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PubMed2006

Validation of endogenous reference genes for expression profiling of RAW264.7 cells infected with Mycobacterium avium subsp. paratuberculosis by quantitative PCR.

Reference genes are frequently used to normalize between different biological samples the levels of mRNA measured using quantitative PCR (qPCR). The expression level of many commonly used reference genes has been shown to vary between tissues or cells, or following exposure to various treatments including infection with microbes. The selection of an appropriate reference gene for an individual experiment is therefore a crucial step in the process of accurately determining changes in gene expression. For this purpose, we analyzed the expression of nine commonly used reference genes in a murine macrophage cell line, RAW264.7, for their potential use in the analysis of differential gene expression by quantitative polymerase chain reaction (qPCR) following experimental infection with Mycobacterium avium subsp. paratuberculosis. Only one of nine putative reference genes tested, casc3a, was found to be suitable, and combinations of two or more reference genes were disadvantageous. Based on data from the study, we recommend an approach for selection of reference genes, conducting assays with technical replicates in duplicate rather than triplicate, determining decision-limit quality control criteria for technical replicates and assessing the significance of gene expression fold differences using DeltaDeltaC(t) based on knowledge of the variation in the reference gene.

Actins↗
PubMed2005

Effects of seropositivity for bovine leukemia virus, bovine viral diarrhoea virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum on culling in dairy cattle in four Canadian provinces.

The purpose of this research was to determine the effects of seropositivity for exposure to bovine leukemia virus (BLV), bovine viral diarrhoea virus (BVDV), Mycobacterium avium subspecies paratuberculosis (MAP) and Neospora caninum (NC) on overall and reason-specific culling in Canadian dairy cattle. Serum samples from, approximately, 30 randomly selected cows from 134 herds were tested for antibodies against BLV, MAP and NC using commercially available ELISA test kits, while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhoea virus (BVDV). For analyzing the time (in days) to culling of cows after the blood testing, a two-step approach was utilized, non-parametric (Kaplan-Meier survival graphs) visualization and then semi-parametric survival modelling (Cox proportional hazards model), while controlling for confounding variables and adjusting for within herd clustering. For all reasons of culling, MAP-seropositive cows had a 1.38 (1.05-1.81, 95% C.I.) times increased hazard of culling compared to MAP-seronegative cows. Seropositivity for the other pathogens was not associated with an increased risk of overall culling. Among cows that were culled because of either decreased reproductive efficiency or decreased milk production or mastitis, MAP-seropositive cows were associated with 1.55 (1.12-2.15, 95% C.I.) times increased hazard compared to MAP-seronegative cows. Among cows that were culled because of reproductive inefficiency, NC-seropositive cows had a 1.43 (1.15-1.79, 95% C.I.) times greater hazard than NC-seronegative cows. Among cows that were culled because of decreased milk production, cows in BVDV-seropositive herds had a 1.86 (1.28-2.70, 95% C.I.) times increased hazard compared to cows in BVDV-seronegative herds. BLV-seropositive cows did not have an increased risk of reason-specific culling as compared to BLV-seronegative cows. No significant interaction on culling among seropositivity for the pathogens was detected, but only a limited number of cows tested seropositive for multiple pathogens. Results from our research will help in better understanding the economic impacts of these pathogens and justification for their control.

Animals↗
PubMed2006

Comparison of IS900 tissue PCR, bacterial culture, johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats.

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.

Animals↗
PubMedDate not supplied

Altered intestinal macrophage phenotype in ovine paratuberculosis.

The expression of macrophage surface markers that are likely to be important in antigen presentation and cell interactions was examined in normal sheep and those with clinical paratuberculosis. Immunohistological studies demonstrated that intestinal macrophages in diseased sheep expressed MHC class II, LFA-1 and CR4 antigens weakly compared with normal tissues. Reverse transcriptase-polymerase chain reaction analysis of MHC class II mRNA in intestinal whole tissue samples showed no significant difference between control and diseased groups. A reduction in molecules such as MHC class II and LFA-1 on the surface of infected macrophages could have implications for survival of the intracellular mycobacteria and the persistence of infection.

Animals↗
PubMed2003

Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells.

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labeling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome.

Animals↗
PubMed2000

A simulated surveillance program for bovine paratuberculosis in dairy herds in Norway.

Monte Carlo simulation models were used to evaluate the feasibility and potential results of a proposed national survey of the prevalence of bovine paratuberculosis (PTB) in dairy herds in Norway. The expected herd prevalence was assumed to be 0.2% in the simulations. The low sensitivity of the ELISA test, the assumed low herd prevalence, the typical low within-herd prevalence of PTB and the small herd sizes all present problems in detection of the disease. Simulations with 500, 1000, 2500 and 6000 herds tested were done. Our results suggest that a national survey would not be feasible at present, due to the low probability of detecting infected herds and because of the high number of false-positive reactions that would be expected to occur.

Animal Husbandry↗
PubMed2002

Maximum-likelihood estimation of sensitivity and specificity of ELISAs and faecal culture for diagnosis of paratuberculosis.

The accuracy of three diagnostic tests for paratuberculosis was evaluated using maximum-likelihood estimation of sensitivity and specificity. We also explored the variety of estimates that can be obtained if the tests are to be used in populations of different composition with regard to infection and disease states. Two enzyme-linked immunosorbent assays (ELISAs) were evaluated separately with the faecal culture (FC). The study was carried out as a cross-sectional field study to cover all likely states of infection with Mycobacterium avium subsp. paratuberculosis.The three basic assumptions for the maximum-likelihood technique were evaluated to validate the results. Our accuracy estimates for the ELISAs were not very different from those previously published, but those for faecal culture differed if a different cut-off value was chosen for the ELISA. If faecal culture was used for screening in a Danish dairy region where the median ELISA reading was a measure of the general disease situation, the sensitivity of the faecal culture was 20-25%. If faecal culture was used as a confirmatory test on cows with a high ELISA reading (and thus high level of antibodies), the sensitivity of the faecal culture would be in the range 60-70%. These results emphasise the importance of the composition of a target population before selecting a specific diagnostic test for a given purpose. We concluded that faecal culture is useful for confirmation but not for screening purposes.

Animals↗
PubMed2003

Prevalence of and carcass condemnation from maedi-visna, paratuberculosis and caseous lymphadenitis in culled sheep from Quebec, Canada.

We determined the prevalence of lung and mammary gland lesions associated with maedi-visna (MV) infection, the prevalence of paratuberculosis (PTB), and the prevalence and lesions distribution of caseous lymphadenitis (CL) in culled sheep. Total of 451 ewes and 34 rams were selected randomly from two slaughterhouses in Quebec, Canada. MV serostatus was determined by recombinant ELISA test. PTB diagnosis was based on characteristic histological lesions in the terminal ileum, ileocecal lymph node and/or ileocecal valve and CL by gross detection of abscesses and isolation of Corynebacterium pseudotuberculosis. Seroprevalence of MV was 44% (95% CI: 40, 48). Seropositivity increased with age and was higher in ewes than in rams. The percentages of lung and mammary gland lesions in seropositive sheep were 14 and 40%, respectively, but mammary gland lesions lack specificity. The prevalence of PTB was 3% (95% CI: 2, 5). PTB increased with age and was lower among sheep with abscesses. The prevalence of CL was >/=21% (95% CI: 17, 24). The most-prevalent site of caseous lymphadenitis lesions was the thoracic cavity. The risk of carcass condemnation was significantly associated with region, body score and abscesses. Only the presence of abscesses was associated with an increase in trimming of carcasses.

Abattoirs↗
PubMed1986

Participation of iron on the growth inhibition of pathogenic strains of mycobacterium avium and M. paratuberculosis in serum.

Serum, which is low in freely-available iron, was bacteriostatic for M. avium and M. paratuberculosis irrespective of the ability of these organisms to produce the iron-chelators mycobactin and exochelin for iron acquisition. Bacteriostasis was overcome when the serum was supplemented with exochelin or mycobactin, but only if the mycobacteria had previously adapted to growth in a low iron environment. The results imply that for studies on mycobacterial pathogenicity the test cultures should be grown under low iron conditions before being used. The role of the exochelins, mycobactin and iron in the development of in vivo growth is discussed.

Animals↗
PubMed2000

Further studies on the GS element. A novel mycobacterial insertion sequence (IS1612), inserted into an acetylase gene (mpa) in Mycobacterium avium subsp. silvaticum but not in Mycobacterium avium subsp. paratuberculosis.

We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

Acetylesterase↗
PubMed2003

Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex.

Mycobacterial interspersed repetitive units (MIRU) comprise short tandem repeat structures found at multiple loci throughout the Mycobacterium tuberculosis genome and have been used for typing these pathogens. We have identified MIRU at 18 conserved loci throughout the common portions of the Mycobacterium avium subspecies paratuberculosis (MAP) and M. avium subspecies avium (MAA) genomes. Six of these loci were found to differ between MAA and MAP in the number of tandem repeat motifs occurring at each MIRU locus. Locus specific PCR at 4 of these loci segregated MAP into two major groups, which could be differentiated from ovine-pigmented strains of MAP and the MAP vaccine strain 316F. The same PCR differentiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other M. avium complex (MAC) tested. PCR at both loci 1 and 4 also distinguished MAP from Mycobacterium intracellulare. MIRU typing may provide an additional simple and rapid procedure for the differentiation between MAP and other MAC.

Amino Acid Sequence↗
PubMed2005

Improved detection of Mycobacterium paratuberculosis in milk.

At present there is no rapid microbiological method for the detection of viable Mycobacterium paratuberculosis in milk. By combining an extensive milk sample pretreatment with solid phase cytometry as the detection technique we were able to demonstrate viable mycobacterial cells in 50 ml of artificially contaminated pasteurized milk in less than one working day.

Animals↗
PubMed2001

Heat inactivation data for Mycobacterium avium subsp. paratuberculosis: implications for interpretation.

AIMS: We discuss several factors that are critical for heat inactivation experiments and which should be taken into account for future research. METHODS AND RESULTS: On the basis of examples from the literature we discuss critical factors influencing the calculated heat inactivation of Mycobacterium avium subsp. paratuberculosis (MAP). Furthermore, using a modelling approach, we show that tailing of the inactivation curve of MAP is caused by the presence of cell clumps and not by a more heat-resistant cell fraction. CONCLUSIONS: The experimental conditions of the MAP heat inactivation studies of different research groups vary significantly and lead to considerable differences in results and conclusions. Therefore, a more consensual approach should be employed in future studies. In addition, our model on clumping of MAP can be used to predict the decimal reduction of MAP during heat treatment and to study the effect of clumping on other lethal effects. SIGNIFICANCE AND IMPACT OF THE STUDY: We discuss several factors that should be carefully considered in heat resistance experiments. This is essential for a thorough interpretation of results from experiments and should be given proper attention in future experiments and publications on this topic.

Animals↗
PubMed2003

Evaluation of culture media for the recovery of Mycobacterium avium subsp. paratuberculosis from Cheddar cheese.

AIMS: The study evaluated the efficacy of four Mycobacterium avium subsp. paratuberculosis (MAP) culture media in suppressing commonly used starter cultures and typical nonstarter microflora present during the manufacture and ripening of Cheddar cheese, with a view to identify a suitable medium for the enumeration of MAP during laboratory-scale Cheddar production. METHODS AND RESULTS: Four Cheddar starter cultures and Cheddar cheese manufactured with these starters were inoculated onto Herrold's egg yolk medium (HEYM); HEYM supplemented with vancomycin, amphotericin B and nalidixic acid (HEYM/VAN); Middlebrook 7H10 agar containing polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) antibiotic supplement; and BACTEC 12B radiometric medium with and without a preliminary decontamination step (0.75% w/v hexadecylpyridinium chloride (HPC), 5 h). The inclusion of a decontamination step inhibited all Cheddar cheese starter and nonstarter micro-organisms. The medium 7H10/PANTA and to a lesser extent HEYM/VAN were effective inhibitors of cheese microflora when no decontamination step was employed. CONCLUSIONS: Middlebrook 7H10 medium, supplemented with PANTA antibiotics, suppressed all micro-organisms associated with ripening Cheddar cheese manufactured with pasteurized milk. SIGNIFICANCE AND IMPACT OF THE STUDY: A MAP culture medium has been identified, which may be used to enumerate this bacterium during the laboratory manufacture and ripening of Cheddar cheese and hence facilitate further research into the persistence of this pathogen in the product.

Animals↗
PubMed2006

Sensitivity and specificity of the agar-gel-immunodiffusion test, ELISA and the skin test for detection of paratuberculosis in United States Midwest sheep populations.

Our objective was to estimate the sensitivity and specificity of the agar-gel-immunodiffusion test (AGID), the ELISA, and the skin test for the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in sheep using Bayesian methods without a gold standard. Fourteen flocks (2 465 sheep) were used. Five flocks (450 sheep) were considered MAP non-infected and 9 flocks (2 015 sheep) had sheep infected with MAP. Sheep were skin tested and blood was collected for AGID and ELISA testing. Results were analyzed using a Bayesian 3-test in 1-population model fitted in WinBUGS. The model allowed for dependence (correlation) between the two serologic tests, but these two tests were assumed to be conditionally independent of the skin test. The estimated specificity was 99.5% (95% PI of 98.9-99.9%) for the AGID; 99.3% (98.4-99.8%) for the ELISA using an optical density measured cutoff of 0.20; 99.2% (98.1-99.8%) using a cutoff of 0.15; 97.5% (95.8-98.7%) using a cutoff of 0.10; and 98.7% (97.3-99.5%) for the skin test. The estimated sensitivities were 8.3% (6.2-10.7%) for the AGID; 8.0% (6.0-10.4%), 10.6% (8.3-13.1%), and 16.3% (13.5-19.4%) for the ELISA using the cutoffs 0.20, 0.15, and 0.10 respectively; and 73.3% (62.3-85.8%) for the skin test. The skin test was specific in non-infected populations and sensitive in infected populations, although in some cases a positive skin test might represent MAP exposure rather than infection. The AGID and ELISA were specific but lacked sensitivity. The AGID and ELISA consistently identified two different populations of infected sheep with only moderate overlap between positive test results.

Animals↗
PubMed2000

Effect of monensin sodium on histological lesions of naturally occurring bovine paratuberculosis.

Of 19 adult cows naturally infected with paratuberculosis, 13 were treated with monensin sodium and six remained untreated. At the beginning of the study, the severity of the histological lesions was assessed from biopsy samples of ileum, liver, mesenteric lymph node and rectal mucosa. From the data acquired it was possible to assign the animals so that the lesions in the two groups were similar (P=0. 8323). Monensin was administered in the feed, which contained 147.5 mg/kg, and each treated cow received 450 mg of monensin daily for 120 days. At the end of this period all cows were killed and histopathological findings in the ileum, liver, mesenteric lymph node and rectal mucosa were compared with the initial findings. A scoring system showed that monensin had a beneficial effect in the ileum (P=0.077), liver (P=0.0322) and rectal mucosa (P=0.0578), but under the conditions of the experiment no such effect could be demonstrated in mesenteric lymph node (P=0.3599). There appeared to be an overall effect on all tissues taken together (P=0.1335). The effect of monensin may have been due to both a halting and a reversal of the pathological process. In all but one of the six untreated cows, the lesions worsened during the course of the experiment.

Animals↗
PubMed2005

The complete genome sequence of Mycobacterium avium subspecies paratuberculosis.

We describe here the complete genome sequence of a common clone of Mycobacterium avium subspecies paratuberculosis (Map) strain K-10, the causative agent of Johne's disease in cattle and other ruminants. The K-10 genome is a single circular chromosome of 4,829,781 base pairs and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon. In silico analysis identified >3,000 genes with homologs to the human pathogen, M. tuberculosis (Mtb), and 161 unique genomic regions that encode 39 previously unknown Map genes. Analysis of nucleotide substitution rates with Mtb homologs suggest overall strong selection for a vast majority of these shared mycobacterial genes, with only 68 ORFs with a synonymous to nonsynonymous substitution ratio of >2. Comparative sequence analysis reveals several noteworthy features of the K-10 genome including: a relative paucity of the PE/PPE family of sequences that are implicated as virulence factors and known to be immunostimulatory during Mtb infection; truncation in the EntE domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence of Map; and Map-specific sequences that are likely to serve as potential targets for sensitive and specific molecular and immunologic diagnostic tests. Taken together, the availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology in Map and enables the development of new generations of diagnostic tests for bovine Johne's disease.

Animals↗
PubMed2006

Lesions in sheep following administration of a vaccine of a Freund's complete adjuvant nature used in the control of ovine paratuberculosis.

CASE HISTORIES: Occurrences of adverse reactions in seven sheep flocks in Australia following vaccination against paratuberculosis where veterinary attention was requested are reviewed. All cases occurred within the 3-year period following commencement of use of a vaccine of a Freund's complete adjuvant nature, at a time when approximately six million doses of vaccine had been administered. CLINICAL FINDINGS: In the first case, 26/58 (45%) Merino sheep vaccinated as adults had palpable tissue reactions at or near the site of vaccination; enlarged prescapular lymph nodes were palpated in 17 (29%), and nine (16%) sheep had both palpable lesions at the site of vaccination and enlarged prescapular lymph nodes. The reactions included caseous nodules up to 5.5 cm in diameter. In the other cases, fistulating or granulomatous wounds were occasionally found at the recommended site of injection behind the ear, and myiasis was rare. Occurrences of inappropriate choice of injection site were recorded, including injection into the axilla of two Merino rams, and lesions in the tissues of the maxilla and nose of almost 50% of 350 Border Leicester lambs. Four outbreaks of progressive paralysis due to injection into cervical musculature were reported, described as "OJD staggers" by producers. DIAGNOSIS: Granulomatous cellulitis and lymphadenitis associated with oil droplets typical of "oil granulomata". Injection of vaccine into the dorsal cervical area resulted in progressive paralysis due to myonecrosis and suspected granulomatous leptomeningitis. CLINICAL RELEVANCE AND CONCLUSIONS: Although lesions at and near the site of injection are common, adverse reactions to vaccination were rare and included mortality from cervical spinal injection, production losses from injection in the maxilla or axilla or if myiasis resulted, and potential marketing losses if animals or carcasses are discounted as a result of the lesions. Risk factors for adverse reactions included inadequate restraint of sheep, breed of sheep, experience of the operator, poor injection technique, and inappropriate placement of vaccine. Increasing attention to the proper restraint of animals, restricting vaccination to the recommended site behind the ear, careful placement of the vaccine into subcutaneous tissue to avoid drainage of vaccine material into tissues such as the spinal cord, and post-vaccination supervision to address welfare concerns should adverse reactions occur are recommended.

Animals↗