Sero-prevalence of bovine Johne's disease in buffaloes and cattle population of North India using indigenous ELISA kit based on native Mycobacterium avium subspecies paratuberculosis 'Bison type' genotype of goat origin

Present pilot study is the first attempt in the country to estimate sero-prevalence of Bovine Johne's disease (BJD) by screening cattle and buffaloes representing large population belonging to farmer's and farm herds in the home tracts (Uttar Pradesh (UP) and Punjab) of Hariana cattle and Murrah buffaloes in North India. Indigenous and in-house plate ELISA kit (using protoplasmic antigen from native Mycobacterium avium subsp. paratuberculosis 'Bison type' strain of goat origin), originally developed for goats and sheep was standardized in bovines and used for screening. For this study, 33 villages of south and west UP were randomly selected and surveyed from 2001 to 2003. There were 7943 farmer's families having 38,251 livestock, including cattle, buffaloes, goats and sheep (per family 4.8% livestock). Numerically, buffaloes and cattle were 54.7% and 22.1%, respectively. Serum samples were collected from 726 animals (4.2% of 16, 981 livestock with 4375 farmer's families) located in 33 randomly surveyed villages. Serum samples (699), submitted to Epidemiology Department of Veterinary College (Punjab Agricultural University, Ludhiana), in the year 2004 by farmer's and organized farm herds (Buffaloes, 372, Cattle, 327), were screened by this ELISA kit. Soluble protoplasmic antigen was prepared from Map (S 5) 'Bison type' strain isolated from a terminally sick goat with Johne's disease. Of the total 1425 bovine (Buffaloes and cattle) serum samples screened using indigenous ELISA kit, sero-prevalence of Johne's disease was 29.0% (28.6% in buffalo and 29.8% in cattle) in Northern India. State-wise sero-prevalence was 31.9% and 23.3% in UP and Punjab, respectively. In UP, of the 601 randomly sampled buffaloes, sero-prevalence was 40.3% (16.6% in young and 40.9% adults) and 25.5% (10.5% in young and 26.3% adults) in south and west UP, respectively. Of the 125 cattle screened, sero-prevalence was 42.6% (nil in young and 44.4% adults) and 30.0% (nil in young and 30.6% adults) in south and west UP, respectively. Of the 699 serum samples screened from Ludhiana, Punjab, sero-prevalence of BJD was 23.0%. Sero-prevalence was 23.3% (12.1% in young and 24.4% in adults) and 26.9% (27.2% in young and 26.8% in adults) in buffaloes and cattle, respectively. High prevalence of BJD in buffaloes in native tract of Murrah breed, and Hariana breed of cattle correlated with poor per-animal productivity and BJD may be the major cause. Indigenous ELISA kit was rapid, economic and sensitive test for large-scale screening of buffaloes and cattle population against incurable BJD.     

Pathogenic 'Bison-type' Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India

Despite low per-animal productivity of ruminants in developing countries, Johne's disease has not been investigated in buffaloes, which are primarily found in these countries. This is due to lack of expertise, diagnostic kits and priority to production diseases like Johne's disease. Presence of pathogenic Mycobacterium avium subspecies paratuberculosis (Map) was investigated by screening of target tissues (mesenteric lymph nodes and large intestine) by culture and IS 900 PCR, in 50 sacrificed buffaloes. Indigenous ELISA kit originally developed for goats and sheep was standardized in buffaloes and used to estimate sero-presence of Map in 167 serum samples representing population of buffaloes in Agra region of North India. In culture, 48.0% buffaloes were positive from 50 tissues each from mesenteric lymph nodes (34.0%) and large intestine (36.0%). IS 900 PCR was standardized using specific primers (150 C and 921) and 229 bp-amplified product was characteristic for Map. Of the 25 mesenteric lymph nodes, 40.0% were positive in IS 900 PCR. Genomic DNA from Map cultures was successfully amplified from all the 24 isolates (100.0%). Map was further genotyped as 'Bison type' using IS 1311 PCR-REA. Culture of tissues showed high presence of Map in target tissues, despite high culling rate in buffalos in view of high demand of buffalo meat. Specific tissue-PCR provided rapid confirmation of Map infection in sacrificed buffaloes. In tissue-PCR, all the cultures were positive as compared to 40.0% detected directly from tissues. ELISA kit using indigenous protoplasmic antigen was highly sensitive as compared to commercial antigen in detecting Map infection therefore, could be used as 'Herd Screening Test' in buffaloes against Johne's disease. This pilot study first time reports a highly pathogenic 'Bison-type' genotype of M. avium subspecies paratuberculosis from the riverine buffaloes (Bubalus bubalis) of Agra region in North India.     

Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry

Mycobacterium avium subspecies paratuberculosis is considered as one of the most serious problems affecting the world’s ruminant industry due to its significant impact on the global economy and the controversial issue that it may be pathogenic for humans. M. avium subspecies paratuberculosis is the causative agent of Johne’s disease in animals and might be implicated in cases of human Crohn’s disease. We provide an insight into M. avium subspecies paratuberculosis from some bacteriological, clinical, and molecular epidemiological perspectives.     

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.     

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.     

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.     

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.     

Seroprevalence of Mycobacterium avium subspecies paratuberculosis in Korean black goats (Capra hircus aegagrus)

In total, 582 sera from 116 black goat herds were analyzed by a commercially available ELISA kit to monitor the seroprevalence of Mycobacterium avium subspecies paratuberculosis (Mpt) in Korean black goats (Capra hircus aegagrus). The mean number of goats sampled per herd was 5.11, 4.66, and 5.38 for the northern, central, and southern regions of Korea, respectively. The apparent regional prevalence of Mpt was estimated at 18.2-38.2% and 4.6-15.3% for herds and goats, respectively. The Mpt-positive goats were predominantly detected in the south (n=28), compared to either the northern (n=9) or central (n=11) regions (chi=14.459, P<0.05). Our findings indicate that Mpt is prevalent among the goat population, but regional variation exists.     

Molecular identification and characterization of Mycobacterium avium subspecies paratuberculosis in free living non-human primate (Rhesus macaques) from North India

In recent years, Mycobacterium avium subspecies paratuberculosis (MAP) has emerged as major animal pathogen with significant zoonotic concerns, worldwide. MAP infection is endemic in domestic and wild ruminant population in India. However, information on MAP infection in free ranging animal species and non human primates is limited. Present study aimed to estimate the status of MAP infection in free living Rhesus macaques suffering with multiple clinical conditions (coughing and loose stool). A total of 25 stool samples were collected from six colonies of Rhesus macaques from Mathura region (North India) and screened for the presence of MAP, using microscopic examination and IS900 PCR, directly from stool samples. PCR positive DNA samples were further genotyped using IS1311 PCR-restriction enzyme analysis. Of the 25 stool samples, 10 (40.0%) and 2 (8.0%) were positive for MAP using microscopic examination and direct IS900 PCR, respectively. IS900 PCR positive DNA samples were genotyped as ‘Indian Bison type’, which is a major MAP genotype infecting domestic and wild ruminant species and human beings in India. Prevalence of MAP in Rhesus macaques (Indian monkeys) was moderately high and confirmed interspecies sharing of MAP between domestic livestock and non-human primates. Presence of MAP in non-human primates, support the etiological role of MAP in inflammatory bowel disease patients. Indian monkeys may serve as model for understanding the role of non-human primates in sustenance, transmission and pathogenesis of MAP infection.     

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.     

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.     

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.     

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.     

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.