Mycobacterium paratuberculosis and milk

The possibility that milk from cattle with Johne's disease could be a potential vehicle of transmission of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) to humans has been the focus of a UK government-funded research programme at Queen's University, Belfast since 1993. The main findings of this research programme are reported and practical advice about the most appropriate methods for the isolation/detection of this organism in milk is given. The findings of several milk surveys during which optimised sensitive detection methods were employed (decontamination with 0.75% cetyl pyridinium chloride for 5 h prior to culture and a novel immunomagnetic PCR technique) have revealed that detectable levels of M. paratuberculosis are present in bulked raw cows' milk in the UK at both the farm level and at dairy processing plants prior to pasteurisation. Furthermore, results of three different experimental approaches to assess the effect of pasteurisation time/temperature conditions on the viability of M. paratuberculosis (laboratory pasteurisation studies, a national survey of commercially pasteurised milk, and processing of naturally infected milk through commercial-scale pasteurising plant) provide firm evidence that this organism is capable of surviving commercial milk pasteurisation on occasion. Hence, both raw and pasteurised cows' milk are potential vehicles of transmission of M. paratuberculosis to humans.     

Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method.

The ability of Mycobacterium avium subsp. paratuberculosis to survive in bovine monocytes was studied using radiometric (BACTEC) culture, standard plate counting and microscopic counting of acid-fast stained monocyte monolayers. Results of microscopic counts sharply contrasted with results of viable counts determined both by plate counting and radiometric counting. We observed an early phase (the first 6 d after in vitro infection) of intracellular bacillary growth, followed by a later phase of mycobacteriostasis or killing (up to 12 d after in vitro infection) in monocytes from non-infected cows. The data suggest that multiplication and death of M. avium subsp. paratuberculosis occur simultaneously in bovine monocytes infected in vitro. Using the BACTEC method, we compared the ability of bovine monocytes from normal cows and cows infected with M. avium subsp. paratuberculosis and showing evidence of a strong Thl-like cellular immune response to ingest and inhibit the intracellular growth of M. avium subsp. paratuberculosis. There was a trend toward greater phagocytosis and faster killing of Mycobacterium avium subsp. paratuberculosis by monocytes from the infected, immune responder cows. However, the observed numbers of viable M. avium subsp. paratuberculosis at each time after monocyte infection were not significantly different between normal and infected cows.     

Antigenic relationship between Mycobacterium paratuberculosis and Mycobacterium avium

Four prototype strains of Mycobacterium paratuberculosis contained the type-specific glycopeptidolipid antigen of serovar 8 of the M avium complex. This glycolipid was distinguished by a 4,6-(1'-carboxyethylidene)-3-O-methyl-beta-D-glucopyranosyl terminal unit. Of 59 low-passage, field isolates of M paratuberculosis, 2 contained this antigen, and these 2 isolates were indistinguishable from M avium serovar 8. However, most M paratuberculosis isolates had no characteristic surface glycopeptidolipid. Seemingly, M paratuberculosis, long regarded as a single species and the causative agent of bovine paratuberculosis, is not a homogeneous taxon. Most isolates obtained from infected ruminants may be antigenically defective, variants of M avium and, thereby, more successful pathogens.     

Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle.

Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical paratuberculosis in dairy cattle using fecal specimens from a repository of paratuberculosis specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis, by conventional or radiometric culture, from fecal samples or internal organs of dairy cattle without diarrhea or chronic weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. Among 182 infected cattle, RCM and HEY fecal culture and the DNA probe had test sensitivities of 54.4%, 45.1% and 33.5%, respectively. Fecal samples from only 111 of the M. paratuberculosis-infected cows tested positive by at least one of the three tests and these cows were designated as fecal shedders; the remaining 71 were considered to have prepatent infections. Among the 111 M. paratuberculosis fecal shedders, RCM, HEY and the probe detected the organism in 89.2%, 73.8% and 55.0% of the fecal specimens, respectively. Herd prevalence significantly affected the sensitivity of all three diagnostic tests (p less than 0.05) but only affected the fecal shedder detection efficiency of the DNA probe (p less than 0.01). No positive DNA probe results were found on 100 randomly selected fecal samples from cows in four certified paratuberculosis-free herds, thus the DNA probe was 100% specific. Probe analyses could be performed in 24 h or less. Time to complete the culture-based tests was 12 wk for HEY and 7 wk for RCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.     

Comparison of different media for the isolation of small ruminant strains of Mycobacterium paratuberculosis

Two different egg based media, with and without the incorporation of sodium pyruvate, were used to isolate M. paratuberculosis from sheep, goat and cattle samples obtained at our laboratory for two years. Both media were adequate for bovine material, with a slightly improved isolation rate for Herrold's egg yolk medium incorporating sodium pyruvate; however, most of the small ruminant strains grew only on L?wenstein-Jensen medium without sodium pyruvate. Those results point out the need to use different media when working with small ruminant samples and provide further evidence for the existence of different varieties of M. paratuberculosis causing paratuberculosis in cattle and in small ruminants.     

Detection methods of Mycobacterium paratuberculosis

Mycobacterium paratuberculosis is receiving increasingly wider interest of scientific groups worldwide. This slow-growing mycobacterium does not only evoke paratuberculosis--an infectious cattle disease that brings huge economic losses--but it is also regarded as a potential cause of human Crohn;s disease. It is very difficult to diagnose precisely this kind of animal infection in its very early stages, as well as to detect occurrence of M. paratuberculosis cells in the environment, including food of animal origin. This paper reviews currently known and employed diagnostic techniques for M. paratuberculosis cell detection and identification.     

Use of BACTEC radiometric culture method and polymerase chain reaction for the rapid screening of faeces and tissues for Mycobacterium paratuberculosis

SUMMARY The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC - PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.     

The GroES antigens of Mycobacterium avium and Mycobacterium paratuberculosis.

The GroES antigen provokes a strong immune response in human beings with tuberculosis or leprosy. We cloned and sequenced the Mycobacterium avium and Mycobacterium paratuberculosis GroES genes. M. avium and M. paratuberculosis have identical GroES sequences which differ from other mycobacterial species. This supports the current formal designation of M. paratuberculosis as M. avium subsp. paratuberculosis. Immunodominant epitopes from Mycobacterium tuberculosis GroES are conserved in M. avium, but some Mycobacterium leprae epitopes are distinct. GroES is unlikely to be specific as a serologic or skin test reagent, but may be an appropriate component of a broad mycobacterial vaccine.     

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.     

Characterization of Mycobacterium paratuberculosis antigenic proteins

The characterization of a purified antigen from Mycobacterium paratuberculosis, recently made commercially available for use in serodiagnosis by enzyme-linked immunosorbent assay (ELISA), of paratuberculosis in cattle was described. This assay had 89% specificity and 83% sensitivity for M paratuberculosis infection. The protein/polypeptide composition of the purified antigen was compared with that of a crude protoplasmic extract of strain 18 M paratuberculosis used in the agar-gel immunodiffusion test and ELISA and with that of sonicated strain 19698 M paratuberculosis organisms grown on Dorset-Henley synthetic liquid medium. The sonicated M paratuberculosis contained 27 major proteins/polypeptides; the crude protoplasmic extract, 18; and the purified antigen contained 14 proteins/polypeptides, using sodium dodecyl-sulfate polyacrylamide-gel electrophoresis analysis. The serologic reactivity of these proteins/polypeptides were defined, using the enzyme-linked immuno-electrotransfer blot technique. The sonicated M paratuberculosis contained 20 serologically reactive proteins/polypeptides (34,000 to 84,000 daltons); the crude protoplasmic extract contained 3 (37,000 to 45,000 daltons); and the purified extract contained a diffuse polypeptide band (34,000 to 38,000 daltons). Identification by enzyme-linked immuno-electrotransfer blot technique of M paratuberculosis antigens reactive in the ELISA will allow us to further study these antigens in the ELISA to improve sensitivity and specificity of the diagnostic test.     

Mycobacterium paratuberculosis in dairy herds in Alberta

Fifty dairy herds in Alberta were tested for the presence of Mycobacterium paratuberculosis by fecal culture and serum enzyme linked immunosorbent assay (ELISA). Individual sera (1500) were tested for antibodies to M. paratuberculosis by ELISA. Fecal samples were combined in pools of 3 (10 pools/herd) for a total of 500 pools that were cultured for M. paratuberculosis. Thirty cultures, including all 10 pools from 1 herd, were not readable due to fungal contamination. The remaining 470 cultures, representing 49 herds, yielded 16 positive pools (3.4% +/- 2.1%) from 10 herds (20.4% +/- 11.3%). The ELISA of each of the 1500 sera detected 105 (7.0% +/- 2.4%) positive sera and 20 (40.0% +/- 13.6%) positive herds, based on 2 or more individual positive sera in the herd. The true herd-level prevalence, as determined by ELISA, was 26.8% +/- 9.6%. The true herd-level prevalence, as determined by M. paratuberculosis fecal culture, ranged from 27.6% +/- 6.5% to 57.1% +/- 8.3%, depending on whether 1, 2, or all 3 individual fecal samples in the positive fecal pool were culture positive.     

Disseminated Mycobacterium paratuberculosis infection in a cow

A cow with chronic diarrhea and weight loss caused by localization of Mycobacterium paratuberculosis in the intestinal tract (Johne's disease) had gross and microscopic changes indicative of a disseminated infection. A direct association between the remote lesions and the intestinal infection was shown by isolation of M paratuberculosis from renal tissue, detection of intracellular M paratuberculosis antigen(s), using an indirect immunoperoxidase method, and by the characteristic granulomatous nature of the lesions. This case illustrates the potential for extra-intestinal lesions in M paratuberculosis infection of cattle and should cause veterinarians to consider mycobacterial disease when confronted with multinodular lesions of the bovine kidney. The immunoperoxidase method was useful in determining the cause of the inflammatory lesion in which intact organisms were not evident.     

Mycobacterium paratuberculosis and the bovine immune system

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a deadly intestinal ailment of ruminants. Johne's disease is of tremendous economic importance to the worldwide dairy industry, causing major losses due to reduced production and early culling of animals. A highly controversial but developing link between exposure to M. paratuberculosis and human Crohn's disease in some individuals has led to the suggestion that M. paratuberculosis is also a potential food safety concern. As with many other mycobacteria, M. paratuberculosis is exquisitely adapted to survival in the host, despite aggressive immune reactions to these organisms. One hallmark of mycobacteria, including M. paratuberculosis, is their propensity to infect macrophages. Inside the macrophage, M. paratuberculosis interferes with the maturation of the phagosome by an unknown mechanism, thereby evading the host's normal first line of defense against bacterial pathogens. The host immune system begins a series of attacks against M. paratuberculosis-infected macrophages, including the rapid deployment of activated gammadelta T cells, CD4+ T cells and cytolytic CD8+ T cells. These cells interact with the persistently infected macrophage and with each other through a complex network of cytokines and receptors. Despite these aggressive efforts to clear the infection, M. paratuberculosis persists and the constant struggle of the immune system leads to pronounced damage to the intestinal epithelial cells. Enhancing our ability to control this important and tenacious pathogen will require a deeper understanding of how M. paratuberculosis interferes with macrophage action, the cell types involved in the immune response, the cytokines these cells use to communicate, and the host genetic factors that control the response to infection.     

Transitions in immune responses to Mycobacterium paratuberculosis

The host immune response to infection with Mycobacterium paratuberculosis is paradoxical, with strong cell-mediated immune responses during the early, subclinical stages of infection and strong humoral responses during the late clinical stages of the disease. Cell-mediated immune responses modulated by various T cell subsets are essential to provide protective immunity and prevent progression of the disease. Secretion of cytokines by T cell populations serves to activate macrophages to kill ingested M. paratuberculosis as well as activate other T cell subsets to contain the infection. This paper reviews the current knowledge of T cell immune responses in M. paratuberculosis infection based upon clinical studies and research using mouse models.