Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by culture and serology

Faecal samples from 186 dairy cows representing ten commercial dairy herds with sporadic clinical paratuberculosis (group A), and from 100 dairy cows from herds without a history of paratuberculosis (group B) were cultured for Mycobacterium avium subspecies paratuberculosis (MAP). Two different decontamination methods, a NaOH/oxalic acid method and treatment with 0.75% hexadecylpyridinium chloride (HPC) were performed prior to inoculation of Loewenstein-Jensen agar slants with and without mycobactin. Cultures were incubated for 16 weeks. Acid-fast staining bacteria were identified as MAP on the basis of mycobactin dependency and by PCR-RFLP analysis of the IS 1311-insertion element of M. avium. MAP was grown from 15 out of 186 group A animals (8.1%) whereas faecal culture for MAP was consistently negative in group B. The growth rate of MAP was significantly higher (8.1% vs. 1.6%) and the contamination rate of cultures was significantly lower (17.6% vs. 21.5%) in faecal samples decontaminated with NaOH/oxalic acid than with HPC-treated faecal samples (p<0.01, McNemar's test). Atypical mycobacteria which were grown from 46.8% of NaOH/oxalic acid treated specimens were not obtained from any of the HPC-treated samples. A commercial ELISA with MAP-lipoarabinomannan as the antigen was used to detect MAP-antibodies in unabsorbed sera from all animals. The percentage of ELISA-positive cows was 16.8%. Overall agreement between antibody detection and MAP-positive faecal culture was 15.4%.     

T lymphocyte responses during early enteric Mycobacterium avium subspecies paratuberculosis infection in cattle

Johne's disease (JD) is a costly intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map), which is transmitted to perinatal calves by the fecal-oral route. Disease control efforts focus on identification and culling of infected cattle from herds; therefore failure to identify animals early is a major obstacle to reducing transmission. Development of host immunity during early JD remain incompletely characterized so detecting subclinical JD using immunologic techniques is a substantial challenge in the field. Development of a test with high sensitivity and specificity is a major research goal with significant implications for the cattle industry. The objectives of this study were to compare early Map-specific T lymphocyte responses in naive, experimentally Map infected and Map vaccinated calves using a subcutaneous matrigel biopolymer-based assay. We examined the phenotype of recruited lymphocytes and local interferon gamma (IFNγ) production within subcutaneously placed matrigel containing Map antigen 30 days after experimentally induced intestinal Map infection or Map vaccination. We show that IFNγ-secreting CD4+ T cells are recruited to matrigel sites in vaccinated but not infected or naïve calves. γδ T cells recruited to matrigel sites of Map-infected calves were mostly WC1-, while γδ T cells recruited to matrigel sites of Map-vaccinated calves were predominantly WC1+. IFNγ at matrigel sites was a discriminating factor between infected calves, naïve calves and vaccinated calves. These data contribute to our understanding of early anti-Map immunity, and may be useful for detecting early intestinal Map infections in calves or for enhancing our ability to discriminate between Map-infected and Map-vaccinated calves.     

Proteome-determined type-specific proteins of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is a pathogen of ruminants, causing paratuberculosis (characterized by severe emaciation). The disease is endemic in many countries including the UK and places a severe economic burden on the global livestock industry. Two types of M. a. paratuberculosis can be classified by pulsed-field electrophoresis (I/III and II), which are phenotypically distinct and appear to have different host preferences. Proteomes of Type I and Type II M. a. paratuberculosis were analyzed by 2-D gel electrophoresis to determine if any significant differences existed between the subtypes. Seven different strains of Type I and 18 strains of Type II were analyzed and compared to detect type-specific differences. These 'type-specific' differences existed regardless of growth phase and were also exhibited in cells isolated directly from pathogenic lesions. Twenty-three spots predominated on the Type I profile, from which 17 proteins were identified. Twenty-one spots predominated on the Type II profile, from which 16 proteins were identified. None of the proteins identified as differentially represented on the profiles of Type I or Type II corresponded to open reading frames of the defining genomic regions as previously described for the Type I (sheep) and Type II (cattle). Sequence polymorphisms existing in Type I and II strains were identified in some open reading frames or regulatory regions of genes that correspond to proteins expressed in a type-specific fashion. The consequence of these is discussed in relation to protein expression and their impact on the type phenotype is discussed.     

Progress in molecular typing of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is responsible for paratuberculosis or Johne's disease, a chronic inflammation of the gastrointestinal tract in different animal species. Some studies have also established a link between this microorganism and Crohn's disease in humans. Although, M. a. paratuberculosis is a difficult microorganism to cultivate in the laboratory (occasionally is non-cultivable), a proper molecular characterization of M. a. paratuberculosis is necessary to better understand the epidemiology of the disease, and design strategies to eradicate it. In the present review, we compile and discuss the recent progress attained in the diagnostic and characterization of this pathogen.     

Factors affecting the herd level of antibodies against Mycobacterium avium subspecies paratuberculosis in dairy cattle

A case-control study was made of Norwegian dairy herds with high and low herd levels of antibodies against Mycobacterium avium subspecies paratuberculosis. A high proportion of the herds had a considerable number of seropositive cows, and environmental and management factors were examined for possible associations with the high serological levels of antibodies. The most important appeared to be: geographical location, red deer (Cervus elaphus) gaining access to the pastures for cattle, the observation of wild birds in the feed storage, and herds sharing common pasture with other herds of cattle. However, diagnostic tests showed that none of the animals in the case herds was infected with M a paratuberculosis.     

Within-herd contact structure and transmission of Mycobacterium avium subspecies paratuberculosis in a persistently infected dairy cattle herd

Within-herd transmission of pathogens occurs either by direct or by indirect contact between susceptible and infected animals. In dairy herds that are structured into groups, the way in which animals encounter each other and share an environment can affect pathogen transmission. Dairy cattle are heterogeneous in terms of susceptibility and infectivity with respect to Mycobacterium avium subspecies paratuberculosis (Map) transmission. It is mainly young animals that are susceptible and adults that are infectious. Both vertical and horizontal transmission through the ingestion of Map shed into the environment by adults and transiently infected calves can occur. Our objective was to assess the effect of contact structure on Map transmission in persistently infected dairy herds and to examine the effect of isolating calves from other calves or from adults before weaning. We developed a stochastic compartmental model of Map transmission in a closed dairy herd. The model reflects the Map infection process and herd management characteristics. Indirect transmission via the environment was modelled explicitly. Six infection states (susceptible, resistant, transiently infectious, latently infected, subclinically infected, and clinically affected) and two contaminated farm area environments (whole farm and calf area) were modelled. Calves were housed in hutches, individual indoor pens, or group indoor pens. Two different levels of exposure of calves to a farm environment contaminated by adults were possible: no exposure and indirect exposure through fomites. Three herd sizes were studied. We found that contacts between calves before weaning did not influence Map transmission in a herd, whereas the level of exposure of calves to an environment contaminated by adults and the starting age of exposure of calves to adults were pivotal. Early culling of clinically affected adults led to a lower prevalence of infectious adults over time. The results were independent of herd size. Despite the many transmission routes that are known, the best control approach is to limit the exposure of calves to adult faeces through the systematic separation of adults and calves in combination with hygiene measures. Reducing contact between calves does not appear effective.     

Genomic analysis of local isolate of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD or paratuberculosis) in animals and has also been implicated in Crohn's disease of humans. It has been shown that MAP is endemic in animal population of India. Understanding of heterogeneity among MAP strains is important both for diagnosis and design control measures. Genotyping and epidemiological investigations revealed that MAP 'Bison type' was the predominant strain infecting domestic ruminant population in India. MAP 'Bison type' has also been reported from USA. A number of comparative genomics studies have been conducted to understand 'Cattle type' and 'Sheep type' strains. However, present study was the first attempt to characterize MAP 'Bison type' S5 using different markers including IS900, ISMAP02, IS1311, LSPs and SSRs. Study showed that MAP S5 is similar to MAP K10 in terms of number of IS900, IS1311 and ISMAP02 elements. There was high sequence similarity for IS900 and ISMAP02 between MAP K10 and MAP S5. However, this study also reported genetic differences between two strains. In some IS1311 loci, TG gap at 64th and 65th position was observed in MAP S5. Further sequencing of few more MAP isolates confirmed that this gap was specific to indigenous MAP 'Bison type' and can be further used as molecular signature. ISMAP02 locus 1 was observed at polymorphic position in MAP S5 compared to MAP K10. MAP 'Bison type' S5 also showed polymorphic profile for LSP(P)4. Polymorphism was also observed in SSRs. This pilot study may form the basis for future epidemiological investigations.     

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.     

Bayesian kriging of seroprevalence to Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta beef and dairy cattle

Identifying spatial patterns of risk is important in the study of diseases with ecologic causes. Furthermore, relatively complex hierarchical modeling is required to determine how factors that are organized across levels interact, such as how an ecologic cause interacts with farm management and with animal characteristics. The objective of this study was to map the risk for Mycobacterium avium subspecies paratuberculosis (MAP - the causative agent of Johne's disease) and Neospora caninum (NC - the cause of neosporosis) infections in Alberta beef and dairy cattle. This objective utilized Bayesian generalized linear kriging to partition herd effects into a portion attributable to location and a portion that was independent of location. Seropositivity to NC in beef cattle showed strong support for spatial covariance, suggesting that ecologic causes were important for beef cattle but not dairy cattle. There was little evidence of spatial covariance for MAP seropositivity in either beef or dairy cattle.     

Characterization of Mycobacterium avium subspecies paratuberculosis disseminated infection in dairy cattle and its association with antemortem test results

Mycobacterium avium subspecies paratuberculosis (MAP) disseminated infection in dairy cattle affects animal health and productivity and is also a potential public health concern. The study objectives were to characterize MAP disseminated infection in dairy cattle and to determine the role of antemortem tests in detecting cattle with disseminated infection. Forty culled dairy cows representing a variety of serum enzyme-linked immunosorbent assay (ELISA) results and body conditions were selected for the study. The physical condition of the cows was assessed via clinical examination prior to euthanasia and blood and feces were collected and tested by serum ELISA and fecal culture, respectively. Fifteen tissues were aseptically collected from each cow during necropsy and cultured for isolation of MAP. Disseminated infection was diagnosed when MAP was isolated in tissues other than the intestines or their associated lymph nodes (LNs) and was distinguished from infection found only in the gastrointestinal tissues and from absence of infection. Of the 40 cows in the study, 21 had MAP disseminated infection. Results showed that 57% (12/21) of cows with disseminated infection had average to heavy body condition and no diarrhea. Cows with disseminated infection had no to minimal gross pathologic evidence of infection in 37% (8/21) of cases. Only 76% (16/21) of cows with disseminated infection had positive historical ELISA results and only 62% (13/21) had a positive ELISA at slaughter. Thus, antemortem evidence of MAP infection was lacking in a high proportion of cows where MAP disseminated infection was confirmed.     

Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis

Recent publications reported the existence of IS900 like sequences in mycobacteria different from Mycobacterium avium subspecies paratuberculosis (Map). The primers used for IS900 detection of Map have amplified these sequences causing false positive results. In this study, we have developed two new PCR assays for the detection of Map. The first assay is based on the IS900 sequence using primers different from the ones previously reported, the second assay on the f57 sequence. The specificity of the tests was checked by analysis of 190 mycobacterial isolates (74 Map and 116 non-Map isolates). All Map strains were positive and all non-Map strains were negative. Serial dilutions of Map bacteria were used to assess the sensitivity of the assays. We achieved a sensitivity of 1 CFU per PCR for both assays. In addition, a PCR-simulating computer programme was used to evaluate the specificity of the new IS900 primers.

The combination of the two PCR assays has proven to be useful for the identification of Map but validation on a large range of clinical samples still needs to be done.     

Mycobacterium avium subspecies paratuberculosis: a review of current knowledge.

Although Mycobacterium avium subsp. paratuberculosis infection has had its greatest effect on domestic agricultural animal species, it can also have a significant impact on wildlife species. More cases of infection are being reported, and because of its ability to elude immunologic control and to persist in the environment, M. paratuberculosis may spread within and among captive and free-ranging wildlife populations in the absence of organized control programs. Studies to improve our ability to detect the organism in biologic samples such as milk, blood, and manure through immunomagnetic separation, automated culture methods, and improved polymerase chain reaction procedures are underway in several countries. Studies of the organism's genetic components, virulence factors, and antigens support the development of new diagnostic tools and vaccines.     

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.     

Differences in intermittent and continuous fecal shedding patterns between natural and experimental Mycobacterium avium subspecies paratuberculosis infections in cattle

The objective of this paper is to study shedding patterns of cows infected with Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large scale meta-analysis of both natural and experimental infections. Large difference in shedding patterns between experimentally and naturally infected cows were observed. Experimental infections are thus probably driven by different pathological mechanisms. For further evaluations of shedding patterns only natural infections were used. Within such infections, the transition to high shedding was studied as a proxy to the development of a clinical disease. The majority of studied cows never developed high shedding levels. Those that do, typically never reduced their shedding level to low or no shedding. Cows that eventually became high shedders showed a pattern of continuous shedding. In contrast, cows with an intermittent shedding pattern had a low probability to ever become high shedders. In addition, cows that start shedding at a younger age (less than three years of age) have a lower hazard of becoming high shedders compared to cows starting to shed at an older age. These data suggest the presence of three categories of immune control. Cows that are intermittent shedders have the infection process under control (no progressive infection). Cows that start shedding persistently at a young age partially control the infection, but eventually will be high shedders (slow progressive infection), while cows that start shedding persistently at an older age cannot effectively control the infection and become high shedders rapidly.