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.     

Fecal shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows

Between 1982 and 2000, fecal samples were obtained from 786 cows that were shedding Mycobacterium avium subsp. paratuberculosis (Map). These cows were resident on 93 Pennsylvania dairies (mean herd size, 64 milk cows) that had no or minimal previous testing for Map. Feces were cultured on four tubes of Herrold's egg yolk medium and the distribution of mean Map colony forming units (CFU) was evaluated. Most cows were light (< 10 CFU/tube, 51.4%) or high (> 50 CFU/tube, 30.8%) fecal shedders with fewer cows in the moderate category (10-50 CFU/tube). Of the 786 cows, 192 (24.4%) had colonies in only one of four tubes. In the multivariable negative binomial model, there were significant associations between mean CFU/tube and prevalence, herd size, and season and an interaction between herd size and season. The linear mixed model of continuous tube counts with a random herd effect yielded similar findings with associations with herd size as a continuous variable, season, and an interaction between categorized prevalence and continuous herd size. Variability in CFU/tube was greatest among cows in the same herd, intermediate for replicate tubes from the same cow, and smallest among cows in different herds. Reduction in the number of replicate tubes from four would have reduced the sensitivity of fecal culture for Map by approximately 6% (for three tubes) to 12% (for two tubes).     

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.     

Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be used as a tool in understanding the level of MAP infection in a herd or pen, it did not appear to be a sensitive diagnostic method for herd positivity in these low prevalence herds, and its use may require caution.     

Cytokine secretion by peripheral blood mononuclear cells from cows infected with Mycobacterium paratuberculosis

OBJECTIVE: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis. ANIMALS: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis. PROCEDURE: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS. RESULTS: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.     

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.     

Distribution of Mycobacterium avium subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, has particular importance in cattle due to the resulting chronic diarrhoea, weight loss, decreased production, infertility and eventual death. While faecal oral route of infection is generally recognised, reports about semen-derived infection are rare. The objective of this work was to assess whether M.a. paratuberculosis may disseminate from the gastrointestinal tract to reproductive organs, and compare this event between naturally infected bull-calves and breeding bulls. Ten bull-calves, aged 6-28 weeks and four breeding bulls were tested by serology, faecal and tissue culture, IS900 PCR and RFLP. In seven bull-calves M.a. paratuberculosis was isolated predominantly from mesenteric lymph nodes (75%); isolates from mucosa of the intestine constituted 25%. In three breeding bulls, M.a. paratuberculosis was isolated both from intestinal mucosa and mesenteric lymph nodes. Head and mediastinal lymph nodes, liver, spleen and semen of bull no. 1 (Holstein-Friesian); testes and epididymis of bull no. 2 (Piemonte); testes, epididymides and seminal vesicle of bull No. 3 (Hereford); and seminal vesicle of bull No. 4 (Simmental) tested positive by culture. Hot-start PCR revealed M.a. paratuberculosis in semen, seminal vesicle and intestinal tissue where culture isolation was difficult. Isolates from bull-calves and breeding bulls were of RFLP types B-C9 and B-C1, respectively. Bull-calves born in infected herd can be sources of infection when later used for natural mating or artificial insemination. Sub-clinically infected bulls release M.a. paratuberculosis into semen, consequently infecting the uterine environment of cows.     

Augmentation of secreted and intracellular gamma interferon following johnin purified protein derivative sensitization of cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Measurement of secreted interferon (IFN)-gamma has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-gamma assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-gamma were observed. Intradermal injection resulted in significant increases in secreted IFN-gamma for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosis antigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-gamma was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-gamma was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-gamma, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.     

Tactics of Mycobacterium avium subsp. paratuberculosis for intracellular survival in mononuclear phagocytes

Johne''s disease is a condition that refers to chronic granulomatous enteritis in ruminants. It is believed that survival and replication of Mycobacterium (M.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of Johne''s disease. However, it is not clear how M. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of Johne''s disease. Investigating the intracellular fate of M. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). Existing animal models also have limitations. Despite those obstacles, there has been progress in understanding the intracellular survival tactics of M. paratuberculosis and the host response against them. In this review, we compare known aspects of the intracellular survival tactics of M. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes.     

Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp. paratuberculosis

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.     

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.     

Evaluation of four commercial serum ELISAs for detection of Mycobacterium avium subsp. paratuberculosis infection in dairy cows

During the first 3 months of 2006, a study was performed on four dairy cattle herds with a history of clinical paratuberculosis, to evaluate different enzyme-linked immunosorbent assays (ELISAs). Serum samples obtained from 326 animals were analysed using four ELISAs to detect antibodies to Mycobacterium avium subsp. paratuberculosis (MAP). Kappa (kappa) concordance coefficients in pairwise comparisons of the ELISA outcomes ranged up to 0.22 (linear kappa) and 0.25 (quadratic kappa). When the borderline positives obtained were considered as negatives, kappa values remained low (kappa up to 0.19). Having performed the serological tests, faecal samples were then obtained from 55 animals (including all animals testing positive in two or more ELISAs) from the same herds. Faecal culture and faecal polymerase chain reaction (PCR) for the detection of MAP were negative in all cases. The results indicate that neither the currently available serum ELISAs nor faecal culture and PCR are effective for the early detection of MAP in dairy cattle.     

Agreement between three ELISAs for Mycobacterium avium subsp. paratuberculosis in dairy cattle

During a 10-month period in 1999, 994 serum and tissue samples were collected from dairy cows at slaughter in eastern Canada. The sources of these cattle were from all four Atlantic Canadian provinces along with some cows from the state of Maine. The sera were used to assess the agreement of three commercially available ELISAs for Mycobacterium avium subsp. paratuberculosis. Two ELISAs were indirect absorbed ELISAs licensed for use in North America, the third was an indirect non-absorbed ELISA licensed for use in Europe. Overall, there was poor agreement between the three ELISAs. The highest and lowest kappa values were 0.33 and 0.18, which is fair and poor agreement, respectively. However, when only tissue culture-positive cattle were compared, the ELISAs had better agreement (kappa=0.37-0.51). The proportions of positive tests, however, were significantly different among the three ELISAs. The poor agreement among the three ELISAs is as concerning as the fact that these tests have low sensitivity. The implications are greatest when the tests are used at the cow level to make individual animal decisions, which is not the recommended method on the product labels. At the cow level, if the result obtained from one ELISA is positive, using a different ELISA in a pre-clinical animal has a high likelihood of giving a different result due to low predictive values of positive test results.     

Shedding patterns of dairy calves experimentally infected with Mycobacterium avium subspecies paratuberculosis

Although substantial fecal shedding is expected to start years after initial infection with Mycobacterium avium subspecies paratuberculosis (MAP), the potential for shedding by calves and therefore calf-to-calf transmission is underestimated in current Johne’s disease (JD) control programs. Shedding patterns were determined in this study in experimentally infected calves. Fifty calves were challenged at 2 weeks or at 3, 6, 9 or 12 months of age (6 calves served as a control group). In each age group, 5 calves were inoculated with a low and 5 with a high dose of MAP. Fecal culture was performed monthly until necropsy at 17 months of age. Overall, 61% of inoculated calves, representing all age and dose groups, shed MAP in their feces at least once during the follow-up period. Although most calves shed sporadically, 4 calves in the 2-week and 3-month high dose groups shed at every sampling. In general, shedding peaked 2 months after inoculation. Calves inoculated at 2 weeks or 3 months with a high dose of MAP shed more frequently than those inoculated with a low dose. Calves shedding frequently had more culture-positive tissue locations and more severe gross and histological lesions at necropsy. In conclusion, calves inoculated up to 1 year of age shed MAP in their feces shortly after inoculation. Consequently, there is potential for MAP transfer between calves (especially if they are group housed) and therefore, JD control programs should consider young calves as a source of infection.     

Evaluation of Mycobacterium avium subsp paratuberculosis infection of dairy cows attributable to infection status of the dam

OBJECTIVE: To estimate the extent to which infection with Mycobacterium avium subsp paratuberculosis (MAP) of cows in a large dairy was attributable to the infection status of their dams. DESIGN: Retrospective longitudinal study. ANIMALS: 625 dam-daughter pairs of Holstein cows. PROCEDURE: Serologic test results were compared between cows and their dams. Logistic regression was used to assess whether a cow's serologic status was associated with its dam's serologic status. Infection with MAP attributable to being born to a seropositive dam was estimated for individual cows and for the herd. RESULTS: Cows with seropositive dams were 6.6 times as likely to be seropositive, compared with cows of seronegative dams. For seropositive cows born to seropositive dams, 84.6% of seropositivity was attributable to being born to a seropositive dam and 15.4% to other exposures, including exposure as calves to flush water that contained feces of adult cattle. For the herd as a whole, the seropositive status in 34% of seropositive cows was attributable to being born to a seropositive dam. CONCLUSIONS AND CLINICAL RELEVANCE: For dairy herds that breed seropositive cows, subsequent transmission of MAP to their daughters, either congenitally or via exposure to feces and colostrum of the dam shortly after birth, can contribute substantially to maintaining prevalence of MAP in a herd. Removal of seropositive, clinically unaffected cows and their daughters would be necessary to reduce infection with MAP attributable to congenital or peri-parturient transmission from dam to daughter.     

Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep

Immunoperoxidase (IPX) labelling for CD4, CD8, TCR-gammadelta, WC1, CD1b, IFN-gamma, CD45R, CD56 and lysozyme was used to investigate changes in cell mediated immune effector cell populations in the intestinal Peyer's patches (PP) and mesenteric lymph nodes of lambs, 2 and 4 months after experimental infection with low doses of sheep strain Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). The organism was cultured from the tissues of each infected lamb, but histological lesions were not present. This infection model was considered to be more representative of natural M. a. paratuberculosis infection than previous studies. Infected sheep had significantly more CD4+ cells in the mucosa, domes and interfollicular areas of the terminal ileum, and in the interfollicular areas of the jejunal Peyer's patch. Infected sheep also had significantly increased numbers of TCR-gammadelta+ cells in the mucosa and interfollicular areas of the jejunal Peyer's patch, and increased numbers of WC1+ cells in the ileal Peyer's patch. These findings are consistent with previous findings in sheep given higher doses of cattle strain M. a. paratuberculosis. Significantly fewer CD1b+ cells were present in the paracortical areas of the mesenteric lymph nodes of infected sheep, and the reduction was greater in sheep infected for 4 months compared to sheep infected for only 2 months. Down-regulation of CD1b expression may be important for the continued survival and multiplication of M. a. paratuberculosis as specific adaptive immunity develops. Across all sheep, jejunal Peyer's patches had higher numbers of CD4+, CD8+, TCR-gammadelta+, WC1+ and CD45R+ cells, and lower numbers of CD56+ fibres compared to ileal Peyer's patches. These findings confirm and extend the peculiarities of the terminal ileal Peyer's patch in the young ruminant, with possible implications for the early establishment of M. a. paratuberculosis infection.     

Seroprevalence of Mycobacterium avium subsp paratuberculosis infection among dairy cows in Colorado and herd-level risk factors for seropositivity

OBJECTIVE: To estimate seroprevalence of Mycobacterium avium subsp paratuberculosis (MAP) infection among adult dairy cows in Colorado and determine herd-level factors associated with the risk that individual cows would be seropositive. DESIGN: Cross-sectional observational study. ANIMALS: 10,280 adult (> or = 2 years old) dairy cows in 15 herds in Colorado. PROCEDURE: Serum samples were tested with a commercial ELISA. A herd was considered to be infected with MAP if results of mycobacterial culture of > or = 1 individual cow fecal sample were positive or if > or = 1 culled cow had histologic evidence of MAP infection. RESULTS: 424 of the 10,280 (4.12%) cows were seropositive. Within-herd prevalence of seropositive cows ranged from 0% to 7.82% (mean, 2.6%). Infection was confirmed in 11 dairies. Cows in herds that had imported > or = 8% of their current herd size annually during the preceding 5 years were 3.28 times as likely to be seropositive as were cows in herds that imported < 8%. Cows in herds with > or = 600 lactating cows were 3.12 times as likely to be seropositive as were cows in herds with < 600 lactating cows. Cows in herds with a history of clinical signs of MAP infection were 2.27 times as likely to be seropositive as were cows in herds without clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE: Annual importation rate, herd size, and whether cows in the herd had clinical signs typical of MAP infection were associated with the risk that individual cows would be seropositive for MAP infection.