Risk factors for ovine Johne's disease in infected sheep flocks in Australia

We conducted a cross-sectional study in 2004-05 to investigate risk factors for ovine Johne’s disease (OJD) involving 92 infected Merino sheep flocks in Australia. In each enrolled flock we collected pooled faecal-samples from 3- to 5-year-old sheep and cultured them for Mycobacterium avium subsp. paratuberculosis (MAP) to determine their OJD status. Based on pooled faecal-culture (PFC) results, three outcome variables representing different facets of disease biology were derived: pool OJD status (binomial: positive or negative), log pool MAP number (continuous) and cohort OJD prevalence level (ordinal: low (10%) prevalence). We used these outcomes in three separate multivariable analyses to identify risk factors, which were based on a questionnaire administered during a face-to-face interview with the farmer. We found higher OJD infection in sheep whose dams had been in poor condition and kept at a high stocking rate during lambing and in sheep which had experienced a longer period of growth retardation during their lifetime. Flocks that had vaccinated for >2 years (rather than only 1 to 2 years) with a killed MAP vaccine had significantly lower OJD infection. In addition, practices including culling low body-weight sheep or selling sub-flocks experiencing high losses, sharing of roads between neighbouring farms, and greater frequency of application of super-phosphate fertilizers were associated with higher OJD. Of the confounders investigated, infection was higher in flocks experiencing high mortalities; in wethers compared to ewes; and in 3-year-old sheep compared to 4-year-old sheep. Keywords: risk factors, paratuberculosis, ovine Johne’s disease, Australia, Mycobacterium, epidemiology, cross-sectional studyMeat and Livestock Australi

Association of farm soil characteristics with ovine Johne's disease in Australia

Speculation about the association of soil characteristics with the expression of ovine Johne’s disease (OJD) prompted this cross-sectional study. We enrolled 92 sheep flocks in Australia during 2004-05 and in each enrolled flock collected pooled faecal samples from an identified cohort (group of same age and sex) of sheep and soil samples from the paddocks grazed by this cohort of sheep. Faecal pools were cultured to create three outcome variables: positive or negative status of faecal pools (pool OJD status, binary); the log number of viable Mycobacterium avium subsp. paratuberculosis (MAP) organisms per gram of faeces (log pool MAP number, continuous); and the prevalence of faecal shedders (cohort OJD prevalence level, ordinal: low 10%). Separate statistical models were then developed to investigate the association between soil characteristics and each outcome variable. Sheep raised on soils with a higher percentage of organic carbon and clay had a higher OJD prevalence whereas, sheep grazing on soils with a higher content of sand and nitrogen had a lower OJD prevalence. Iron content of the soil was positively associated with OJD infection but the association between soil pH and OJD was inconclusive. Parent soil type, the only farm level factor, was not significant in any of the final models. Study results indicate a higher risk of OJD in sheep raised on soils with greater organic matter and clay content. We hypothesise that this is due to adsorption of MAP to clay and the consequent retention of the bacteria in the topsoil, thus making them available in higher numbers to grazing sheep. Keywords: risk factors, paratuberculosis, mycobacterium, cross-sectional study, organic carbon, clay, sand, pH, iron.Meat and Livestock Australi

Comparative study of the commonly used virulence tests for laboratory diagnosis of ovine footrot caused by Dichelobacter nodosus in Australia.

Footrot in sheep and goats is expressed as a spectrum of clinical entities ranging from benign, which is a self limiting interdigital dermatitis to highly virulent, in which severe under running of the horn of the hoof occurs. Interactions between the host, the virulence of the causative strain of Dichelobacter nodosus and environmental conditions determine the severity of the disease. Clinical diagnosis of virulent footrot, which a notifiable disease in some states of Australia, is not always straightforward. Therefore, the Gelatin Gel and Elastase tests for protease activity, and the intA PCR test for an inserted genetic element in D. nodosus are commonly used to support or to confirm a clinical diagnosis. A comparative study of these laboratory tests with a large number of samples collected from 12 flocks of sheep with clinically virulent footrot was conducted. Based on the elastase test, 64% of the isolates tested were classified as virulent compared to 91% on the gelatin gel test and 41% according to the intA test. The agreement between the elastase and the gelatin gel test was low (kappa =0.12) as were the agreements between other tests. Only about 21% of the isolates were virulent in all 3 tests. Therefore these tests on their own may not provide standard and reliable results and are likely to remain as supplementary tests for clinical diagnosis of the disease. Keywords: Footrot, virulence, elastase test, gelatin gel test, intA PCR test

Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

Mycobacterium avium subsp. paratuberculosis causes Johne’s disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be ex- pressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced

Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

Mycobacterium avium subsp. paratuberculosis causes Johne’s disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be ex- pressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced

Comparison of pre- and post-vaccination ovine Johne's disease prevalence using a Bayesian approach

postprintThis study was conducted to evaluate the effectiveness of GudairTM vaccine in decreasing the prevalence of shedding of Mycobacterium avium subsp. paratuberculosis (MAP) in flocks of varying initial prevalence. Thirty seven self-replacing Merino flocks from New South Wales and Victoria (Australia) that had been vaccinating lambs with GudairTM for at least five years were enrolled in the study. These flocks had been tested prior to or at commencement of vaccination using pooled faecal culture, agar gel immunodiffusion or both tests. These pre-vaccination test results were used to estimate pre-vaccination prevalence. Post-vaccination prevalence was estimated from culture of usually 7 pools of 50 sheep collected from the enrolled flocks in 2008-2009, approximately five or more years after commencement of vaccination. A Bayesian model was developed to estimate and compare the pre- and post-vaccination prevalences for the enrolled flocks. Apparent pre- and post-vaccination prevalences for flocks were modelled as functions of the true pre- and post-vaccination prevalences, respectively, and the sensitivities and specificities of the respective diagnostic tests. Logit-normal models were specified on pre- and post-vaccination true prevalences and were then used to make inferences about the median and 90th percentile of the prevalence distributions and their differences. Priors were mostly specified based on published literature or analysis of abattoir surveillance data for this population of flocks. The analysis found a significant decline in ovine Johne’s disease prevalence from a pre-vaccination median prevalence of 2.72% [95% probability interval (PI): 1.40; 6.86%] to a post-vaccination median prevalence of 0.72% (0.39; 1.27%). However 30 of the 37 flocks still contained sheep that were shedding MAP in their faeces. The results suggest that vaccination with Gudair™ is usually effective in reducing the prevalence of faecal shedding but the response to vaccination is variable among flocks. This approach could be implemented in similar situations to compare prevalences where information from multiple diagnostic tests with varied sensitivities and specificities is available. Keywords: Ovine Johne’s disease; Gudair; Vaccination; Abattoir surveillance; Faecal culture; Agar gel immune-diffusion test

Comparison of pre- and post-vaccination ovine Johne's disease prevalence using a Bayesian approach

This study was conducted to evaluate the effectiveness of GudairTM vaccine in decreasing the prevalence of shedding of Mycobacterium avium subsp. paratuberculosis (MAP) in flocks of varying initial prevalence. Thirty seven self-replacing Merino flocks from New South Wales and Victoria (Australia) that had been vaccinating lambs with GudairTM for at least five years were enrolled in the study. These flocks had been tested prior to or at commencement of vaccination using pooled faecal culture, agar gel immunodiffusion or both tests. These pre-vaccination test results were used to estimate pre-vaccination prevalence. Post-vaccination prevalence was estimated from culture of usually 7 pools of 50 sheep collected from the enrolled flocks in 2008-2009, approximately five or more years after commencement of vaccination. A Bayesian model was developed to estimate and compare the pre- and post-vaccination prevalences for the enrolled flocks. Apparent pre- and post-vaccination prevalences for flocks were modelled as functions of the true pre- and post-vaccination prevalences, respectively, and the sensitivities and specificities of the respective diagnostic tests. Logit-normal models were specified on pre- and post-vaccination true prevalences and were then used to make inferences about the median and 90th percentile of the prevalence distributions and their differences. Priors were mostly specified based on published literature or analysis of abattoir surveillance data for this population of flocks. The analysis found a significant decline in ovine Johne’s disease prevalence from a pre-vaccination median prevalence of 2.72% [95% probability interval (PI): 1.40; 6.86%] to a post-vaccination median prevalence of 0.72% (0.39; 1.27%). However 30 of the 37 flocks still contained sheep that were shedding MAP in their faeces. The results suggest that vaccination with Gudair™ is usually effective in reducing the prevalence of faecal shedding but the response to vaccination is variable among flocks. This approach could be implemented in similar situations to compare prevalences where information from multiple diagnostic tests with varied sensitivities and specificities is available. Keywords: Ovine Johne’s disease; Gudair; Vaccination; Abattoir surveillance; Faecal culture; Agar gel immune-diffusion test

Enzyme-linked immunospot: an alternative method for the detection of interferon gamma in Johne's disease

Abstract. To date, the sensitivity of the interferon gamma (IFN-c) enzyme-linked immunosorbent assay (ELISA) to detect Johne’s disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN-c detection in the early stages of infection, an alternate assay needs to be developed. The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive technique for the detection of cytokines and has the potential to improve the diagnosis of JD. Of the variables examined, choice of capture antibody and the method by which the peripheral blood mononuclear cells were isolated significantly affected the ability to enumerate IFN-c–secreting cells. The ELISPOT assay was as sensitive as or better than the IFN-c ELISA at detecting ovine JD and could also detect disease at early time points postinoculation. The IFN-c ELISPOT could distinguish infected from unexposed animals; however, neither the IFN-c ELISA nor the ELISPOT assay could distinguish between sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis and those exposed to the bacterium but diagnosed as uninfected at necropsy. Key words: Enzyme-linked immunosorbent assay; enzyme-linked immunospot assay; interferon gamma; Johne’s disease; paratuberculosis; sheep

Optimization of a whole blood gamma interferon assay for the detection of sheep infected with Mycobacterium avium subspecies paratuberculosis

The capacity of a commercially available gamma interferon (IFNγ) assay to detect infected sheep early in the pathogenesis of Johne's disease enables the removal of such animals from the flock before bacterial shedding and pasture contamination. However, nonspecific IFNγ responses in the assay have meant that to achieve high-test specificity, there has been a reduction in sensitivity. Although the optimal conditions for the use of the assay in cattle have been well documented, there have been few studies optimizing the assay for use in sheep. The current study details the effect of anticoagulant, duration of incubation, cell concentration, blood storage temperature, time of stimulation of cells with antigen relative to time of sample collection, and temperatures during transit on IFNγ synthesis. Maximal IFNγ synthesis occurred with incubation periods of 48 hr in blood collected into heparinized tubes. Decreasing the leukocyte population by diluting the total peripheral blood leukocyte concentration was associated with a decreasing IFNγ response. Conversely, concentrating the peripheral blood concentration 2-fold resulted in an increase in the IFNγ production. In field studies, immediate incubation of blood samples with antigen at 37°C resulted in larger IFNγ responses; however, significantly lower IFNγ values were obtained if the samples were transported at ambient temperature. The results of this study indicate that optimization of the IFNγ assay may enable increased synthesis of IFNγ during the stimulation phase of the assay and that future work may determine whether this translates to increased sensitivity of the assay in detecting early infections in sheep. Bovigam assay, gamma interferon, Johne's disease, paratuberculosis, sheepResearch was funded by Meat and Livestock Australia (MLA

Can early host responses to mycobacterial infection predict eventual disease outcomes?

Diagnostic tests used for Johne’s disease in sheep either have poor sensitivity and specificity or only detect disease in later stages of infection. Predicting which of the infected sheep are likely to become infectious later in life is currently not feasible and continues to be a major hindrance in disease control. We conducted this longitudinal study to investigate if a suite of diagnostic tests conducted in Mycobacterium avium subspecies paratuberculosis (MAP) exposed lambs at 4 months post infection can accurately predict their clinical status at 12 months post infection. We tracked cellular and humoral responses and quantity of MAP shedding for up to 12 months post challenge in 20 controls and 37 exposed sheep. Infection was defined at necropsy by tissue culture and disease spectrum by lesion type. Data were analysed using univariable and multivariable logistic regression models and a subset of variables from the earliest period post inoculation (4 months) was selected for predicting disease outcomes later on (12 months). Sensitivity and specificity of tests and their combinations in series and parallel were determined. Early elevation in faecal MAP DNA quantity and a lower interferon gamma (IFNγ) response were significantly associated with sheep becoming infectious as well as progressing to severe disease. Conversely, early low faecal MAP DNA and higher interleukin-10 responses were significantly associated with an exposed animal developing protective immunity. Combination of early elevated faecal MAP DNA or lower IFNγ response had the highest sensitivity (75%) and specificity (81%) for identifying sheep that would become infectious. Collectively, these results highlight the potential for combined test interpretation to aid in the early prediction of sheep susceptibility to MAP infection. KEYWORDS: Paratuberculosis; diagnostic tests; Mycobacterium; faecal DNA; Johne’s disease; interferon gamma.This work was supported by Meat and Livestock Australia and by Cattle Council of Australia, Sheepmeat Council of Australia and WoolProducers Australia through Animal Health Australia