Association between risk-assessment scores and individual-cow Johne's disease-test status over time on seven Michigan, USA dairy herds

To evaluate the effectiveness of management practices implemented to control the spread of Johne's disease (JD), we conducted a 5-year observational study (January 2003 to December 2007) on seven Michigan, USA dairy herds containing cows infected with Mycobacterium avium subsp. paratuberculosis (MAP; the causative agent of the disease). The JD incidence and prevalence was monitored in each herd annually by serum ELISA and/or fecal culture of all adult cows. A JDCP was designed specifically for each herd based on the results of an initial risk-assessment. The risk-assessment was repeated annually and the control program updated as needed. Herd risk-assessment scores were used to measure compliance with the control program and create JD-risk profiles for individual cows raised on the farms. The association between specific risk-assessment scores and the JD-test status of individual cows was evaluated using logistic regression. We accounted for clustering of cows within herds using generalized estimating equations (GEE). Multivariable models were built with purposeful selection of risk factors assessed on univariable analyses. The dataset analyzed consisted of 3707 cows raised on the respective farms, of which 616 were classified as infected with MAP based on testing positive on fecal culture or serum ELISA. Of the cows that were not exposed to the control program, 20% were classified as infected, while only 7% of cows that were exposed to the control program were infected. The final multivariable model consisted of two factors: exposure to adult cows other than dam at birth (OR=1.09, 95% CI: 1.06, 1.13), and feeding colostrum from one cow to multiple calves (OR=1.10, 95% CI: 1.09, 1.12). Based on this study, implementing practices that minimize the exposure of newborn calves to MAP being shed by infected adult cows should take priority.     

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.     

Estimated within-herd prevalence (WHP) of Mycobacterium avium subsp. paratuberculosis in a sample of Minnesota dairy herds using bacterial culture of pooled fecal samples

The objective of this study was to describe the estimated within-herd prevalence (WHP) of Mycobacterium avium subsp. paratuberculosis (Map) in a sample of infected dairy herds in Minnesota (N = 66) using test results from bacterial culture of pooled fecal samples. Fecal samples were collected from up to 100 cows in each herd and were tested using bacterial culture in pools of 5 cows based on age order. The mean herd size was 222 (44 to 1500) milking cows; the cows were predominantly Holstein. Using a frequentist approach, the within-herd mean individual fecal prevalence was 10% [95% confidence interval (CI) = 4% to 16%] assuming 70% test sensitivity and 99.5% test specificity. Using Bayesian methods, the estimated true within-herd individual cow prevalence was 14% (95% CI = 7% to 27%). Within-herd prevalence was higher in larger dairy herds than in herds with fewer cows. As Map is the causative agent of Johne's disease (JD), the results of this study could contribute to the success of a nationwide control program for this disease.     

Proteomic analysis of plasma from cows affected with milk fever using two-dimensional differential in-gel electrophoresis and mass spectrometry

Milk fever is an important metabolic disorder of dairy cows after calving, and is characterized by hypocalcemia, tetany, lateral recumbency, and eventual coma. To date, there have been many reports about the pathogenesis and pathophysiology of milk fever, but the plasma protein profile in milk fever has not been reported. The aim of our study was to investigate novel pathophysiological changes in the plasma proteome of cows affected with milk fever. Plasma samples were collected from eight Holstein cows with milk fever (T), and eight control Holstein cows without milk fever (C), at an intensive Holstein dairy farm in Heilongjiang province, China. Samples were analyzed by fluorescence two-dimensional (2D) differential in-gel electrophoresis (DIGE), followed by in-gel digestion, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for peptide mass fingerprinting of selected protein spots. Eight of the 23 differential protein spots in the plasma of T and C cows were isolated and identified by 2D-DIGE and MALDI-TOF-MS. The protein spots represented five unique proteins, and had significant alterations in spot volume as determined by DeCyder differential in-gel analysis (DIA) software. The upregulated proteins were identified as serpin peptidase inhibitor (angiotensin), which regulates blood pressure and maintains fluid and electrolyte homeostasis, and endopin 2B which is involved in neural regulation. The downregulated proteins were serum albumin, which acts as a transport protein, fibrinogen beta chain which is involved in blood coagulation, and IgG heavy-chain C-region (IgG-C(H)) which participates in the immune response. In conclusion, we were able to use proteomic technologies to identify several novel plasma proteins in cows affected with milk fever. These findings may reveal new pathophysiological changes that occur in cows with milk fever.     

Modulation of cytokine gene expression and secretion during the periparturient period in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more than 22% of US dairy herds. Periods of immunosuppression may contribute to the transition from the subclinical to the clinical stage of infection. Understanding the effects of stressors such as parturition on the escalation of disease may provide information that will help to manage JD. The objective of this study was to characterize cytokine gene expression and secretion in periparturient dairy cows naturally infected with MAP. Blood was collected from the jugular vein of healthy noninfected, and subclinically and clinically infected dairy cows for 3 weeks pre-calving to 4 weeks post-calving. Real-time PCR was performed to evaluate the expression of the following cytokine genes by peripheral blood mononuclear cells: IFN-gamma, TNF-alpha, IL-12p35, IL-10, TGF-beta, and IL-4. To assess the effects of parturient immunosuppression on cytokine gene expression, RT-PCR data were analyzed by using 2(-ddCt) values calibrated to dCt value at +1 day relative to calving for each animal. Overall, cytokine gene expression was not influenced by infection status of the cows in this study. However, significant effects in cytokine gene expression were noted across sampling days within the periparturient period. Expression of IFN-gamma by NS and ConA-stimulated PBMCs declined at calving compared with prepartum values in both control and infected cows. Similarly, a decline in expression of IL-4 and IL-10 was observed for cells isolated from subclinically infected cows after stimulation with ConA. ConA-stimulated PBMCs isolated from infected cows secreted higher concentrations of IFN-gamma compared with the controls. A significant decline in IFN-gamma secretion was noted for MPS-stimulated cells for clinical cows from -21 days to +1 day. Stimulating cells with MPS resulted in greater secretion of IL-10 by infected cows during the postpartum period. A trend was also observed for higher TGF-beta secretion by NS PBMCs isolated from clinical cows in the postpartum period. Cells isolated from clinically infected cows and stimulated with MPS secreted higher levels of nitric oxide throughout the periparturient period when compared to control or subclinically infected cows. These data suggest that parturition is a very dynamic time period for host immunity, with potential for altered immunity to hinder the ability of dairy cows to thwart infectious diseases.     

In silico screened Mycobacterium avium subsp. paratuberculosis (MAP) recombinant proteins upregulated under stress conditions are immunogenic in sheep

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants. MAP is known to enter a dormant phase outside the host, typically on soil. In vitro experiments have reported regulation of certain MAP genes when exposed to stressors similar to what is thought to produce dormancy. It is believed that in vivo regulation of dormancy genes and associated proteins by MAP may play a role in evading the host defence mechanisms and induce the host immune response against these dormancy-related proteins. Five proteins encoded by dormancy-related genes that were previously found to be upregulated under stress conditions and predicted through in silico analysis to possess immune epitopes (three hypothetical proteins and two proteins involved in fatty acid metabolism) were selected. Recombinant proteins were produced, purified and evaluated by indirect enzyme-linked immunosorbent assay (ELISA) for immunogenicity using a panel of sera obtained from sheep unexposed and exposed to MAP. The antibody levels of the exposed group were significantly higher than the unexposed group (P<0.001). Individually, the five proteins were found to discriminate between sera from sheep exposed to MAP compared to unexposed sheep. At 91% diagnostic specificity, the diagnostic sensitivity of the recombinant antigen ELISA ranged from 24% to 42% and AUC(ROC) from 0.7015 to 0.8405.     

Comparison of the Johne''s absorbed EIA and the complement-fixation test for the diagnosis of Johne''s disease in cattle

A commercially available absorbed ELISA for the diagnosis of Johne's disease (JD) (paratuberculosis) in cattle, the Johne's Absorbed EIA, was compared with the conventional complement-fixation test (CFT) used in Australia. Stored plasma from 3 Victorian dairy herds with a history of JD, sera from specimens submitted from animals showing clinical signs of JD and sera from the US National Repository for Paratuberculosis Specimens were used to determine the sensitivity of each test. The EIA detected 48.8% of 43 Australian animals with subclinical JD, while the CFT detected only 12 (21.4%) of 56 subclinically affected cattle. Of 150 subclinically infected US cattle, the EIA detected 47.3% and the CFT detected 52.0%. The EIA detected 59.7% of animals which at the time of sampling were shedding Mycobacterium paratuberculosis in their faeces, but showed no clinical signs of JD, while the CFT detected 57.3%. The EIA correctly identified 88.2% of 136 histologically confirmed clinical cases, and the CFT detected 83.4%. The specificity of each test was determined by testing sera collected at slaughter from animals residing in a known JD-free area of Australia, and from samples from the US National Repository of Paratuberculosis Specimens collected from certified-free herds in Wisconsin. The EIA was found to have a specificity of 99.8% when 998 Australian animals were used as the test population, and 99.0% when 196 US animals were used. The specificity of the CFT using Australian samples was 96.9% and 95.2% using American samples.     

Parturition invokes changes in peripheral blood mononuclear cell populations in Holstein dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease (JD) is characterized by a protracted period of subclinical infection. Infected cows may remain in the subclinical state until stressors such as parturition and lactation invoke more clinical signs of disease. The objective of this study was to evaluate changes in the percentages of CD4(+), CD8(+), and gammadelta T-cells, B-cells, monocytes, as well as the expression of the activation marker, CD5, on these cell subpopulations in the peripheral blood of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) during the periparturient period. Peripheral blood mononuclear cells (PBMCs) were collected from 3 wk pre- to 4 wk post-calving and freshly isolated or cultured for 7d. Day 7 cultures were infected with live MAP at a 10:1 MOI (bacteria to adherent PBMC), and cultures were incubated for an additional 24h. Fluorescent antibody labeling of lymphocyte subsets and monocytes was conducted and analyzed with flow cytometry. Freshly isolated PBMCs from subclinical cows expressed a greater (P<0.05) percentage of CD8(+) and gammadelta T-cells compared with clinical cows. The percentage of CD4(+) T-cells increased (P<0.08) in clinical cows as parturition approached. During the postpartum period, clinical cows had greater (P<0.05) CD4:CD8 ratios compared with subclinical and control cows. After 8d, uninfected PBMCs from clinical cows had greater (P<0.05) percentages of CD14(+) cells compared with subclinical cows. When infected with live MAP, there was no effect of infection group or parturition on cell subpopulations. In fresh PBMCs, clinical cows expressed lower percentages of CD4(+)CD5(bright) and CD8(+)CD5(bright) compared with control cows, but greater percentages of CD5(dim) cells for all lymphocyte subsets. These results suggest changes in the percentages of lymphocyte subsets, monocytes, and CD5 markers are modulated by both infection status and the periparturient period.     

Progress in national control and assurance programs for bovine Johne's disease in Australia

Cattle strains of Mycobacterium paratuberculosis are known to infect cattle, goats and alpaca in southeastern Australia, where there are also significant numbers of farmed deer. Although sheep strains have recently been identified in some cattle in Australia, epidemiological evidence to date supports the distinction (between bovine Johne's disease (JD), caused by cattle strains in cattle, goats and alpaca, and ovine JD, caused by sheep strains in sheep and goats) for the purposes of control and assurance programs. The National Johne's Disease Control Program is coordinated by the Australian Animal Health Council, working with the livestock industries and with the Commonwealth, state and territory governments. The council also brokers industry and government funding for the program. The National Johne's Disease Market Assurance Program for Cattle was launched in 1996 as the first of a suite of voluntary national market assurance programs (MAPs) to assess and certify herds as negative for JD. By December 1998, over 550 herds had achieved an assessed negative status. A MAP was also launched for alpaca in 1998 and a program for goats should be finalized in early 1999. National standards for state control of JD through zoning, movement controls and procedures in infected and suspect herds have also been developed. The paper covers factors affecting development and implementation, uptake of and improvements to national control and assurance programs for bovine JD in Australia.     

Prevalence of Michigan dairy herds infected with Mycobacterium avium subspecies paratuberculosis as determined by environmental sampling

A cross-sectional, stratified random survey of Michigan dairy herds was conducted to estimate the prevalence of herds infected with Mycobacterium avium paratuberculosis (MAP), the causative agent of Johne's disease, in Michigan using targeted environmental sampling. One pooled sample each from the primary manure storage area and a high-traffic common cow area from each herd was collected and cultured for MAP using the ESP^® culture system II. A herd was classified as positive if at least one sample was culture positive for MAP. State, agricultural district, and herd size stratum prevalence were calculated. Information on past MAP testing and cattle purchase history was collected, and logistic regression was performed to determine their importance to the MAP status of the herd. One hundred twenty-seven herds were contacted, and 94 agreed to participate in the study. The environment of 38 (40.4%) herds cultured positive for MAP. MAP was found in all herds (n = 15) with greater than 200 lactating cows. Herds that had tested for MAP or purchased cattle in the previous 5 years were 4.6 and 3.1 times, respectively, more likely to be infected than herds that had not. MAP continues to be prevalent on Michigan dairy farms, especially those with greater than 200 lactating cows. The environmental sampling protocol used in this study is an economically attractive alternative for monitoring herd level prevalence and the progress of Johne's disease control programs at the state or national level. Implementation of such a program would aid states in monitoring Johne's control program progress, and guide changes over time.     

Altered patterns of toll-like receptor gene expression in cull cows infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteric disease of cattle. The mechanism how MAP can co-exist in the gastro-intestinal tract despite a massive infiltration of immune cells is not known. Toll-like receptors (TLRs) are known to play an important role in both innate and acquired immune responses but it is unclear what role different TLRs play in response to MAP. In this study, 38 cull cows from herds infected with MAP were classified into four groups, based on MAP culture from gut tissues and histopathological lesion scores. The expression of TLR1, 2 and 4 mRNA from MAP antigen-stimulated mesenteric lymph node (MLN) cultures and peripheral blood mononuclear cells (PBMCs) and in the MLN and ileum tissues of these animals was determined. MAP antigen-specific expression of TLR1 in MLN and PBMC was significantly lower in the MAP-infected groups than the non-infected control group, suggesting that in MAP-infected animals there is impairment in the up-regulation of TLR1 in response to MAP antigen. TLR4 expression in MLN tissues was significantly higher in the severely infected group than the control group suggesting up-regulation of endogenous TLR4 expression at a site of MAP infection in animals severely affected with Johne's disease. A preliminary screening of TLR1, 2 and 4 in the cull cows revealed the presence of polymorphisms in TLR1 and TLR2. In summary, one mechanism how MAP may subvert the immune system is that there is an apparent lack of recognition of MAP antigens as foreign by TLR1 in MAP-infected cows.     

Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of Mycobacterium tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n = 9) and naturally infected cows in the subclinical (n = 10) and clinical (n = 13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-γ) release assay as well as immunoblots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore the identification of antigenic proteins with potential as diagnostic targets was characterized.     

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).     

Demographics of cattle positive for Mycobacterium avium subspecies paratuberculosis by faecal culture,from submissions to the Cork Regional Veterinary Laboratory

The demography of bovine infections caused by Mycobacterium avium subspecies paratuberculosis (MAP) in Ireland is poorly defined. The objective of this study was to describe the demographics of cattle positive to MAP on faecal culture, based on submissions to the Cork Regional Veterinary Laboratory (Cork RVL) from 1994 to 2006. The study focused on all available faecal samples from adult cattle with non-responsive chronic diarrhoea that were submitted by private veterinary practitioners to Cork RVL for MAP culture. For each MAP-positive by faecal culture animal, data were collated from Cork RVL and Cattle Movement Monitoring Scheme (CMMS) records. Johne's disease (JD) was confirmed in 110 animals from 86 herds by the Cork RVL between 1994 and 2006, with a rate of positive cases between 15% and 18% over last four years of the study. Two breeds (Holstein/Friesian or Limousin) made up 78% of submissions. Movements were assessed for the 57 study animals with available movement information, 90% died within one year of the test and 26% tested positive in the herd they were born into. The study provides preliminary information about movement trends and demographics of animals with MAP positive submissions. Although the study area is restricted, it includes the most intensive (and economically-important) dairy region in Ireland. The demographics of JD infection from the study area are in agreement with international reports. Further work is required to determine demographic trends, incidence and prevalence of JD throughout Ireland. It is hoped this work may contribute to the development of a surveillance strategy for MAP by regional veterinary laboratories.     

Efficacy of monensin sodium for the reduction of fecal shedding of Mycobacterium avium subsp. paratuberculosis in infected dairy cattle

Reducing the quantity of Mycobacterium avium subsp. paratuberculosis (MAP) being shed by cows with Johne's disease should decrease the risk of spread of this disease to young stock. Previous work has suggested that monensin sodium decreases the pathologic lesions associated with Johne's disease, but the impact on shedding of viable MAP remains unknown. After serologic screening of 32 dairy herds in southwestern Ontario, 228 cows from 13 of these herds were enrolled into a randomized clinical trial. Fecal culture and PCR were used to identify 114 cows as potential fecal shedders, while another 114 cows were enrolled as ELISA negative, herd and parity matched controls. All cows were randomized to receive either a monensin controlled release capsule (CRC) or a placebo capsule. Serial fecal and blood samples were collected for fecal culture and serum ELISA testing over a 98-day period. On day 98 of the study, treatments were switched for all cows continuing in the trial. These remaining cows were followed for another 98 days with a similar sampling protocol. Mixed effect models were used to measure the impact of treatment on the number of colony forming units identified on fecal cultures over time. During the first 98 days of the study, cows treated with a monensin CRC were found to shed 3.4 cfu per tube less than placebo treated cows (P = 0.05). The serum ELISA S/P ratio was reduced by 1.39 units in cows given monensin (P = 0.06). However, treatment with monensin did not reduce the odds of testing positive on serology. Only the cows shedding MAP on day 0 were found to have a reduced odds of testing positive on fecal culture when treated with monensin (OR = 0.27; P = 0.03). Monensin sodium administered to infected animals at 335 mg/day marginally reduced fecal shedding of MAP in mature dairy cattle, but the biological significance of this reduction is unknown.     

Protein expression in dairy cows with and without subclinical hypocalcaemia

AIM: To determine differences in plasma proteomic profiles between healthy cows and those with subclinical hypocalcaemia within 12 hours after calving, and thereby explore the underlying biological mechanism of subclinical hypocalcaemia in dairy cows.

METHODS: Plasma samples were collected within 6 hours of calving from Holstein cows on a farm in Heilongjiang, China; 32 with subclinical hypocalcaemia (plasma calcium concentration 1.38–2.00?mmol/L and no clinical signs) and 59 control cows (plasma calcium concentration 2.10–2.8?mmol/L). Plasma samples were applied to weak cationic exchange protein chips for protein profiling by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS), and the data were analysed using the PBS-IIC system. The amplitude of peaks for the two groups were compared using the Wilcoxon sum-rank test, and the mass-to-charge ratio of the peaks that differed was used to identify peptide fragments using the Swiss-Prot protein database.

RESULTS: Seven peaks were identified in the subclinical hypocalcaemia group that differed from those of the control group (p<0.001), that represented six unique proteins. Expression of serum albumin, fibrinogen alpha chain, amyloid beta A4 proteins and neurosecretory protein VGF were increased, and expression of apolipoprotein A-II and serum amyloid A proteins were decreased in the subclinical hypocalcaemic cows compared with control cows.

CONCLUSION: Use of SELDI-TOF-MS technology can effectively identify differences in plasma protein expression patterns in cows with subclinical hypocalcaemia. Neurosecretory protein VGF and amyloid beta A4 protein might represent useful biomarkers for diagnosis of subclinical hypocalcaemia.     

Relationships between clinical signs, pathological changes and tissue distribution of Mycobacterium avium subspecies paratuberculosis in 21 cows from herds affected by Johne's disease

Twenty-one cows from eight herds affected by Johne's disease were assigned to four groups: seven were not thriving and had persistent diarrhoea, six were not thriving and had intermittent diarrhoea, four were not thriving but did not have diarrhoea, and four were clinically normal. Postmortem, macroscopic lesions consistent with Johne's disease were identified in 17 of the cows and Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from all of them. However, except for the fact that diarrhoea was correlated with the presence of lesions in the large intestine there was little correlation between the presence or absence of clinical signs and the lesions associated with Johne's disease. The tissue distribution of MAP was also poorly correlated with either the clinical signs or the lesions. The organism was widely distributed in 17 of the 21 cows, including three of the clinically normal animals, and was present in the mammary tissues of seven cows including two of the clinically normal animals. Three distinct histopathological patterns were observed in the affected intestines: infiltration of the lamina propria with giant cells, tuberculoid lesions, and lepromatous lesions; the lepromatous lesions were associated with extensive pathological changes.     

Osteopontin immunoreactivity in the ileum and ileocecal lymph node of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Osteopontin (Opn), a highly acidic glycoprotein, promotes cellular adhesion and recruitment and has been shown to be upregulated in the granulomas of mycobacterial infections. Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is associated with granulomatous enteritis. The objective of this experiment was to identify Opn in the ileum and ileocecal lymph node (ICN) of dairy cows naturally infected with MAP and to compare the frequency and intensity of staining between noninfected healthy controls, subclinical and clinical cows. Sections from these three groups of animals were selected from a tissue archive. Immunohistochemical analysis was used to determine the location and expression of Opn. The frequency and intensity of staining was also reported. Confirmation of acid-fast bacilli in the tissue sections was achieved by the Ziehl-Neelsen method. Within the ileal tissue, macrophages, lymphocytes, and plasma cells stained positive for Opn. Clinical cows expressed Opn at a greater frequency in the lamina propria. Control and subclinical cows did not have areas of granulomatous inflammation but cells staining for Opn were equally intense for the three groups. The frequency of staining for Opn in the ICN was not affected by MAP infection. Results of this study confirm for the first time, the expression of Opn in the ileum and ICN of MAP-infected cattle.     

Herd-level risk factors for infection with Mycobacterium paratuberculosis in US dairies and association between familiarity of the herd manager with the disease or prior diagnosis of the disease in that herd and use of preventive measures

OBJECTIVE: To evaluate associations among herd infection status, herd management practices, and familiarity of the herd manager with Mycobacterium paratuberculosis (Johne's disease) or prior disease diagnosis in that herd to support development of Johne's disease-control programs. DESIGN: Population-based cross-sectional study. SAMPLE POPULATION: 1,004 US dairies, each with > or = 30 cows, representing 79.4% of US dairy cows. PROCEDURE: Questionnaires were administered to dairy managers, and blood samples were collected from cows during herd visits. Sera were tested for antibodies to M paratuberculosis, using a commercially available ELISA. Multivariable logistic regression analyses were used to evaluate associations between use of management practices, herd disease status, and familiarity of the manager with Johne's disease or prior diagnosis of Johne's disease in that herd. RESULTS: Results from serologic testing revealed that 3.4% of cows and 21.6% of dairy herds were infected with M paratuberculosis. Factors associated with infection included number of cows in herd, region of country, percentage of cows born at other dairies, group housing for periparturient cows, and group housing for preweaned calves. Few preventive practices were positively associated with prior diagnosis of Johne's disease (time of separation of newborn calf from dam) or familiarity of the manager with the disease (teats and udder washed before colostrum collected). CONCLUSIONS AND CLINICAL RELEVANCE: Risk factors associated with Johne's disease in this study confirmed those management practices generally recommended for disease control. An educational problem, however, is the finding that herd managers familiar with Johne's disease generally use management practices similar to those used by managers unfamiliar with the disease.