Prevalence and characteristics of Shiga toxin-producing Escherichia coli in Swiss raw milk cheeses collected at producer level

The aim of this study was to describe the prevalence, serotypes, and virulence genes of Shiga toxin-producing Escherichia coli (STEC) isolated from raw milk cheese samples collected at the producer level with the purpose of determining whether raw milk cheeses in Switzer-land represent a potential source of STEC pathogenic for humans. Raw milk cheese samples (soft cheese, n = 52; semihard and hard cheese, n = 744; all produced from Swiss cows’, goats’, and sheep's milk) collected at the producer level throughout Switzerland within the national sampling plan during the period of March 2006 to December 2007 were analyzed. Of the 432 cheese samples obtained in the year 2006 and the 364 samples obtained in the year 2007, 16 (3.7%) and 23 (6.3%), respectively, were found to be stx positive. By colony dot-blot hybridization, non-O157 STEC strains were isolated from 16 samples. Of the 16 strains, 11 were typed into 7 E. coli O groups (O2, O15, O22, O91, O109, O113, O174), whereas 5 strains were nontypeable (ONT). Among the 16 STEC strains analyzed, stx₁ and stx₂ variants were detected in 1 and 15 strains, respectively. Out of the 15 strains with genes encoding for the Stx2 group, 4 strains were positive for stx₂, 6 strains for stx_2d2, 2 strains for stx_2-O118, 1 strain for stx_2-06, 1 strain for stx_2g, 1 strain for stx₂ and stx_2d2, and 1 strain for stx₂ and stx_2g. Furthermore, 3 STEC strains harbored E-hlyA as a further putative virulence factor. None of the strains tested positive for eae (intimin). Results obtained in this work reinforce the suggestion that semihard raw milk cheese may be a potential vehicle for transmission of pathogenic STEC to humans.     

Application of IS900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk

A polymerase chain reaction (PCR)-based assay was developed for detection of insertion sequence 900 (IS900) of Mycobacterium avium subsp. paratuberculosis in raw milk. This IS900 PCR assay included DNA extraction and PCR assay using commercially available kits. The DNA extraction and PCR assay were optimized to detect the IS900 sequence directly from raw milk. The IS900 PCR assay was evaluated by inoculating raw bulk milk and Middlebrook's 7H9 broth with 0 to 10(8) cfu/ml of each of four American Type Culture Collection strains of M. paratuberculosis. Under experimental conditions, both milk culture on Herrold's egg yolk medium slants, and IS900 PCR could detect 10 to 100 cfu/ml of M. paratuberculosis. Detection of M. paratuberculosis by IS900 PCR was consistent (24/24 PCR assays) when about 100 cfu/ml were present, whereas detection was variable (12/24 PCR assays) at concentrations as low as 10 cfu/ml. Based on the findings of the experimental study, IS900 PCR was further evaluated with pooled quarter milk samples from 211 cows from five herds with known history of Johne's disease. Out of 211 animals examined, nine (4%) and 69 (33%) were positive for M. paratuberculosis by milk culture and IS900 PCR from milk, respectively. A total of 20 bulk tank milk sample aliquots (one sample, four aliquots from each herd) were also examined, of which 10 (50%) were positive for M. paratuberculosis by IS900 PCR. By contrast, only one out of 20 (5%) bulk tank milk sample aliquots was positive by culture. The IS900 PCR amplified product of 229-bp obtained on testing of quarter milk and bulk tank milk samples was confirmed to be the IS900 of M. paratuberculosis by DNA sequence analysis. The results of this study suggest that M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR.     

Osteopontin: a novel cytokine involved in the regulation of Mycobacterium avium subspecies paratuberculosis infection in periparturient dairy cattle

Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by Mycobacterium avium ssp. paratuberculosis (MAP) have a devastating effect on the dairy industry. We sought to characterize Opn at the level of gene and protein expression in periparturient dairy cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMC) were isolated from control, subclinical, and clinical periparturient dairy cows naturally infected with MAP beginning 3 wk precalving to 5 wk postcalving and incubated with medium alone (non-stimulated: NS), concanavalin A (ConA), or a whole-cell sonicate of MAP (MPS). Real-time PCR was performed to evaluate expression of Opn and classical Th1 and Th2 cytokines. Results demonstrated greater Opn expression in nonstimulated PBMC isolated from subclinical cows compared with control and clinical cows. For clinical cows, there was a strong correlation between Opn expression and expression of the Th1 cytokines IFN-γ and IL-1α for nonstimulated PBMC and IFN-γ and IL-12 for PBMC stimulated with MPS. Expression of tumor necrosis factor-α was greater in clinical cows than the other groups. Nonstimulated, ConA, and MPS-stimulated PBMC from subclinical cows secreted more IFN-γ, and MPS-stimulated PBMC from clinical cows secreted more IL-4 compared with the other groups. Immunoblot analysis of PBMC detected 4 Opn proteins at 60, 52, 34, and 27 kDa. This is the first study to evaluate the role of Opn on the immune response of dairy cows naturally infected with MAP, and results suggest Opn may be a key regulator against MAP infection.     

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.     

Effects of seasonal climatic conditions on the diagnosis of Mycobacterium avium subspecies paratuberculosis in dairy cattle

Validity of Johne's disease programs and control protocols that rely on established cut points [e.g., specified sample-to-positive (S/P) ratios] for ELISA serological tests depends on interpreted results that are not susceptible to variable test accuracy. It was hypothesized that seasonal variability exists in serological response to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Further, a reciprocal response may occur, resulting in greater risk of fecal shedding in subclinically infected animals. A testing regimen was invoked that included multiple testing of individual adult cows during the 4 seasons. Serum was collected on a cyclic, monthly basis from 3 randomly selected cohorts of dairy cows, and fecal samples were collected from the 20% of cows with the greatest ELISA test S/P ratios. Staggered, quarterly sampling was continued for 1 yr, and at the conclusion, serum was analyzed en masse. The ELISA outcome values (i.e., S/P ratio) were treated both as categorical and continuous variables. The potential lagged effects of temperature-related seasonality on S/P ratio, as well as the potential for a change in test result caused by temperature were assessed. Results for fecal culture were analyzed on a categorical scale and compared with the ELISA results to explore the possibility of reciprocal fecal shedding. No significant seasonal effects on either S/P ratios or the proportion of cows seropositive to MAP were observed. Furthermore, no evidence was found linking temperature-related seasonality to a reciprocal increase in the risk of fecal culture positivity for MAP.     

Detection of Mycobacterium avium subspecies paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. The disease causes diarrhea, reduced milk production, poor reproductivity, emaciation, and eventually death. Culture on Herrold's egg yolk agar is considered to be the definitive test for diagnosis of Johne's in cattle. This method has moderate sensitivity (30 to 50%) and is 100% specific; however, it can take up to 16 wk due to the slow growth of MAP. Currently, serum ELISA is used to screen herds for Johne's disease, but positive tests must be confirmed culturally or by PCR. The current research sought to evaluate an in-house direct fecal PCR procedure and directly compare it to ELISA using culture as the gold standard. Serum and fecal samples were collected from cows (n = 250) with unknown Johne's status. Fecal samples were processed for culture on Herrold's egg yolk agar and direct PCR. Serum samples were tested using the Parachek serum ELISA. Overall, 67/250 [26.8%, 95% confidence interval (CI) 21.4 to 32.8] animals were culturally confirmed to be shedding MAP. The PCR and ELISA detected 74/250 (29.6%, 95% CI 24 to 35.7) and 25/250 (10%, 95% CI 6.6 to 14.4), respectively. Culture and PCR were able to detect more positive animals than ELISA. Overall, direct fecal PCR was 70.2% sensitive and 85.3% specific when using culture as the gold standard. The ELISA method was 31.3% sensitive and 97.8% specific. When culture reported <10 cfu, the sensitivity and specificity of PCR and ELISA were 57.1 and 85.3%, and 4.8 and 97.8%, respectively. When culture reported 10 to <40 cfu, the sensitivity of PCR and ELISA were 75 and 50%, respectively. When culture reported ≥40 cfu, the sensitivity of PCR and ELISA were 100 and 88.2%, respectively. Specificity could not be calculated at these levels because there were no negative samples. The direct PCR outperformed the ELISA in detecting animals potentially infected with MAP and was not significantly different when compared with culture. The direct fecal PCR method described here provides faster results than traditional culture and is more sensitive than ELISA at detecting animals suspected of Johne's disease. These data support the use of PCR as an alternative method for screening herds for prevalence and diagnosis of Johne's disease.     

An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces

There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk Adiapure and accompanying PCR detection kit Adiavet alongside 'in-house' molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml(-1) raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for 'in-house' Map detection were observed when the Adiapure extraction kit was combined with 'in-house' detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900ml(-1) raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml(-1) raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.     

Reliability of environmental sampling to quantify Mycobacterium avium subspecies paratuberculosis on California free-stall dairies

The reliability of environmental sampling to quantify Mycobacterium avium ssp. paratuberculosis (MAP) based on collector and time was evaluated. Fecal slurry samples were collected using a standardized protocol simultaneously by 2 collectors of different experience levels. Samples were collected from 30 cow pens on 4 dairies every other day on 3 occasions while cow movements between pens were minimal. The 4 study herds had moderate MAP seroprevalence and were housed in free-stall dairies in central California. Results of testing the environmental samples for MAP using PCR and culture were strongly correlated. The reliability of environmental sampling simultaneously by different collectors as estimated by the intraclass correlation coefficient was excellent (81%) for PCR and good (67%) for culture and may justify comparison of quantitative results of samples collected by different investigators. The reliability of environmental sampling over a 5-d period was good (67 and 64% for PCR and culture results, respectively), which justifies the utility of environmental sampling to identify pens with a high MAP bioburden between routine cow pen changes on a dairy. Environmental sampling of free-stall pens using the standardized sampling protocol yielded comparable PCR and culture results across collectors with different experience levels and at different times within a 5-d period.     

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.     

Scenario analysis of changes in consumption of dairy products caused by a hypothetical causal link between Mycobacterium avium subspecies paratuberculosis and Crohn's disease

Johne's disease is an infectious disease of cattle caused by Mycobacterium avium subspecies paratuberculosis (MAP). Crohn's disease (CD) is a human disease of unknown etiology that is characterized by chronic bowel inflammation. No causal link has been scientifically established between MAP and CD, but it is important to understand possible impacts on society should such a causal link be established. The goal of this paper is to analyze the implications and the possible economic impacts that finding such a link would have on milk demand in the dairy industry and to provide a framework for further discussion among stakeholders. Three scenarios were developed based on the effectiveness of possible risk-mitigation strategies. In the first scenario, it was assumed that an effective strategy exists; therefore, a negligible demand decrease in the consumption of dairy products was expected. In the second scenario, it was assumed that new risk mitigation would need to be implemented to minimize the health hazard for humans. In this case, a small milk demand decrease was expected, but larger demand decreases were also possible. The third scenario assumed that no fully effective risk mitigation was available, and this resulted in a considerable demand decrease and a potential reduction in milk supply as a result of regulatory measures. A milk demand reduction of 1 or 5% resulted in a reduction in consumer surplus of $600 million and $2.9 billion, and a reduction in dairy farm income of $270 million and $1.3 billion, respectively. A decrease in milk supply would cause a slight increase in total losses, but would cause the greatest losses to test-positive dairy farms. Given the current scientific knowledge about MAP and CD, we conclude that if a link were established, it is most likely that the first or second scenario would occur. Thus, consumer response and economic consequences to the discovery of such a link are expected to be limited, but could be large if the consumer's perception of risk is large or if risk-mitigation strategies were ineffective.     

Effect of pasteurization on survival of Mycobacterium paratuberculosis in milk

Mycobacterium paratuberculosis (Mptb) is the causative agent of Johne's disease of ruminant animals including cattle, goats, and sheep. It has been suggested that this organism is associated with Crohn's disease in humans, and milk is a potential source of human exposure to this organism. A total of 18, including 7 regular batch and 11 high temperature short time (HTST) pasteurization experiments, were conducted in this study. Raw milk or ultra-high temperature pasteurized milk samples were spiked at levels of 10(3), 10(5), and 10(7) cfu of Mptb/ml. Escherichia coli and Mycobacterium bovis BCG strains at 10(7) cfu/ml were used as controls. Pasteurization experiments were conducted using time and temperature standards specified in the Canadian National Dairy Code: regular batch pasteurization method: 63 degrees C for 30 min, and HTST method: 72 degrees C for 15 s. The death curve of this organism was assessed at 63 degrees C. No survivors were detected after 15 min. Each spiked sample was cultured in Middlebrook 7H9 culture broth and Middlebrook 7H11 agar slants. Samples selected from 15 experiments were also subjected to BACTEC culture procedure. Survival of Mptb was confirmed by IS900-based PCR of colonies recovered on slants. No survivors were detected from any of the slants or broths corresponding to the seven regular batch pasteurization trials. Mptb survivors were detected in two of the 11 HTST experiments. One was by both slant and broth culture for the sample spiked to 10(7) cfu/ml of Mptb, while the other was detected by BACTEC for the sample spiked to 10(5) cfu/ml. These results indicate that Mptb may survive HTST pasteurization when present at > or = 10(5) cfu/ml in milk. A total of 710 retail milk samples collected from retail store and dairy plants in southwest Ontario were tested by nested IS900 PCR for the presence of Mptb. Fifteen percent of these samples (n = 110) were positive. However, no survivors were isolated from the broth and agar cultures of 44 PCR positive and 200 PCR negative retail milk samples. The lack of recovery of live Mptb from the retail milk samples tested may be due to either the absence of live Mptb in the retail milk samples tested or the presence of low number of viable Mptb which were undetected by the culture method used in this study.     

Short communication: Application of an N-acetyl-l-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subspecies paratuberculosis from milk of naturally infected cows

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne’s disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5 h or N-acetyl-l-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold’s egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds.     

Clinical disease and stage of lactation influence shedding of Mycobacterium avium subspecies paratuberculosis into milk and colostrum of naturally infected dairy cows

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n = 39) and symptomatic clinical (n = 29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrold's egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrold's egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0–60), with negligible positive samples observed in mid (DIM 60–240) and late (DIM 240–305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd.     

Case-control study: Productivity and longevity of dairy cows that tested positive for infection with Mycobacterium avium ssp. paratuberculosis as heifers compared to age-matched controls

A case-control study was performed to determine if dairy heifers testing positive for Mycobacterium avium ssp. paratuberculosis (MAP) before 2 yr of age by either fecal culture or serum ELISA had decreased productivity and longevity as cows compared with age-matched herdmates. Cases were individually matched with 4 controls. Survival analysis was conducted to determine differences in longevity between cases and controls. Conditional logistic regression was used to assess differences in mean 3.5% fat-corrected 305-d mature-equivalent milk, milk fat, and milk protein production, linear somatic cell count, and MAP test and clinical status as mature cows. No significant difference was found between cases and controls for any parameter assessed. Herd production performance and longevity did not appear to be impaired; therefore, testing immature dairy heifers for MAP is not economically justifiable, using currently available culture methods and commercial serum ELISA tests.     

Association between Mycobacterium avium subspecies paratuberculosis infection and milk production in two California dairies

The association between Mycobacterium avium ssp. paratuberculosis (MAP) and milk production was estimated on 2 California dairies using longitudinal data from 5,926 cows. Both study herds had moderate MAP seroprevalence, housed cows in freestalls, and had Johne's disease control programs. Cow MAP status was determined using both serum ELISA and fecal culture results from cows tested at dry-off and from whole-herd tests. Potential confounders were evaluated based on a causal diagram. Mixed models with 2 functions (splines) for days in milk (DIM) representing milk production pre- and postpeak used in similar studies were further modified to use each cow's observed DIM at peak and lactation length. Cows that were seropositive produced 2.5 kg less 4% fat-corrected milk (FCM) per day than their seronegative herdmates. In addition, cows that were fecal-culture positive by liquid culture and confirmed by PCR produced 2.2 kg less 4% FCM per day than their fecal-culture negative herdmates. The decrease in milk production in MAP test-positive compared with test-negative cows started in the second lactation. A switch in MAP status in either ELISA or fecal culture results from positive to negative had no significant association with milk production. Modified DIM functions that used the observed DIM at peak had better model fit than another function that assumed a fixed peak at 60 DIM. Cows that tested positive for MAP on serum ELISA or fecal culture produced less milk than cows that tested negative, and the association between MAP and milk production was not confounded by mastitis, elevated somatic cell counts, or uterine or metabolic cow conditions.     

Pasteurization of colostrum reduces the incidence of paratuberculosis in neonatal dairy calves

In the present study, the potential benefits of feeding pasteurized colostrum were demonstrated in calves born to dams naturally infected with Mycobacterium avium ssp. paratuberculosis. Calves were separated at birth from their dams and randomly allocated into a group fed either the colostrum of their dam (DC; n = 6), followed by feeding the milk of the dam for 3 wk and then milk replacer, or into a group fed pooled pasteurized colostrum (PC; n = 5) from healthy noninfected dams, followed by milk replacer. At 6 wk of age, calves were weaned onto calf starter, housed together, and fed in a similar manner throughout the rest of the 12-mo study. Calves were necropsied at the end of the study, and 25 tissue sites were sampled from each animal and cultured for M. avium ssp. paratuberculosis. Sixteen of the 25 tissue sites were positive for calves across both treatment groups, with 14 of the 16 tissue sites positive for DC calves and 9 of the 16 tissue sites positive for PC calves. The degree of colonization within a tissue was low and variable for calves within treatment groups, and fecal shedding of M. avium ssp. paratuberculosis was minimal during the 12-mo study. As a measure of the early immune response to infection, blood obtained from calves was stimulated in vitro with M. avium ssp. paratuberculosis antigen preparations, and IFN-_Y secretion was measured. Antigen-specific IFN-_Y was consistently greater throughout the study in DC calves (0.95 ± 0.19) compared with PC calves (0.43 ± 0.10). Although long-term benefits are unknown, these results indicate that feeding a source of colostrum from paratuberculosis-free dams may decrease the initial exposure of neonates to M. avium ssp. paratuberculosis, perhaps decreasing dissemination of infection over time.     

The distribution of Mycobacterium avium ssp. paratuberculosis in the environment surrounding Minnesota dairy farms

The objective of this study was to characterize the distribution of Mycobacterium paratuberculosis (Map) in the environment of infected and uninfected Minnesota dairy farms. Eighty herds known to be infected from Minnesota's Johne's Disease Control Program (JDCP) and 28 herds known to be uninfected from Minnesota Voluntary Johne's Disease Herd Status Program (VJDHSP) were sampled. Fecal samples from up to 100 cows in each herd were cultured in pools of 5 cows. Two environmental samples were obtained from each farm from various locations. All samples were tested using bacterial culture for Map. Eighty percent of the JDCP herds had at least one positive pool. Environmental samples were cultured positive in 78% of the JDCP herds. Two (7%) of the VJDHSP herds had one positive pool, and one herd had one positive environmental sample. Environmental samples were cultured positive in cow alleyways (77% of the herds), manure storage (68%), calving area (21%), sick cow pen (18%), water runoff (6%), and postweaned calves areas (3%). There was an association between maximum level of colonies per tube from cow alleyways and manure storage and fecal pool prevalence. Herds with both areas cultured negative were estimated to have 0.3 to 4% fecal pool prevalence. Herds with both areas having a heavy load of bacteria were estimated to have 53 to 73% fecal pool prevalence. The study results indicate that targeted sampling of cow alleyways and manure storage areas appears to be an alternative strategy for herd screening and Johne's infection status assessment and for estimating herd fecal prevalence.     

Short communication: Detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohn's disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohn's disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil.     

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S–23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.     

Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on large California dairies

Environmental samples collected from each of 3 locations on 23 large California dairies were cultured to evaluate the utility of this approach for identifying herds infected with Mycobacterium avium ssp. paratuberculosis. Results were compared with concurrent ELISA testing of ≥ 60 animals in each herd, and with previously performed individual and pooled fecal cultures of 60 animals. The estimated proportions of infected herds did not differ significantly among the testing methods (environmental sampling, 74%; previous fecal culture, 70%; and concurrent ELISA testing, 65%). Measures of agreement between environmental sampling and the results of previous fecal cultures were 70% (observed agreement), 85% (positive agreement), 62% (negative agreement), and 0.47 (kappa), whereas agreement between environmental sampling and concurrent ELISA testing was 65, 75, and 43%, and 0.19, for the same measures, respectively. The proportion of positive environmental samples on each farm was significantly correlated with the proportion of seropositive animals (r = 0.53), suggesting that environmental sampling may also provide a qualitative estimate of within-herd prevalence. Of the sampling locations that were evaluated, samples of lagoon water (15/23; 65%) were significantly more likely to yield a positive result than were composite manure samples (8/22; 36%) collected from the sick/fresh cow pen or from the alleyway (9/23; 39%) where cows exited from the milking parlor. Environmental sampling was an effective and inexpensive method of identifying herds infected with Mycobacterium avium ssp. paratuberculosis.