Campylobacter spp. in conventional broiler flocks in Northern Ireland: epidemiology and risk factors.

Risk factors for Campylobacter infection in conventional broiler flocks in the time period up to the first removal of birds to slaughter were investigated over a maximum of five consecutive production cycles in a cohort of 88 broiler farms in Northern Ireland. Samples for Campylobacter culture, which consisted of 14 cloacal swabs per flock, were collected from one house on each farm prior to the first depopulation of birds. In total 388 flocks were sampled, of which 163 tested positive for Campylobacter spp. (42.0%; 95% CI 35.1-48.9%). Data on farm and flock variables were obtained from questionnaires and random-effects logistic regression modelling used to investigate the association between these and the Campylobacter status of flocks. Six variables, all of which were significant at p<0.05, were included in the final multivariable model. These included a combined variable on the presence of rodents on farms, which showed an increased odds of infection in flocks where the farmer reported having observed rodents during the production cycle (OR=2.1) and/or where rodent droppings were observed at the sampling visit (OR=2.9). Other variables that were significantly associated with an increased odds of infection included the age of the birds at sampling (odds ratio for its linear effect=1.16 for each day of increase in age), season (summer versus other seasons OR=2.0), farms with three or more broiler houses (OR=2.9 compared to those with one house), the frequency of footbath disinfectant changes (OR=2.5 for once weekly and OR=4.0 for less than once weekly compared to twice weekly changes) and a categorical variable on the standard of tidiness and cleanliness of the broiler house ante-room (OR=2.0 and OR=4.9 for flocks from houses with poorer standards). There was no significant evidence of direct carry-over of infection from one production cycle to the next, neither was there evidence of other farm species acting as a source of infection.

Use of Hugh and Leifson's medium as a simple screening test to aid in the differentiation of Arcobacter spp. from background flora during their isolation from foodstuffs.

AIMS: To investigate the suitability of Hugh and Leifson's medium (HLM) as the basis of a simple screening test to differentiate between contaminants and Arcobacter spp. during their isolation from foodstuffs. METHODS AND RESULTS: Characterized Arcobacter spp. were obtained from recognized culture collections. Wild-type isolates of Arcobacter spp. and contaminants were obtained using published isolation protocols. Retail packs of red meats were used as the source of the isolates. Eighteen defined Arcobacter spp. gave no reaction on HLM, as did 10 local wild-type isolates. Overall 163 contaminants were studied for oxidative reactions on HLM and 86% of isolates demonstrated this property. CONCLUSIONS: HLM can usefully serve as a simple and effective screening test to differentiate between Arcobacter spp. and contaminants. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacter isolation procedures are still being developed, and no effective diagnostic media currently exist. Rapidly excluding most contaminants can markedly increase the efficiency of isolation procedures by removing the need for extensive biotyping or the requirement to isolate DNA and conduct PCR tests.

Salmonella in slaughter pigs in Northern Ireland: prevalence and use of statistical modelling to investigate sample and abattoir effects.

A cross-sectional survey of pigs at slaughter in Northern Ireland was undertaken to determine the overall prevalence of Salmonella infection. In total 513 pigs were sampled across four abattoirs, with Salmonella spp. isolated from the caecal contents of 31.4% (95% confidence interval [CI] 27.4%-35.4%) and from 40.0% (95% CI 35.8%-44.3%) of swabs taken from the surface of carcasses post-evisceration. Two serovars, S. Typhimurium and S. Derby, were predominant and accounted for 52% and 35% respectively, of isolates from caecal contents. Antimicrobial resistance was most common amongst isolates of S. Typhimurium with 63.9% multiresistant compared to 10.8% of S. Derby isolates and 8.0% of other Salmonella spp. The proportion of pigs showing serological evidence of infection was significantly lower, with 11.5% (95% CI 8.9%-14.6%) and 10.1% (95% CI 7.7%-13.1%) of meat-juice samples giving positive and suspect reactions, respectively. The ratio of caecal positive to serologically positive animals is higher than in a number of other studies and may suggest recent infection, such as infection occurring during transport or lairage, in a proportion of animals. Statistical (logistic regression) modelling was used to investigate the association between the risk of Salmonella on carcasses and the isolation of Salmonella from caecal contents, and/or the serological status of the animal, while adjusting for other possible explanatory and confounding variables such as abattoir, season, day and time of sampling. The occurrence of Salmonella in caecal contents (odds ratio [OR] 2.39; 95% CI 1.52-3.77) or a suspect/positive serological reaction (OR 2.15; 95% CI 1.28-3.61) were both independently associated with the occurrence of Salmonella on carcasses in homebred, but interestingly not in imported animals. In most multivariable models there were also significant differences in carcass contamination between seasons with the highest odds of carcass contamination occurring in the April to June quarter and the lowest in the October to December quarter. Differences between sampling days were also evident with the highest odds of carcass contamination at the end of the week (Fridays) and the lowest at the start of the week (Mondays). These associations, after adjusting for the caecal or serological result, would suggest the occurrence of abattoir effects, such varying residual levels of abattoir contamination, which are independent of the individual pig status.

Molecular analyses of mycobacteria other than the M. tuberculosis complex isolated from Northern Ireland cattle.

Mycobacteria other than the Mycobacterium tuberculosis complex (MOTT), isolated from Northern Ireland cattle, were identified by PCR amplification of the 16S rRNA gene, and subsequent reverse cross blot hybridisation and sequence analyses. Elucidation of the MOTT species was to facilitate specificity testing of new and existing diagnostic test reagents for bovine tuberculosis. The presence of the genes for potential diagnostic antigens: MPB70, MPB64, ESAT-6 and CFP-10 in the isolated MOTT species was investigated. Molecular analyses of cultured isolates from bovine lymph node specimens of 48 cattle identified a wide variety of mycobacterial species including Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium bohemicum, Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium holsaticum, Mycobacterium palustre, Mycobacterium sp. IWGMT 90210, Mycobacterium sp. LIV-2129, a potentially novel mycobacterial species (EMBL/GenBank/DDBJ Accession Number AJ617495) and Rhodococcus equi. Apart from M. kansasii, the results of traditional (standard phenotypic and biochemical) and molecular identification methods did not correlate well, with traditional methods identifying fewer species. Most of the species identified were either recognised pathogenic or potential pathogenic species. The genes for ESAT-6, CFP-10 and, unusually, MPB64 were detected in M. kansasii only. The MPB70 gene was not detected in any of the species. This study supported restricted species distribution of these genes as well as identifying a different range of MOTT species that could be included in specificity testing of new diagnostic reagents for bovine tuberculosis.

Bovine tuberculosis: an old disease but a new threat to Africa.

Bovine tuberculosis (TB) is a disease characterised by progressive development of specific granulomatous lesions or tubercles in lung tissue, lymph nodes or other organs. Mycobacterium bovis is the causative agent of the disease. Bovine species, including bison and buffaloes, are susceptible to the disease, but nearly all warm-blooded animals can be affected. All species are not equally susceptible to the disease; some are spill-over (end) hosts and others maintenance hosts. In Africa, bovine TB primarily affects cattle; however, infection in other farm and domestic animals, such as sheep, goats, pigs, dogs and cats, is not uncommon. Wild ruminants and carnivores are also affected and are the natural reservoirs of the infectious agent in the wild. Man is also susceptible to the disease, the highest risk groups being individuals with concomitant HIV/AIDS infection. In Africa, human TB is widely known to be caused by M. tuberculosis; however, an unknown proportion of cases are due to M. bovis. This infection in humans is under-reported as a result of the diagnostic limitations of many laboratories in distinguishing M. bovis from M. tuberculosis. None of the national reports submitted to the OIE and WHO by African member states mention the importance of M. bovis in human TB cases. Consumption of unpasteurised milk and poorly heat-treated meat and close contact with infected animals represent the main sources of infection for humans. This review attempts to examine the impact of bovine TB on the health of animals and humans.

PCR studies of feline leprosy cases.

16S rRNA gene sequence analysis provided evidence for two different mycobacterial species, Mycobacterium lepraemurium and a potentially novel species, as causative agents of 'feline leprosy'. Comparison of 16S rRNA gene sequence data obtained for M. lepraemurium and the potentially novel species indicated 12 nucleotide differences over a 446 bp region encompassing the V2 and V3 hypervariable regions. From available 16S rRNA gene sequence data, M. lepraemurium shared greatest nucleotide identity with M. avium subsp paratuberculosis and M. avium. The novel species had a long helix 18 in the V3 region and shared greatest nucleotide identity with M. leprae, M. haemophilum and M. malmoense. The novel species had an additional 'A' nucleotide at position 105 of the aligned 16S rRNA gene sequence, the only other mycobacterial database sequence having this same extra nucleotide being M. leprae. This nucleotide variation was exploited to develop specific PCR assays for the two species. These were found to be effective and specific when tested against a panel of mycobacteria including species found in feline leprosy lesions and closely related mycobacteria and also when applied directly to formalin-fixed, paraffin-embedded tissues from feline leprosy cases.

Mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis.

Skin test negative cattle from a herd containing an unusually high proportion (194/382) of tuberculin skin test positive cattle were investigated for remaining Mycobacterium bovis infected animals. Blood samples from the skin test negative cattle, analysed by an antibody ELISA and an interferon-gamma assay, were mostly test negative for M. bovis. Radiometric culture of nasal mucus samples from 48 of the cattle yielded 22 culture positives with acid-fast bacilli and cording in 6 of these. Subculture on solid media was successful for 7, including 2 with cording of the 22 radiometric culture positives. Mycobacterium tuberculosis complex DNA probe testing using the Accuprobe (Gen-Probe, Inc.) and M. tuberculosis complex-specific PCR amplification, performed on the solid media subcultures, were negative. 16S rRNA PCR and sequence analysis were successful for 6 of the 7 solid media subcultures obtained and revealed the presence of Mycobacterium nonchromogenicum in all 6 subcultures. This is the first report of M. nonchromogenicum in nasal mucus of cattle. The observation highlights the importance of integrating definitive tests such as the PCR for diagnosis of bovine tuberculosis and indicates a possible zoonotic risk.

Antimicrobial susceptibility of Campylobacter spp. isolated from broiler chickens in Northern Ireland.

Between February 2000 and October 2001, cloacal swabs were collected from 387 broiler chicken flocks in Northern Ireland. Campylobacter isolates from the 262 positive flocks were tested with common antimicrobial agents using a disc diffusion method and by Etests. Resistance to erythromycin, gentamicin and chloramphenicol was <1%, whereas for ampicillin, nalidixic acid and tetracycline, resistance was 33%, 10% and 13%, respectively. Ciprofloxacin resistance was 3%, one of the lowest in recent reports from studies on human or poultry isolates. Sequence data of the quinolone resistance-determining region of the gyrA gene showed a mutation leading to Thr-86 to Ile substitution among highly resistant ciprofloxacin isolates. Only 0.8% of the isolates studied were resistant to four or more antibiotics.

Mycobacterium bovis infection and tuberculosis in cattle.

This review considers the possible events that can occur when cattle are exposed to Mycobacterium bovis and, where appropriate, draws on principles accepted for tuberculosis infection in humans and laboratory animal models. Consideration is given to the many complex factors which influence the outcome of challenge with tubercle bacilli. These include features inherent to the mycobacterium, the host and the environment. It is apparent that clinical disease probably occurs only in a relatively small, but undetermined, proportion of cattle that are exposed to Al. bovis. The majority of animals may clear infection or control the bacilli, possibly in a condition of latency. It is concluded that a better understanding of the dynamics of the events following M. bovis exposure and subsequent infection in cattle would be of significant benefit in developing new tools appropriate for disease control and to designing optimal approaches for their application.

Putative regulatory molecules in plants: evaluating melatonin.

Numerous classes of chemicals have been considered as regulators of various aspects of plant growth and development. In evaluating these putative regulatory molecules, plant biologists have encountered a number of challenges, including: the problem of quantifying substances present at trace levels in extremely complex mixtures; difficulty in obtaining and interpreting phenotypic responses to exogenous applications; and, until recently, the inability to selectively alter endogenous levels of these substances. Melatonin (N-acetyl 5-methoxytryptamine), a methoxylated indoleamine, is a potential regulatory molecule found in plants. Although no specific phenotype is currently associated with melatonin or its analogs in higher plants, it has important and unique biological activity in many other taxa, from algae to primates. In these organisms, melatonin functions as a night signal, coordinating responses to diurnal and photoperiodic environmental cues. We assess the process by which melatonin has been evaluated in plants so far and find that many of the methods for melatonin analysis, which have been adopted from animal studies, are inappropriate for use with plant materials. Thus, despite some interesting preliminary reports, research supporting the case for melatonin as a plant regulator is still in its infancy.

Melatonin in plant organs.

The indoleamine melatonin, a well-known animal chemical, has been identified in extracts from several plant species. The function of melatonin in plants is unknown. Two major functions of melatonin in animals are dark signaling and antioxidant protection. Fruit ripening was used as a model physiological process that involves changes in the oxidative status of an organ. Tomato fruits at various stages of ripeness were sampled. Morning glory (Pharbitis nil Choisy, cv. Violet) and tomato (Lycopersicon esculentum Mill., cv. T5 and Castlemart) organs were collected throughout a light/dark cycle to determine whether melatonin levels increased during the night. No consistent evidence was found that melatonin increased significantly in organs of these plants during the night, as it does in many animals. The melatonin content of the fruits generally increased during ripening up to the mature ripe stage and thereafter as the fruit became over ripe.

Pathogenesis of tuberculosis in cattle.

There has been a renewed interest in the pathogenesis of bovine tuberculosis in many countries, in an attempt to understand better its transmission, to improve diagnosis and assess the potential of vaccination. This paper, which overviews current knowledge of aspects of the pathogenesis of bovine tuberculosis, draws from studies of field cases and experimental infections and highlights deficiencies in current understanding. The pathogenesis of bovine tuberculosis has not received the same level of attention as with human tuberculosis, and in many instances, the processes involved in bovine tuberculosis have been drawn from studies of human tuberculosis or from small animal models of infection. This paper however, considers the successful emulation of naturally acquired tuberculosis using experimental cattle models and identifies the complex and integrated nature of microbiological, immunological and pathological events involved. Current understanding of the initiation of infection, immune responses, and subsequent pathology, which can vary significantly in individual animals are discussed. Whilst there are aspects of M. bovis that still remain elusive to scientific investigation, further studies on the pathogenesis of bovine tuberculosis are advocated as necessary to provide a better scientific basis on which to review control and eradication strategies, which are currently less than effective in many regions.

Immune responses in bovine tuberculosis.

Knowledge of the immune responses which develop in cattle following infection with Mycobacterium bovis is essential both to the understanding of disease pathogenesis and to the logical development of immune-dependent tools, such as diagnostic tests and vaccines, which can be used to combat the disease. Studies of field cases of bovine tuberculosis (TB) and of experimental bovine models of M. bovis infection have indicated that cell-mediated immune responses (CMI) predominate within a spectrum of immunity which exists. This paper reviews aspects of recent research and indicates how knowledge of T-cell antigenic targets in bovine TB along with increasing knowledge of T-cell subpopulations and their interactions with M. bovis -infected macrophages provides opportunities for the development of better methods for disease control.

Molecular epidemiology of Mycobacterium bovis: exploiting molecular data.

'Molecular epidemiology' is defined as the integration of conventional epidemiological approaches with molecular techniques to track specific strains of pathogens in order to understand the distribution of disease in populations. It has become a very powerful tool in the study of Mycobacterium tuberculosis and human tuberculosis, where it has been exploited to provide 'added value' to conventional epidemiological approaches (contact tracing) and has often challenged accepted dogmas. It has been used to confirm epidemiologically suspected transmission, to detect epidemiologically unsuspected transmission, to identify risk factors and environments where transmission is occurring, to detect laboratory errors and to monitor the efficacy of tuberculosis control programmes. For Mycobacterium bovis and bovine tuberculosis, molecular epidemiology has a key role to play in providing more precise epidemiological data on the issues of interbovine transmission and the role of wildlife reservoirs in disease maintenance and transmission. M. bovis strains may also differ in key biological properties, such as virulence, transmissibility, stability and antigenic variation, which may help to explain field observations. There may be correlation between strain type and 'herd level' factors such as breakdown size etc. Molecular 'strain typing' studies have provided useful information in several countries, notably New Zealand, where strain typing data is used as an integral part of M. bovis control schemes, to influence the level of herd testing or wildlife control and to define the extent and spread of infected wildlife. This presentation will review the methods and approaches currently appropriate for M. bovis strain typing and will review selected applications as well as discussing future perspectives and challenges for the application of molecular epidemiology to bovine tuberculosis.

Lymphocyte subtypes in experimentally induced early-stage bovine tuberculous lesions.

The identity of the lymphocyte subtypes constituting the lymphocytic mantle within developing early-stage lesions of bovine tuberculosis was investigated immunohistochemically in calves inoculated intranasally with 2 x 10(7) colony-forming units of a field isolate of Mycobacterium bovis. Pulmonary lesions were examined 7, 14, 21, 28 and 42 days after inoculation, and bronchial lymph node lesions at 35 days. The immunolabelling results reported were obtained with monoclonal antibodies against two T-cell epitopes (WC1+ gamma delta and CD2+) and against B-cell epitopes. Large numbers of CD2+ T-lymphocytes were observed around developing areas of necrosis throughout the study; WC1+ gamma delta cells, however, were more numerous at these sites up to and including day 21. On the other hand, aggregates of B lymphocytes did not become prominent in areas adjacent to lesions until day 42. The results suggest that these lymphocyte phenotypes play a role in the pathogenesis of early-stage lesions.

Detection of Mycobacterium bovis in bovine clinical specimens using real-time fluorescence and fluorescence resonance energy transfer probe rapid-cycle PCR.

Nucleic acid sequence capture extraction was coupled with LightCycler PCR amplification and product detection using real-time fluorescence for rapid, definitive detection of Mycobacterium bovis in lymph node specimens from 38 cattle with bovine tuberculosis lesions. PCR amplification of sequence-captured DNA using both a conventional heating block thermocycler and a LightCycler thermocycler was compared with culture and histopathological analyses. Conventional PCR enabled detection of 26 of 28 culture-positive specimens (93%) in approximately 9 h, and the LightCycler PCR detected 20 of 28 culture-positive specimens (71%) in only 30 min. Specific confirmation of Mycobacterium tuberculosis complex DNA was achieved by LightCycler PCR amplification using Syb Green 1 and an M. tuberculosis complex-specific Cy5-labeled fluorescence resonance energy transfer probe. The system described here enabled rapid and specific laboratory confirmation of bovine tuberculosis, and this is the first report of the detection of M. bovis in tissues using LightCycler PCR. The fluorescence technology used in the study has potential to allow development of a high-throughput molecular diagnostic test for bovine tuberculosis.

Cattle-to-cattle transmission of bovine tuberculosis.

In developed countries, Mycobacterium bovis infection in cattle is now mostly confined to the respiratory system, which reflects transmission and establishment of infection mainly by this route. A single bacillus transported within a droplet nucleus is probably sufficient to establish infection within the bovine lung. Infected cattle should always be considered as potential sources of infection, since studies have demonstrated that a significant proportion of tuberculous cattle excrete M. bovis. In general, the dynamics of M. bovis transmission are poorly understood and the conditions under which a tuberculous animal becomes an effective disseminator of infection are currently not defined although environmental contamination appears to be a less effective method of disease transmission. Field studies indicate a wide spectrum of transmission rates but generally the spread of M. bovis infection is still considered to be a relatively slow process. Slaughter of diseased cattle detected by tuberculin testing and at meat plant inspection has been shown to be an effective policy for tuberculosis eradication, provided there are no other reservoirs of infection and all involved in the cattle industry are committed to a policy of eradication. Epidemiological approaches, particularly case-control studies, seem to provide the best method for quantifying the relative importance of the various sources of M. bovis transmission to cattle and modelling techniques can be used to assist in the design of cost-effective control measures that may lead to tuberculosis eradication.

In vitro T-cell activation of monocyte-derived macrophages by soluble messengers or cell-to-cell contact in bovine tuberculosis.

The macrophage plays a dual role in tuberculosis, promoting not only protection against mycobacteria, but also survival of the pathogen. Macrophages inhibit multiplication of mycobacteria but also act in concert with lymphocytes through presentation of antigens to T cells. Studies in animal and human infections have suggested a correlation of in vitro growth rates of mycobacteria with in vivo virulence, using uracil uptake to assess mycobacterial metabolism. This study found that blood-derived, non-activated bovine macrophages were capable of controlling Mycobacterium bovis bacillus Calmette-Gurin growth for up to 96 hr, but were permissive to intracellular growth of virulent M. bovis. The present investigation compared the in vitro modulation of these macrophage activities by cytokine-rich T-cell supernatants or cell-to-cell contact. On the one hand, treatment of cultured monocytes with mitogen-produced T-cell supernatants promoted morphological changes suggestive of an activation status, enhanced the antigen presentation capabilities of monocytes and up-regulated major histocompatibility complex class II expression. However, this activation was not associated with enhanced anti-M. bovis activity. On the other hand, incubation of infected monocytes with T-cell populations resulted in proportionally increased inhibition of M. bovis uracil uptake. This inhibition was also seen using cells from uninfected animals and indicated the necessity for cell-to-cell contact to promote antimycobacterial capability.