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PubMed2005

IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep.

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.

Animals↗
PubMed2006

Agreement between three ELISAs for Mycobacterium avium subsp. paratuberculosis in dairy cattle.

During a 10-month period in 1999, 994 serum and tissue samples were collected from dairy cows at slaughter in eastern Canada. The sources of these cattle were from all four Atlantic Canadian provinces along with some cows from the state of Maine. The sera were used to assess the agreement of three commercially available ELISAs for Mycobacterium avium subsp. paratuberculosis. Two ELISAs were indirect absorbed ELISAs licensed for use in North America, the third was an indirect non-absorbed ELISA licensed for use in Europe. Overall, there was poor agreement between the three ELISAs. The highest and lowest kappa values were 0.33 and 0.18, which is fair and poor agreement, respectively. However, when only tissue culture-positive cattle were compared, the ELISAs had better agreement (kappa=0.37-0.51). The proportions of positive tests, however, were significantly different among the three ELISAs. The poor agreement among the three ELISAs is as concerning as the fact that these tests have low sensitivity. The implications are greatest when the tests are used at the cow level to make individual animal decisions, which is not the recommended method on the product labels. At the cow level, if the result obtained from one ELISA is positive, using a different ELISA in a pre-clinical animal has a high likelihood of giving a different result due to low predictive values of positive test results.

Animals↗
PubMed2006

Secreted antigens of Mycobacterium avium subspecies paratuberculosis as prominent immune targets.

We here describe the identification and characterization of three novel secreted Mycobacterium avium subsp. paratuberculosis antigens of 9, 15 and 34 kDa (Map2609, Map2942c and Map0210c, respectively) by screening a genomic expression library with a serum of a naturally infected clinical cow. The 9, 15 and 34 kDa antigens display strong homology to previously described M. tuberculosis antigens, TB8.4, MPT53 and Erp, respectively. Furthermore, these antigens were shown to be recognized by antibodies from infected cattle, when tested with a limited number of sera from subclinical (n=7) and clinical (n=3) infected cattle.

Amino Acid Sequence↗
PubMed2006

Evaluation of a Pourquier ELISA kit in relation to agar gel immunodiffusion (AGID) test for assessment of the humoral immune response in sheep and goats with and without Mycobacterium paratuberculosis infection.

The present study was designed to evaluate a commercial ELISA kit (Institut Pourquier) for the diagnosis of ovine and caprine paratuberculosis under Australian conditions and to compare its accuracy with the existing AGID test. The sensitivity of the ELISA in sheep and goats was 34.9% and 56.4%, with a specificity of 98.8% and 100.0%, respectively. Sensitivity of AGID was 13.8% for sheep and 39.5% for goats, with specificity of 100.0% for both species. The sensitivity of the ELISA in sheep depended on the category of histological lesions. AGID and ELISA were conditionally independent, and appeared to detect overlapping but distinct subgroups of infected animals. The ELISA was significantly more sensitive than the AGID. The ELISA was simple to perform, robust and repeatable. Coefficients of variation of <12.0% were observed for positive and negative controls included on 193 plates over a 10-month period and there was a high level of intraassay repeatability with 12.0% of the duplicate samples having CV of >15.0%.

Animals↗
PubMed2006

Efficacy of spheroplastic and cell-wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats.

A Mycobacterium avium subspecies paratuberculosis (MAP) vaccine that reduced the incidence of clinical disease or reduced fecal shedding of MAP would aid control of Johne's disease (JD). The objective of the present study was to evaluate the efficacy of four MAP vaccine combinations, including cell-wall competent (CWC) alum adjuvant, CWC-QS21 adjuvant, cell-wall deficient (CWD) alum adjuvant and CWD-QS21 adjuvant vaccines. Eighty baby goats were vaccinated at 1 and 4 weeks of age with one of these vaccines or a sham control vaccine consisting of alum adjuvant. Kids were challenged orally with approximately 6.0x10(9) organisms in four divided doses of 1.5x10(9) organisms using a goat isolate of MAP. Vaccinated challenged and challenged control groups had 10 and 6 kids per group, respectively. Half of the kids within each group were necropsied at either 6 or 9 months post-challenge. Gross and microscopic lesions and relative number of acid-fast bacilli were evaluated and scored at necropsy. Results indicated all challenged kids had some lesions compatible with JD suggesting none of the vaccines prevented infection. Three vaccines (CWC-alum, CWC-QS21 and CWD-QS21) reduced lesion scores by 46-51% at 9 months. CWD-alum vaccine resulted in a more severe (+33.5%) lesion score than sham-vaccinated challenged control. Lesion scores were greater at 9 months than at 6 months post-challenge in the sham-vaccinated challenged group and CWD-alum vaccinated group, while lesion scores were generally stable with remaining vaccines. Mean fecal CFU/g were significantly different across time from challenge to 9-month necropsy (p=0.043) and the CWC-QS21 vaccine group had a marked reduction in fecal CFU/g at all time points post-challenge. A reduction in MAP CFU/g was also detected in necropsy tissues from kids given the CWC-alum, CWC-QS21 and CWD-QS21 vaccines, and increased CFU/g were detected in tissues from kids given the CWD-alum vaccine. Immunological tests evaluated included, humoral response evaluation by AGID, ELISA and Western blot, and cell mediated response by comparative PPD skin testing (M. avium, Old Johnin, M. bovis and Lot 2 Johnin PPD's), and production of MAP induced gamma-interferon. Vaccination also resulted in false-positive PPD skin test reactions for M. avium PPD, Old Johnin PPD and gamma-interferon tests. When a 2-mm cutoff above normal skin thickness was used to define positive skin test reactions, false-positive reactions for M. bovis were detected in only 2 of 32 kids given a vaccine with QS21 adjuvant.

Animals↗
PubMed2003

Comparison of histopathology, cultivation of tissues and rectal contents, and interferon-gamma and serum antibody responses for the diagnosis of bovine paratuberculosis.

The diagnosis of infection with Mycobacterium avium subsp. paratuberculosis was studied in 12 cattle from infected dairy herds and four from non-infected herds. A comparison was made of (1) histopathological examination and bacterial culture of tissues, (2) culture of serial samples of rectal contents, and (3) examination of repeated blood samples for interferon-gamma (IFN-gamma) and antibody responses. Tissue samples were taken from the small and large intestine and corresponding mesenteric lymph nodes, and from the pharyngeal tonsil and other lymphoid nodes (retropharyngeal, mediastinal, hepatic and supramammary). Histopathological examination and bacterial culture of tissues each revealed that six of the 16 cattle were infected, whereas repeated culture of rectal contents revealed only five infected animals. Except for the hepatic lymph node of a single animal, culture-positive tissues were confined to the intestinal tract and related lymph nodes. Bacterial culture of tissues from the ileum, caecum and lymph nodes draining the jejunum revealed the highest number of infected animals. Moreover, slightly greater numbers of positive tissues were revealed by culture than by histopathological examination. For both the IFN-gamma response and the antibody response, the means of the values for the final three samples before slaughter were significantly higher in infected than in non-infected cattle. However, these immunological responses were too variable to provide a reliable indication of infection.

Animals↗
PubMed2003

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle.

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80-96% at the cut-off values of 4, 7 and 17 OD%.

Animals↗
PubMed2003

Prevalence estimates for paratuberculosis adjusted for test variability using Bayesian analysis.

The ELISA tests that are available to detect an infection with Mycobacterium avium subsp. paratuberculosis (MAP) have a limited validity expressed as the sensitivity (Se) and specificity (Sp). In many studies, the Se and Sp of the tests are treated as constants and this will result in an underestimation of the variability of the true prevalence (TP). Bayesian inference provided a natural framework for using information on the test variability (i.e., the uncertainty) in the estimates of test Se and Sp when estimating the TP. Data from two prevalence studies for MAP using an ELISA in several regions in two locations were available for the analyses. In location 1, all cattle of at least 3 years of age were sampled in approximately 90 randomly sampled herds in each of the four regions of the country. In location 2, in 30 randomly sampled herds in each of three regions, approximately 30 randomly selected cows were sampled. Information about the unknown test Se and Sp and MAP prevalence was incorporated into a Bayesian model by joint prior probability distributions. Posterior estimates were obtained by combining the actual likelihood with the prior distributions using Bayes' formula. The corrected cow-level TP (proportion of infected cows in a herd) was low, 5.8 and 3.6% in locations 1 and 2, respectively. Certain regions within a location differed significantly in herd-level TP (proportion of infected herds). The herd-level TP was 54.3% in location 1 (95% credible interval (CI) 46.1, 63.3%) and 32.9% in location 2 (95% CI: 14.4, 73.3%). The variation in the herd-level TP estimate for location 2 was more than three times as large as the variation in location 1 mainly because of the relatively small number of investigated herds in location 2. In future prevalence studies for MAP, sample size calculations should be based on a very low cow-level prevalence. Approximately 50 and 90% of the herds in the current study had an estimated cow-level TP below 4 and 10%, respectively.

Animals↗
PubMed2002

A real time PCR assay for the detection and quantitation of Mycobacterium avium subsp. paratuberculosis using SYBR Green and the Light Cycler.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, and may also contribute to the onset and development of Crohn's disease in humans. Due to its reported heat resistance, isolation from pasteurised milk and the potential for transmission of MAP through environmental sources, rapid detection is imperative. Here, we present a rapid real time PCR assay for MAP that can simultaneously detect and quantify this organism in the laboratory using SYBR Green and the Lightcycler (LC). This method uses the highly characterised and specific P90, P91 primers, which amplify a 400-bp region of the IS900 element. Using quantified standard DNA, this assay can accurately detect as few as 20 copies (equivalent to approximately 1.5 organisms). The method is effective using a variety of templates including isolated MAP DNA, pure colonies or liquid culture sources, and can also work in the presence of contaminating bacteria. A useful application of this assay is the ability to accurately and rapidly quantitate the number of MAP cells in liquid culture as determined by comparison to previously enumerated standards.

Animals↗
PubMed2003

Evaluation of two recovery methods for detection of Mycobacterium avium subsp. paratuberculosis by PCR: direct-dilution--centrifugation and C(18)-carboxypropylbetaine processing.

A duplex polymerase chain reaction (PCR)-hybridization assay based on Mycobacterium avium subsp. paratuberculosis (MAP)-specific IS900 integration sites was used to evaluate two mycobacterial recovery methods from bovine feces: a direct-dilution-centrifugation method and a C(18)-carboxypropylbetaine (CB-18)-based method. All MAP PCR results were confirmed for absence of inhibitors using a novel PCR system based on the rpoB gene of plant chloroplasts as an internal control. The detection limits of both MAP recovery methods when coupled with PCR were determined to be between 100 and 1000 organisms. Using culture as a 'gold standard' PCR following the direct-dilution-centrifugation protocol was 92.6% sensitive and 83.7% specific, whereas PCR following the CB-18 method was 100% sensitive and 53.5% specific. Both methods were 100% specific when 60 'true' negatives from two uninfected herds were tested. Both the CB-18 and direct processing methods coupled with a target-specific amplification technique may provide greater sensitivity to diagnose subclinical animals as they were able to detect more positives, on samples derived from infected herds, than conventional culture methods; however, more extensive investigation and follow-up of suspect animals will be required to fully validate the MAP recovery and molecular detection protocols described.

Animals↗
PubMed2000

Paratuberculosis in sheep: an emerging disease in South Africa.

During a serological survey for ovine paratuberculosis a total of 145934 ovine serum samples from 2019 farms throughout South Africa were tested by means of the AGID assay. Fifty-two infected farms were identified in the Western Cape and Eastern Cape provinces. Links between infected farms in the two provinces were established. Examination of the distribution of infected farms in the Western Cape indicated a positive correlation between acid soils and occurrence of infection. In an attempt to increase the sensitivity and facilitate screening of large numbers of sera two commercial ELISA systems were evaluated for their potential use in a future monitoring program. Sera from histologically positive sheep and known negative sheep flocks were used. The highest sensitivity (50. 9%) was found if both ELISA systems were run concurrently and the results of both systems combined.

Animal Husbandry↗
PubMed2000

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR.

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.

Animals↗
PubMed2000

Differences in the immune responses in lambs and kids vaccinated against paratuberculosis, according to the age of vaccination.

In order to evaluate and compare the peripheral immune responses induced by the vaccination against paratuberculosis in relation with the age of immunization, two groups of lambs and goat kids were vaccinated at 15 days and 5 months old, respectively. A heat-killed commercial vaccine was inoculated subcutaneously and humoral and cellular immune responses were measured by an ELISA and IFN-gamma assay, respectively, at 0, 30, 90, 180, 270 and 360 dpv in the lambs and 0, 30, 90 and 180 dpv in the caprine. IFN-gamma values did not show statistically significant differences between both groups, but when compared to the unvaccinated controls, this cytokine response tend to disappear earlier in animals vaccinated at 15 days old. The antibody response was always higher and more persistent in animals vaccinated at 5 months. The possibility of the incomplete degree of maturation of the immune system in 15 days old animals as the cause of the differences in the immune response to vaccination is suggested.

Age Factors↗
PubMed2003

The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces.

The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated in BACTEC cultures from clinical samples was assessed by spiking tissues and faeces at various points during the decontamination procedure. Routine protocols in the laboratory were shown to decrease the number of organisms isolated per sample by about 2.7 log(10) and 3.1 log(10) for faeces and tissues, respectively. These findings are important for the interpretation of negative culture results and may be useful in epidemiological studies. Addition of a centrifugation step to the tissue protocol increased the recovery by about 1 log, but resulted in increased contamination of BACTEC cultures. These studies may also facilitate future improvements to decontamination procedures.

Animals↗
PubMed2003

Preliminary characterization of chemically generated Mycobacterium avium subsp. paratuberculosis cell wall deficient forms (spheroplasts).

Cell wall deficient forms (CWD, spheroplasts) genetically indistinguishable from M. avium subsp. paratuberculosis (MAP) have been isolated from patients with Crohn's disease and sarcoidosis. These MAP CWD may be important in the pathogenesis of these diseases and in Johne's Disease in other animal species. CWD forms are extremely difficult to isolate and generally revert to cell wall competent forms (CWC) when cultured in vitro. Cultured MAP strain 19698 were chemically treated to generate sufficient CWD to compare to CWC organisms by electron microscopy, chemotype profile (matrix solid-phase dispersion and thin layer chromatography), silver-stained SDS-PAGE gels with and without periodic acid treatment and Western blots with antigen recognition by sera from confirmed Johne's positive and Johne's negative cattle. On electron microscopy, CWD organisms were larger and rounder than cell wall competent forms and had lost the majority of their cell walls, being bounded only by a plasma membrane. Chemotype profiles of CWD lacked bands generally associated with cell wall glycolipids. Silver-stained SDS-PAGE gels of CWD demonstrated loss of bands that migrate in the same region as lipoarabinomannan (LAM) and some bands likely representing proteins and weakening of bands that migrate similarly to phosphatidylinositol mannosides (PIM). Western blots of CWD demonstrated bands with loss or attenuation of signal that migrate similarly to LAM and other constituents. In summary, CWD and CWC forms of MAP 19698 had marked differences in morphology, chemotype profile, cell wall constituents, and antigens recognized by Johne's disease positive and negative bovine sera.

Animals↗
PubMed1996

Immunology: resistance to paratuberculosis.

Disease caused by Mycobacterium paratuberculosis involves a complex interaction of lymphoid and phagocytic cells of the peripheral and mucosal immune responses. For resistance to develop, animals must generate an effective cellular immune response to primary infections as well as multifocal exogenous and endogenous reinfections. If an effective immune response does not develop, infected animals transgress through a complex immunologic spectrum in which the immunologic reactions themselves are responsible for the disease manifestations.

Animals↗
PubMed1996

Paratuberculosis: a potential zoonosis?

Available literature on the controversial role of Mycobacterium paratuberculosis as an etiologic agent in human Crohn's disease is reviewed. Despite almost 15 years of investigation, the question of causal or consequential association between Johne's disease and Crohn's disease continues to linger.

Animals↗
PubMed2003

Activation of macrophages and interference with CD4+ T-cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium.

Mycobacterium avium subspecies paratuberculosis (M. ptb) and M. avium subspecies avium (M. avium) are closely related but exhibit significant differences in their interaction with the host immune system. The macrophage line, J774, was infected with M. ptb and M. avium and analysed for cytokine production and stimulatory capacity towards antigen-specific CD4+ T cells. Under all conditions J774 cells were activated to produce proinflammatory cytokines. No influence on the expression of major histocompatibility complex (MHC) class II, intracellular adhesion molecule-1 (ICAM-1), B7.1, B7.2 or CD40 was found. However, the antigen-specific stimulatory capacity of J774 cells for a CD4+ T-cell line was significantly inhibited after infection with M. ptb, but not with M. avium. When a T-cell hybridoma expressing a T-cell receptor identical to that of the T-cell line was used, this inhibition was not observed, suggesting that costimulation which is essential for the CD4+ T-cell line is influenced by the pathogenic bacterium M. ptb.

Animals↗