The immunogenicity and tissue reactivity of Mycobacterium avium subsp paratuberculosis inactivated whole cell vaccine is dependent on the adjuvant used.

Johne's disease (JD) is a chronic enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current commercial vaccines are effective in reducing the occurrence of clinical disease although vaccinated animals can still become infected and transmit MAP. Many vaccinated sheep develop severe injection site lesions. In this study a range of adjuvants (Montanide TM ISA 50V, ISA 50V2, ISA 61VG, ISA 70 M VG, ISA 71 VG, ISA 201 VG and Gel 01 PR) formulated with heat-killed MAP were tested to determine the incidence of injection site lesions and the types of immune profiles generated in sheep. All the novel formulations produced fewer injection site lesions than a commercial vaccine (Gudair®). The immune profiles of the sheep differed between treatment groups, with the strength of the antibody and cell mediated immune responses being dependant on the adjuvant used. One of the novel vaccines resulted in a reduced IFN-γ immune response when a second "booster" dose was administered. These findings have significance for JD vaccine development because it may be possible to uncouple protective immunity from excessive tissue reactivity, and apparently poorly immunogenic antigens may be re-examined to determine if an appropriate immune profile can be established using different adjuvants. It may also be possible to formulate vaccines that produce targeted immunological profiles suited to protection against other pathogens, i.e. those for which a bias towards cellular or humoral immunity would be advantageous based on understanding of pathogenesis.

Sheep and cattle exposed to Mycobacterium avium subspecies paratuberculosis exhibit altered total serum cholesterol profiles during the early stages of infection.

Pathogenic mycobacteria such as Mycobacterium tuberculosis are capable of utilising cholesterol as a primary carbon-based energy source in vitro but there has been little research examining the significance of cholesterol in vivo. Johne's disease is a chronic enteric disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). This study sought to evaluate the levels of total serum cholesterol in the host following exposure to MAP. Blood samples were collected from both sheep and cattle prior to experimental challenge with MAP and at monthly intervals post-challenge. Total serum cholesterol levels in sheep challenged with MAP were significantly elevated at 9 weeks post-inoculation (wpi) in comparison to controls. When stratified based on disease outcome, there was no significant difference in serum cholesterol at the timepoints examined between MAP exposed sheep that were susceptible and those that were resistant to Johne's disease. There was a similar elevation in serum cholesterol at 9 wpi in cattle with histopathological gut lesions associated with disease or those with an early high IFN-γ response. Total serum cholesterol in exposed cattle was significantly lower when compared to controls at 13 wpi. Taken together, these results demonstrate changes in serum cholesterol following MAP exposure and disease progression which could reflect novel aspects of the pathogenesis and immune response associated with MAP infection in both sheep and cattle.

Case definition terminology for paratuberculosis (Johne's disease).

Paratuberculosis (Johne's disease) is an economically significant condition caused by Mycobacterium avium subsp. paratuberculosis. However, difficulties in diagnosis and classification of individual animals with the condition have hampered research and impeded efforts to halt its progressive spread in the global livestock industry. Descriptive terms applied to individual animals and herds such as exposed, infected, diseased, clinical, sub-clinical, infectious and resistant need to be defined so that they can be incorporated consistently into well-understood and reproducible case definitions. These allow for consistent classification of individuals in a population for the purposes of analysis based on accurate counts. The outputs might include the incidence of cases, frequency distributions of the number of cases by age class or more sophisticated analyses involving statistical comparisons of immune responses in vaccine development studies, or gene frequencies or expression data from cases and controls in genomic investigations. It is necessary to have agreed definitions in order to be able to make valid comparisons and meta-analyses of experiments conducted over time by a given researcher, in different laboratories, by different researchers, and in different countries. In this paper, terms are applied systematically in an hierarchical flow chart to enable classification of individual animals. We propose descriptive terms for different stages in the pathogenesis of paratuberculosis to enable their use in different types of studies and to enable an independent assessment of the extent to which accepted definitions for stages of disease have been applied consistently in any given study. This will assist in the general interpretation of data between studies, and will facilitate future meta-analyses.

Variation in susceptibility of different breeds of sheep to Mycobacterium avium subspecies paratuberculosis following experimental inoculation.

Exposure to Mycobacterium avium subspecies paratuberculosis (MAP) does not always lead to Johne's disease. Understanding differences in disease susceptibility of individual animals is a key aspect to controlling mycobacterial diseases. This study was designed to examine the susceptibility or resistance of various breeds of sheep to MAP infection. Merino, Suffolk first cross Merino, Border Leicester, and Poll Dorset sheep were orally inoculated with MAP and monitored for 14 months. Clinical disease occurred more frequently in the Merino (42%) and Suffolk first cross Merino (36%) compared to the Border Leicester (12%) and Poll Dorset (11%) breeds. Infection risk, as determined by culture of gut and associated lymphoid tissues, ranged from 75% for the Suffolk first cross Merino to 47% for the Poll Dorset sheep. Significant differences were identified in the site in the intestines of the most severe histopathological lesions and the immune responses to infection between the breeds. However, there was no difference in faecal MAP shedding by clinical cases between breeds. All breeds tested were susceptible to MAP infection, as determined by infection and clinical disease development, although there were differences in the proportions of diseased animals between the breeds. Poll Dorset and Border Leicester sheep were more resilient to MAP infection but there was evidence that more animals could have developed disease if given more time. These findings provide evidence of potential differential disease susceptibility between breeds, further our understanding of disease pathogenesis and risks of disease spread, and may have an influence on control programs for paratuberculosis.

Specific faecal antibody responses in sheep infected with Mycobacterium avium subspecies paratuberculosis.

Many studies have examined the serum antibody response to Mycobacterium avium subspecies paratuberculosis (MAP) infection in cases of Johne's disease (JD), but there are no reports on the mucosal antibody response. Faecal immunoglobulin (Ig) G and IgA ELISA responses were examined from sheep experimentally inoculated with MAP for up to 23 months post inoculation (PI). Corresponding serum IgG responses and the presence of viable MAP shed in faeces were also examined. The sheep were divided into three groups: (i) "un-inoculated controls" (n=10), (ii) "clinical cases" (n=8) which were inoculated animals that developed clinical disease and had moderate to high levels of MAP shedding and (iii) "survivors" (n=11) which were inoculated animals from which MAP could not be cultured from tissues at the conclusion of the trial. Serum IgG responses gradually increased in all inoculated animals, peaking at 12-16 months PI. A significant increase in the levels of MAP-specific faecal IgG and IgA was measured in the survivors at 16 and 17 months PI, while levels in the un-inoculated controls and clinical cases remained at baseline levels. The detection of faecal Ig in the survivors coincided with the removal of sheep that developed clinical disease. The data suggest that some sheep produced MAP-specific IgG and IgA in the intestinal mucosa, which was released into their faeces. We hypothesise that the survivors produced faecal Ig as a direct response to ingestion of MAP associated with environmental contamination from clinical cases. Thus MAP specific mucosal antibodies may play a previously unreported role as part of a protective response triggered by environmental exposure.

Histopathological Characterization of Cutaneous Delayed-type Hypersensitivity and Correlations with Intestinal Pathology and Systemic Immune Responses in Sheep with Paratuberculosis.

Cell-mediated immunity has been exploited historically in the diagnosis of mycobacterial diseases through elicitation of a delayed-type hypersensitivity (DTH) reaction following intradermal injection of an antigen. Here we describe the histopathological features of the cutaneous DTH reaction and its association with intestinal pathology and systemic immune responses in sheep with Mycobacterium avium subspecies paratuberculosis (MAP) infection. A mixed mononuclear cellular infiltrate dominated the DTH reaction and was present in perivascular and periadnexal patterns. Multiple multinucleate giant cells were present in the cellular infiltrate in one sheep while plasma cells were an obvious feature in six others. Sheep with paucibacillary intestinal lesions had the greatest degrees of cutaneous induration, more severe cellular infiltration in DTH lesions and high systemic interferon (IFN)-γ production. In contrast, sheep with multibacillary intestinal lesions, and particularly those with dissemination of MAP to extra-intestinal tissues, had minimal cutaneous induration, nil to mild cellular infiltration in DTH lesions and high serum anti-MAP antibody levels. Systemic IFN-γ production generally was augmented following skin sensitization. In general, the gross and histopathological features of the cutaneous DTH response matched the stage of paratuberculosis reflected by intestinal pathology and systemic measures of humoral and cellular immunity.

Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep.

An enzyme-linked immunosorbent assay (ELISA) was developed and optimised using a Mycobacterium avium subspecies paratuberculosis (MAP) antigen prepared from a C strain (316v) passed through a French press. The optimised assay was evaluated with a panel of sera from MAP infected (n = 66) and uninfected (n = 1,092) sheep. Animals in the MAP infected category were positive on either tissue culture or histopathology but were of unknown serum antibody status. The diagnostic performance and cost of the assay were compared with those of a commercial ELISA (IDEXX). At 99.8% diagnostic specificity the assay showed a diagnostic sensitivity of 23% (95% CI: 15.1-35.8) compared with 36.4% (95% CI: 25.8-48.4) for the commercial ELISA (McNemar's test: chi-square 5.82, p < 0.05). The sensitivities were 5.9% (95% CI: 1-26.9), 27.9% (95% CI: 14.7-45.7) and 35% (95% CI: 18.1-56.7), for low grade, paucibacillary and multibacillary lesion grades, respectively. The cost of the commercial assay kit was 2.7 to 5.2 times greater than that of the 316v ELISA for an equivalent number of tests, the multiple depending on the number of plates processed per run. For flock-level surveillance, to account for the lower sensitivity of the 316v ELISA compared with the commercial ELISA, sample sizes would be increased but the test cost would still be lower. The 316v assay will be useful for diagnosis of Johne's disease in sheep flocks, particularly in developing countries where labour costs are low relative to the cost of consumables.

Biomarker discovery for ovine paratuberculosis (Johne's disease) by proteomic serum profiling.

Paratuberculosis (Johne's disease) is a chronic granulomatous enteritis affecting ruminants and other species. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI TOF-MS) was used as a platform to identify candidate biomarkers from sheep serum. Multivariate biomarker models which aimed to differentiate sheep with paratuberculosis and vaccinated-exposed sheep from unexposed animals were proposed based on classification and regression tree (CART) and linear discriminant analysis (LDA) algorithms from two array types. The accuracy of classification of sheep into unexposed or exposed groups ranged from 75 to 100% among models. SELDI was used to monitor protein profile changes over time during an experimental infection trial by examining sera collected at 4-, 8- and 13-months post infection. Although three different SELDI instruments were used, nine consistent proteomic features were observed associated with exposure to MAP. Two of the putative serum biomarkers were purified from serum using chromatographic methods and were identified as transthyretin and alpha haemoglobin by tandem mass spectrometry. They belong to highly abundant, acute phase reactants in the serum proteome and have also been discovered as serum biomarkers in human inflammatory conditions and cancer. Their relationship to the pathogenesis of Johne's disease remains to be elucidated.

Does a Th1 over Th2 dominancy really exist in the early stages of Mycobacterium avium subspecies paratuberculosis infections?

The immune response of ruminants to Johne's disease has been long associated with a cell mediated immune (CMI) response in the early stages of infection with a switch to an antibody response later as the disease manifests. This study examines the immune response in sheep to Mycobacterium avium subspecies paratuberculosis (Map) infections, specifically the antigen-specific interferon gamma (IFN-γ) and antibody responses as surrogates of T helper-1 (Th1) and Th2 immunity. The difference in IFN-γ production between paucibacillary and multibacillary diseased animals was also examined. The results show that sheep are more likely to have a combined antibody and IFN-γ response (seen in 50% of the animals) rather than a switch from an IFN-γ to antibody response (39%). Multibacillary diseased animals were found to have a decrease in functional ability to produce IFN-γ from cells stimulated with MAP-specific antigens and non-specific mitogens. This indicates that the immune responses to Map infections are more complex than thought, where both antibody and cellular immunity may play key roles in the early stages of disease manifestation or resistance. The loss of the cellular response in multibacillary animals may be an indication that the entire immune response is dysfunctional, with the cell mediated responses becoming affected first.

Experimental infection model for Johne's disease in sheep.

Johne's disease in ruminants results in chronic enteritis caused by the pathogenic bacterium Mycobacterium avium subsp. paratuberculosis. This study examined two M. avium subsp. paratuberculosis strains (JD3 and W), using different doses and routes of infection, to establish the optimal time postchallenge when predictable levels of infection, gut lesions, and clinical disease occur in a large proportion of sheep. While a small proportion (25%) of sheep challenged with a low-passage-number laboratory culture of M. avium subsp. paratuberculosis (strain W) became infected, no infection was found in animals exposed to a high-passage-number culture isolate of strain W. In contrast, a primary tissue homogenate of M. avium subsp. paratuberculosis (JD3) resulted in high (90%) infection rates and gut histopathology following oral or intratonsillar challenge. The optimal conditions necessary to produce Johne's disease involve oral inoculation of 3-month-old lambs with four doses of 5 x 10(8) CFU of M. avium subsp. paratuberculosis isolated directly from the gut lymphatic tissues of clinically affected sheep. This resulted in consistent gut histopathology at 9 months and the onset of clinical disease by 11 months postchallenge.

Vaccination of sheep against M. paratuberculosis: immune parameters and protective efficacy.

Johne's disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis (Map). Currently available Map commercial vaccines protect against clinical disease but not infection. In this study, the proprietary Johne's vaccine Neoparasec and an aqueous formulation of Map 316F (AquaVax) were tested in sheep. Detailed immunological examination of blood and gut-associated lymphoid tissues was carried out on animals after vaccination and challenge with virulent Map to identify markers of protective immunity. Neoparasec vaccination provided significant protection against disease while AquaVax did not. Immune animals had stronger cell-mediated responses and altered proportions of CD4+, CD8+, CD25+ and B cells in blood, spleen and the gut lymphatics, than diseased animals.

Personal and situational influences on drink driving and sober driving among a cohort of young adults.

OBJECTIVES: To compare personal and situational influences on incidents involving drink driving with those involving sober driving. METHODS: Information on a range of road safety practices was sought in face to face interviews conducted with 969 members of the Dunedin Multidisciplinary Health and Development Study cohort at age 26 years. A total of 750 study members reported an incident that involved the opportunity to consume alcohol and also travel by motor vehicle. Of these, 87 were classified as "drink drive incidents" and 663 as "sober drive incidents". RESULTS: Study members who were male, of lower socioeconomic status, had no school qualifications, or were dependent on alcohol or marijuana at age 21 were significantly more likely to report a drink drive incident at age 26. Compared with the sober drive incidents, the drink drive incidents were more commonly associated with driving alone, drinking at bars, and no advanced planning. For drink drive incidents the amount of alcohol consumed was influenced by the conviviality of the occasion, whereas for sober drive incidents it was the need to drive. One quarter of those reporting drink drive incidents stated they had used marijuana and/or LSD at the event at which they drank. CONCLUSIONS: Drink drive and sober drive incidents differed, particularly with regard to decisions made before the event. Prevention efforts could usefully be targeted toward these decisions.

Impact of graduated driver licensing restrictions on crashes involving young drivers in New Zealand.

OBJECTIVE: To determine the impact on young driver crashes of the three main driving restrictions in the New Zealand graduated driver licensing (GDL) system: night-time curfew, no carrying of young passengers, and a blood alcohol limit of 30 mg/100 ml. METHOD: The database for this study was created by linking police crash reports to hospital inpatient records (1980-95). Multivariate logistic regression was used to compare car crashes involving a young driver licensed before GDL (n=2,252) with those who held a restricted graduated licence (n=980) and with those who held a full graduated licence (n=1,273), for each of the main driving restrictions. RESULTS: Compared with the pre-GDL group, the restricted licence drivers had fewer crashes at night (p=0.003), fewer involving passengers of all ages (p=0.018), and fewer where alcohol was suspected (p=0.034), but not fewer involving young casualties (p=0.980). Compared with the pre-GDL drivers, those with the full graduated licence had fewer night crashes (p=0.042) but did not differ significantly for any of the other factors examined. CONCLUSION: These results suggest that some of the GDL restrictions, especially the night-time curfew, have contributed to a reduction in serious crashes involving young drivers.

Validity of self reported crashes and injuries in a longitudinal study of young adults.

OBJECTIVES: The aim of this study was to determine the validity of self report as a source of information on crashes and injuries. SETTING: This study was part of the Dunedin Multidisciplinary Health and Development Study (DMHDS), which is a longitudinal study of the health, development, and behaviour of a cohort of young New Zealanders. METHOD: At the age 21 assessment DMHDS study members were asked to report serious injury and motor vehicle traffic crashes experienced over the previous three years. The self reported injuries were compared with the New Zealand Health Information Service (NZHIS) public hospital discharge file to determine the completeness of the self reported data. The traffic crashes were compared with the police traffic crash reports to determine the accuracy of self reported crash details. RESULTS: Twenty five (86%) of the 29 unintentional injuries, six (67%) of the nine assaults, and one (14%) of the six self inflicted injuries on the NZHIS file were self reported. The level of agreement between the self reported crash details and those recorded on the traffic crash report was high. CONCLUSIONS: The results show that self reports can be a useful and valid source of injury and crash data.

A longitudinal study of lifestyle factors as predictors of injuries and crashes among young adults.

This study was part of the Dunedin Multidisciplinary Health and Development Study. This is a longitudinal study of the health, development and behaviour of a cohort of 1037 young people born in Dunedin, New Zealand between 1 April 1972 and 31 March 1973. Explanatory measures covering background, behavioural and personality factors were obtained at ages 15 and 18 and were used as potential predictors of outcomes reported at age 21. Four outcomes were considered: any crash, injury crash, non-injury crash, and serious injury (not motor vehicle related). Overall, very few lifestyle factors were important predictors of any of these outcomes. Factors that were shown to predict injury crashes differed from those that predicted non-injury crashes. Also, those that predicted a traffic crash differed from those that predicted a serious non-traffic injury. These results suggest that focusing injury prevention efforts on changing the lifestyles of young adults is unlikely to reduce overall crash risk, and would have little impact on the risk of serious injury.

Road traffic practices among a cohort of young adults in New Zealand.

AIM: To describe the road safety practices of young adults in New Zealand. METHOD: Face to face structured interviews, seeking information on a range of road safety practices, were conducted with 948 members of the Dunedin Multidisciplinary Health and Development Study cohort when they were 21 years old. RESULTS: In the 30 days before the interview, 49% of the males and 32% of the females reported driving within two hours of drinking alcohol; 19% males and 8% females reported driving after drinking too much to perhaps be able to drive safely: and 25% males and 9% females reported driving after using marijuana. Also, 25% males and 6% females reported that they "often" drove fast just for the thrill of it, and 38% males and 11% females reported "often" driving faster than 120 kph on the open road. Seatbelt use as a driver was reported as "always" or "nearly always" by 87% of the males and 95% of the females, but as a rear seat passenger it was 34% for males and 47% for females. CONCLUSIONS: Unsafe road traffic practices, especially among males, were unacceptably high. Continued efforts are required to find new ways of addressing these issues.

Inattentive and hyperactive behaviors and driving offenses in adolescence.

OBJECTIVE: The associations between symptoms of attention-deficit hyperactivity disorder (ADHD), conduct disorder, anxiety, or depression and no disorder in relation to driving offenses were examined in 916 adolescents. METHOD: Self-report and parent report were used to assess a birth cohort of New Zealand adolescents' mental health status at age 15 years. Adolescents who scored 1.5 SD above the mean on the DSM-III total ADHD symptom scale were identified as reporting significant ADHD symptomatology. Self-report data and official traffic conviction records were used to identify adolescents who had committed driving offenses between ages 15 and 18 years. RESULTS: ADHD symptomatology and conduct disorder were strongly associated with driving offenses. ADHD symptomatology in females was significantly associated with driving offenses and more traffic crashes compared with other disorder or no disorder. CONCLUSIONS: Adolescents with a history of ADHD and conduct problems are significantly more likely than their peers to commit traffic offenses. Research in ADHD and risky driving should include female adolescents, as those with attentional difficulties are at a high risk for being involved in traffic crashes than females who do not experience attentional difficulties.

Sport and delinquency: an examination of the deterrence hypothesis in a longitudinal study.

OBJECTIVE: To determine whether involvement in sporting activity in mid-adolescence would deter delinquent behaviour in late adolescence. METHODS: Members of a longitudinal cohort study were interviewed at ages 15 and 18 years and, among other topics, were asked questions relating to involvement in physical activity and delinquent behaviour. Logistic regression models were used to examine the relation between sports involvement and delinquency at age 15 years and delinquency at age 18. RESULTS: After controlling for delinquent behaviour and psychosocial factors at age 15, females with moderate or high levels of sporting activity, and males with high levels of sporting activity, were significantly more likely to be delinquent at age 18 years than those with low levels of sporting activity. No significant association was found between sporting activity and aggressive behaviour, team sport participation and delinquency, and team sport participation and aggressive behaviour. CONCLUSIONS: This study did not support the deterrence hypothesis and showed that high involvement in sporting activity, but not team sport, was associated with a subsequent increase in delinquent behaviour.

An evaluation of the New Zealand graduated driver licensing system.

Young drivers have a disproportionately high risk of experiencing a road traffic crash. On 1 August 1987 a Graduated Driver's Licensing System (GDLS) was introduced in New Zealand. This system was designed to give young drivers (i.e. 15-24 years inclusive) experience in driving while being excluded from high risk driving situations. We sought to determine the impact of the GDLS on serious injury crashes. The source of the injury crash data was New Zealand's Health Information Services' national public hospital inpatient morbidity data files for the years 1979-1992 inclusive. We disaggregated the occupant data into three age groups 15-19 years, 20-24 years, and persons 25 years of age or older and compared their trends in injury. In order to determine whether the incidence of motor vehicle crashes was simply following trends in other injury events we also included two 15-19 year old non-traffic injury comparison groups. Using time series analyses we showed that the introduction of the GDLS was closely followed by substantial reductions in car crash injuries for all age groups, especially 15-19 year olds (23% reduction). After considering effects for older occupants we speculate that the effect is likely to be substantially less than 23%. An analysis of licensure data suggests that the reduction in crashes may, in large part, be attributable to an overall reduction in exposure.

The New Zealand graduated driver licensing system: teenagers' attitudes towards and experiences with this car driver licensing system.

OBJECTIVES: This study examined the attitudes of teenagers towards the New Zealand graduated driver licensing system (GDLS), and the extent to which it affected them. METHOD: Teenagers, who are members of a longitudinal study of a birth cohort, were interviewed at 15 years of age when the GDLS was first introduced and before they had begun licensure, and again at 18 years of age after they had experience with this licensing system. RESULTS: At both ages the majority (over 70%) agreed with the driving restrictions imposed by this system. After experience with the restrictions, however, significantly more reported being affected a lot by them, than had expected to be at age 15. This was especially true of the restrictions on the carrying of passengers and the night time curfew (10 pm - 5 am). However, few reported that they were affected by the alcohol restriction. Sixty eight per cent of those with a graduated licence reported breaking at least one of the conditions, most frequently carrying passengers. Very few were penalised by the police for this. CONCLUSIONS: Generally these young drivers were positively disposed towards the driving restrictions, but noncompliance was common. A full evaluation of all aspects of this licensing system is recommended.