Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis

OBJECTIVE: To assess the protective value of a live-attenuated vaccine in sheep already exposed to Mycobacterium avium subsp paratuberculosis and to investigate the progression of a systemic immune response in experimentally infected sheep. STUDY DESIGN: Twenty-eight lambs, aged 1 to 1.5 months, were dosed via stomach tube with approximately 4.4 x 10(8) M a paratuberculosis organisms. Two weeks later, 14 of these 28 animals received subcutaneous injections of 1 mL of a live-attenuated vaccine. Thirteen additional lambs were neither dosed nor vaccinated (negative controls). Antigen-induced production of IFN-gamma in blood, and antibody concentrations in serum were sequentially monitored in vaccinated, unvaccinated and control animals for 1 year. Each sheep was examined for infection by an IS900-based PCR test on samples of ileum and ileocaecal lymph node and histological examination at the time of necropsy. RESULTS: Seven of 14 unvaccinated and two of 14 vaccinated sheep developed clinical paratuberculosis that was later confirmed by histological examination and/or the IS900-based PCR test. The granulomatous inflammation in the jejunal and ileal mucosa was less severe in vaccinated than in unvaccinated sheep. Acid-fast organisms were detected only in the unvaccinated group. The PCR assay on ileal samples gave positive reactions in two vaccinated and eight unvaccinated sheep. Both the antibody response and IFN-gamma response were detected earlier and were more substantial in vaccinated than in unvaccinated sheep. Furthermore, in experimentally infected but unvaccinated sheep, the IFN-gamma concentrations were higher in those animals without acid-fast organisms than in those with them. CONCLUSIONS: Vaccination of lambs with live-attenuated vaccine 2 weeks after oral inoculation with M a paratuberculosis stimulated the host response against the organism and led to a reduced mycobacterial burden. The diminished IFN-gamma responses in experimentally infected sheep with acid-fast organisms suggest a positive relationship between the magnitude of the systemic cell-mediated immune response and an animal's ability to control infection.     

Nested PCR on blood and milk for the detection of Mycobacterium avium subsp paratuberculosis DNA in clinical and subclinical bovine paratuberculosis

OBJECTIVE: To determine the potential of PCR on blood and milk to detect cattle infected with Mycobacterium avium subsp paratuberculosis. PROCEDURE: A nested PCR method probing for IS900 was developed and compared to ELISA serology in 11 clinically infected and 46 subclinically infected, lactating Holstein cows from a herd with confirmed paratuberculosis (Johne's disease). RESULTS: When compared to serum ELISA the nested blood- and milk PCRs were equal in identifying DNA from clinically infected animals. The PCR procedures also gave positive DNA results with some subclinically infected animals when these only gave suspicious or negative results in the ELISA test. Most clinically and subclinically infected animals were detected with milk PCR. CONCLUSION: Since there may well be a haematological phase in paratuberculosis, nested PCR testing of blood and milk samples shows potential to detect animals subclinically infected with M a paratuberculosis. More subclinically infected animals need to be tested and confirmed infected before estimates of sensitivity and specificity can be made.     

Temporal patterns of diagnostic results in serial samples from cattle with advanced paratuberculosis infections.

As part of investigating diagnostic strategies for Mycobacterium avium subsp. paratuberculosis (Map), serial results from polymerase chain reaction (PCR) on extraintestinal tissues (blood, milk, and liver) were compared with those from more conventional detection methods including serum enzyme-linked immunosorbent assay (ELISA), fecal culture, and fecal PCR. Three cows previously identified as being subclinically infected with Map were selected for the study. Blood, milk, and feces were collected daily and liver biopsies were obtained weekly for a 30-day period. Unexpectedly, a substantial daily variation in serum ELISA sample to positive (S/P) ratios was observed in all 3 cows. In contrast, fecal culture results were consistently positive. However, whereas fecal culture colony counts were consistently high for 2 cows throughout the study, colony counts from the third cow varied from day to day. Diagnostic sensitivity of PCR for fecal, blood, milk, and liver samples in these advanced subclinically infected cows was 87%, 40%, 96%, and 93%, respectively.     

Antigen-induced production of interferon-gamma in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis.

The production of interferon-gamma (IFN-gamma) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-gamma assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-gamma test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-gamma assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-gamma assay on samples incubated overnight, respectively. The AGID and IFN-gamma assay on blood samples incubated for 48h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-gamma assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-gamma assay on samples incubated overnight and for 48h or the PLN IFN-gamma assay on samples incubated overnight, but one gave a positive result in the PLN IFN-gamma assay on samples stimulated for 48h. It is likely that the positive reactions obtained by the PLN IFN-gamma assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.     

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.     

Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease.     

The influence of multiple liver biopsies on hematologic and serum biochemical values of sheep

A rapid, simple liver biopsy technique for sheep was developed to obtain in situ liver samples for evaluation of histological tissue, enzyme assay, and ultrastructural changes that might occur during toxicological testing. The study was designed because repeated biopsies in the same individual could induce bias in hematologic, histological or serum biochemical parameters caused by the trauma of the biopsy procedure. In Experiment I, sheep were divided into a biopsied group (B), sham-operated group (SO), and a nonsurgical, untreated control group (C). Three liver biopsy samples per week were removed from each subject in Group B for five weeks. Biweekly blood samples were drawn for evaluation of hematologic and serum biochemical parameters. In Experiment II, Groups B and SO were dosed with CCl4 in order to validate the applicability of the biopsy procedure. Weekly biopsies were continued in Group B. Biweekly blood samples were evaluated for any hematologic or serum biochemical variations. Significant elevations of enzyme activities confirmed CCl4 hepatotoxicity in treated groups (B and SO) versus C group. Histological lesions in biopsy samples correlated closely with serum biochemical data in treated groups. It was concluded that the serial liver biopsy procedure causes negligible alterations in the hematologic, histological or serum biochemical parameters measured; is simple and rapid to perform, and provides a method for possible early detection of liver disease.     

Contribution to the Diagnosis of Johne’s Disease in Cattle.Comparative Studies on the Validity of Ziehl-Neelsen Staining,Faecal Culture and a Commercially Available DNA-Probe® Testb in Detecting Mycobacterium paratuberculosis in Faeces from Cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infectedcattle from dairy herds known to be affected by Johne’s disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNAProbe ® test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe® test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe® test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne’s disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNAProbe® test has to be enhanced to enable a quick and reliable diagnosis of Johne’s disease.     

Intrauterine and transmammary transmission of Mycobacterium avium subsp paratuberculosis in sheep

OBJECTIVE: To investigate intrauterine infection of foetuses with Mycobacterium avium subsp paratuberculosis and the presence of infection in mammary secretions of sheep. DESIGN: A study of 142 late-pregnant ewes and their foetuses from two heavily infected flocks. PROCEDURE: Infection of ewes was determined at necropsy by histopathology and culture of tissues and mammary secretions. Antemortem tests (clinical assessment, faecal culture and serology) were also applied. Foetuses from 59 infected ewes and 47 apparently uninfected ewes were examined by culture and histopathology. RESULTS: Five of five ewes with clinical ovine Johne's disease had infected foetuses. Only one of 54 subclinically affected ewes, and none of 47 uninfected ewes had an infected foetus. M a paratuberculosis was cultured from mammary secretions or mammary glands of only two of 76 ewes, both of which were clinical cases and had infected foetuses. CONCLUSION: Although intrauterine or transmammary transmission of Mycobacterium avium subsp paratuberculosis may occur frequently in clinically affected sheep, these are less common in subclinically infected ewes. Therefore these modes of transmission are unlikely to compromise existing control programs for ovine Johne's disease on most farms, especially if programs include the immediate culling of clinically affected sheep.     

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.     

Cytokine secretion by peripheral blood mononuclear cells from cows infected with Mycobacterium paratuberculosis

OBJECTIVE: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis. ANIMALS: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis. PROCEDURE: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS. RESULTS: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.     

Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene

The aim of this study was to determine if fluorescent PCR could be used instead of nested PCR, for the detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in clinical specimens, to improve the sensitivity without increasing the risk for cross-contamination. We investigated and compared the sensitivity of single PCR, fluorescent PCR and nested PCR for the detection of IS900, an insertion sequence specific for M. paratuberculosis. A previously described extraction method for clinical specimens, based on xylene, was evaluated regarding its suitability for routine diagnostic work. The sensitivity of each PCR system was assessed by analysing a serial dilution of M. paratuberculosis DNA. To improve the reliability of the PCR and to facilitate the interpretation of the PCR results, a positive internal control molecule ("mimic") was developed and used for single and fluorescent PCR. In nested PCR, an existing mimic was used. The efficiency of recovering DNA of M. paratuberculosis from clinical specimens by the extraction method and detection of the organism by PCR was studied by analysing spiked ileum mucosa specimens. The final evaluation was performed on seventeen ileum mucosa specimens, previously found positive for M. paratuberculosis by bacterial culture. Twelve of the samples were positive by fluorescent PCR and nested PCR, and 10 samples were positive by single PCR. The use of mimics showed inhibition in specimens harbouring few M. paratuberculosis organisms, illustrating the effect of inhibitory substances in combination with small amounts of M. paratuberculosis DNA. We conclude that the extraction method was not adequate to recover small amounts of M. paratuberculosis and that inhibitory substances were still present in the processed specimens, but that the method is useful for identifying positive samples. Fluorescent PCR was a suitable alternative to both single PCR and nested PCR for the detection of M. paratuberculosis.     

Incidence of Mycobacterium paratuberculosis in raw sheep and goats' milk in England, Wales and Northern Ireland

A blind survey of 104 raw sheep and goats' milk samples (90 goat, 14 sheeps) from bulk tanks on farms throughout England, Wales and Northern Ireland was carried out over a 5-month period (January-May 1998) in order to determine the incidence of Mycobacterium paratuberculosis. Each milk sample (100 ml) was divided into two 50ml portions. One portion was decontaminated with 0.75% hexadecylpyridinium chloride for 5h before culture on slopes of Herrold's egg yolk medium and in BACTEC radiometric medium. The second portion was subjected to immunomagnetic separation followed by IS900 PCR (IMS-PCR). The IMS-PCR assay was employed in order to provide a more rapid indication of the presence of M. paratuberculosis in each milk sample than is possible by culture. Information on the Johne's disease status of the sheep and goat herds that took part in the survey was not sought at the time of milk sampling. However, it subsequently emerged that at least some of the herds whose bulk milk was tested during this study were previously or currently infected with Johne's disease. Overall, during this survey one raw goats' milk sample tested positive for the presence of M. paratuberculosis by IMS-PCR (<1% of milk samples tested) but no viable M. paratuberculosis were isolated by culture. The results of this study suggest that bulk raw sheep and goats' milk from these regions of the UK may not represent significant vehicles of transmission of M. paratuberculosis to humans.     

Augmentation of secreted and intracellular gamma interferon following johnin purified protein derivative sensitization of cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Measurement of secreted interferon (IFN)-gamma has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-gamma assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-gamma were observed. Intradermal injection resulted in significant increases in secreted IFN-gamma for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosis antigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-gamma was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-gamma was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-gamma, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.     

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.     

Isolation of Mycobacterium avium subsp paratuberculosis from environmental samples collected from farms before and after destocking sheep with paratuberculosis

OBJECTIVE: To determine whether Mycobacterium avium subsp paratuberculosis could be isolated from soil-pasture, faecal, water and sediment samples on farms before and after removal of sheep with paratuberculosis. A feasibility study and subsequent field survey. PROCEDURE: First the analytical sensitivity of radiometric culture of the organism from two types of soil was determined relative to faeces. Then soil-pasture, faecal, water and sediment samples were collected for culture from a range of sites from 6 farms with paratuberculosis affected sheep and goats. Similar samples were collected from 20 farms at least 9 months after removal of infected stock. RESULTS: The analytical sensitivity of culture of M a paratuberculosis from soil samples was 2 orders of magnitude less than that from faeces, and environmental samples required longer incubation periods to yield significant growth in radiometric culture (BACTEC) medium. However, the organism was recovered from approximately 20% of 163 soil-pasture, water and sediment samples from 6 properties with clinically-affected animals with paratuberculosis. The positive samples were from a range of topographic sites, including open exposed and dry areas, however, low lying areas tended to have larger numbers of organisms. When the same sites were sampled again about 5 months later, only 1 was culture positive, and none were culture positive > 12 months later. Of 17 water and dam sediment samples collected from farm 6, which had long-standing high prevalence OJD infection, only one water sample and one sediment from the same dam were culture positive. None of the 5 water samples from the other farms were culture positive. Of 96 water samples, 90 sediment samples and 93 soil samples from farms that had been destocked of infected sheep/goats for 9 to 24 months, one sediment sample from a farm in Victoria (destocked for 12 months) and two sediment samples from a farm in New South Wales (10, 19 months) were culture positive. Recontamination from cattle or water could not be excluded as a cause of the positive cultures from the second farm. CONCLUSION: M a paratuberculosis can be detected by radiometric culture in environmental samples from farms grazed by sheep or goats with paratuberculosis. There is a relatively low likelihood of recovery of the organism from water samples from such farms, and at 5 or more months after removing stock with paratuberculosis the likelihood of positive cultures from environmental samples is very low. Although the analytical sensitivity of culture from environmental samples is less than that from faeces, surveys of environmental sites are nevertheless feasible. However, improved culture methods are needed for critical surveys and to study the movement and fate of the organism in the environment.     

Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.     

Molecular epidemiology of Types I/III strains of Mycobacterium avium subspecies paratuberculosis isolated from goats and cattle

Molecular characterization of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolates classifies them into three groups: cattle or Type II, sheep or Type I, and intermediate or Type III. To avoid problems associated with characterization of extremely slow growth strains, PCR-based techniques that divide the M. a. paratuberculosis strains in two main groups (cattle or Type II, and sheep or Types I/III) can be performed. The objectives of this study were to characterize the M. a. paratuberculosis isolates identified by different PCR-based tests (IS1311-PCR and restriction endonuclease analysis, PCR test based on a DNA sequence difference, and a PCR aimed at three Type I-specific loci), and to determine the clinical and epidemiological implications of Types I/III M. a. paratuberculosis strains in livestock. One hundred and fifty-eight M. a. paratuberculosis strains from domestic ruminants were analyzed. One hundred and six M. a. paratuberculosis isolates (61 from goats and 45 from cattle) were classified as Type II strains; and 52 (29 from cows, 20 from goats, and three from sheep) were included in the Types I/III. The Types I/III M. a. paratuberculosis strains were associated to Spanish native breeds. The majority of these animals had not been in direct or indirect contact with sheep flocks infected with M. a. paratuberculosis. This fact should be taken into account when implementing paratuberculosis control programs.     

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCR TaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838-843]. The pCS20 quantitative real-time PCR TaqMan probe was compared to the currently used pCS20 PCR and PCR/(32)P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCR TaqMan probe was the most sensitive assay detecting seven copies of DNA/mul of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/(32)P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCR TaqMan probe assay was the most sensitive and can be performed within 2h it is an effective assay for epidemiological surveillance and monitoring of infected animals.     

The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy

The detection of Mycobacterium paratuberculosis organisms in bovine faeces by isolation was compared with that by the microscopical examination of Ziehl-Neelsen stained faecal smears for the presence of clumps of acid-fast M. paratuberculosis organisms. Faeces were obtained from cattle naturally or experimentally infected with M. paratuberculosis as well as from uninfected cattle. Microscopical examination was an unreliable method for the detection of M. paratuberculosis organisms, since the organisms were only detected in 99 (=55.9%) of 177 culturally positive faecal samples. 1111 addition, clumps of acid-fast organisms indistinguishable from M. paratuberculosis were also observed iin three of 18 samples from cattle free from Johne's disease and in 18 of 37 culturally negative samples from paratuberculous cattle. When M. paratuberculosis organisms were added to faeces from an uninfected cow, results showed that isolation attempts should be positive when 15 or more M. paratuberculosis organisms per gram of faeces are present.