DETECTION OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN ROCK HYRAXES ( PROCAVIA CAPENSIS) IMPORTED FROM SOUTH AFRICA.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic, progressive, and consecutively fatal enteritis, especially in ruminants. MAP distribution among wildlife is not yet clear. In this study, three wild-born rock hyraxes ( Procavia capensis) had been imported from South Africa to a German zoological garden. During the quarantine period, four young animals were born. The wild-born animals showed symptoms of mild diarrhea shortly after their arrival in the zoological garden, but all routine parasitological and bacteriologic tests performed were negative. Therefore, the animals were additionally tested for MAP infection. MAP DNA was detected by seminested PCR (snPCR) in a pooled fecal sample of the seven animals. Subsequent PCR analysis of the individual feces samples confirmed the excretion of MAP in two rock hyraxes (one wild-born and one born in captivity). Sequence analysis of the corresponding 278-bp amplicons revealed 100% homology to the reference MAP-K10 IS900 sequence. No antibody response against MAP was detected in the individual serum samples. MAP-specific postmortem lesions were not observed by gross pathology and histology, neither after death nor after euthanization of the animals. Nevertheless, MAP was detected by snPCR and culture in the gastrointestinal tract, urogenital tract, cardiovascular system, and/or respiratory system of three other animals of the group (one wild-born and two born in captivity). This study is the first report confirming MAP occurrence in rock hyraxes. Therefore, it is recommended that veterinarians and zoo employees consider rock hyraxes as a possible source of MAP infection for domestic livestock in South Africa and the valuable animal stock of zoological facilities.

Bayesian semiparametric modeling for stochastic precedence, with applications in epidemiology and survival analysis.

We propose a prior probability model for two distributions that are ordered according to a stochastic precedence constraint, a weaker restriction than the more commonly utilized stochastic order constraint. The modeling approach is based on structured Dirichlet process mixtures of normal distributions. Full inference for functionals of the stochastic precedence constrained mixture distributions is obtained through a Markov chain Monte Carlo posterior simulation method. A motivating application involves study of the discriminatory ability of continuous diagnostic tests in epidemiologic research. Here, stochastic precedence provides a natural restriction for the distributions of test scores corresponding to the non-infected and infected groups. Inference under the model is illustrated with data from a diagnostic test for Johne's disease in dairy cattle. We also apply the methodology to the comparison of survival distributions associated with two distinct conditions, and illustrate with analysis of data on survival time after bone marrow transplantation for treatment of leukemia.

Aspectos clínico-patológicos e controle da paratuberculose em rebanho bovino leiteiro / Clinic-pathological aspects and control of paratuberculosis in a dairy cattle herd

A paratuberculose ou doença de Johne é uma enterite granulomatosa causada por Mycobacterium avium subsp. paratuberculosis. Descrevem-se os aspectos epidemiológicos, clínico-patológicos e laboratoriais da paratuberculose em rebanho bovino leiteiro no município de Rio Claro, região Sul do Estado do Rio de Janeiro. No período de 2006 a 2009, oito vacas adultas da raça Girolanda apresentaram diarreia crônico-intermitente e perda progressiva de peso. À necropsia, observaram-se linfonodos mesentéricos aumentados de volume e úmidos ao corte, vasos linfáticos subserosos das alças intestinais proeminentes, serosa do intestino com aspecto anelado e cerebroide e a mucosa espessada, pregueada e com aspecto microgranular. À microscopia havia, desde o duodeno até o reto, inflamação granulomatosa difusa, marcada dilatação dos vasos linfáticos no ápice das vilosidades, linfangiectasia e linfangite granulomatosa na submucosa, muscular e serosa. A inflamação granulomatosa também foi vista nos linfonodos mesentéricos. A coloração de Ziehl-Neelsen revelou variável quantidade de bacilos álcool-ácido resistentes no interior de macrófagos, de células gigantes de Langhans e livres na mucosa e submucosa dos intestinos delgado e grosso e em linfonodos mesentéricos. Em alguns animais, a lâmina própria da mucosa, principalmente do jejuno e íleo exibia acentuada hipertrofia. Mycobacterium avium subsp. paratuberculosis foi isolado em cultivo bacteriano de Herrold com micobactina, a partir de amostras de fezes, de raspado de mucosa intestinal e de leite e identificado pela técnica de PCR IS900. Através da avaliação sorológica semestral, foram analisadas 298 vacas do mesmo rebanho a partir de três anos de idade, observou-se cerca de 40 por cento de animais reagentes ao teste ELISA indireto no período estudado. O diagnóstico da paratuberculose foi baseado nos dados clínico-patológicos, na sorologia, no isolamento e identificação do agente através de cultivo bacteriano e PCR IS900. Após implementação de medidas de controle, tais como eliminação de animais doentes, abate seletivo dos animais soropositivos, separação dos bezerros ao nascer e utilização de banco de colostro, observou-se, nos três anos de estudo, diminuição da ocorrência de casos clínicos no rebanho, de seis casos por ano para cerca de um caso por ano.

Paratuberculosis (Johne's disease) is a granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis. Epidemiology, clinic-pathological and laboratorial aspects of paratuberculosis in a dairy cattle herd are described. The disease was diagnosed from 2006 to 2009 in eight cows that presented chronic-intermittent diarrhea and chronic weight loss, in the Rio Claro municipality, Rio de Janeiro. At necropsy, the subserosal lymphatic vessels were proeminent and dilated, mesenteric nodes were enlarged and intestinal mucosa was corrugated, thickened and of microgranular aspect. From duodenum to the rectum, histopathology revealed severe and diffuse granulomatous inflammation of the lamina propria and submucosa, broadened and distorted villi, dilatation of the lymphatic vessels in their apex, lymphangioectasia and granulomatous lymphangitis in the submucosa. Ziehl-Neelsen stain showed variable amounts of acid-fast bacilli in macrophages, in Langhans giant cells and freely in the mucosa and submucosa of the small intestine, colon and lymphnodes. In some cows, the lamina propria presented severe hypertrophy, mainly in the jejunum and ileum. Mycobacterium avium subsp. paratuberculosis was isolated through bacterial cultivation of samples taken from feces, intestinal mucosa and milk, and identified through IS900 PCR. From 298 cows older than three years, the percentage of reactive animals was 40 percent by indirect ELISA test. The diagnosis of paratuberculosis was based on clinic-epidemiological data, serology, bacterial isolation in Herrold egg yolk medium with micobactin and on IS900 PCR. After the adoption of control measures, as slaughter of cows with clinical signs, selective slaughter of seropositive cows, removal of the calf from the dam at birth, and use of the colostrum bank, we observed a reduction from six clinical cases to only one case per year, in the last three years of the study.

Age at which dairy cattle become Mycobacterium avium subsp. paratuberculosis faecal culture positive.

Age at which cattle become faecal culture positive for Mycobacterium avium subsp. paratuberculosis (Map) can be used as a proxy parameter for age at onset of faecal shedding, which is an important parameter in the control of Map in cattle herds. To investigate the age at becoming faecal culture positive, survival analysis methods were applied. The analyses were carried out on asynchronous interval censored data of faecal culture results of samples collected from 18,979 female Holstein-Frisian cattle in 353 Dutch herds between 1996 and 2002. The data were analysed with a Weibull proportional hazards model. The results indicate that the distribution of age at onset of faecal shedding in Holstein-Frisian dairy cattle in infected herds is associated with the within-herd prevalence. In higher classes of apparent prevalence, cattle started to shed Map at younger age on average. In herds with an apparent prevalence <0.05, 0.05-0.1, 0.1-0.2 and ≥0.2, the proportion (95% CI) of cattle with onset of faecal shedding before 2 years of age was estimated at 1% (0.5%; 2%), 4% (3%; 5%), 8% (5%; 10%) and 20% (11%; 32%), respectively. This study indicates that a considerable proportion of young stock is shedding Map, especially in high prevalence herds. Therefore, infectious young stock should be a major concern in the control of paratuberculosis.

Evaluation of diagnostic tests for Johne's disease (Mycobacterium avium subspecies paratuberculosis) in New Zealand dairy cows.

To quantify the sensitivity and specificity of a serum enzyme-linked immunosorbent assay (ELISA) and fecal culture (FC) tests and to estimate the prevalence of Johne's disease (JD) in New Zealand dairy herds using Bayesian methods, 4 New Zealand dairy herds were tested simultaneously by ELISA and FC 5 times over 3 lactations. Test results were dichotomized. A Bayesian regression model was developed that considered test sensitivity as a function of the covariates parity, lactation stage, and prevalence of JD, which is expected to vary between herds. It was applied to a cross-sectional subset of the data and the full, repeated measures data set. Results were compared with frequentist pseudo gold standard results of the full data. Using the regression model, sensitivity of the ELISA was higher in older animals, but the sensitivity of the FC test showed no trend across age groups. Both FC and ELISA sensitivity were lower in late lactation. Estimated prevalence was lower and FC sensitivity higher when analyzing the complete data. The regression model enabled a more accurate diagnosis of JD to be made because it incorporated cow-specific information in the diagnosis, such as age and lactation stage. The model also enabled the incorporation of previous test results for an individual when diagnosing disease. The trends in results from the regression model support the current understanding of the disease process. The advantage of repeated testing of individuals in the assessment of test performance is discussed in the current study.

A Bayesian Weibull survival model for time to infection data measured with delay.

Survival analysis methods can be used to identify factors associated with the time to induction of infection. In the absence of a perfect test, detection of infection is generally delayed and depends on the duration of the latent infection period. We assess, via simulations, the impact of ignoring the delayed detection of infection on estimated survival times and propose a Bayesian Weibull regression model, which adjusts for the delayed detection of infection. The presence of non-differential detection delay seriously biased the baseline hazard and the shape of the hazard function. For differential detection delay, the associated regression coefficients were also biased. The extent of bias largely depended on the longevity of the delay. In all considered simulation scenarios our model led to corrected estimates. We utilized the proposed model in order to assess the age at natural infection with Mycobacterium avium subsp. paratuberculosis (MAP) in Danish dairy cattle from the analysis of available time to milk-seropositivity data that detected infection with delay. The proposed model captured the inverse relationship between the incidence rate of infection and that of seroconversion with time: susceptibility to infection decreases with time (shape parameter under the proposed model was rho=0.56<1), while older animals had a higher probability of sero-converting (rho=2.67>1, under standard Weibull regression). Cows infected earlier in their lives were more likely to subsequently shed detectable levels of MAP and, hence, be a liability to herd-mates. Our approach can be particularly useful in the case of chronic infections with a long latent infection period, which, if ignored, severely affects survival estimates.

Financial modelling of the potential cost of ovine Johne's disease and the benefit of vaccinating sheep flocks in southern New South Wales.

OBJECTIVE: To develop an enterprise gross margin (GM) model that predicts the on-farm financial impact of ovine Johne's disease (OJD) for various sheep enterprises in Australia. In addition, to estimate the benefits and costs of control through the use of the Gudair vaccination, including a breakeven point. DESIGN AND POPULATION: Data for the model was gained from an observational study conducted over a 3-year period from 2002 to 2004 using sheep from 12 OJD-infected flocks from southern New South Wales. Flocks ranged between 3500 and 20,000 sheep, with owner estimates of 5% or greater OJD mortality at the start of the study. PROCEDURE: A GM model was developed to predict the on-farm financial impact of OJD for various sheep enterprises in Australia, comparing non-infected, infected (status quo) and infected (vaccination) disease scenarios. RESULTS: Vaccination breakeven points are achieved within 2 to 3 years for breeding enterprises if OJD mortalities are high, rising towards 7 years for a Merino ewe enterprise if OJD mortalities are low. CONCLUSION: The GM model demonstrates the returns to investment of vaccination for Australian sheep producers with OJD-infected flocks.

Estimate of the direct production losses in Canadian dairy herds with subclinical Mycobacterium avium subspecies paratuberculosis infection.

The objective of this study was to estimate the annual losses from Mycobacterium avium subspecies paratuberculosis (MAP) for an average, MAP-seropositive, Canadian dairy herd. A partial-budget simulation model was developed with 4 components of direct production losses (decreased milk production, premature voluntary culling, mortality, and reproductive losses). Input values were obtained primarily from a national seroprevalence survey of 373 Canadian dairy farms in 8 of 10 provinces. The model took into account the variability and uncertainty of the required input values; consequently, it produced probability distributions of the estimated losses. For an average Canadian dairy herd with 12.7% of 61 cows seropositive for MAP, the mean loss was $2992 (95% C.I., $143 to $9741) annually, or $49 per cow per year. Additional culling, decreased milk production, mortality, and reproductive losses accounted for 46%, 9%, 16%, and 29% of the losses, respectively. Canadian dairy producers should use best management practices to reduce these substantial annual losses.

The associations between culling due to clinical Johne's disease or the detection of Mycobacterium avium subsp. paratuberculosis fecal shedding and the diagnosis of clinical or subclinical diseases in two dairy herds in Minnesota, USA.

Our objectives were to identify associations between clinical or subclinical diseases and subsequent culling because of clinical Johne's disease (JD) or the detection of fecal shedding of Mycobacterium paratuberculosis (Map) after 305 days in milk (DIM). A total of 1297 cows from two Minnesota dairies were enrolled in the study. From study cows, fecal samples were obtained prior to calving (close-up period) and after at least 305 DIM or at the time of leaving the herd (sold/dead). Between 3 and 21 DIM, blood samples were obtained for serum betahydroxybutyrate (BHB) and serum total-protein testing. Body-condition score (BCS) was evaluated during the close-up period, between 3 and 21 DIM, and at the end of lactation. The diagnosis time (DIM) of clinical disease events (culling because of JD clinical signs, ketosis, lameness, mastitis, displacement abomasum, injury, metritis, milk fever, pneumonia, and retained placenta) was recorded. Sixty-six cows were culled because of JD clinical signs (CCDJ) with average DIM of 209. CCDJ was associated with event of pneumonia (n=131) (OR=3.0, 95% CI=1.0-6.0) and level of fecal shedding (light: OR=13.0, 95% CI=5.3-30.0; moderate: OR=34.0, 95% CI=13.0-89.0; heavy: OR=66.0, 95% CI=26.0-171.0). Detection of fecal shedding at the end of the lactation (n=79) was associated only with event of pneumonia (OR=2.0, 95% CI=1.0-4.0).

The association between Mycobacterium avium subsp. paratuberculosis fecal shedding or clinical Johne's disease and lactation performance on two Minnesota, USA dairy farms.

Lactation performance of cows infected with Mycobacterium avium subsp. paratuberculosis (Map) was previously studied using only serum ELISA as a diagnostic method. This study evaluated on two dairy farms in Minnesota, USA the lactation performance (measures of health, production, reproduction, and survival) of cows shedding Map in feces before calving and of cows culled with clinical signs consistent with Johne's disease (JD) during the subsequent lactation. Fecal samples were collected from 1052 cows within 21 day before calving and tested for Map with bacterial culture. Producers' observed signs of clinical disease (milk fever, retained placenta, metritis, ketosis, displaced abomasum, lameness, mastitis, pneumonia, and JD) and production and reproduction data were recorded for each cow. The association between fecal shedding or clinical JD and lactation performance was evaluated. Logistic regression was used to evaluate the association with any clinical and subclinical diseases as the outcome. General linear model was used to evaluate the association with milk production, and survival analysis techniques were used to evaluate the association with days in the study before culling and days from calving to conception. In 84 cows (8% of 1052 cows) fecal samples were positive for Map (46% light, 26% moderate, and 28% heavy shedders). In multivariable analysis, light, moderate, and heavy fecal shedding cows produced on average 537, 1403, and 1534 kg, respectively, less milk per lactation and 1.4, 5.2, and 7.5 kg, respectively, less milk per day than fecal negative cows. Fecal culture positive cows were less likely to be bred and conceive. In the multivariable analysis the 56 cows culled with presumed JD produced approximately 1500 kg/lactation or 5 kg/day less than all other cows. The negative economic impact implied by decreased lactation performance in cows shedding Map or with clinical JD may motivate producers to implement programs to control Map infection and subsequent JD.

The effect of Johne's disease on production traits in Romney, Merino and Merino x Romney-cross ewes.

AIM: To quantify the effects of clinical Johne's disease on the performance of Romney, Merino and Merino x Romney-cross ewes. METHODS: The performance of ewes was compared using eight birth cohorts (1971-1978). Merino and Merino-cross genotypes included New Zealand Merino and Australian Superfine Merino sources. Intensive monitoring of Johne's disease was undertaken over the production years 1975-1982. Positive diagnostic evidence of Johne's disease was established post mortem from lesions of granulomatous enteritis associated with high numbers of acid-fast bacilli. Over years, data on a total of 2,341 Romney ewes and 1,292 Merino and Merino x Romney-cross ewes were recorded, consisting of annual records of liveweight (LWT), greasy fleece weight (FWT), number of lambs born per ewe per year (NLB), and lifetime productivity of ewes. RESULTS: A total of 82 (3.5%) Romney ewes and 62 (4.8%) Merino and Merino x Romney-cross ewes were diagnosed with clinical Johne's disease over the 8-year monitoring period, equivalent to 0.9% and 1.2% annual cases for these breeds, respectively, of the ewes present at mating. The percentage of clinical cases (p<0.04) and the age at death from Johne's disease (p<0.02) were lower for Romneys than for Superfine Merinos. The mean age of death from Johne's disease was 3.41 (standard error (SE) 0.06) years, lower than the mean disposal age from the flock of 5.03 (SE 0.02) years for clinically normal ewes (p<0.001). In their final year of production, ewes with clinical Johne's disease had lower LWT by 5.3 kg (10.5% of the mean; p<0.001), lower annual FWT by 0.54 (SE 0.10) kg (14.2%; p<0.001), fewer NLB by 0.15 (SE 0.07) lambs (13%; p<0.05), and lower litter weaning weights by 3.6 (SE 1.3) kg (15%; p<0.01) compared with clinically normal ewes. The size of the production losses associated with Johne's disease depended on the age to which ewes survived. Considering all production years of ewes (up to 8 years), the total weight of lambs weaned by ewes with clinical Johne's disease was 30.9 (SE 3.4) kg lower (46%; p<0.001) than the total from clinically normal ewes. CONCLUSIONS: Clinical Johne's disease led to significant losses in LWT, FWT, NLB, and in the lifetime production of ewes, amounting overall to a 46% reduction in productivity (p<0.001). CLINICAL RELEVANCE: Productivity losses from clinical cases of Johne's disease would be of considerable economic importance in flocks with a high incidence of the disease. The lack of good diagnostic tests for Johne's disease in the live animal, and the lack of active surveillance programmes, has made it difficult to establish the true prevalence of Johne's disease in sheep flocks in New Zealand, and its economic consequences.

Losses of adult sheep due to ovine Johne's disease in 12 infected flocks over a 3-year period.

OBJECTIVE: To measure the biological and financial impact of ovine Johne's disease (OJD) mortalities on 12 infected flocks within the endemic area of southern New South Wales over a 3-year period. DESIGN AND POPULATION: An observational study was conducted over a 3-year period from 2002 to 2004 on sheep from 12 OJD-infected flocks from southern NSW. Flocks ranged from between 3,500 and 20,000 sheep. At the start of the study owner estimates of OJD mortality were 5% or greater. METHOD: Annual mortality rates were estimated from farm records provided by owners. The proportion of OJD mortalities was assessed after histological examination of tissues collected from dead and moribund sheep during 5-day necropsy inspections conducted in autumn, winter, spring and summer in 2002. The financial impact was estimated using a gross margin analysis for each of the three study years and by placing a financial value on the necropsied sheep. RESULTS: On the 12 farms, the average OJD mortality rate was 6.2% (range 2.1% to 17.5%) in 2002, 7.8% (range 1.8% to 14.6%) in 2003 and 6.4% (range 2% to 11.9%) in 2004. The average decrease in gross margin due to OJD infection on a farm in 2002 was 6.4% (range 2.2% to 15.4%), 8.5% (range 3.1% to 15.8%) in 2003 and 7.4% (range 1.5% to 15.4%) in 2004. This equates to an average reduction in annual income of $13,715 per farm per year. OJD losses accounted on average for two thirds of the total estimated financial loss associated with sheep deaths. CONCLUSION: This study demonstrates the significant biological and financial impact of OJD on sheep flocks. These findings are of relevance to all Australian sheep flocks infected or at risk of OJD infection.

Efficacy of a killed vaccine for the control of paratuberculosis in Australian sheep flocks.

A field trial was undertaken from 1999 until 2004 to determine the efficacy of a killed M. a. paratuberculosis vaccine, Gudair, for the control of ovine Johne's disease (OJD) in merino sheep run under Australian pastoral conditions. On each of three farms experiencing significant OJD losses (5-15% per annum), 200 merino lambs (age 1-4 months) were vaccinated, and 200 lambs were left unvaccinated. Animal assessments and sample collections were conducted twice yearly until 4 or 5 years of age. The impact of vaccination on mortality rate, faecal shedding of M. a. paratuberculosis (by pooled and individual faecal culture), liveweight, wool productivity, vaccine injection site lesions and cellular (BOVIGAM) and humoral (PARACHEK) immunity was examined. Vaccination stimulated cell-mediated and humoral immune responses, reduced mortalities due to OJD by 90% and delayed faecal shedding for the first year post-vaccination. Thereafter, the prevalence of shedders among vaccinates was reduced by 90%. The numbers of M. a. paratuberculosis excreted by the vaccinated groups were also reduced by at least 90% at most sampling times. However, high levels of excretion by vaccinates occurred on some occasions, and although only 7 of 600 vaccinates died from OJD, all 7 had multibacillary lesions. Thus there remains a risk that some vaccinated sheep will transfer the disease. Small reductions in liveweight were found in vaccinated lambs in the first year, but there was little effect on wool production. Vaccine injection site lesions were detected in almost 50% of sheep after 2 months, and these persisted for at least 4 years in 20-25% of vaccinates. Data from this trial enabled the registration of Gudair in Australia in 2002 and underpins the pivotal role of vaccination in the current management of OJD.

Effect of paratuberculosis on culling, milk production, and milk quality in dairy herds.

OBJECTIVE: To determine the effect of paratuberculosis on culling, milk production, and milk quality in infected dairy herds. DESIGN: Cross-sectional study. ANIMALS: 689 lactating dairy cows in 9 herds. PROCEDURE: Milk, blood, and fecal samples were obtained from all cows. Fecal samples were evaluated via mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium avium subsp paratuberculosis, and preserved milk samples were tested with an ELISA for antibodies against M paratuberculosis. Mixed effect and proportional hazards models were used to determine the effect of paratuberculosis on 305-day milk, fat, and protein production; somatic cell count linear score; and the risk of culling. RESULTS: Cows with positive results of bacteriologic culture of feces and milk ELISA produced less milk, fat, and protein, compared with herdmates with negative results. No difference in 305-day milk or fat production was detected in cows with positive results of serum ELISA, compared with seronegative cows. The 3 survival analyses revealed that cows with positive results of each test were at higher risk of being culled than cows with negative results. Paratuberculosis status, as determined by use of all 3 diagnostic tests, was not associated with milk somatic cell count linear score. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for the 9 herds in this study, paratuberculosis significantly decreased milk production and cow longevity.

Clinical paratuberculosis as a cause for higher culling rate in cows with left displaced abomasum and diarrhoea in a holstein dairy herd.

The present report identifies a statistically significant (P < 0.001) higher postoperative culling rate in cows with left displaced abomasum (LDA) exhibiting diarrhoea, compared with cows with LDA and normal faeces from a large Holstein dairy herd (odds ratio = 34). Paratuberculosis was histopathologically confirmed in 77.0% of cows with LDA exhibiting diarrhoea.

Johne's disease in alpacas (Lama pacos) in Australia.

Johne's disease was diagnosed in 10 alpacas (Lama pacos) in Australia between February 1993 and May 1994. Eight of the animals were between 12 and 24 months of age, one was a 6-year-old female, and one was a 4-year-old male. Five, including the 6-year-old and the 4-year-old alpacas, showed weight loss and diarrhoea before death or slaughter. The other cases showed no clinical signs of Johne's disease but 4 gave a positive result on faecal culture and one gave a positive result on testing with the caprine AGID assay and had acid-fast organisms in its faeces. At necropsy, all cases had grossly enlarged mesenteric lymph nodes. Johne's disease was diagnosed after histological examination of the lymph nodes with conventional culture and polymerase chain reaction testing of tissue samples. This report outlines the clinical, epidemiological, and pathological findings in these cases.