The pathogenesis and persistence of Mycoplasma bovis (Mb) infection of the respiratory tract is incompletely understood. Cyclooxygenase (COX)-2 is overexpressed during inflammatory responses by different cell types in the lung. This study evaluated COX-2 expression immunohistochemically in the inflammatory lesions of calves with naturally occurring and experimentally induced Mb pneumonia. Experimentally infected lungs showed catarrhal bronchointerstitial pneumonia and varying degrees of peribronchiolar mononuclear cell cuffing. Lesions in calves with spontaneously arising disease included exudative bronchopneumonia and extensive foci of coagulative necrosis surrounded by inflammatory cells. Mb antigen was located in epithelial and inflammatory cells in the airway lumina and surrounding areas of necrosis. COX-2 protein was detected in the lung of all infected calves and was localized to goblet cells, bronchial, bronchiolar and alveolar epithelial cells and macrophages. COX-2 protein was overexpressed during Mb infection and was always associated with areas of pneumonia and with the presence of Mb antigen.

Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.

Aims: To develop a sensitive detection method for Mycobacterium avium ssp. paratuberculosis (Map) in water by modifying and optimizing an existing immunomagnetic separation polymerase chain reaction (IMS-PCR) technique.Methods and Results: Sterile distilled water (50 ml) spiked with 10(6) Map ml(-1) was subjected to either filtration (0.45 mum pore size) followed directly by IS900 PCR (method 1) or centrifugation (2500 g for 20 min) followed by IMS and IS900 PCR (method 2). Method 2 permitted the detection of Map, whereas method 1 did not. Method 2 was then optimized by adding different concentrations of Tween 80 (0.05, 0.1, 0.2, 0.4 and 0.6% v/v) to water samples spiked with Map (10(6)-1 CFU ml(-1)) prior to centrifugation, and assessing the impact of this action on the detection sensitivity of subsequent IMS-PCR. The optimum Tween 80 concentration was found to be 0.4%, which permitted the detection of 10 Map CFU ml(-1) in spiked water samples by IMS-PCR.Conclusions: This method will be used to determine the incidence of Map in water destined for domestic use in future studies.Significance and Impact of the Study: A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.

A large-scale survey of commercially pasteurised cows' milk, and pasteurisation trials using commercial-scale plant (2,000 l/h with turbulent flow) and naturally infected milk, were carried out to determine the efficacy of HTST pasteurisation applied to milk naturally infected with Mycobacterium avium subsp. paratuberculosis (Map). During the milk survey a total of 567 commercially pasteurised milk samples from approved dairy processing establishments throughout the UK were tested for the presence of viable Map. In the pasteurisation trials, naturally infected milk from two local farms was subjected to four different heat treatments (73 degreesC for 15 and 25 s, with and without prior homogenisation) on twelve separate occasions. In both studies, milk samples were subjected to decontamination with 0.75 % (w/v) cetylpyridinium chloride for 5 h at room temperature before culture on Herrold's egg yolk medium and in BACTEC 12B medium. Overall, viable Map was cultured from 10 (1.8 %) of 567 pasteurised milk samples tested during the UK milk survey and from 10 (6.7 %) of 144 pasteurised milk samples tested during the commercial-scale pasteurisation experiments. Surviving Map were isolated from pasteurised milk samples that had been heated at greater than or equal to 72 degreesC for both 15 and 25 s. The results of these two studies provide clear evidence that Map bacteria in naturally infected milk are capable of surviving commercial HTST pasteurisation on occasion. It should be noted that the above studies differ from previous laboratory-scale pasteurisation studies in several respects: no Map were artificially added to milk prior to heat treatment; a larger volume (50 ml) of pasteurised milk was tested thereby increasing the chances of detecting survivors; and 24-72 h elapsed between heat treatment and milk testing potentially giving sub-lethally heat-injured Map cells time to recover viability.

ABSTRACT Over a 17-month period (March 1999 to July 2000), a total of 814 cows' milk samples, 244 bulk raw and 567 commercially pasteurized (228 whole, 179 semiskim, and 160 skim), from 241 approved dairy processing establishments throughout the United Kingdom were tested for the presence of Mycobacterium paratuberculosis by immunomagnetic PCR (to detect all cells living and dead) and culture (to detect viable cells). Overall, M. paratuberculosis DNA was detected by immunomagnetic PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. Confirmed M. paratuberculosis isolates were cultured from 4 (1.6%; 95% confidence interval, 0.04 to 3.1%) and 10 (1.8%; 95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, following chemical decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. The 10 culture-positive pasteurized milk samples were from just 8 (3.3%) of the 241 dairy processing establishments that participated in the survey. Seven of the culture-positive pasteurized milk samples had been heat treated at 72 to 74°C for 15 s; the remainder had been treated at 72 to 75°C for the extended holding time of 25 s. When typed by restriction fragment length polymorphism and pulsed-field gel electrophoresis methods, some of the milk isolates were shown to be types distinct from those of laboratory strains in regular use within the testing laboratory. From information gathered at the time of milk sample collection, all indications were that pasteurization had been carried out effectively at all of the culture-positive dairies. That is, pasteurization time and temperature conditions complied with the legal minimum high-temperature, short-time process; all pasteurized milk samples tested phosphatase negative; and postprocess contamination was considered unlikely to have occurred. It was concluded that viable M. paratuberculosis is occasionally present at low levels in commercially pasteurized cows' milk in the United Kingdom.

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin-biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.

Aims: One possible route of transmission of Mycobacterium paratuberculosis from cattle to humans is via contaminated water supplies. The aim of this work was to determine whether this organism can survive standard water treatment processes.Methods and Results: Two strains of M. paratuberculosis (bovine strain, NCTC 8578 and human strain Linda, ATCC 43015) were subjected to various chlorine concentrations (0.5, 1.0 and 2.0 mug ml(-1)) for 15 and 30 min. Chlorine test solutions were made up in two types of water, sterile water that had been deionized and subjected to reverse osmosis (DRO) and DRO water containing MgCl2, CaCl2, NaHCO3 and bovine serum albumin (0.3% w/v), the latter to mimic conditions the organism would experience in commercial water treatment operations.Conclusions: The data showed that when initial inoculum levels were high (10(6) Cfu ml(-1)) neither M. paratuberculosis strain was completely killed at the free chlorine concentrations and contact times applied. Log(10) reductions in the range 1.32-2.82 were observed. The greatest log(10) reduction in cell numbers (2.82 and 2.35 for the bovine and human strains, respectively) was observed at the highest chlorine concentration (2 mug ml(-1)) and longest contact time (30 min).Significance and Impact of the Study: This work highlights the need for further research into the survival of M. Paratuberculosis during water treatment.

The distribution of cells containing lysozyme, S-100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus-associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3-5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1-6), M. mycoides ssp. capri (goats no. 7-12) and M. capricolum ssp. capricolum (goats no. 13-18). Microscopically, infected animals showed a moderate broncho-interstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3+ T lymphocytes, and the ratio of CD4+:CD8+ cells was > 2. The BALT showed large germinal centres mainly composed of IgG+ B lymphocytes, with numerous S-100+ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG+ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.

AIMS:Four chemical decontamination protocols for milk were compared with respect to mean percentage recovery of spiked Mycobacterium avium subsp. paratuberculosis, minimum detection limit and ease of application. METHODS AND RESULTS:Raw milk spiked with 106 cfu M.a. paratuberculosis was decontaminated prior to culture by: (1) treatment with 0.75% (w/v) hexadecylpyridinium chloride (HPC) for 5 h; (2) and (3) Cornell methods employing brain heart infusion broth containing 0.75% (w/v) and 0.9% (w/v) HPC, respectively; and (4) a C18-carboxypropylbetaine (CB-18) METHOD:The 0.75% HPC method yielded the highest mean percentage recovery of M.a. paratuberculosis (28.7%) and was capable of detecting the lowest number of cells (30 cfu/40 ml). CONCLUSION:Treatment of milk with 0.75% HPC for 5 h was shown to be superior to the other methods for decontaminating milk prior to culture for M.a. paratuberculosis. SIGNIFICANCE AND IMPACT OF STUDY:Certain chemical decontamination protocols are too harsh for application to milk. The "best" decontamination protocol only recovered a fraction of the M.a. paratuberculosis cells present in a milk sample.

Mycobacterium avium subsp. paratuberculosis (MAP) is the known cause of Johne's disease in cattle and has been implicated as a possible cause of Crohn's disease in humans. When present in milk in high numbers the organism has been shown to survive laboratory pasteurisation treatments. This apparent heat resistance was investigated by: (1) using a combined acid fast/viability stain to visualise viable MAP in milk at different stages during pasteurisation; (2) comparing the thermal resistance of clumped and declumped MAP cells; and (3) assessing the lethality of a range of time/temperature heat treatments. The results provide circumstantial evidence that the tendency for the organism to clump contributes to its apparent heat resistance. Processing strategies to reduce the possibility of its survival in commercially pasteurised milk are suggested.

Goats aged 3 months were inoculated with a recent isolate of Mycoplasma agalactiae (five animals) or Mycoplasma bovis (five animals) by a combined (intratracheal+intranasal) route. Two control goats were inoculated by the same route with sterile mycoplasma broth. Animals were killed 14 or 21 days after infection. At necropsy, tracheal and lung tissue was taken for pathological and immunohistochemical examination to determine changes in the lymphocyte subpopulations in the bronchus-associated lymphoid tissue (BALT). Consolidation of the lungs was not observed in any animal. M. agalactiae or M. bovis was recovered from the respiratory tract and lung of all but two infected animals. Both Mycoplasma spp. induced a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells into the alveolar walls. The predominant phagocytic cell in the pulmonary parenchyma and the airways was the macrophage. The main cellular type in the BALT was the CD3(+)T lymphocyte, and the ratio of CD4(+): CD8(+)cells was >1. It is likely that cellular immune mechanisms, through the activation of CD4(+)T lymphocytes, plays a prominent role in the acute and subacute phase of these infections.

AbstractMycobacterium paratuberculosis is the known cause of Johne's disease in cattle and has been implicated as a cause of Crohn's disease in humans. Concern has been expressed that the organism, which is excreted in milk and faeces of infected cattle, may be transmitted via pasteurised milk. This is largely from work that has shown an unacceptable risk of survival of the organism when it is present in raw milk at numbers exceeding 102cfu/ml. Three possible reasons for this apparent heat resistance were investigated viz. use of a milk heating menstruum, presence of a heat resistant sub–population, and the tendency of the organism to form clumps. Heat resistance studies using a combined acid–fast/viability stain, and a comparison of the relative heat sensitivities of clumped and de-clumped M. paratuberculosis cells provided circumstantial evidence that it is the organism's tendency to form clumps which confers the apparent heat resistance. This work casts doubt on the efficacy of current commercial pasteurisation heat treatments (72°C/15s) for the inactivation of M. paratuberculosis. This would be of concern if a link between M. paratuberculosis and Crohn's disease is eventually established.

Molecular biology methods have the potential to be valuable tools in the diagnosis of Johne's disease. However, the direct application of IS900 PCR for the detection of Mycobacterium avium subsp. paratuberculosis (M. ptb) in faeces is hindered by the presence of faecal constituents which are inhibitory to the Tag polymerase enzyme. An immunomagnetic PCR (IMS-PCR) technique recently developed at Queens University to facilitate the selective recovery of M. ptb cells from milk and, at the same time, remove the constituents of milk inhibitory to the PCR reaction, was adapted for use on faecal samples. The sensitivity and specificity of the IMS-PCR assay were evaluated and compared to ZN staining and the 'gold standard' i.e. faecal culture. Inhibitory components in faeces were subsequently removed prior to PCR by diluting faeces (1:5) in PBS supplemented with 0.05%Tween 20 and subjecting it to low speed centrifugation at 300xg/3min, IMS-PCR signals were obtained from faeces spiked with as few as 10 CFU ofM. ptb/g. A blind trial was carried out on faeces (106 samples) from animals of unknown disease status to compare IMS-PCR, faecal smear and faecal culture results. Results showed that IMS-PCR detected more infected samples than the faecal smear and a commensurate number of infected samples as found using the 'gold standard' faecal culture. It is, therefore, suggested that IMS-PCR may aid in the diagnosis of Johne's disease due to its rapid nature and its high specificity and sensitivity.

Raw cow's milk spiked with 10(6) cfu ml(-1) of Mycobacterium paratuberculosis was subjected to heat treatments of 72, 75, 78, 80, 85 or 90 degrees C for 15 s, and 72 degrees C for 20 and 25 s, using laboratory pasteurizing units. Three bovine strains of Myco. paratuberculosis were studied (NCTC 8578, B2 and DVL 943). Each strain was subjected to all the heat treatments indicated on three separate occasions. Although each of the heat treatments achieved a substantial (5-6 log(10)) reduction in numbers of viable Myco. paratuberculosis, small numbers of the organism (4-16 cfu 10 ml(-1)) survived in a proportion of the milk samples at each of the higher temperatures investigated, right up to 90 degrees C for 15 s. A longer holding time of 25 s at 72 degrees C was found to be more effective at inactivating Myco. paratuberculosis. Only one of the three strains studied, B2, yielded small numbers of survivors after heating at 72 degrees C for 20 s, but it was completely inactivated by extending the holding time at 72 degrees C by a further 5 s to 25 s. It was concluded that a longer holding time is more likely to achieve the complete inactivation of Myco. paratuberculosis in milk than a higher pasteurization temperature.

The potential use of a novel immunomagnetic PCR (IMS–PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS–PCR sensitivity could be further increased by initial centrifugation (2500g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100°C for 15 min. It was estimated that the IMS–PCR assay could detect approximately 103 CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 105 CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (106 CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS–PCR and conventional IS900 PCR. IMS–PCR correctly identified 97.5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS–PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.

The efficacy of high-temperature, short-time (HTST) pasteurization (72 degrees C/15 s) when low numbers (< or = 10(3) cfu ml-1) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10(3) cfu ml-1, 10(2) cfu ml-1, 10 cfu ml-1, and 10 cfu 50 ml-1) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14.8% and 10% of HTST-pasteurized milk samples at the 10(3) and 10(2) cfu ml-1 inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3.7% and 6.7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis. No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml-1 or 10 cfu 50 ml-1.

ABSTRACT An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium paratuberculosis cells from milk. Rabbit polyclonal antibodies against radiation-killed intact M. paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti- M. paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. paratuberculosis as determined by slide agglutination, and microscopic examination confirmed that M. paratuberculosis cells attached to the IMB. The IMB were found to have a maximum binding capacity of 10 4 to 10 5 CFU of M. paratuberculosis . Studies showed that IMS selectively recovered M. paratuberculosis from inoculated milk containing as few as 10 CFU of M. paratuberculosis per ml when 10 μl of IMB (ca. 10 6 beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline–0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation of M. paratuberculosis from a milk sample relies on chemical decontamination, followed by culturing on Herrold’s egg yolk medium, which must be incubated at 37°C for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of M. paratuberculosis in milk when it is used in conjunction with end point detection methods, such as IS 900 PCR or an enzyme-linked immunosorbent assay.

A pool of three monoclonal antibodies, 3H12, 6D11 and 2A3, inhibited the in vitro growth of 12 Mycoplasma mycoides subspecies mycoides small colony (MmmSC) strains and seven Mycoplasma mycoides subspecies mycoides large colony (MmmLC) strains. This test did not cross-react with other 16 Mycoplasma mycoides cluster and nine non-Mycoplasma mycoides cluster strains representing 13 different species. Although MmmSC was not differentiated from MmmLC, the monoclonal antibodies distinguished both M mycoides subspecies mycoides biotypes from the other Mycoplasma species tested in contrast to results with polyclonal antisera.

A novel staining method employing tetrazolium reduction in combination with the auramine O fluorescent acid-fast stain is described. It was developed in order to assess the viability and clumping of Mycobacterium paratuberculosis cells in buffer (PBS) or milk at different stages during heating at holder pasteurization temperature (63.5 degrees C). The staining procedure was as follows: (1) an aliquot of milk or PBS (1 ml) containing M. paratuberculosis was added to 0.5 ml of a pre-warmed 0.2% (w/v) solution of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) and incubated in a waterbath at 37 degrees C for 5 hr (2) three drops of the INT-treated milk or PBS were transferred to a microscope slide and heat-fixed; (3) smears were stained with auramine O, decolorized with acid-alcohol and counterstained with methylene blue. This staining procedure represents ct simple and rapid method of assessing the viability of M. paratuberculosis which is notoriously slow-growing by conventional methods. Pt was observed that M. paratuberculosis cells which occurred as single cells in PBS or milk were inactivated more readily during heating than cells present in clumps. At heating times corresponding to the 'tail' regions of the thermal inactivation curves, any viable cells observed were always located within clumps of predominantly dead cells. These observations suggest that M. paratuberculosis cells within clumps may be being protected from the lethal effects of heat by other cells surrounding them, and provide some evidence that the non-linear thermal inactivation kinetics observed for M. paratuberculosis during pasteurization may result because of cell clumping.