Individual faecal and boot swab sampling to determine John's disease status in small cattle herds.

Individual faecal samples were collected from adult animals in 275 cattle farms previously positive for Mycobacterium avium subsp. paratuberculosis (MAP). In addition, boot swab samples were collected in 30 randomly chosen farms. Faecal samples were tested for MAP by a combination of bacterial culture and PCR. A logistic regression and the Pearson Correlation were used to calculate the relation between the number of MAP­positive cows and boot swab results. In 66.9% of all previously tested herds, no positive individual faecal sample was detected, indicating possible fadeout of the infection. In 9 (30.0%) of the 30 selected farms, at least one MAP­shedding animal was detected in faecal samples individually collected, while only 5 (16.7%) of these farms were found positive when the boot sampling method was used. The sensitivity of the boot swab sampling increased up to 92% (95% CI: 41%­99%), if at least 12 animals were faecal MAP­shedders in a herd. The current study shows possible fadeout of JD in a substantial percentage of previously infected herds. Furthermore, in small herds, a relatively high within­herd prevalence of MAP­shedding animals is needed to assure reliable positive boot swab results.

Short communication: Risk factors associated with Mycobacterium avium ssp. paratuberculosis introduction into dairy herds in Galicia, northwestern Spain.

This study assessed potential risk factors associated with introduction of Mycobacterium avium ssp. paratuberculosis (MAP) into dairy cattle herds in the Galicia region, northwestern Spain. The study was carried out with data collected from 93 dairies enrolled in a voluntary MAP control program. Information on potential risk factors was obtained through personal interviews with the farmers and veterinarians in charge of the control program of each farm. In addition, blood samples were taken annually over 2 years from cows on the farms in the program, and analyzed with a commercial ELISA to detect antibodies to MAP. Fecal samples of all ELISA-positive cows were analyzed using PCR. Based on χ2 test and Fisher's exact test, purchase practices, shared manure truck, shared materials, and visitors per month who contacted animals were found to be significantly associated with farm MAP infection status. Multiple logistic regression indicated that purchase practices and herd size (included as a potential confounder) are the variables that best predict MAP status.

An assessment of the association between soil pH and ovine Johne's disease using Australian abattoir surveillance data.

There has long been discussion in the literature about the role of soil on ovine Johnes disease (OJD). This is especially true of soil pH, however there is very little research to support an association between pH and OJD prevalence. The primary objective of this study was to examine the hypothesis that there is an association between soil pH and OJD. Several additional hypotheses were also assessed. Sheep properties that were surveyed by the Australian National Sheep Health Monitoring Project where classified as OJD reactor positive or otherwise. A variety of explanatory variables such as soil (especially soil pH), environmental and management factors were examined. Spatial regression models were assessed using information theory to examine support for various hypotheses and to examine associations; especially that soil pH is associated with OJD. A total of 1213 properties from 10,578 were classified as OJD positive (11.5%, 95% CI: 10.9-12.1). Within the limitations of the study, only modest support was found for an association between soil pH and the presence or absence of OJD. Instead, OJD prevalence was affected by several factors concurrently, a so called multi-factorial model (hypothesis). In this supported multifactorial hypothesis soil pH was marginally associated with OJD (p=0.04) and had a relatively weak effect (OR 0.91, 95% CI 0.82 to 1.00). OJD was strongly associated with a number of biosecurity and environmental factors such as the time since infection arrived in a region, absence of biosecurity programs (such as regional biosecurity programs or state based programs) and, to a lesser extent, solar irradiation. Soil pH may play a relatively small role in explaining OJD prevalence when evaluated as part of a multifactorial model. Biosecurity and other environmental factors appear to be more strongly associated with the presence of OJD in Australia.

Paratuberculosis in sheep and goats.

Paratuberculosis is a chronic insidious, often serious, disease of the global small ruminant industries, mainly causing losses from mortalities and reduced productivity on-farm, interference in trading and, in Australia, profound socio-economic impacts that have periodically compromised harmony of rural communities. The pathogenesis, diagnosis, impacts and disease management options for ovine and caprine paratuberculosis are reviewed, comparing current controls in the extensive management system for sheep in wool flocks in Australia with the semi-intensive system of dairy flocks/herds in Greece. Improved understanding of the immune and cellular profiles of sheep with varying paratuberculosis outcomes and the recognition of the need for prolonged vaccination and biosecurity is considered of relevance to future control strategies. Paratuberculosis in goats is also of global distribution although the prevalence, economic impact and strategic control options are less well recognized, possibly due to the relatively meagre resources available for goat industry research. Although there have been some recent advances, more work is required on developing control strategies for goats, particularly in dairy situations where there is an important need for validation of improved diagnostic assays and the recognition of the potential impacts for vaccination. For all species, a research priority remains the identification of tests that can detect latent and subclinical infections to enhance removal of future sources of infectious material from flocks/herds and the food chain, plus predict the likely outcomes of animals exposed to the organism at an early age. Improving national paratuberculosis control programs should also be a priority to manage disease risk from trade. The importance of strong leadership and communication, building trust within rural communities confused by the difficulties in managing this insidious disease, reflects the importance of change management considerations for animal health authorities. Although concerns of vaccine efficacy, safety and issues with diagnosis and administration persist, vaccination is increasingly recognized as providing a robust strategy for managing paratuberculosis, having made important contributions to the health of Australian sheep and the lives of producers with affected properties, and offering a mechanism to reduce risk of infection entering the food chain in ovine and caprine products.

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

Expression of genes associated with cholesterol and lipid metabolism identified as a novel pathway in the early pathogenesis of Mycobacterium avium subspecies paratuberculosis-infection in cattle.

Johne's disease (JD) is a chronic disease affecting ruminants and other species caused by the pathogenic mycobacterium, Mycobacterium avium subsp. paratuberculosis (MAP). MAP has developed a multitude of mechanisms to persist within the host, and these in turn are counteracted by the host through various immune pathways. Identifying and characterising the different strategies employed by MAP to alter the host immune system in its favour, and thereby persist intracellularly, could hold the key to developing strategies to fight this disease. In this study we analysed a subset of bovine microarray data derived from early time points after experimental infection with MAP. A specifically developed integrated approach was used to identify and validate host genes involved in cholesterol homeostasis (24DHCR, LDLR, SCD-1), calcium homeostasis and anti-bacterial defence mechanisms, (CD38, GIMAP6) which were downregulated in response to MAP exposure. A trend for upregulation of granulysin gene expression in MAP-exposed cattle in comparison to unexposed cattle was also observed. From these analyses, a model of potential pathogen-host interactions involving these novel pathways was developed which indicates an important role for host lipids in mycobacterial survival and persistence.

In vivo and in vitro expression pattern of Toll-like receptors in Mycobacterium avium subspecies paratuberculosis infection.

Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic infectious disease of ruminants. Activation of the Toll-like receptors (TLR) in response to microbial stimuli, including MAP, initiates responses in immune cells of the blood and within peripheral tissues. TLR2, 4 and 9 are believed to play a critical role in the initiation of immune responses against mycobacteria. In this study we report on the in vivo expression pattern of these receptors in sheep and cattle experimentally exposed to MAP. Experiments using the mouse macrophage cell line, RAW 264.7, and on isolated bovine monocytes were also carried out to assess the expression pattern of TLR2 and 4 in response to MAP and the non-pathogenic mycobacterial strain, M. smegmatis. Results from the in vivo study showed that there was a significant upregulation of TLR2 (P<0.05) at early time-points post-inoculation in the peripheral blood cells of sheep exposed to MAP S strain that went on to develop severe (multibacillary) disease. However, in the cattle during the initial months post-exposure to MAP C strain, TLR2 was significantly downregulated (P<0.05). TLR4 was significantly upregulated (P<0.05) at later stages (12 months post-inoculation) in MAP-exposed sheep with multibacillary disease; however significant differences in TLR4 expression were not observed in cattle. Expression of TLR9 was unchanged in MAP-exposed sheep and cattle. In vitro studies on mouse macrophages supported the findings of in vivo TLR2 gene expression increases seen in the sheep, in that the TLR2 receptor expression in response to MAP-infection was significantly increased in comparison to cells infected with a non-virulent mycobacterium, M. smegmatis. A likely role for TLR2 in the pathogenesis of Johne's disease is proposed.

Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry.

Mycobacterium avium subspecies paratuberculosis is considered as one of the most serious problems affecting the world's ruminant industry due to its significant impact on the global economy and the controversial issue that it may be pathogenic for humans. M. avium subspecies paratuberculosis is the causative agent of Johne's disease in animals and might be implicated in cases of human Crohn's disease. We provide an insight into M. avium subspecies paratuberculosis from some bacteriological, clinical, and molecular epidemiological perspectives.

Producer survey of bird-livestock interactions in commercial dairies.

The objective of this producer survey was to identify and estimate damage caused by bird-livestock interactions in commercial dairies. The interactions between birds and livestock have previously been implicated in causing economic damage while contributing to the environmental dissemination of microorganisms pathogenic to livestock and humans. Very little research exists to help producers understand what bird species use dairies, why they use dairies, or the scope and nature of damage created as a result of bird-livestock interactions. To better characterize these interactions, we surveyed dairy operators within Pennsylvania, New York, and Wisconsin. Survey results suggest that the most common and destructive bird species found on commercial dairies are invasive to North America, and their use of dairies is associated with the loss of cattle feed, increased operating costs, and an increase in dairies self-reporting Salmonella spp. and Mycobacterium avium ssp. paratuberculosis. Cattle feed loss estimates generated from this survey were used to parameterize an input-output (IO) economic model using data from 10 counties in the state of Pennsylvania (Bedford, Berks, Blair, Bradford, Chester, Cumberland, Franklin, Lancaster, Lebanon, and Somerset). This IO model allowed us to estimate direct, indirect, and induced economic effects of feed loss from bird damage to dairies within these counties. The IO model output suggests that feed loss costs Pennsylvania between $4.11 and $12.08 million (mean $10.6 million) in total economic damage, with approximately 43 to 128 jobs (mean 112) forgone statewide in 2009.

Epidemiology and economics of paratuberculosis.

Johne's disease is the clinical manifestation of Mycobacterium avium subsp. paratuberculosis (MAP) infection and has become widespread since it was first observed in the United States in the early 1900s. MAP is primarily spread through the fecal-oral route, and herds generally become infected by unknowingly purchasing infected animals. The economic losses from the disease are primarily due to decreased milk production, decreased weaning weights in nursing young stock, increased replacement costs, and decreased slaughter value.

Detection of Mycobacterium avium subspecies paratuberculosis in naturally exposed dairy heifers and associated risk factors.

An observational prospective study was conducted to identify risk factors associated with fecal shedding of Mycobacterium avium ssp. paratuberculosis (MAP) in naturally exposed dairy heifers. The study population consisted of heifers from 8 dairy herds in Michigan participating in a MAP control demonstration project. Ten heifers from 4 age groups (0 to 3, 4 to 6, 7 to 14, and 15 to 24 mo) were selected from each herd every 4 mo for 28 mo and tested for the presence of MAP by fecal culture (FC). Heifers from dams testing positive for MAP by serum ELISA or FC were preferentially selected, with the remainder of the age cohort filled with randomly selected heifers. Logistic regression using generalized estimating equations to account for clustering of data within herds and repeated measures across heifers was used to evaluate the relationship between MAP FC status of heifers and herd risk factors. In total, 1,842 fecal samples were collected from 1,202 heifers. Thirty-six (2%) fecal samples, representing 27 individual heifers, cultured positive for MAP. Heifers shedding MAP were more likely to occur in herds with adult-cow MAP ELISA prevalence >10% (odds ratio = 4.7; 95% confidence interval: 2.0-11.1) and herds milking >300 cows (odds ratio = 5.7; 95% confidence interval: 2.4-13.4). Mycobacterium avium ssp. paratuberculosis can be cultured from the feces of naturally infected dairy heifers. The future performance of these MAP FC-positive heifers is unknown and needs to be explored.

Paratuberculosis in a dairy Gyr herd in the State of Paraíba, Brazil / Paratuberculose em um rebanho Gir leiteiro no Estado da Paraíba Brasil

This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (Johne's disease) in a dairy Gyr herd in the State of Para�ba, northeastern Brazil. An eight years old cow with chronic unresponsive diarrhea was clinically examined and euthanized for pathological evaluation. Fecal samples from all 160 animals over 12 months of age from the herd were collected for isolation of Mycobacterium avium subsp. paratuberculosis. Clinically, the index case cow was severely dehydrated, cachectic, with profuse mucous diarrhea. The main post-mortem findings were emaciation and thickened intestinal wall. Microscopically, the intestinal lamina propria and submucosa were infiltrated by macrophages, epithelioid cells, and Langhans giant cells with numerous alcohol-acid resistant bacilli in the cytoplasm. Two fecal samples displayed growth in slants of Herrold's egg-yolk agar supplemented with mycobactin J, 150 days after incubation. No growth was noticed in slants without mycobactin J. Microscopic examination of the isolated microorganisms stained by Ziehl-Neelsen revealed considerable amounts of alcohol-acid resistant bacilli, morphologically compatible with Mycobacterium spp. Based on the clinical signs, gross and histological lesions, growth time, bacterial morphology in Ziehl-Neelsen staining, and dependence of mycobactin J, the first diagnosis of paratuberculosis in Zebu cattle was made.

Objetivou-se descrever os aspectos clínicos, anátomo-histopatológicos e microbiológicos da paratuberculose em um rebanho Gir leiteiro no Estado da Paraíba. Uma vaca de oito anos que apresentava diarréia persistente, refratária a tratamento foi necropsiada para estudo anátamo-histopatológico. Também foram coletadas amostras de fezes de todos os 160 animais do plantel, com idade superior a 12 meses, para tentativa de isolamento de Mycobacterium avium subsp. paratuberculosis. Ao exame clínico, o animal caso índice apresentou caquexia, diarréia profusa e desidratação grave. À necropsia, o animal apresentou-se emaciado e, ao exame detalhado do trato digestivo, foi observado espessamento da parede e superfície mucosa do íleo e intestino grosso. À microscopia, verificou-se intensa infiltração de macrófagos espumosos associado a raras células epiteliódes e gigantes do tipo Langerhans na lâmina própria e submucosa. À coloração de Ziehl-Neelsen foram observadas miríade de bacilos álcool-ácido resistentes no citoplasma destas células. Houve crescimento de colônias bacterianas em duas das 160 amostras de fezes após 150 dias de incubação em tubos com meio Herrold's egg-yolk suplementados com micobactina J e ausência de crescimento nos tubos com mesmo meio, mas sem suplementação. Os microrganismos isolados foram corados pelo Ziehl-Neelsen observando-se presença de grande quantidade de bacilos álcool-ácido resistente, com morfologia compatível ao gênero Mycobacterium. Baseado na história clínica, achados anátomo-histopatológicos e histoquímicos (Ziehl-Neelsen), e microbiológicos, firmou-se o primeiro diagnóstico de paratuberculose em Zebu na Paraíba.

Is alpha-dystroglycan the missing link in the mechanism of enterocyte uptake and translocation of Mycobacterium avium paratuberculosis?

Mycobacterium avium paratuberculosis (MAP) is an important animal pathogen with a potential role in human disease. MAP is recognized to cause severe diarrheas and wasting syndrome in patients infected by the human immuno-deficiency virus. Recently, there is also growing evidence that MAP is somehow involved into the patho-mechanisms of Crohn's disease. However, the mechanism how MAP binds to the intestinal mucosa and consecutively invades and translocates the intestinal epithelial cells is still unknown. Here it is suggested, that MAP enters the intestinal cells via the dystrophin-glycoprotein-complex (DGC) in a similar manner as known from Mycobacterium tuberculosis in peripheral Schwann cell invasion. Recent approaches to identify the mechanism of intestinal MAP uptake revealed several molecules which are therefore thought to be involved in MAP cell invasion. Nevertheless, there is no comprehensive connection so far to link all identified mechanisms together. Since the DGC has a direct association to all identified molecules and mechanisms and therefore seems to be the missing link, it is hypothesized now, that MAP binds to alpha-dystroglycan and exploits an endogenous recycling mechanism to control the dystroglycan expression levels to enter and translocate the intestinal cells. Since there are options to modify dystroglycan this might be a potential new target to prevent or even treat intestinal MAP infections.

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

We conducted a cross-sectional study in 2004-2005 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 (<2%), medium (2-10%) and high (>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 1-2 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.

Diagnostic validity and costs of pooled fecal samples and individual blood or fecal samples to determine the cow- and herd-status for Mycobacterium avium subsp. paratuberculosis.

Two tests are used on a regular basis to detect Mycobacterium avium subsp. paratuberculosis (Map): ELISA and fecal culture. Fecal culture is considered more sensitive and specific but is costly and requires 3-4 months for results. Pooling of fecal samples of individual animals may reduce the high costs of fecal culture. The objective of the study was to investigate the diagnostic validity and costs for pooling of fecal samples in dairy farms relative to culture or an ELISA on individual samples to determine the cow- or herd-status for Map. Fifty fecal and blood samples per herd were collected in 12 Chilean dairy herds. The sensitivity of pooling was estimated given the pool-size, amount of shedding in the pool and the prevalence in the herd. The sensitivity of the pools relative to individual fecal culture was 46% (95% CI 29-63%) and 48% (28-68%) for pools of 5 and 10 cows, respectively. The sensitivity of the pools was lower in pools with low shedders (26 and 24% for pools of 5 and 10, respectively) than in pools with moderate or heavy shedders (>75% sensitivity). Pools of 10 cows are the better option to determine or monitor the herd status. A whole-herd ELISA is the least expensive way to determine the status of individual cows but has a lower Se and Sp than individual culture.

Environmental mycobacteria in soil and water on beef ranches: association between presence of cultivable mycobacteria and soil and water physicochemical characteristics.

Exposure to environmental mycobacteria has been reported to be a factor contributing to false-positive results on bovine serological tests detecting antibodies to Mycobacterium avium subsp. paratuberculosis (Mptb). This study was conducted to investigate the association between recovery of mycobacteria from the environment of cattle and both (i) historically high or low seroprevalence to Mptb, and (ii) soil and water physicochemical characteristics. Eighty-two samples (soil and water) from nine beef cattle ranches in South-central and South Texas were assessed for the presence of mycobacteria. Twelve mycobacterial species were cultured from soil and water from four herds; no Mptb were detected in environmental samples. A positive culture of environmental mycobacteria from soil was significantly associated with lower pH and calcium as well as higher iron, zinc and manganese contents. Beef cattle are likely to be exposed to environmental mycobacteria that may contribute to false-positive results on ELISAs for Mptb infection. Exposure rates to these mycobacteria likely vary across small geographical areas and may be related to soil and/or water physicochemistry.

Longterm investigation in an Austrian dairy herd with low prevalence of paratuberculosis detection of antibodies in blood and milk.

Paratuberculosis (Johne's disease), which is widely distributed throughout the world, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Diagnosis of subclinically infected cattle is challenging and is especially problematic in herds with low prevalence of MAP. The aim of this long-term study was the comparison of different diagnostic tests for MAP and specific antibodies in a herd with low prevalence of MAP. Three different commercially available serum-ELISA (Svanovir-ELISA, Svanova, Uppsala, Sweden; IDEXX-ELISA, IDEXX Laboratories, Maine, USA; Pourquier-ELISA, Institut Pourquier, Montpellier, France) and two milk ELISA (Svanovirm-ELISA Svanova, Uppsala, Sweden; Pourquier-ELISA, Institut Pourquier, Montpellier, France) were compared. Apart from these indirect diagnostic tests, two methods for the detection of the etiologic agent (bacteriologic culture and real-time PCR of faecal samples) were performed. In January 2005 the first and in April 2005 the second herd investigation of all animals older than 2 years (n=335) were carried out. Blood, milk and faecal samples were taken. From November 2005 until April 2006 follow up investigations were performed. For this purpose, blood-, milk- and faecal samples were monthly taken from 63 selected animals. The highest number of blood- and milk samples with a detectable antibody-level was found by the Svanovir-ELISA. There was a significant correlation between serum- and milk- Svanovir-ELISA results, whereas the agreement between ELISA and faecal culture/PCR was low. Significant correlations between Svanovir-serum-ELISA results and milk somatic cell counts could be registered. Moreover, there was significant agreement between IDEXX-serum-ELISA results with the age and number of lactations of the cows, as well as the mother's MAP-status.