistical analysis. The average prevalence of antibodies to M. avium subspecies paratuberculosis was 1.99% in Austria. The highest prevalence was seen in 6-year-old cattle (2.84%) and Holstein Frisian cattle (3.51%). Sero-positive animals were found on 6.96% of farms tested, and the prevalence was highest in Vorarlberg, followed by Salzburg, the Tyrol, Styria and Carinthia. This study is unique in Europe in the use of an adequate random sampling plan for an investigation of this magnitude.

Age Factors↗
PubMed2001

Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study.

In the present study, we compared the utility of immunohistochemistry with serological and histological results for the characterization of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) in tissues of affected red deer. Bacterial isolation was considered the standard reference. Samples were taken from seven clinically affected animals with typical macroscopic lesions. The enzyme linked immunosorbent assay (ELISA) and the gel diffusion tests (GD) were used for serological determinations. Samples from intestine and mesenteric lymph nodes were processed for bacterial isolation and histology. M. paratuberculosis was isolated from all the animals. Histologically, lymph nodes displayed necrosis and mineralization at the cortical and medullar areas. Ziehl-Neelsen stained bacteria were numerous inside macrophages and Langhans-type giant cells. Giant and epithelioid cells and lymphocytes were prominent at the ileal mucous membrane. The immunostaining of M. paratuberculosis was very clear inside epithelioid and giant cells. Image analysis was carried out to determine the immunostained area. There was total agreement among the methods employed. Immunohistochemistry can be very useful when the microorganism cannot be recovered from tissues or faeces.

Animals↗
PubMed2001

The grazing response of cattle to pasture contaminated with rabbit faeces and the implications for the transmission of paratuberculosis.

Transmission of Mycobacterium avium subspecies paratuberculosis, the organism responsible for paratuberculosis (or Johne's disease) in ruminants, occurs through the faecal-oral route. As M. a. paratuberculosis has been isolated from rabbit faeces, cattle grazing rabbit faecal contaminated pasture may thus be at risk.A herd of 57 beef cattle was monitored on a farm in Perthshire, throughout the 1999 'grazing year', to investigate whether the cattle avoided rabbit faecal contaminated pasture and thus the potential for disease transmission. Grazing was measured every two days over eight rotations by sward heights on 40 marked treatment plots (0.5 m x 0.5 m) to which 0, 10, 50 and 250 rabbit faecal pellets were added. Cattle were also monitored by an active transponder system which enabled individual animals contacting two plots per field rotation (one control and one contaminated) to be recorded. During the monitored grazing year, grazing pressure was low with a net mean sward offtake of 18% of sward height per rotation. There were no significant differences between rabbit faecal treatments (0, 10, 50 and 250 pellets) with respect to the height or proportion of sward removed, or between the numbers of contacts made by cattle on contaminated and uncontaminated plots. Over 90% of all the cattle contacted contaminated plots, indicating that the potential for disease transmission was widespread among the herd. To our knowledge, this is the first reported instance of a lack of avoidance by grazing cattle towards swards contaminated with faeces, and implies that the potential for transmission of paratuberculosis from rabbit contaminated pasture is high.

Animal Husbandry↗
PubMed2002

Paratuberculosis with special reference to cattle. A review.

Paratuberculosis is a chronic, granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis affecting domestic and wild ruminants. The symptoms of clinical paratuberculosis are chronic diarrhoea and progressive weight loss while subclinically infected animals mainly have decreased production. The infection is widespread throughout the world and causes substantial financial losses for the farming industry. One of the major obstacles in the control of this disease, is the difficulty of identifying subclinically infected animals. This review gives a summary of several aspects of paratuberculosis including clinical importance, pathology, immunology and properties of the infectious agent. Special emphasis will be on the available diagnostic methods, their use and limitations.

Animals↗
PubMed2000

Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing.

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohn's disease in humans. It differs from other members of the M. avium complex in having 14-18 copies of IS900 inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the Mycobacterium tuberculosis and M. avium subsp. avium genomes were identified. These included regions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at locus 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS900 sited two bases closer to the initiation codon. IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp. paratuberculosis isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. avium, from one M. avium subsp. silvaticum and from six other mycobacterial species. A multiplex PCR of IS900 loci (MPIL) typing method was developed which was able to discriminate 10 different types of M. avium subsp. paratuberculosis from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/Bst:EII RFLP type, suggesting that this method may be applicable to typing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstEII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS900 loci.

Animals↗
PubMed1994

Cloning and expression in Escherichia coli of DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis, the causative agent of Johne's disease.

Polymerase chain reaction (PCR) was used to generate DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis using primers complementary to sequences at the 5' and 3' ends of 60 kDa stress protein genes (encoding the '65 kDa antigens') of M. leprae and M. tuberculosis. The predicted PCR product of 1.8 kb contained the entire coding sequence of an M. paratuberculosis 60 kDa stress protein, with non-coding regions of 124 bp and 1 bp at the 5' and 3' ends, respectively. DNA encoding the entire ORF for the 60 kDa stress protein, as well as thrombin and Factor Xa proteolytic cleavage sites, was ligated into the bacterial expression vector pGEX-2T and used to transform Escherichia coli strain JM83. Transformed bacteria, induced by IPTG, expressed an 85 kDa fusion protein comprising glutathione S-transferase (GST) and M. paratuberculosis 60 kDa stress protein. This fusion protein was purified by adsorption to glutathione-agarose beads and shown to cross-react in Western blot analysis with an anti-mycobacterial 60 kDa stress protein monoclonal antibody. Recombinant M. paratuberculosis 60 kDa stress protein was liberated from GST by proteolytic cleavage with either thrombin or Factor Xa enzyme. Authenticity of liberated recombinant stress protein was confirmed by N-terminal amino acid sequencing.

Amino Acid Sequence↗
PubMed1995

Use of BACTEC radiometric culture method and polymerase chain reaction for the rapid screening of faeces and tissues for Mycobacterium paratuberculosis.

The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC-PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.

Animals↗
PubMed1997

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis.

OBJECTIVES: To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis. DESIGN: Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates. PROCEDURE: DNAs from 35 Australian isolates of M paratuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared. RESULTS: The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales. CONCLUSION: Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis, and could be used to study the transmission of strains in Australia.

Animals↗
PubMed2000

Comparison of three serological tests and an interferon-gamma assay for the diagnosis of paratuberculosis in experimentally infected sheep.

OBJECTIVE: To compare the diagnostic performance of a complement fixation test, an agar gel immunodiffusion test, an enzyme-linked immunosorbent assay, and a whole-blood interferon-gamma assay for paratuberculosis in 14 sheep experimentally infected with Mycobacterium avium subsp paratuberculosis. EXPERIMENTAL DESIGN: Longitudinal study. RESULTS: The IFN-gamma assay detected more experimentally infected sheep, and earlier, than any of the serological tests. None of the antibody assays was able to detect all sheep with histologically confirmed paratuberculosis. CONCLUSIONS: The superior performance of the IFN-gamma assay in detecting infected sheep in this small experimental population warrants its further evaluation in a larger population of sheep naturally exposed to M a paratuberculosis.

Animals↗
PubMed2006

Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle.

A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-"gold standard" analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI]=98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI=24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle.

Animals↗