Gary F. Yates

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk.

ABSTRACT A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity ofMycobacterium avium subsp.paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72°C in duplicate trials. No strains survived at 72°C for 15 s; and only one strain survived at 69°C. Means of pooled Dvalues (decimal reduction times) at 63 and 66°C were 15.0 ± 2.8 s (95% confidence interval) and 5.9 ± 0.7 s (95% confidence interval), respectively. The mean extrapolatedD72°C was <2.03 s. This was equivalent to a >7 log10 kill at 72°C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6°C. These five strains showed similar survival whether recovery was on Herrolds egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johnes disease. After heating at 72°C for 15 s, the minimum M. avium subsp.paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosisin retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2° above 72°C.

Paratuberculosis in Farmed Deer: Case Reports and DNA Characterization of Isolates of Mycobacterium Paratuberculosis

Mycobacterium paratuberculosis was isolated from farmed deer in New Zealand on 21 occasions over a 7-year period. The number of cases increased during the last 3 years of the study. Clinical paratuberculosis was observed in 5 deer, 3 other cases came from grossly normal animals that reacted to a tuberculin skin test, and the remaining 13 animals had tuberculous lesions that were identified at meat inspection. Pathologic features of the lesions in these 13 cases included necrosis and mineralization in lymph nodes draining the gastrointestinal tract. The histologic changes in these lesions were very similar to those caused by Mycobacterium bovis and members of the M. avium complex. Characterization of 20 of the isolates of M. paratuberculosis by restriction endonuclease analysis and DNA hybridization revealed that 3 of these isolates were identical to New Zealand sheep isolates and 17 were the same as cattle isolates. The source of the cervine infections was not determined.

Route of BCG administration in possums affects protection against bovine tuberculosis.

The Australian brushtail possum (Trichosurus vulpecula) is the major wildlife reservoir of Mycobacterium bovis in New Zealand. Control of bovine tuberculosis in farmed animals requires measures to reduce the transmission of M. bovis from wildlife. Possums were vaccinated with BCG intranasally by aerosol spray, orally or subcutaneously to compare the efficacy of these three routes on protection against challenge with virulent M. bovis. Possums vaccinated with BCG by the intranasal or subcutaneous routes had a marked reduction in severity of disease compared to possums which had been unvaccinated or orally vaccinated. The severity of the disease was assessed by changes in body weight and pathology. BCG vaccination by all three routes resulted in reduced dissemination of M. bovis to the spleen and liver following challenge. Intranasal and oral BCG vaccination induced lower mean peripheral blood lymphocyte blastogenic responses to bovine PPD than subcutaneous vaccination, indicating that these responses did not correlate well with protection from the disease. Given a suitable delivery system, aerosol vaccination of possums, used in conjunction with other control measures, may be a suitable method of reducing the spread of M. bovis from wildlife to domestic animals.

The emergence of Mycobacterium paratuberculosis in farmed deer in New Zealand — a review of 619 cases

Abstract AIM: To review cases in which Mycobacterium paratuberculosis was identified in farmed deer in New Zealand. METHODS: Case histories were reviewed where M. paratuberculosis was identified in deer by either culture or a polymerase chain reaction (PCR) test using primers from IS900. RESULTS: Between 1986 and 2000, M. paratuberculosis was identified by bacterial culture and/or PCR in 619 farmed deer from 299 herds, representing approximately 6% of deer herds in New Zealand. Over 85% of cases were identified during the last 6 years. In 60% of the infected herds, only one infected animal was identified. The maximum number of cases identified in a single deer herd was 47, and these were identified over a period of 8 years. Only 36 (5.8%) cases came from clinically affected animals identified on farms by veterinarians. The majority (89.7%) of the 619 cases were identified from lesions in mesenteric lymph nodes, including the ileocaecal lymph nodes, identified at meat inspection as being macroscopically either typical or equivocal of bovine tuberculosis (M. bovis). While the overwhelming majority of lesions were identified in mesenteric lymph nodes, M. paratuberculosis was also identified in 27 lesions in lymph nodes of the head, especially the retropharyngeal lymph node. CONCLUSIONS: The figures presented underestimate the true prevalence of infection with M. paratuberculosis, especially since not all suspect cases were submitted for culture or PCR. However, they do show that M. paratuberculosis appears to be spreading in farmed deer in New Zealand and highlight the possibility that Johnes disease is emerging as a potential major problem affecting this species. Identification of the organism by bacterial culture or PCR is required in many cases to distinguish lesions in mesenteric lymph nodes and lymph nodes of the head caused by M. paratuberculosis from those caused by M. bovis and M. avium.

Isolation of Mycobacterium bovis and other mycobacterial species from ferrets and stoats

As part of wildlife surveillance for bovine tuberculosis, pooled lymph nodes from 21,481 ferrets, 1056 stoats and 83 weasels were cultured for mycobacteria. A total of 268 isolates of Mycobacterium bovis were obtained from ferrets, 2 from stoats and none from weasels, demonstrating the presence of a wildlife reservoir of infection in ferrets. DNA typing by restriction endonuclease analysis (REA) of 48 selected isolates of M. bovis revealed 23 REA types. Twenty-one of these types had previously been isolated from cattle and farmed deer, demonstrating a complex cycle of infection involving wildlife and domestic animals. Apart from M. bovis, a further 208 mycobacterial isolates were obtained, the majority of which (178) were members of the M. avium complex. Speciation of the remaining 30 mycobacterial isolates by DNA sequencing of the 16s rRNA gene, identified half the isolates as M. triplex. Other species identified included M. fortuitum, M. florentinum, M. interjectum, M. intracellulare, M. holsaticum, and M. septicum/M. peregrinum.

Surveillance of wildlife for Mycobacterium bovis infection using culture of pooled tissue samples from ferrets (Mustela furo)

Abstract AIM: To compare culture results of homogenates of pooled lymph nodes from individual ferrets with and without macroscopic lesions of bovine tuberculosis for the presence of Mycobacterium bovis, and to determine whether homogenates from 10–30 ferrets could be combined and cultured without loss of sensitivity as a possible method for improving cost-effectiveness of surveillance for M. bovis infection in wildlife populations. METHODS: Numbers of colony forming units (cfu) of M. bovis present in cultures of homogenates of pooled lymph nodes from individual ferrets known to be infected and having no visible lesions (NVL) or macroscopic lesions consistent with bovine tuberculosis were determined. Prevalences of M. bovis infection in populations of ferrets in the Marlborough region of the South Island of New Zealand were determined by culturing homogenates of pooled lymph nodes from individual animals. Samples from homogenates from North Canterbury were combined to form pools representing 10, 20 and 30 animals and also cultured for M. bovis. RESULTS: Fewer M. bovis cfu were isolated from ferrets with NVL (mean=0.77 log10) compared with ferrets with macroscopic lesions (mean=3.22 log10; p<0.05). The mean prevalence of infection in eight different surveys involving 427 ferrets from the Marlborough region was 18% (range 8–44%), which included a small number of animals with macroscopic lesions of tuberculosis. Pooling of samples from up to 30 different ferrets with NVL did not reduce the sensitivity of detecting M. bovis-infected populations. CONCLUSION: Culturing of pools of lymph node samples detected a significant proportion of M. bovis-infected ferrets that would otherwise have gone unnoticed based on samples that had only macroscopic lesions. Culturing of samples pooled from up to 30 different ferrets could provide significant cost savings in surveys of wildlife for the presence of M. bovis infection without any apparent loss of sensitivity.

Sustained protection against tuberculosis conferred to a wildlife host by single dose oral vaccination

BACKGROUND Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation. METHODS Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis. RESULTS Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens. DISCUSSION The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible.

Case Report and DNA Characterization of Mycobacterium Avium Isolates from Multiple Animals with Lesions in a Beef Cattle Herd

The Mycobacterium avium complex includes the closely related species M. avium, M. intracellulare,and M. paratuberculosis. In animals, M. paratuberculosis is undoubtedly the most important member of the M. avium complex as far as economic losses due to reduced production and morbidity are concerned. However, other members of the M. avium complex also cause veterinary problems by infecting a wide range of different mammalian hosts, including pigs, farmed deer, and cattle. Members of the M. avium complex apart from M. paratuberculosiscan cause lesions in cattle that are grossly and microscopically indistinguishable from those caused by Mycobacterium bovis. 2 Infection with members of this complex, including M. paratuberculosis, can also cause cattle to respond to bovine tuberculin in a skin test. Evidence from the use of avian tuberculins in comparative skin tests suggests that in some areas, there is a high prevalence of infection in cattle. 7 However, many of these animals do not have visible lesions on necropsy, and it is rare to find generalized infections causing clinical disease by members of the complex other than M. paratuberculosis. The epidemiology of infections in cattle caused by members of the M. avium complex other than M. paratuberculosis has not been well defined. While members of this complex have been isolated not only from birds, but also from a wide range of environmental sites, including soil and water, 5 the source of M. avium complex infections in cattle has rarely been established. 1 The advent of DNA typing methods offers new tools to investigate the epidemiology of infections caused by this group of organisms. A compulsory control scheme for bovine tuberculosis has been in effect in New Zealand for over 25 years. 10 This scheme has been very successful in eradicating M. bovis from cattle herds in all areas of the country except where there are reservoirs of infection in wildlife. As part of the control scheme, all animals are carefully examined at slaughter for the presence of lesions resembling bovine tuberculosis. Samples from suspect cases are taken for histologic and bacteriologic confirmation of the macroscopic findings. Bacteriological examinations have shown a low prevalence of tuberculous lesions caused by the M. avium complex (Table 1). Apart from the episode described in this paper, no cattle herds were found in New Zealand in 1994 or 1995 that contained more than 3 animals with lesions caused by the M. avium complex. In 1992, M. bovis was isolated from 10 animals from a beef herd in the Hawkes Bay region of New Zealand. The

Transmission of Mycobacterium orygis (M. tuberculosis Complex Species) from a Tuberculosis Patient to a Dairy Cow in New Zealand

ABSTRACT Mycobacterium orygis, previously called the oryx bacillus, is a member of the Mycobacterium tuberculosis complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow.

Use of a polymerase chain reaction to subtype Mycobacterium avium subspecies paratuberculosis, an increasingly important pathogen from farmed deer in New Zealand.

Abstract AIMS: To review the number of microbiologically-confirmed cases of Johnes disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. METHODS: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. RESULTS: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. CONCLUSIONS: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johnes disease in farmed deer and the number of infected herds. Johnes disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johnes disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer.