Risk factors for the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection on 59 Irish dairy herds

Since 1994, Irish cattle have been exposed to greater risks of acquiring Mycobacterium avium subspecies paratuberculosis (MAP) infection as a consequence of the importation of over 70,000 animals from continental Europe. In recent years, there has been an increase in the number of reported clinical cases of paratuberculosis in Ireland. This study examines the prevalence of factors that promote the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) on selected Irish dairy farms in the Cork region, and the association between these factors and the results of MAP screening tests on milk sock filter residue (MFR). A total of 59 dairy farms, selected using non-random methods but apparently free of endemic paratuberculosis, were enrolled into the study. A questionnaire was used to collect data about risk factors for MAP introduction and transmission. The MFR was assessed on six occasions over 24 months for the presence of MAP, using culture and immunomagnetic separation prior to polymerase chain reaction (IMS-PCR). Furthermore, blood samples from all entire male and female animals over one year of age in 20 herds were tested by ELISA. Eighteen (31%) farms had operated as closed herds since 1994, 28 (47%) had purchased from multiple sources and 14 (24%) had either direct or indirect (progeny) contact with imported animals. Milk and colostrum were mixed on 51% of farms, while 88% of farms fed pooled milk. Thirty (51%) herds tested negative to MFR culture and IMS-PCR, 12 (20%) were MFR culture positive, 26 (44%) were IMS-PCR positive and seven (12%) were both culture and IMS-PCR positive. The probability of a positive MFR culture was significantly associated with reduced attendance at calving, and with increased use of individual calf pens and increased (but not significantly) if mulitiple suckling was practised. There was poor agreement between MFR culture and MFR IMS-PCR results, but moderate agreement between MFR culture and ELISA test results. This study highlights a lack of awareness among Irish dairy farmers about the effect of inadequate biosecurity on MAP introduction. Furthermore, within-herd transmission will be facilitated by traditional calf rearing and waste management practices. The findings of viable MAP in the presence of known transmission factors in non-clinically affected herds could be a prelude to long-term problems for the Irish cattle and agri-business generally.     

Culture of Mycobacterium avium subspecies paratuberculosis (MAP) from blood and extra-intestinal tissues in experimentally infected sheep

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants, and has been suggested to play a role in Crohn's disease in humans. While Johne's disease is primarily expressed in the gastrointestinal tract, isolation of MAP from extra-intestinal tissues indicates that microbial dissemination via the haematogenous route may occur during the infection. Consequently, the occurrence of mycobacteraemia and dissemination to the liver and hepatic lymph node was investigated in 111 sheep. Disseminated infection was detected in 18 of the 53 sheep that were confirmed to be infected following oral exposure to MAP while the bacterium was isolated from the blood of only 4 of these animals. Disseminated infection was detected more frequently from animals with a positive compared to a negative faecal culture result, multibacillary compared to paucibacillary lesions, and clinical compared to subclinical disease. Detection of MAP in blood by culture was significantly associated with increased time post-exposure and clinical disease, with trends for increased detection in animals with multibacillary lesions and positive faecal culture results. Isolation of MAP from blood was difficult in the early stages of the disease and in paucibacillary animals as the bacteraemia may be intermittent, below the limit of detection or MAP may be present in a dormant non-culturable form. Prolonged incubation periods prior to growth in BACTEC were consistent with inhibition of growth or dormancy in some blood cultures.     

Prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) infection in suspected diarrhoeic buffaloes and cattle reporting at Veterinary University in India

Murrah buffaloes, best breed for milk production are native of Haryana state. They contributes significantly to the farmer’s income, livelihood and food (milk and meat) security, in the semi-tropical regions of North India. Johne’s disease though endemic in the domestic livestock of the country, but reports are not available in the buffaloes suffering from morbidity due to progressive weakness and diarrhoea. We estimated the status of JD in diarrhoeic buffaloes and cattle reporting at Veterinary Clinical Complex of Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India, using conventional, serological and PCR assays.141 buffaloes suffering from chronic diarrhoea were screened to estimate sero-prevalence of MAP and 50.0 % young and 53.52 % adult animals were positive. Of 14 cattle screened, none of the young and 66.6 % adult cows were positive. In buffaloes, 66.1 and 6.77 %, fecal samples were positive in microscopy and IS900 PCR, respectively. Sero-prevalence of JD was very high in diarrhoeic buffaloes and cattle from Haryana state of India.Buffaloes positive for Mycobacterium avium subspecies paratuberculosis (MAP) infection had reduced total leukocyte count and lymphocytes.     

Association between cattle herd Mycobacterium avium subsp. paratuberculosis (MAP) infection and infection of a hare population

Paratuberculosis has long been considered a disease of domestic and wild ruminants only. The known host range of Mycobacterium avium subsp. paratuberculosis (MAP) was recently extended to include non-ruminant wildlife species believed to be exposed to spillover of MAP from infected domestic cattle herds. The aim of the present study was to assess the association between cattle herd MAP infection pressure level and the infection level of a hare population in two dairy farms of southern Chile. Fifty hares from a herd A and 42 hares from herd B were captured and sampled for MAP culture. The results showed a statistically significant association between the cattle herds’ infection prevalence and the hare infection prevalence.     

Reduction in milk yield associated with Mycobacterium avium subspecies paratuberculosis (Map) infection in dairy cows

To assess the profitability of control schemes for Mycobacterium avium subspecies paratuberculosis (Map)-infection implemented in dairy herds, accurate estimates of its production effects are needed. This study aimed at quantifying the variation in milk yield of dairy cows according to their Map-infection status. The cow-status was determined by combining (i) its testing(s)-result(s) (serum ELISA, faecal culture (FC), PCR, Ziehl staining), (ii) the Map-status of its herd, and (iii) its possible vaccination against Map. A total of 15 490 cows in 569 herds located in western France was considered. The effect on test-day milk yield (TDMY) of the cow-status to Map was assessed separately in parity 1, 2 and 3 or more, using mixed linear models, after adjustment for herd-season (random), days in milk and breed. Average TDMY was significantly lower in cows from herds with at least one Map-infected cow (defined as positive herds). Individual TDMY showed a reduction ranging from 1.58 to 3.30, 2.03 to 2.51, 5.36 to 7.20 kg/day (P < 0.001) depending on parity for unvaccinated cows and testing ELISA-positive, PCR- or FC-positive, and Ziehl-positive, respectively, compared to cows in Map-free herds. The loss in milk yield increased with increased parity in ELISA-positive and Ziehl-positive cows. Cows that were both tested ELISA-positive and vaccinated had a smaller loss in TDMY than those that were unvaccinated. The estimates from this study can be used to further assess the economic impact associated with Map-infection in dairy herds or to help in the culling decisions regarding infected cows.     

In vivo kinetics of peripheral cellular immune responses in Mycobacterium avium subspecies paratuberculosis (MAP) infected and vaccinated goats

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (ParaTB) also known as Johne’s disease (JD) in ruminants, which is characterized by chronic intestinal inflammation. A similar counterpart has been observed in the form of Crohn's disease in humans. The present study is the first trail in goats to understand the peripheral cellular immune responses following experimental MAP infection and vaccination. Fifteen apparently healthy male kids (3–6 months old) of Barbari breed were included in this study. In the experimental study, 5 kids were infected with ‘S 5’ strain of MAP (“Indian Bison Type”), 5 were vaccinated (Indigenous Vaccine) against MAP infection (Singh et al., 2007) and the remaining 5 kids were uninfected and non-vaccinated controls. Kids were observed for a period of 180 days post exposure (infection and vaccination) and were tested for development of infection. Cellular immune responses (in blood) were recorded post-exposure by three assays. We measured the frequencies of CD4 and CD8T cells, estimated plasma IFNγ and TNα and in the third assay, in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) from vaccinated, infected and controls were examined in response to polyclonal stimulation. The frequencies of peripheral CD4 and CD8T cells were comparable in control, infected and vaccinated animals except around day 49 post-infection where MAP infected animals showed a trend towards significantly reduced frequencies of CD4 T cells compared to apparently healthy controls. Significantly reduced plasma TNFα levels were also observed in infected animals compared to vaccinated animals,during the course of infection. Diminished levels (although non significant) of TNFα were observed in the supernatants from polyclonally stimulated PBMCs at around day 49 post infection. It is conceivable that the diminished cellular immune responses may coincide with an impairment (immune exhaustion) of perhaps antigen-specific CD4T cells that might, in the course of infection, contribute to the progressive nature of caprine paratuberculosis.     

Goat paratuberculosis in Chile: first isolation and confirmation of Mycobacterium avium subspecies paratuberculosis infection in a dairy goat.

In October 2004, 41 goats > 2 years old from a Saanen dairy goat herd located in Purranque County, 10th Region, Chile, were sampled and tested for paratuberculosis. While collecting samples it was observed that several goats were thin and emaciated. One goat was sufficiently debilitated to warrant humane euthanasia. This animal was brought to the Veterinary School at the Universidad Austral de Chile for necropsy. The goat selected for necropsy was a 12-year-old doe. The animal showed classical clinical signs of caprine paratuberculosis: emaciation despite willingness to eat, dry and rough hair coat, and no evidence of diarrhea. Gross pathology and histopathology of the necropsied goat were consistent with paucibacillary paratuberculosis. Bacteriology, serology, and PCR confirmed the diagnosis. This is the first published report of goat paratuberculosis in Chile confirming a case of caprine paucibacillary paratuberculosis.     

No evidence that wild red deer (Cervus elaphus) on the Iberian Peninsula are a reservoir of Mycobacterium avium subspecies paratuberculosis infection

The potential role of red deer (Cervus elaphus) as a reservoir of Mycobacterium avium subspecies paratuberculosis (MAP) infection is largely unknown. A total of 332 wild red deer were investigated using post-mortem examination, bacteriology and serology. Only three animals (1.12%) were found to have lesions on histopathological examination and no MAP bacteria were recovered on culture. The results suggest it is unlikely that wild red deer make a significant contribution to the maintenance of MAP infection in the region. The cross-reactivity of the ELISAs used indicates this diagnostic modality is ineffective in the detection of MAP infection in this species. The implications of these results for the control of this important pathogen in both livestock and wildlife are discussed.     

Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review

Mycobacterium avium subspecies paratuberculosis (Map) is an important pathogen that causes a chronic, progressive granulomatous enteritis known as Johne’s disease or paratuberculosis. The disease is endemic in many parts of the world and responsible for considerable losses to the livestock and associated industries. Diagnosis and control are problematic, due mostly to the long incubation period of the disease when infected animals show no clinical signs and are difficult to detect, and the ability of the organism to survive and persist in the environment. The existence of phenotypically distinct strains of Map has been known since the 1930s but the genetic differentiation of Map strain types has been challenging and only recent technologies have proven sufficiently discriminative for strain comparisons, tracing the sources of infection and epidemiological studies. It is important to understand the differences that exist between Map strains and how they influence both development and transmission of disease. This information is required to develop improved diagnostics and effective vaccines for controlling Johne’s disease. Here I review the current classification of Map strain types, the sources of the genetic variability within strains, growth characteristics and epidemiological traits associated with strain type and the influence of strain type on infection and pathogenicity.     

Immune response to and faecal shedding of Mycobacterium avium ssp. paratuberculosis in young dairy calves, and the association between test results in the calves and the infection status of their dams

The objectives of this study were to: (i) evaluate the results of an intradermal skin test, a modified IFN-gamma test, and a commercial ELISA in commercially raised dairy calves at 2, 4, 6 and 8 months of age relative to faecal shedding of Mycobacterium avium ssp. paratuberculosis (MAP); (ii) determine the proportion of 8-month-old calves shedding MAP in faeces as detected by culture and One Tube Nested Polymerase Chain Reaction (OTSN-PCR) and (iii) explore the association between results of tests described above in the calves and the Paratuberculosis (PTB) status of their dams as determined by faecal culture and/or serology. The study calves belonged to two dairy herds with different risk of exposure to MAP (high and low) and were enrolled based on their dam's ELISA results prior to calving. Approximately 3% of the calves were shedding MAP in faeces at 8 months of age. No agreement was observed among the evaluated immunity-based tests or between the immunity-based tests and the detection of MAP in faeces. Although no association was observed between the infection status of the dam and the results from the IFN-gamma and skin tests on the calves, there is an indication that calves born from dams that were faecal shedders might be at a higher risk of testing positive to the IFN-gamma test at 8 months of age. The disagreement among all tests evaluated in this calf cohort suggests that the detection of MAP infection in young stock requires the use of combined multiple tests. The early detection of PTB in calves is a challenge that requires further exploration of new methods to confirm infection status. These new testing methods should be both affordable and compatible with regular husbandry practices.     

First report of bovine viral diarrhea virus and Mycobacterium avium subspecies paratuberculosis infection in Tibetan sheep (Ovis aries) in Tibetan Plateau,China

Tropical Animal Health and Production - Bovine viral diarrhea virus (BVDV) and Mycobacterium avium subspecies paratuberculosis (MAP) are important pathogens, which cause serious disease in animals....     

Mycobacterium avium subspecies paratuberculosis cultured from the feces of a Southern black rhinoceros (Diceros bicornis minor) with diarrhea and weight loss

Abstract: Mycobacterium avium paratuberculosis (Map) was cultured from the feces of a wild-caught, female, adult Southern black rhinoceros. The animal, which presented with a 4-mo history of diarrhea and weight loss, was prescribed a course of antimycobacterial drugs. The clinical signs resolved, and the feces were repeatedly culture negative thereafter. Although the Rhinocerotidae are likely to be resistant to Johne's disease, this case raises the possibility that they can become transiently infected with the causative organism.     

Evaluation of two absorbed enzyme-linked immunosorbent assays and a complement fixation test as replacements for fecal culture in the detection of cows shedding Mycobacterium avium subspecies paratuberculosis.

Control of paratuberculosis in dairy herds is based on preventing the transmission of Mycobacterium avium subsp. paratuberculosis (Mptb) from cows to calves by management measures, supported by removal of cows excreting these bacteria by the fecal route (Mptb shedders). Fecal culture is the most accurate test for identifying Mptb shedders, but this technique is expensive and takes up to 16 weeks for results to be available. Serologic tests are inexpensive, rapid, and easy to perform. Of serologic tests, the complement fixation test (CFT) and absorbed enzyme-linked immunosorbent assay (ELISA) are the serologic tests used most frequently; the CFT is considered less accurate than the ELISA with respect to sensitivity and specificity. The commonly accepted absorbed ELISA is from the Australian Central Serum Laboratory. However, a European supplier has marketed a second ELISA that is supposed to be more sensitive in detecting Mptb shedders. These 2 absorbed ELISAs, designated ELISA-A and ELISA-B, and an in-house CFT were compared with data from 2 serum panels. The Mptb shedding panel consisted of sera from 198 culture-positive cows from 53 infected herds. The method used for culture of fecal samples was a modified J?rgensen method on individual samples. The Mptb shedder detection rate by the 3 serologic tests ranged from 29.8% to 39.4%. Detection rate for ELISA-A was lower than that for ELISA-B and CFT. For all 3 tests, detection rate was dependent on the level of Mptb shedding and the age of the animals. Detection rates increased as cattle age increased to 4 years. The specificity panel was initially composed of sera from 811 cows randomly selected from 41 herds without clinical paratuberculosis that were negative for Mptb based on whole-herd fecal culture. The modified J?rgensen method for culture was used on pooled fecal samples. Serologic test specificity ranged from 93.4% to 99.8%. The specificity of ELISA-A was higher than that of ELISA-B and CFT. Specificity of ELISA-B between herds was 75-100%. Specificity of CFT between herds was 62-100%. The low specificity of ELISA-B and CFT could not be explained by a higher sensitivity for Mptb-infected cows before onset of shedding, because in the 19 herds with 8 more subsequent negative whole-herd fecal cultures in the 4 years after sampling, specificity was not improved. The insufficient specificity of ELISA-B was not corrected sufficiently by heightening the cutoff value because Mptb shedder detection rate was lowered to 28.9%, equal to that of ELISA-A, and specificity only rose to 97%, much lower than that of ELISA-A. Taking into account the different test characteristics, serologic tests are a cost-effective alternative to fecal culture in high-prevalence herds. For certification programs, only ELISA-A is recommended because in a large number of nonsuspect herds specificity remained almost 100%.     

Crohn's disease and Mycobacterium avium subsp. paratuberculosis: the need for a study is long overdue

The initial suggestion that Mycobacterium avium subsp. paratuberculosis (Map) might be involved in the pathogenesis of Crohn's disease (CD) was based on the apparent similarity of lesions in the intestine of patients with CD with those present in cattle infected with Map, the etiological agent of Johne's disease (JD). Recent investigations have now revealed the presence of Map or Map DNA in blood or lesions from adults and children with CD. Of special interest, Map has also been found in patients with other diseases as well as healthy subjects. The latter observations indicate all humans are susceptible to infection with Map and that, like with other mycobacterial pathogens such as Mycobacterium tuberculosis, infection does not invariably lead to development of clinical disease but rather development of a persistent latent stage of infection where an immune response controls but does not eliminate the pathogen. Limited information has been obtained on the immune response to Map in healthy subjects and patients with CD. Understanding how Map may be involved in the pathogenesis of CD will require a better understanding of the immune response to Map in one of its common hosts as well as healthy humans and patients with CD.     

Comparison of two enzyme-linked immunosorbent assays for serologic diagnosis of paratuberculosis (Johne's disease) in cattle using different subspecies strains of Mycobacterium avium.

Serologic diagnosis of bovine paratuberculosis (Johne's disease) with currently available tests may give false-positive results due to cross-reactions with avian and bovine tuberculosis viruses and other infectious agents. Indirect enzyme-linked immunosorbent assays (ELISA) for detection of antibodies against paratuberculosis based on antigens from Mycobacterium avium subsp. avium (A-ELISA) and M. avium subsp. paratuberculosis (P-ELISA) were compared. Despite an expected higher specificity for M. a. paratuberculosis in the P-ELISA, the 2 antigens were equally suitable for demonstration of antibody to M. a. paratuberculosis in cattle. Receiver operating characteristic (ROC) curves was used to demonstrate the possible antigenic relationship. The area under the curve (AUC) was calculated for each of the 2 ROC curves. The AUC for the P-ELISA ROC curve was 0.9197, and the AUC for the A-ELISA ROC curve was 0.9149, demonstrating a negligible difference in efficiency of the 2 tests (z = 0.182).     

Specificity of four serologic assays for Mycobacterium avium ss paratuberculosis in llamas and alpacas: a single herd study.

An investigation was conducted for Mycobacterium avium ss paratuberculosis infections in a research herd of llamas and alpacas. Herd culture-negative status was established over a 23-month period by screening any individuals with any signs compatible with paratuberculosis (n = 1), high serology values (n = 8), or other health and research related reasons (n = 24). There were no M. avium ss paratuberculosis isolates from radiometric cultures of multiple tissue and fecal samples from these individuals and no known sources of exposure. Paratuberculosis is uncommon in North American llamas and alpacas: only 5 cases were identified after an extensive search of the Veterinary Medical Data Base, diagnostic laboratory records, publication databases, and personal communications. Therefore, serum samples from llamas (n = 84) and alpacas (n = 16) in the culture-negative herd were used to obtain preliminary estimates of test specificity for 3 enzyme-linked immunoassays (ELISAs) and an agar gel immunodiffusion (AGID) assay kit for detecting serum antibodies to M. avium ss paratuberculosis in South American camelids. The ELISAs were modifications of established bovine assays for antibody detection. With provisional cutoffs, ELISA-A had 52 false positives (specificity 48%), ELISA-B had 8 false positives (specificity 92%), ELISA-C had two false positives (specificity 98%), and the AGID had 0 false positives (specificity 100%). The range of ELISA values for culture-positive llamas and alpacas (n = 10) from other herds overlapped the range of values for culture-negative llamas and alpacas. The accuracy of the ELISAs may be improved by using age- and sex-specific cutoffs because uninfected male llamas and alpacas that were older than 1 year had higher values for some tests. These tests can be used for either llamas or alpacas; the protein-G conjugate ELISA (ELISA-B) may be useful for multispecies applications. These assays are best used for rapid presumptive diagnoses of llamas and alpacas with diarrhea and weight loss and as a screening tool for herds known to be exposed to infection. All seropositive results should be confirmed with culture.