Isolation of Mycobacterium paratuberculosis from sheep and cattle in Iceland.

Isolation of Mycobacterium paratuberculosis from sheep and cattle in Iceland. Acta vet. scand. 1979, 20, 191–199. — Culture experiments concerning the Icelandic variant of Mycobacterium paratuberculosis are described. Various decontaminating agents and culture media were employed and the colonial morphology of freshly isolated strains on different media described. The growth rate and culture requirements are compared with those of the Norwegian goat-pathogenic variant of M. paratuberculosis. For primary isolation modified Herrold’s medium gave the best results. However, on all the various culture media used, the growth of the Icelandic variant was much more sporadic than that of the Norwegian goatpathogenic variant. It is concluded that bacteriological culture is not useful for the diagnosis of Johne’s disease caused by the Icelandic variant of M. paratuberculosis.     

Granulomatous enteritis following oral inoculation of newborn rabbits with Mycobacterium paratuberculosis of bovine origin.

To assess the rabbit as a model for the study of paratuberculosis infection, two groups of newborn rabbits were orally inoculated at one to two days of age with Mycobacterium paratuberculosis (ATCC 19698 or field strain 22206) and compared to uninoculated controls. Nine of thirteen rabbits (69%) inoculated with ATCC 19698, and all three rabbits inoculated with 22206, experienced episodes of intermittent diarrhea starting four months after inoculation. Multifocal granulomas containing acid-fast organisms were observed in the sacculus rotundus and vermiform appendix of the cecum in three of nine rabbits (all with diarrhea) that had been inoculated with ATCC 19698. Although M. paratuberculosis was not recovered from inoculated rabbits when fecal cultures were incubated three months in vitro, a slow-growing mycobactin-dependent form of Mycobacterium was recovered from feces and ileal tissue after incubation for 11-15 months. Reduced feed intake, body weight loss and reduced abdominal fat at necropsy, were not observed. Epithelial transport function across the distal ileum in vitro was not altered nine months subsequent to inoculation. Diarrhea and the histological lesions indicate that the rabbit may be a useful model for the study of paratuberculosis infection.     

Granuloma development in cattle after intratonsilar inoculation with Mycobacterium bovis

OBJECTIVE: To examine the temporal development of tuberculous lesions in cattle inoculated with Mycobacterium bovis. ANIMALS: 15 mature crossbred cows obtained from a herd with no history of M bovis infection. PROCEDURE: Inoculation of cattle was done by intratonsilar instillation of 1.48 X 10(5) to 5.4 X 10(7) colony-forming units of M bovis strain 2045T. At 3 to 4 hours, 4 weeks, 6 weeks, and 8 weeks after inoculation, tissues were examined for gross and microscopic lesions and processed for isolation of M bovis. RESULTS: Retropharyngeal lymph nodes from cattle examined 4 weeks after inoculation contained microgranulomas consisting of aggregates of macrophages with few neutrophils. Retropharyngeal lymph nodes from all cattle examined 6 and 8 weeks after inoculation contained multiple, large, coalescing granulomas consisting of central areas of necrosis with mild fibrosis, numerous macrophages, lymphocytes, plasma cells, multinucleated giant cells, and neutrophils. Three of 8 cattle examined 6 or 8 weeks after inoculation had lesions in nonretropharyngeal sites with morphologic characteristics similar to that seen in retropharyngeal lymph node granulomas from cattle examined 4 weeks after inoculation. CONCLUSION: Granulomas can develop in draining lymph nodes of cattle in as little as 4 weeks after inoculation via intratonsilar instillation of M bovis. Intralesional morphologic changes between 4 and 6 weeks after inoculation indicate an increase in cellular chemotaxis and differentiation. Dissemination of bacteria to distant sites most likely was by lymphatic and hematogenous routes after establishment of the primary infection in retropharyngeal lymph nodes.     

Isolation of Mycobacterium paratuberculosis from washed bovine ova after in vitro exposure

To establish whether Mycobacterium paratuberculosis could be cultured from Dulbecco phosphate-buffered saline solution (DPBSS) and to test 3 sampling methods, DPBSS supplemented with 2% fetal bovine serum was inoculated with M paratuberculosis at concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units/ml. The inoculated media was sampled after mixing, after centrifugation, and after centrifugation and decontamination with 0.75% hexadecylpyridinium chloride. The samples were inoculated onto 3 slants of Herrolds egg yolk medium supplemented with sodium pyruvate and mycobactin J and 1 slant without mycobactin J. Mycobacterium paratuberculosis was isolated following all 3 sampling methods for all concentrations. Treatment with hexadecylpyridinium chloride decreased the number of colonies isolated. To test the efficacy of a 10-step wash procedure for removing M paratuberculosis from bovine ova, washed zona pellucida intact bovine ova were incubated in DPBSS supplemented with 2% fetal bovine serum containing concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units of M paratuberculosis/ml for 12 hours at 22 C. Ten zona pellucida intact ova were removed from each concentration and washed by passing through 10 changes of DPBSS supplemented with 15% fetal bovine serum. The media from each wash step was inoculated onto slants of Herrolds egg yolk medium. The ova were included with the tenth wash step. Mycobacterium paratuberculosis was isolated from 1 of 10 tenth-wash steps at the 10(4) concentration and 5 of 10 tenth-wash steps at 10(3).     

Mycobacterium avium subspecies paratuberculosis in bioaerosols after depopulation and cleaning of two cattle barns

Settled dust samples were collected on a commercial dairy farm in the Netherlands with a high prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) (barn A) and on a Dutch experimental cattle farm (barn B) stocked with cattle confirmed to be MAP shedders. Barns were sampled while animals were present, after both barns were destocked and cleaned by cold high-pressure cleaning, and after being kept empty for two weeks (barn A) or after additional disinfection (barn B). MAP DNA was detected by IS900 real-time PCR and viable MAP were detected by liquid culture. MAP DNA was detected in 78 per cent of samples from barn A and 86 per cent of samples from barn B collected while animals were still present. Viable MAP was detected in six of nine samples from barn A and in three of seven samples from barn B. After cold high-pressure cleaning, viable MAP could be detected in only two samples from each barn. After leaving barn A empty for two weeks, and following additional disinfection of barn B, no viable MAP could be detected in any settled dust sample.     

Immune reactions in cattle after immunization with a Mycobacterium paratuberculosis vaccine and implications for the diagnosis of M. paratuberculosis and M. bovis infections

After immunization of four calves with a live modified Mycobacterium paratuberculosis vaccine the course of the humoral and cell mediated immune reactions was studied during a 2-year clinical investigation. Furthermore, the possibility of shedding of the vaccine strain and the influence of the vaccination on the tuberculin skin test was determined. In addition to standard procedures recently developed diagnostic methods (antibody enzyme-linked immunosorbent assay, interferon-gamma test, polymerase chain reaction) were used. A cell-mediated immune reaction, reflected in an increased, specifically induced, interferon-gamma production developed much earlier (1-2 weeks post-immunization) than humoral immunity (8-16 weeks post-gamma immunization). While the increase in antibody titres was transient, declining to extremely low levels 48-60 weeks post-immunization, cell-mediated immunity remained detectable until the end of the investigation. Spread of the vaccine strain into the body and shedding were never detected during the whole course of the study except for one colon site in one calf. As late as 2 years after vaccine application positive or doubtful skin reactions against M. bovis purified protein derivative were measured, reflecting possible interference of the immunization with the diagnosis of bovine tuberculosis. At the end of the investigation, a positive cell-mediated immune reaction was detected the control animal although clinical, pathological and bacteriological examinations gave no indication for a mycobacterial infection.     

Paratuberculosis in pigs. Experimental infection by oral administration of Mycobacterium paratuberculosis

Oral administration of Mycobacterium paratuberculosis to 61 pigs resulted in the development of caseous lesions of the mesenteric lymph nodes in 27.9 % of the animals. Positive results were found by bacterioscopy in 31.1 %, by culture in 60.7 % and by histological examination in 42.6 % of the animals.Histological changes typical of infection with M. paratuberculosis were found in the ileum in eight pigs. These changes, which consisted of infiltrations with epithelioid cells, were usually limited to the Peyer patches, but in one case such infiltrations were also present in the propria mucosae, similarly as in paratuberculosis in cattle.     

Agreement between three ELISAs for Mycobacterium avium subsp. paratuberculosis in dairy cattle

During a 10-month period in 1999, 994 serum and tissue samples were collected from dairy cows at slaughter in eastern Canada. The sources of these cattle were from all four Atlantic Canadian provinces along with some cows from the state of Maine. The sera were used to assess the agreement of three commercially available ELISAs for Mycobacterium avium subsp. paratuberculosis. Two ELISAs were indirect absorbed ELISAs licensed for use in North America, the third was an indirect non-absorbed ELISA licensed for use in Europe. Overall, there was poor agreement between the three ELISAs. The highest and lowest kappa values were 0.33 and 0.18, which is fair and poor agreement, respectively. However, when only tissue culture-positive cattle were compared, the ELISAs had better agreement (kappa=0.37-0.51). The proportions of positive tests, however, were significantly different among the three ELISAs. The poor agreement among the three ELISAs is as concerning as the fact that these tests have low sensitivity. The implications are greatest when the tests are used at the cow level to make individual animal decisions, which is not the recommended method on the product labels. At the cow level, if the result obtained from one ELISA is positive, using a different ELISA in a pre-clinical animal has a high likelihood of giving a different result due to low predictive values of positive test results.     

Different Mycobacterium avium subsp. paratuberculosis MIRU-VNTR patterns coexist within cattle herds

A better understanding of the biodiversity of Mycobacterium avium subsp. paratuberculosis (MAP) offers more insight in the epidemiology of paratuberculosis and therefore may contribute to the control of the disease. The aim of this study was to investigate the genetic diversity in bovine MAP isolates using PCR-based methods detecting genetic elements called Variable-Number Tandem Repeats (VNTRs) and Mycobacterial Interspersed Repetitive Units (MIRUs) to determine if multiple MAP strains can coexist on farms with endemic MAP infection. For 52 temporal isolates originating from infected cattle from 32 commercial dairy herds with known trading history, MIRU-VNTR analysis was applied at 10 loci of which six showed variation. Within the group of 52 isolates, 17 different MIRU-VNTR patterns were detected. One MIRU-VNTR pattern was found in 29 isolates, one pattern in four isolates, one pattern in three isolates, two times one MIRU-VNTR pattern was found occurring in two isolates, and 12 patterns were found only once. Eleven herds provided multiple isolates. In five herds a single MIRU-VNTR pattern was detected among multiple isolates whereas in six herds more than one pattern was found. This study confirms that between dairy farms as well as within dairy farms, infected animals shed MAP with different MIRU-VNTR patterns. Analysis of trading history and age within herds indicated that cows born within the same birth cohort can be infected with MAP strains exhibiting variations in the number of MIRU-VNTR repeats. These data indicate that such multiple genotypes of MAP can coexist within one herd.     

Survival of Mycobacterium avium subsp paratuberculosis in amitraz cattle dip fluid

OBJECTIVE: To determine the survival time of Mycobacterium avium subsp paratuberculosis in amitraz-based cattle dip fluid derived from an active dip site in northern New South Wales. PROCEDURE: Following inoculation of triplicate 5 L containers with faeces (0.5 g/L) from a clinical case of bovine paratuberculosis, samples collected up to 8 weeks after inoculation were examined by conventional and radiometric culture. M a paratuberculosis colonies were enumerated on solid media. RESULTS AND CONCLUSIONS: M a paratuberculosis survived in amitraz cattle dip fluid for up to 2 weeks, but not 3 weeks. Where 1% of solids in dip fluid is derived from a clinical case of paratuberculosis, dip fluid may contain viable M a paratuberculosis for at least 2 weeks. These findings have implications for the management of cattle dip sites.     

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

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

Isolation of Mycobacterium paratuberculosis from the uterine flush fluids of cows with clinical paratuberculosis

Mycobacterium paratuberculosis was isolated from the uterine flush fluids obtained from 3 to 4 cows with clinical paratuberculosis. Four cows with clinical paratuberculosis were subjected to uterine lavage, using an established embryo recovery technique, and the recovered fluids were cultured for M paratuberculosis. Mycobacterium paratuberculosis has previously been demonstrated to adhere to bovine ova. Embryos within the uterus of a superovulated cow infected with M paratuberculosis could be exposed to the organism.     

Mycobacterium avium subsp. paratuberculosis in wild animal species and cattle in Styria/Austria

Infections with Mycobacterium ovium ssp. paratuberculosis (M. paratuberculosis) are increasingly recognised worldwide. In addition to an increased prevalence of paratuberculosis in Austrian cattle herds, recent years have also shown a rise in infections with M. paratuberculosis in wild red and roe deer, chamois and mouflon. During the period from June 2002 to September 2004, mesenteric lymph nodes were taken from a total of 483 wild animals hunted or found dead and from 338 deceased cattle. Samples were analysed using PCR and cultivation methods. In the case of pathomorphological changes or anamnestic indications, investigations also included an analysis of organ samples (e.g. liver, lung) or foetuses. The tests revealed that 129 wild animal samples (red deer, roe deer, chamois, mouflon, fallow deer, ibex, foxes, mountain hare, yellow-necked field mouse, and capercaillie) contained M. paratuberculosis. The major symptoms in the wild aninodes. Evidence of diarrhoea was only observed in about 15% of the positive cases. The study for the first time provided evidence of intrauterine transmission of M. paratuberculosis in red deer (3 cases) and chamois (1 case) and succeeded in the isolation of the pathogen from the liver, lung and subcutaneous granulomas of wild animals. Of the total of 338 mesenteric lymphnodes of cattle from 303 herds, 80 samples from 77 herds tested positive for paratuberculosis. Twenty-two wild animal and 3 cattle isolates have so far been molecularly typed using IS900-RFLP and RAPD analyses in order to prove epidemiological relationships between occurrences in cattle and wild animals. The increase of paratuberculosis in wild animal species is assumed to have been caused by the purchase of animals, a strong increase in suckler cow farming (cow-calf herds) with a concentration of pathogens in the environment and by inadequate feed hygiene for wild animals.     

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis was evaluated. The effects of infection with M. paratuberculosis on cytokine production may influence immune regulation at the site of colonization, resulting in the chronic inflammatory state associated with the latter stages of this disease. Ileal samples were obtained at necropsy from noninfected control cows (n=8) and clinically infected cows (n=7) and processed for immunohistochemistry and in situ hybridization. Cows infected with M. paratuberculosis were in the latter stages of disease with clinical signs such as weight loss, watery diarrhea, and inappetence. Among cytokines we studied, interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma) were expressed significantly more in infected animals than in noninfected control animals. The expression of tumor necrosis factor-alpha (TNF-alpha), however, was not different between the two groups of cattle. In addition, immunohistochemical staining demonstrated that the number of resident macrophages in the ileum of infected animals was three times greater than that of noninfected cows. In contrast to this, ileal tissues from noninfected control animals contained 1.5 times more neutrophils than the ileal tissues from cows infected with M. paratuberculosis. These data demonstrate that localized ileal cytokine production is different between cows chronically infected with M. paratuberculosis and noninfected control cows.     

Survival of Mycobacterium paratuberculosis in slurry.

Cattle and swine slurry and a mixture of equal parts of both, was mixed with a culture of M. paratuberculosis, 0.1 mg per ml (1 mg = 33 X 10(6) viable units) and stored under anaerobic conditions at 5 degrees and 15 degrees C. At 5 degrees C the survival time for M. paratuberculosis was 252 days in all 3 kinds of slurry, and at 15 degrees C it was 98 days in cattle slurry, 182 days in swine slurry, and 168 days in mixed slurry.     

Transitions in diagnostic tests used for detection of Mycobacterium avium subsp. paratuberculosis infections in cattle

Diagnosis of infections with Mycobacterium avium subsp. paratuberculosis (MAP) is difficult due to a long incubation period and lack of tests which can accurately predict the future status of animals. Early detection of infectious animals is necessary to reduce transmission of MAP. The objective of this study was to determine the time from first detection of MAP-antibodies in milk ELISA to start of MAP shedding, for animals with various shedding patterns. An observational longitudinal study was carried out over 3 years. A total of 24,076 milk and 10,074 faecal samples were obtained from 1906 cows and tested using ELISA and FC, respectively. Cows were classified into 5 shedding groups based on their repeated FC: non-shedders (NS; n=1512 cows, 79.3% of total), transient (TS; n=36, 1.9%), intermittent (IS; n=137, 7.2%), low (LS; n=143, 7.5%), and high shedders (HS; n=78, 4.1%). Results showed that 5% of TS, 30% of IS, 60% of LS and 70% of HS were ELISA-positive at the date of first positive FC, and many HS (28%) and LS (14%) were positive >or=1 year prior to first detection of shedding. FC confirmed shedding within the first year after the positive ELISA in 10% of 328 cows with fluctuating ELISA compared with 35% of 445 cows with the last 2 or more ELISAs positive. To conclude, MAP-antibodies were generally detected prior to start of bacterial shedding, with difference between the various patterns of shedding, and a positive ELISA was useful for predicting that an animal would subsequently become infectious.