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PubMed2000

Prevalence and regional distribution of paratuberculosis in dairy herds in The Netherlands.

In the Netherlands a survey was conducted to estimate the prevalence of paratuberculosis in dairy herds. In total 15822 cows of at least 3 years of age, belonging to 378 herds were tested using an absorbed ELISA. Of these herds, 55% (n=207) had one or more serologically positive cows. Of the positive non-vaccinated herds, most had one (n=98) or two (n=49) positive cows. The percentage positive cows per herd was 2.5+/-3.2%.The true prevalences on cow and herd levels, based on a test sensitivity that ranged from 0.3 to 0.4 and a specificity that ranged from 0.985 and 0.995, were estimated at 2. 7-6.9% and 31-71%. Seven herds had been vaccinated against paratuberculosis and these herds had a significantly higher percentage of serologically positive cows (23%) than the non-vaccinated herds (2.5%). In conclusion, a small percentage of the dairy cows and a high percentage of the dairy herds in the Netherlands is serologically positive. The percentages true infected cows and herds are difficult to estimate precisely due to uncertainties in test sensitivity and specificity.

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PubMed2000

ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method.

The sensitivity and specificity of the ELISA and fecal culture tests for paratuberculosis in dairy cattle are examined. ELISA and fecal culture data from seven dairy herds where both fecal cultures and ELISA testing was done concurrently are included. A cohort of 954 cattle including 697 parturient adults, cultured every 6 months from 10 herds followed over 4 years served as the basis to determine fecal culture sensitivity. The fecal culture technique utilized a 2g sample with centrifugation and double incubation. Of the 954 cattle cohort of all ages (calf to adult) that were fecal sampled on the first herd visit, 79 were culture positive. An additional 131 animals were detected as culture positive over the next seven tests at 6-month intervals. The sensitivity of fecal culture to detect infected cattle on the first sampling was 38%. Of the 697 parturient cattle cohort, 67 were positive on the first fecal culture, while an additional 91 adult cattle were culture positive over the next seven tests, resulting in a sensitivity of 42% on the first culture of the total animals identified as culture positive. Animals culled from the herds prior to being detected as infected and animals always fecal culture negative with culture positive tissues at slaughter are not included in the calculations. Both groups of infected cattle will lower the apparent sensitivity of fecal culture. Infected dairy herds tested concurrently with both fecal culture and ELISA usually resulted in more than twofold positive animals by culture compared to ELISA. The classification of infected cattle by the extent of shedding of Mycobacterium paratuberculosis in the feces helps define the relative proportion of cattle in each group and therefore the likelihood of detection by the ELISA test. ELISA has a higher sensitivity in animals with a heavier bacterial load, i.e. high shedders (75%) compared to low shedders (15%). Repeated testing of infected herds identifies a higher proportion of low shedders which are more likely to be ELISA negative. Thus, the sensitivity of the ELISA test decreases with repeated herd testing over time, since heavy shedders will be culled first from the herds.

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PubMed1999

Comparison of serological tests and faecal culture for the detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and analysis of the antigens involved.

Three hundred and forty-one sera from cattle in Western Australia and 106 sera from Mycobacterium paratuberculosis faecal culture positive cattle were used to evaluate the performance of two absorbed enzyme-linked immunosorbent assays (ELISA) (one locally produced, the other a commercial test) and a complement fixation test (CFT) for the detection of Johne's disease in cattle. The diagnostic sensitivity (47.2%) of the local ELISA was significantly higher than that of the commercial ELISA (31.1%), and significantly higher than that for the complement fixation test (17.9%) and immunoblot (20.8%). Diagnostic specificity for the two ELISAs was 99.7% and 97.9% and similar for CFT and immunoblot (97.1% and 97.7%, respectively). The diagnostic sensitivity rose for both ELISAs and the CFT as the number of M. paratuberculosis isolated from the faeces increased. The ELISA antigen was characterised by polyacrylamide gel electrophoresis and electrophoretic immunoblotting and was found to consist mostly of a carbohydrate-type macromolecule of 32-42 kDa. This macromolecule was identified as lipoarabinomannan (LAM) by using a LAM-specific monoclonal antibody in immunoblots and purified LAM in absorption experiments. By applying more complex antigen preparations in immunoblots, serum antibodies against proteins of 47, 37, 30, 24 and 21 kDa, and against the 32-42 kDa carbohydrate component were frequently found in infected cattle, and of these the 47 kDa protein and the 32-42 kDa antigen were immuno-dominant. Pre-absorption of the sera with M. phlei sonicate indicated that the protein antigens contributed markedly to non-specific serological cross-reactions, while the 32-42 kDa non-protein macromolecule appeared to be specific.

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PubMed1996

Transmission of paratuberculosis.

Infection of the calf soon after birth by oral ingestion of M. paratuberculosis organisms from feces of infected cows is the most important method of transmission of paratuberculosis. The severity and rate of progression of the disease are dependent on the quantity of organisms in the exposure and the age of the animal. Only a small dose of organisms may be required to establish infection in a newborn calf, and overwhelming age-related resistance by introduction of a large dose of organisms to an adult cow is probably possible. Other means of transmission such as transplacental and direct excretion in the milk and colostrum should not be ignored and should be a particular concern primarily in advanced stages of infection when heavy fecal shedding is detected.

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PubMed1996

Diagnosis of paratuberculosis.

The currently available and accurate diagnostic tests for paratuberculosis are reviewed. Technical issues concerning test performance are briefly mentioned and the advantages, disadvantages, and comparative accuracy are described. The article concludes with recommendations on which tests to use in eight different situations requiring laboratory diagnostics for paratuberculosis.

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PubMed1997

Comparative study of Mycobacterium paratuberculosis strains isolated from Crohn's disease and Johne's disease using restriction fragment length polymorphism and arbitrarily primed polymerase chain reaction.

To obtain insights into the pathogenic mechanisms involving Mycobacterium paratuberculosis in Crohn's disease (CD) we questioned if the strains of M. paratuberculosis isolated from CD are distinguishable from those involved in Johne's disease (JD), a chronic granulomatous enteritis in cattle. Accordingly we compared human and animal strains at the DNA level, both by the analysis of restriction fragment length polymorphism (RFLP) in and around the insertion sequence IS 900 and by the arbitrarily primed chain reaction (AP-PCR). Results are in favour of a common clonal origin for the 4 strains isolated from CD and for 8 of the 11 strains isolated from cattle and sheep JD.

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PubMed2001

Innate IFN-gamma production in cattle in response to MPP14, a secreted protein from Mycobacterium avium subsp. Paratuberculosis.

Calves experimentally infected with Mycobacterium avium subsp. paratuberculosis and uninfected calves were tested for interferon(IFN)-gamma production after stimulation with purified protein derivative from M. avium subsp. paratuberculosis (PPDp) or a secreted 14 kDa protein (MPP14) specific for the M. avium-intracellulare-scrofulaceum (MAIS) complex. Several calves in both groups responded strongly up to about 5 months to both antigens. Two uninfected calves responded repeatedly, but not always, to MPP14 and PPDp throughout the study. The responses in the uninfected animals seemed to be independent of cell contact between the antigen presenting cells (APC) and the responding population. The supernatant from adherent cells stimulated with MPP14 induced similar levels of IFN-gamma production in CD14+/B-cell depleted peripheral blood mononuclear cells (PBMC) as when the antigen was used directly on PBMC. In contrast, APC/T-cell contact was necessary to induce the IFN-gamma production in infected animals, suggesting that both innate and adaptive IFN-gamma production in response to MPP14 could occur. CD8+ cells contributed to some of the IFN-gamma production in response to MPP14, but the rest could not be explained, while CD4+ cells were responsible for the adaptive response to PPDp. This study showed that secreted proteins could induce innate IFN-gamma production that interferes with diagnostic testing using the IFN-gamma-test.

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PubMed1999

Effects of subclinical bovine paratuberculosis on in-vitro polymorphonuclear neutrophil migration.

Migration of polymorphonuclear neutrophils (PMNs) [unstimulated or stimulated with zymosan-activated serum (ZAS)] from 18 cows was measured in a microwell filter assay. Of these animals, 10 were subclinically infected with Mycobacterium paratuberculosis and shown by culture to be excreting the organism in the faeces; the remaining eight were clinically normal and negative for M. paratuberculosis on faecal culture. PMN "net migration" (stimulated minus unstimulated cells) of the infected cows was significantly lower than that of the uninfected cows. Migration of unstimulated cells in the infected cows did not differ from that in the uninfected cows. It would therefore appear that the infection influenced only the migratory response of the ZAS-stimulated cells. 1999 Harcourt Publishers Ltd.

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PubMed2006

Intra-uterine transmission of paratuberculosis (Johne's disease) in farmed red deer.

AIM: To determine whether intra-uterine transmission of paratuberculosis (Johne's disease) occurs in farmed red deer (Cervus elaphus) in New Zealand. METHODS: On four different farms, nine late-stage pregnant hinds with Johne's disease were slaughtered and samples were taken from them and their 10 fetuses. Samples of the hepatic, ileocaecal and mesenteric lymph nodes and the posterior ileum were collected from the hinds. The lung, liver, spleen, jejunum and ileum from the fetuses were sampled, as were the placentomes. Blood samples were tested using the 'Paralisa' test, a modified immunoglobulin G1 (IgG1) enzyme-linked immunosorbent assay (ELISA). Tissue samples were cultured using the BACTEC system, and fixed samples were sectioned and histological slides examined. RESULTS: All nine hinds and 9/10 fetuses (one hind had twins) were culture-positive for Mycobacterium avium subsp paratuberculosis (M. ptb). Six hinds had gross lesions of Johne's disease, while all hinds had characteristic histopathological lesions affecting the ileum, ileocaecal valve and associated lymph nodes. The only histopathological change observed in the fetuses was some mild inflammation in the lungs of one individual. Acid fast organisms (AFOs) were seen in histological sections of the lymph nodes and ileum of six hinds, and none were seen in tissues from the fetuses. These six hinds were Paralisa-positive, whereas the remaining hinds and fetuses were serologically-negative. CONCLUSIONS: These results confirm that there is a high risk of transmission of M. ptb from clinically affected hinds to their fetuses during pregnancy. CLINICAL RELEVANCE: Johne's disease is an increasingly important disease responsible for deaths in young red deer. Recognising the influence of intra-uterine transmission on the spread of this disease may be an important step towards improved control of Johne's disease.

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PubMed2005

New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's disease) by flow cytometry.

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.

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PubMed2006

Time delay, temperature effects and assessment of positive controls on whole blood for the gamma interferon ELISA to detect paratuberculosis.

Our objective was to evaluate the effects of time and temperature on whole blood used in the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) for paratuberculosis along with evaluating four potential positive controls, and four different mycobacterial antigens for the ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium ssp. paratuberculosis were used in a randomized complete block design. Forty-nine blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6 and 4.4 degrees C for 0, 4, 8, 12, 18, 24, 32, 48 and 72 h. Each blood tube was tested with four mycobacterial antigens (two johnin PPDs, an avain PPD and a whole cell sonicate) and four potential positive controls [concanavalin A (conA), phytohaemagglutinin A (PHA), pokeweed mitogen (PWM) and Staphylococcus enterotoxin A (SEA)]. After incubation for 24 h, the plasma was assayed with a commercial IFN-gamma ELISA. Blood stored at 21.1 and 15.6 degrees C maintained the highest ELISA optical densities (OD) over time with severe reduction in OD values at or above 37.8 degrees C. None of the potential positive controls exactly mimicked the antigen response. SEA and PWM were able to elicit a response after the whole blood quit responding to the antigen and conA underestimated the responsiveness. Phytohemagglutinin A was similar to the antigens on an average, but there was significant disagreement among samples. The PPDs were more potent at stimulating IFN-gamma production than the whole cell sonicate. In conclusion, whole blood should be stored/transported at ambient room temperature and stimulated within 12 h of collection.

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PubMed1993

Use of rifabutin in treatment of systemic Mycobacterium paratuberculosis infection in mice.

BALB/c mice were infected intraperitoneally with Mycobacterium paratuberculosis and, after allowing the infection to progress for 30 days, were treated with rifabutin at 0, 12.5, 25, and 50 mg/kg of body weight. Rifabutin was administered in drinking water under conditions of water deprivation, whereby the entire daily dose was delivered within a 1-h period. Animals were killed at biweekly intervals from time zero of treatment to 180 days. Spleens and livers from each animal were examined by quantitative bacteriologic culture and histopathology. Restricted water availability was found to be a viable alternative to daily gavage for single-dose bolus administration. Infection, as assessed by bacterial counts, was reduced only in animals that received 50 mg of rifabutin per kg. In these animals, bacterial counts in the liver and spleen were reduced from 7.2 x 10(5) +/- 4.1 x 10(4) and 6.5 x 10(5) +/- 4.1 x 10(4) to 3.0 x 10(3) +/- 1.8 x 10(2) and 3.1 x 10(3) +/- 2.2 x 10(2), respectively, over the 6-month treatment period. Rifabutin may be an appropriate chemotherapeutic drug for long-term treatment of M. paratuberculosis infection and should be considered in any multidrug regimen.

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PubMed2003

Use of growth indices from radiometric culture for quantification of sheep strains of Mycobacterium avium subsp. paratuberculosis.

A simple method for using growth indices from radiometric BACTEC cultures was evaluated for the enumeration of Australian sheep strains of Mycobacterium avium subsp. paratuberculosis. The numbers of viable organisms in inocula were determined by end-point titration in BACTEC cultures. Growth indices were measured by using a BACTEC 460 machine. There was a linear relationship between the number of days taken for the cumulative growth index to reach 1,000 (dCGI1000) and log(10) inoculum size. The use of dCGI1000 was shown to be as effective as the use of growth index data from the entire growth cycle for the estimation of inoculum size. For particular isolates characterized by end-point titration, the dCGI1000 of a single BACTEC vial provided estimates of viable numbers within narrow prediction limits. Predictive relationships were also established for the enumeration of M. avium subsp. paratuberculosis from field samples by using the dCGI1000 of a single BACTEC vial, with prediction limits of +/-1 to 2 log units. Organisms from feces or contaminated soil grew more slowly than those from cultures or tissues, and separate equations were developed for enumeration from these sources.

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PubMed2006

Routes of intraspecies transmission of Mycobacterium avium subsp. paratuberculosis in rabbits (Oryctolagus cuniculus): a field study.

Rabbits have been increasingly linked to the persistence of paratuberculosis (Johne's disease) in domestic ruminants in the United Kingdom. The aims of this study were to determine the routes of intraspecies transmission of Mycobacterium avium subspecies paratuberculosis (MAP) in rabbits and to estimate the probability of transmission via each route, in order to gain understanding of the dynamics of MAP in this host. Rabbits were sampled from two sites where MAP had previously been isolated from the livestock and rabbit populations. No pathology was noted in any animals, but the overall prevalence of MAP in rabbits was high at both sites studied, 39.7% and 23.0%, respectively. MAP was isolated from the testes, uterus, placenta, fetuses, and milk. This is the first time that the bacterium has been isolated from any of these tissues in a nonruminant wildlife species. These results suggest that transmission may occur vertically, pseudovertically, and horizontally. Vertical, i.e., transplacental, and/or pseudo-vertical, i.e., through the ingestion of contaminated milk and/or feces, transmission occurred in 14% of offspring entering the population at 1 month of age. As infection via these routes is only possible from infected adult females, this equates to a probability of infection via this route of 0.326. Probability of infection via horizontal transmission (including interspecies transmission) occurred at up to 0.037 per month. The presence of these routes of transmission within natural rabbit populations will contribute to the maintenance of MAP infections within such populations and, therefore, the environment.

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PubMed1995

Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis.

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from our M. paratuberculosis PTB65K genomic library. A high degree of identity was found between the open reading frame (ORF) of the PTB65K gene and those of Mycobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tuberculosis and M. leprae proteins each differed by 36 amino acid residues and that the M. avium 18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne's disease and patients with Crohn's disease against the recombinant PTB65K antigen. Immunoblot analysis showed that sera from only 3 of 10 clinically ill and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne's disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn's disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with non-inflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne's disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn's disease patients compared with that from noninflammatory bowel disease patients showed a positive correlation with mycobacterial diseases.

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PubMed1998

Detection of Mycobacterium avium subsp. paratuberculosis in infected tissues by new species-specific immunohistological procedures.

We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930-4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947-954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne's disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity.

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PubMed2003

Mycobacterium avium subsp. paratuberculosis infection causes suppression of RANTES, monocyte chemoattractant protein 1, and tumor necrosis factor alpha expression in peripheral blood of experimentally infected cattle.

Blood from cattle with subclinical Mycobacterium avium subsp. paratuberculosis infection was stimulated with M. avium subsp. paratuberculosis antigens, and expression of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), RANTES, monocyte chemoattractant protein 1 (MCP-1), and IL-8 was measured. Expression of TNF-alpha, RANTES, and MCP-1 was lower in infected than in uninfected cattle. The reduced response may weaken protective immunity and perpetuate infection.

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PubMed2006

Genomic comparison of Mycobacterium avium subsp. paratuberculosis sheep and cattle strains by microarray hybridization.

Microarray-based comparisons of three Mycobacterium avium subsp. paratuberculosis isolates, including one sheep strain and two cattle strains, identified three large genomic deletions in the sheep strain, totaling 29,208 bp and involving 24 open reading frames. These deletions may help explain some of the differences in pathogenicity and host specificity observed between the cattle and sheep strains of Mycobacterium avium subsp. paratuberculosis.

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