Evaluation of the association between fecal excretion of Mycobacterium avium subsp paratuberculosis and detection in colostrum and on teat skin surfaces of dairy cows

Patrick Pithua Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Scott J. Wells Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Sandra M. Godden Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Abstract

Objective—To evaluate the association between fecal excretion of Mycobacterium avium subsp paratuberculosis (MAP) by dairy cows in the periparturient period and detection of MAP DNA in colostrum specimens and on teat skin surfaces.

Design—Cross-sectional study.

Animals—112 Holstein cows.

Procedures—Fecal specimens were collected within 48 to 72 hours prior to parturition, and colostrum and teat swab specimens were collected immediately after parturition. Detection of MAP in fecal specimens was performed via microbial culture, and detection of MAP DNA in colostrum and teat swab specimens was achieved via a PCR assay targeting the genetic element ISMAP02. Logistic regression was used to model the relationship between MAP fecal shedding status and detection of MAP DNA in colostrum or teat swab specimens. Population attributable fractions for the proportion of colostrum and teat swab specimens containing MAP DNA were also calculated.

Results—The odds of detecting MAP DNA in colostrum or teat swab specimens in cows with MAP-positive (vs negative) fecal specimens were 2.02 and 1.87 respectively. Population attributable fractions estimates suggested that withholding colostrum from MAP-positive cows could reduce the odds of exposing calves to MAP in colostrum by 18.2%. Not permitting natural suckling by calves could reduce the odds of exposing calves to MAP on the teat surfaces of MAP-positive cows by 19.5%.

Conclusions and Clinical Relevance—Results underscored the need for strict adherence to practices that limit contact of calves with adult cows from the time of birth and promote hygienic colostrum handling to avoid possible contamination with MAP during colostrum harvest, storage, or feeding.

Abstract

Objective—To evaluate the association between fecal excretion of Mycobacterium avium subsp paratuberculosis (MAP) by dairy cows in the periparturient period and detection of MAP DNA in colostrum specimens and on teat skin surfaces.

Design—Cross-sectional study.

Animals—112 Holstein cows.

Procedures—Fecal specimens were collected within 48 to 72 hours prior to parturition, and colostrum and teat swab specimens were collected immediately after parturition. Detection of MAP in fecal specimens was performed via microbial culture, and detection of MAP DNA in colostrum and teat swab specimens was achieved via a PCR assay targeting the genetic element ISMAP02. Logistic regression was used to model the relationship between MAP fecal shedding status and detection of MAP DNA in colostrum or teat swab specimens. Population attributable fractions for the proportion of colostrum and teat swab specimens containing MAP DNA were also calculated.

Results—The odds of detecting MAP DNA in colostrum or teat swab specimens in cows with MAP-positive (vs negative) fecal specimens were 2.02 and 1.87 respectively. Population attributable fractions estimates suggested that withholding colostrum from MAP-positive cows could reduce the odds of exposing calves to MAP in colostrum by 18.2%. Not permitting natural suckling by calves could reduce the odds of exposing calves to MAP on the teat surfaces of MAP-positive cows by 19.5%.

Conclusions and Clinical Relevance—Results underscored the need for strict adherence to practices that limit contact of calves with adult cows from the time of birth and promote hygienic colostrum handling to avoid possible contamination with MAP during colostrum harvest, storage, or feeding.

Caused by infection with MAP, paratuberculosis (Johne's disease) is a chronic and contagious disease of cattle and other ruminants associated with high economic losses.1–4 The disease is endemic in the United States with a reported national herd-level prevalence ≥ 68%.5 Infection with MAP is difficult to control because of lack of an approved drug for treating the condition,6 the long incubation period of infections,7 and the lack of diagnostic tests with satisfactory sensitivity for early detection of infection in cattle.8–16

Susceptibility to infection with MAP is believed to be greatest in calves, with most calves acquiring infections at ≤ 6 months of age.17,18 Transmission occurs mainly via the fecal-oral route.1,3,17–19 Although the most important source of MAP for within-herd transmission is considered to be feces from infected adult cattle,3,17,19–21 MAP has also been detected in colostrum and milk collected from cows shedding MAP in their feces.22–24 Feeding raw colostrum to calves might therefore be one of the earliest means by which calves become exposed to MAP.

Calves fed raw bovine colostrum reportedly have a higher risk of infection with MAP than do calves fed a plasma-derived commercial colostrum replacement product.25 Similarly, cows fed colostrum collected from multiple cows or sources are more likely to test positive for MAP than are herdmates fed colostrum collected from their own dams.26 In addition, herds in which calves are fed colostrum collected from known MAP-infected cows are reportedly 87 times as likely to be infected with MAP as herds in which such colostrum feeding practices are not typical.27

The scientific literature regarding associations between fecal excretion of MAP and possible contamination of colostrum with MAP is limited. In an Ohio study,23 colostrum specimens collected from 36 subclinically infected cows that were shedding MAP in their feces were cultured for MAP isolation. Overall, MAP was present in the colostrum specimens of 22% (8/36) of the cows with MAP in their feces. Thirty-one percent (11/36) of these were classified as heavy fecal shedders (> 3,000 colonies/agar tube), compared with 70% (25/36) that were classified as light shedders (< 3,000 colonies/agar tube) of MAP. The colostrum specimens collected from 36% (4/11) of the heavy fecal shedders were positive for MAP, compared with 16% (4/25) of the colostrum specimens collected from the light shedders. The lack of control cattle (ie, cows with negative fecal test results) in that early study rendered it impossible to quantify the relationship between MAP fecal shedding status and the odds of recovering MAP in colostrum.

Evidence is also limited regarding possible associations between fecal shedding of MAP and contamination of bovine teats and udders with feces, soils, or other environmental contaminants containing MAP, although fecal contamination of teat surfaces is believed to facilitate transmission of MAP in calves permitted to suckle naturally.28 Indeed, the odds of MAP infection has been reported to be twice as high among calves that suckle their dams or are fostered by other cows within the herd than calves fed milk replacer.26 The purpose of the study reported here was to evaluate the association between fecal excretion of MAP by dairy cows in the periparturient period and detection of MAP DNA in colostrum specimens and on teat skin surfaces.

Materials and Methods

Animals—Cows were enrolled from a convenience sample of 4 commercial Holstein dairy herds located in southeast Minnesota. The original herd selection criteria have been described in detail elsewhere.29 Briefly, requisites for herd selection included evidence of the endemic presence of paratuberculosis in the herd and use of group maternity pens. Initially, paratuberculosis was deemed present in the herds when there were records of past tests for MAP and a history of culling due to paratuberculosis during the year prior to the start of the present study. Herd screening tests were later performed to determine true herd infection status. The requirement for using group maternity pens was due to the fact that short- and long-term goals of the study were to evaluate the efficacy of single-cow calving pens (vs group maternity pens) for the prevention of neonatal calf diseases29 and to evaluate the effect of single-cow calving pens (vs group maternity pens) for prevention of MAP transmission in Holstein calves. Additional criteria for herd selection included routine testing for milk production through the Minnesota Dairy Herd Improvement Association, willingness to comply with study protocols, and proximity (≤ 160-km radius) to the College of Veterinary Medicine, University of Minnesota. The original study,29 of which the present study was part, was approved by the Institutional Animal Use and Care Committee at the University of Minnesota.

Sampling strategy—Fecal, colostrum, and teat swab specimens were collected from all cows in the 4 participating herds that calved between January and December 2005 for the recovery and detection of MAP. However, although all collected fecal specimens were tested for MAP (via fecal culture), not all collected colostrum and teat swab specimens were tested to confirm presence of MAP for budgetary reasons. As such, colostrum and teat swab specimens tested for MAP were selected from the stored specimens collected during the original enrollment period.

Pairs of colostrum and teat swab specimens collected from cows that tested positive for MAP on fecal culture (MAP fecal-positive cows) were all tested for presence of MAP DNA. For each fecal-positive cow in the study, approximately 2 cows with negative fecal MAP culture results (fecal negative cows) were randomly selected from the list of fecal-negative cows in the same herd for use as a comparison group. The colostrum and teat swab specimens collected from the comparison group were then tested for presence of MAP DNA.

Specimen collection—For each cow that was about to calve, approximately 10 g of feces was collected manually by the herdsmen from the rectum by use of a disposable plastic examination sleeve within 48 to 72 hours prior to actual parturition. Fecal specimens were stored in a sterile plastic specimen container and labeled with cow identity prior to refrigeration at ≤ 8°C. Newborn calves were separated from their dams within 1 hour after birth, prior to having the opportunity to suckle.

Sterile gauze swabs impregnated with a solution of 0.45% NaCl were prepared and made available on each study farm in advance of an impending teat swabbing event. Teat skin surfaces of the front and rear teats were swabbed with the sterile gauze preparations prior to being washed by the herdsmen. The teat swab specimens were labeled with the respective cow identification and placed in an opaque, airtight plastic specimen tube containing 5 mL of 0.45% NaCl solution and kept in storage at −20°C. The teat surfaces were then disinfected with a 0.05% povidone iodine commercial teat dip and cleansed with 70% ethyl alcohol-impregnated pledgets prior to collection of the colostrum specimens. The fore-stripping colostrum was discarded, and composite colostrum specimens (50 mL) were collected into a sterile plastic tube from all 4 quarters of the udder, labeled with the respective cow identification, and stored at −20°C.

For collecting colostrum specimens and obtaining the teat swab specimens, latex gloves were worn and fresh pairs of gloves were used each time specimens were collected from a cow. All specimens (fecal, colostrum, and teat swabs) were picked up on a biweekly farm visiting schedule and transported on ice to the University of Minnesota. Isolation of MAP from fecal specimens was performed at the Minnesota Veterinary Diagnostic Laboratory. Colostrum and teat swab specimens were shipped to the National Animal Disease Center, USDA Agricultural Research Service in Ames, Iowa, for further processing and analysis.

Isolation of MAP from fecal specimens—Fecal specimens were used for microbial culture of MAP as described elsewhere.10,30 Briefly, sediments derived from the fecal specimens were plated onto Herrold egg yolk agar slantsa containing mycobactin J and an antimicrobial (0.1 mL of 50-mg amphotericin B) and incubated at 37°C for 1 to 3 weeks. Approximately 6 weeks after inoculation, the agar slants were examined weekly for growth of MAP. On the basis of total colony counts per gram of fecal specimen tested, results were reported as negative (0 colonies/tube) or positive-low shedder (1 to 10 colonies/tube), positive-moderate shedder (10 to 50 colonies/tube), positive-high shedder (51 to 100 colonies/tube), and positive-very high shedder (> 100 colonies/tube).

PCR analysis of colostrum and teat swab specimens—The MAP DNA was extracted from colostrum specimens as described elsewhere.31 The DNA in teat swab specimens was extracted by use of a commercially available kit.b

For the first round of the PCR analysis, a conventional thermal cyclerc was used. Specimens were run in triplicate in 96-well plates according to the following protocol: 1 cycle at 94°C for 5 minutes, followed by 20 cycles of 94°C for 45 seconds, 58°C for 1 minute, and 72°C for 2 minutes, and a final extension cycle at 72°C for 7 minutes. The primer sequences for the initial amplification were 5′-GCACGGTTTTTCGGATAACGAG-3′ (forward primer) and 5′-TCAACTGCGTCACGGTGTCCTG-3′ (reverse primer).

The second amplification was performed by use of a real-time PCR assayd and included a FAM-labeled probe specific for the ISMAP02 target sequence for evaluation of the test samples. Primers nested within the first set included 5′-GGATAACGAGACCGTGGATGC-3′ (forward primer) and 5′-AACCGACGCCGCCAATACG-3′ (reverse primer) for this second amplification, yielding a 117-base pair product. One microliter of DNA from the first amplification was added to a reaction mixture consisting of PCR mastermix,e ultrapure distilled water (free of deoxyribonuclease and ribonuclease), 0.05μM primers, and 0.05μM FAM-labeled probe (5′-/56-FAM/CAACCCGCACGCTG/3BHQ-1/-3′). A standard was constructed by amplifying the ISMAP02 target from MAP strain K-10 genomic DNA and cloned into the topoisomerase I expression vectorf followed by transformation in Escherichia coli. The insert was analyzed for accuracy following plasmid digestion with EcoRI and verification of size on a 4% agarose gel.

Further verification of the cloned insert was conducted by sequencing the product with a commercial DNA analyzerg after labeling the product with a cycle sequencing kit.h Primers used for sequencing were M13F: 5′-CGTTGTAAAACGACGGCCAGT-3′ (forward) and M13R: 5′-CAGGAAACAGCTATGAC-3′ (reverse). Optimal concentrations of the plasmid for use as a real-time PCR standard ranged between 1 to 100 ng. Real-time PCR conditions for the amplification of test samples and standards were 1 cycle at 50°C for 2 minutes, 1 cycle at 95°C for 10 minutes, 40 cycles at 95°C for 25 seconds, and 60°C for 1 minute. Positive samples were visualized in the form of sigmoid curves plotted on 2-D grids, with the x-axis representing the PCR cycle number and the y-axis representing the relative fluorescence of the signal. Sample runs with threshold cycles in the range of 15 to 30 cycles were declared positive.

Statistical analysis—All analyses were performed by use of standard statistical software,i and values of P ≤ 0.05 were considered significant. Data were summarized for the distribution of cow lactation category (first vs ≥ second lactation), colostrum and teat swab specimens with positive results for MAP DNA (yes vs no), and MAP fecal shedding category (0 = negative, 1 = low shedder, 2 = moderate shedder, 3 = high shedder, and 4 = very high shedder) between MAP fecal-positive and negative cows. Observations with missing outcomes were excluded from subsequent analysis. In addition, unconditional association between presence of MAP-DNA in colostrum and teat swab specimens was tested by use of contingency tables and simple χ2 analysis.

Data from colostrum and teat swab specimens with positive PCR assay results (vs specimens with negative results) were each used as dependent variables in 2 separate logistic regression models. Initially, the MAP fecal-shedding and lactation number categories were included as independent variables in each fitted model. However, after grouping the MAP fecal shedding categories into a dichotomous variable, the MAP fecal shedding status (positive vs negative) and lactation number variable (first vs ≥ second lactation) were included in the final regression models as independent variables. Because cows in the same herd were more likely to be similar to each other than to cows in other herds with respect to study outcomes, robust estimates of variance32–34 were used to adjust for this potential within-herd clustering effect. Because significance (P < 0.05) was not achieved for the lactation number variable in the fitted models, this variable was excluded from the final models. The ORs expressing the strength of the associations between MAP fecal shedding status and fecal shedding categories and the odds of detecting MAP DNA present in colostrum and teat swab specimens were calculated from each model. Model fit to the data was evaluated by use of the Hosmer-Lemeshow goodness-of-fit test.32

To estimate the proportion of colostrum and teat swab specimens positive for MAP DNA because the cows from which the specimens originated were fecal culture positive for MAP, the AFP was calculated as follows:
article image
in which pd represents the overall proportion of colostrum or teat swab specimens with positive results for MAP DNA that were collected from MAP fecal-positive cows, and OR was the adjusted OR for the association between MAP fecal shedding status or shedding category and odds of detecting MAP DNA in colostrum and teat swab specimens.35 Ninety-five percent confidence intervals for AFp estimates were also calculated.36

Results

Animals—The 4 participating herds ranged in size from 265 to 580 cows (median, 405 cows). To provide context regarding herd size, 3.7% of Minnesota dairy herds had between 200 and 499 milking cows and ≤ 1% had ≥ 500 in 2004.37 Annual herd milk production (ie, rolling herd average) ranged between 9,752 and 13,607 kg/y (median, 10,381 kg/y). Bulk-tank somatic cell counts ranged between 180,000 and 240,000 cells/mL (median, 230,000 cells/mL). The estimated within-herd prevalence for MAP infection as determined through bacterial culture of feces for MAP ranged between 4% and 14% (median, 8%).

The number of cows enrolled per herd ranged from 50 to 229, for a total of 499 cows. The number of cows from which colostrum and teat swab specimens were collected ranged from 7 to 83 cows.

Overall, data from 112 of the 499 (22%) enrolled cows were available for analysis. Thirty-eight (34%) of these had positive MAP fecal culture test results for MAP, whereas 74 (66%) did not. The MAP shedding categories low (1 to 10 MAP colonies/tube), moderate (10 to 50 colonies/tube), and very high (> 100 colonies/tube) were represented by 25 (66%), 5 (13%), and 8 (21%) of the 38 MAP fecal-positive cows, respectively. There were no cows in the high MAP fecal shedding category.

During transportation and storage, 8 colostrum specimens originating from 5 (13%) MAP fecal-positive cows and 3 (4%) MAP fecal-negative cows were lost, as were 21 teat swab specimens originating from 5 (13%) MAP fecal-positive cows and 16 (22%) MAP fecal-negative cows. Therefore, whether MAP DNA existed in those samples could not be determined. For cows with full data sets available, 26 of 33 (79%) MAP fecal-positive cows and 46 of 71 (65%) MAP fecal-negative cows had colostrum specimens that contained MAP DNA, for a total of 72 (69%) MAP-positive colostrum samples. Similarly, of the 55 of 91 (60%) teat swab specimens in which MAP DNA was detected, 23 of 33 (70%) originated from MAP fecal-positive cows and 32 of 58 (55%) originated from MAP fecal-negative cows. The simple χ2 analysis revealed no evidence of a significant association between presence of MAP DNA in colostrum and in teat swab specimens.

Logistic regression models—The goodness-of-fit analyses for the final models suggested that the models fit the data reasonably well. Logistic regression modeling indicated that the overall OR for the association between MAP fecal shedding status (positive vs negative) and detection of MAP DNA in colostrum was 2.02 (Table 1). The OR for the association between MAP fecal shedding status and detection of MAP DNA in teat swab specimens was 1.87.

Table 1

Results of final logistic regression models estimating the association between MAP fecal shedding status and detection of MAP DNA in colostrum (n = 104) and teat swab (91) specimens collected from Holstein cows in the periparturient period.

VariableORSE*95% CIP value
MAP DNA in colostrum specimens    
   Yes2.020.441.32–3.10< 0.001
   NoReferent
MAP DNA in teat swab specimens    
   Yes1.870.441.18–2.970.008
   NoReferent

The SE was calculated adjusting for the cluster effect of herd. CI = Confidence interval. — = Not applicable.

The ORs for the association between the fecal shedding categories low, moderate, and very high and detection of MAP DNA in colostrum specimens were 1.74, 2.17, and 3.26, respectively (Table 2). The estimated ORs for the association between low, moderate, and very high MAP fecal shedding categories and detection MAP DNA in teat swab specimens were 1.63, 1.63, and 4.06, respectively. In both situations, the odds of MAP DNA detection in colostrum and teat swab specimens appeared to increase with increasing MAP fecal shedding category, although these estimates were not significant for the MAP-DNA-positive colostrum outcome.

Table 2

Results of logistic regression models estimating the association between MAP fecal shedding category and detection of MAP DNA in colostrum (n = 104) and teat swab (91) specimens collected from Holstein cows in the periparturient period.

VariableORSE*95% CIP value
MAP DNA in    
   colostrum specimens    
      Low1.740.700.79–3.810.17
      Moderate2.171.500.56–8.380.26
      High
      Very high3.262.700.65–16.50.15
      NegativeReferent
MAP DNA in    
   teat swab specimens    
   Low1.630.341.07–2.460.02
   Moderate1.630.401.00–2.640.05
   High
   Very high4.063.190.87–19.00.07
   NegativeReferent

Results are reported as negative (nonshedder; 0 MAP colonies/agar tube), low shedder (1 to 10 colonies/tube), moderate shedder (10 to 50 colonies/tube), high shedder (51 to 100 colonies/tube), and very high shedder (> 100 colonies/tube).

See Table 1 for remainder of key.

Estimates for AFp—The proportions of MAP-positive colostrum and teat swab specimens attributable to a positive fecal test result for donor cows in the sample population evaluated were 18.2% and 19.5%, respectively (Table 3).

Table 3

Population attributable fraction estimates for the odds of MAP DNA detection in colostrum (n = 104) and teat swab (91) specimens attributable to detection of MAP in the feces of Holstein cows in the periparturient period.

 MAP DNA in colostrum specimensMAP DNA in teat swab specimens
VariableORAFp95% CI (AFp)ORAFp95% CI (AFp)
MAP fecal culture shedding category      
   Low1.740.11−0.17 to 0.351.630.12–0.21 to 0.42
   Moderate2.170.04−0.11 to 0.211.630.02–0.16 to 0.21
   High
   Very high3.260.08−0.10 to 0.254.060.10–0.09 to 0.31
   NegativeReferentReferent
Fecal culture positive      
   Yes2.020.18−0.14 to 0.431.870.19–0.17 to 0.48
   NoReferentReferent

See Tables 1 and 2 for key.

Discussion

In a previous study,23 MAP was recovered from the colostrum specimens of 22% (8/36) of MAP fecal-positive cows. However, it was impossible to quantify the magnitude of the association between positive fecal shedding status and detection of MAP in colostrum because this early study did not include fecal-negative cows as a control group. The present study was undertaken to quantify the impact of MAP fecal shedding on detection of MAP DNA in colostrum and teat swab specimens of cows in MAP-endemic herds.

A major strength of the present study was the fact that the selected herds were heavily infected with MAP (ie, the prevalence of MAP fecal shedders ranged from 4% to 14%), providing the ideal group of herds for addressing the study objectives. However, there were also several limitations. For example, the cross-sectional observational study design made it impossible to infer cause-and-effect relationships. In addition, the nested PCR assay used to detect MAP DNA in colostrum and teat swab specimens was shown in a previous experimental validation study31 to have an analytic sensitivity that improved with increasing concentration of MAP in colostrum (100% between 1 × 107 cells/mL and 1 × 109 cells/mL, 75% between 1 × 103 cells/mL and 1 × 106 cells/mL, and 50% between 1 × 101 cells/mL and 1 × 102 cells/mL) and an analytic specificity that approximated 0.89 (or a false-positive rate of approximately 0.11). Therefore, given the preceding experimental evidence suggestive of an imperfect sensitivity and specificity of the nested PCR assay, it is likely that the MAP status of some colostrum and teat swab specimens in our study were misclassified because of false-positive and false-negative results. Similarly, the culture method used to determine the MAP fecal shedding status of cows in this study is known to have limited and unreliable sensitivity during the early phase of paratuberculosis in cattle.9–11,16 As such, study cows that were shedding MAP at a lower amount than the limit of detection might have been misclassified as MAP negative, particularly cows in their first lactation (ie, 38% of all cows). Such potential misclassification errors may have biased the association between positive MAP fecal shedding status and detection of MAP in colostrum and teat swab specimens toward the null hypothesis (ie, there is no association), lowering the magnitude of the estimated ORs. Lastly, the small number of animals included in this analysis may have compromised the precision of estimates of association.

Despite the aforementioned limitations, a significant association was detected between positive MAP fecal shedding status and detection of MAP DNA in colostrum and teat swab specimens. There was also evidence suggestive of an increase in the strength of the association between detection of MAP DNA in colostrum or teat swab specimens from the low fecal shedding category to the very high category. However, the fecal shedding category estimates of associations were not significant when the MAP-positive colostrum outcome variable was considered. These findings were not surprising given that we had expected an increase in the odds of detecting MAP DNA in colostrum and teat swab specimens with an increase in the quantity of MAP present per gram of feces tested. Although there appeared to be no association between the amounts of MAP excreted in feces and detection of MAP DNA in colostrum, there was an overall significant positive association between detection of MAP in feces and detection of MAP DNA in colostrum.

Although mechanisms by which MAP may contaminate colostrum are still poorly understood, 2 hypotheses have been advanced as potential explanations. First, feces containing MAP may contaminate teat skin, thereby contributing to contamination of colostrum during the collection or harvesting process.28 However, the present study revealed no evidence to support this hypothesis, given that no significant association was evident between MAP DNA detection in colostrum and in teat swab specimens. Perhaps this lack of a significant association was a result of the degree of hygiene (ie, sterile collection of colostrum) practiced during the collection of colostrum specimens in our study.

The second hypothesized mechanism of MAP contamination of colostrum is through the systemic or hematogenous dissemination of MAP in an infected host into the mammary gland.23 By the hematogenous mechanism, freely circulating MAP-infected macrophages are believed to end up in the mammary glands, with colostrum being their final site of deposition. This idea is based on the fact that macrophages constitute the dominant somatic cell type detected in secretions from uninfected mammary glands during the dry and periparturient periods.38,39 Mycobacterium avium subsp paratuberculosis is an obligate intracellular pathogen known to have an affinity for macrophages and the ability to survive inside macrophages for long periods with no detrimental effects on their cellular hosts.40,41

The presence of MAP on teat skin may result from fecal contamination of a cow's teat directly from MAP-containing feces excreted by the same cow (or another cow).8 It is also possible that the environment of MAP-infected herds serves as an important source of MAP by which MAP-containing feces contaminates the udders and teats when cows lie on contaminated bedding or on contaminated floors. In a previous study42 of the environmental distribution of MAP in Minnesota herds, ≥ 78% of pooled fecal specimens collected from surroundings of paratuberculosis endemic herds contained MAP. Indeed, MAP was detected in pooled specimens collected from cow alleys (77% of samples positive), manure storage areas (68%), maternity pens (21%), and sick cow pens (18%). That finding underscores the ubiquitous nature of MAP in MAP-infected herds and the possible role of the environment in MAP transmission.

Interestingly, the proportions of colostrum and teat swab specimens containing MAP DNA potentially attributable to positive MAP fecal shedding status of the donor cows (ie, AFp) were only approximately 18% and 20%, respectively. This finding suggested that up to 82% and 81% of MAP detected in colostrum and teat swab specimens had sources unrelated to the MAP fecal shedding status of the donor cows. Because para-tuberculosis was endemic in the study herds, with an estimated within-herd prevalence of MAP fecal shedders ranging between 4% and 14%, it was possible that the environments of participating herds played a major role as reservoirs for MAP, providing a source for MAP that eventually contaminated the colostrum specimens and teat surfaces. In absolute terms, more than half of the colostrum and teat swab specimens collected from MAP fecal-negative cows in the present study were positive for MAP DNA.

The AFP estimates obtained in the present study have practical implications for within-herd paratuberculosis control efforts. For example, the AFP estimates suggested that by avoiding the feeding of colostrum from known fecal shedders of MAP within an MAP-infected herd, the odds of exposing calves to MAP (assuming the MAP DNA in colostrum was indicative of viable infectious MAP) within 24 to 48 hours after birth through ingestion of colostrum could be reduced by approximately 18%. Similarly, by prompt separation of calves from their dams within 30 minutes after birth, thereby eliminating the possibility of natural nursing by cows shedding MAP in their feces, the odds of exposing calves through ingestion of MAP present on the dam's teats could be reduced by approximately 20%, assuming that the MAP DNA in teat swab specimens was indicative of viable infectious MAP. However, it is important to recognize that while the preceding interventions might achieve some reduction in odds of exposure, they by no means could completely eliminate the odds of calf exposure to MAP through ingestion of MAP in the colostrum or on the cow's teats in paratuberculosis endemic herds because a larger percentage (≥ 81%) of the MAP DNA-positive colostrum and teat swab specimens were collected from MAP fecal-negative cows.

Implementing other strategies that might assist in preventing MAP contamination of colostrum and teats by other sources such as the environment is highly encouraged to reduce the likelihood of calf exposure to MAP. For example, dairy producers might limit contamination of colostrum by pathogens in manure by adequately preparing udders prior to harvesting colostrum and storing or handling colostrum by use of sanitized equipment. Details on prevention of potential bacterial contamination of colostrum during the harvest, storage, and feeding processes have been reported elsewhere.43,44 A recent report27 indicated that herds in which calves were fed colostrum collected from known MAP-infected cows were 87 times as likely to be infected with MAP as herds in which such colostrum feeding practices were not typical. As such, additional effort could also be directed toward identifying individual cows that are fecal shedders of MAP within a herd through regular microbial culture of feces to identify such cows (ie, MAP fecal shedders) for possible removal from the herd to reduce the pool of cows that may be contributing directly to high environmental loads of MAP or are donors of MAP-contaminated colostrum. Permitting natural nursing or fostering of calves by cows other than a calf's dam within a herd is a significant predictor of future positive test results for MAP in the affected calves.26 Consequently, as previously mentioned, newborn calves should not be allowed to suckle their dams or other cows whenever possible through prompt separation from their dams within 30 minutes after birth. Measures that promote proper manure management should be implemented, as well as those that involve use of individual maternity pens that are cleaned and freshly bedded between successive parturitions.

In interpreting the findings of the present study, it should be remembered that the herds involved were not randomly selected from the entire population of dairy herds in Minnesota, and in terms of herd size, the selected herds were medium (100 to 499 cows) to large (≥ 500 cows) in size. According to the National Agricultural Statistics Service, approximately 4% and ≤ 1% of the total Minnesota dairy herds belonged to these herd size categories in 2004, respectively.37 Caution should therefore be exercised when attempting to extrapolate our results to smaller herds (< 100 cows) or to herds with different management systems. Regardless of this limitation, findings of the present study underscore the need for strict adherence to practices that limit contact of calves to adult cattle shedding MAP in their feces at birth in addition to using strategies that limit potential environmental contamination of colostrum as part of a wider paratuberculosis control effort.

ABBREVIATIONS

AFp

Population attributable fraction

FAM

Fluorophore 6-carboxyfluorescein

MAP

Mycobacterium avium subsp paratuberculosis

OR

Odds ratio

a.

HEYM-BBL, Becton-Dickinson, Franklin Lakes, NJ.

b.

ZR Fecal DNA Kit, Zymo Research Corp, Orange, Calif.

c.

Peltier Thermal Cycler, MJ Research, Waltham, Mass.

d.

7500 Real-time PCR System, Applied Biosystems, Foster City, Calif.

e.

TaqMan Universal PCR Master Mix, Applied Biosystems, Foster City, Calif.

f.

Invitrogen Corp, Carlsbad, Calif.

g.

3100 Genetic Analyzer, Applied Biosystems, Foster City, Calif.

h.

BigDye Terminator, version 3.1, Applied Biosystems, Foster City, Calif.

i.

Stata, version 10, Stata Corp, College Station, Tex.

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    Ott SLWells SJWagner BA. Herd-level economic losses associated with Johne's disease on US dairy operations. Prev Vet Med 1999; 40:179192.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    National Animal Health Monitoring System. Johne's disease on US Dairies, 1991–2007. Available at: nahms.aphis.usda.gov/dairy/index.htm#dairy2007. Accessed Dec 22, 2009.

  • 6.

    St Jean G. Treatment of clinical paratuberculosis in cattle. Vet Clin North Am Food Anim Pract 1996; 12:417430.

  • 7.

    Whitlock RH, Hutchinson LT, Glickman LT, et al. Paratuberculosis (Johne's disease) update. Bovine Pract 1986;21:2430.

  • 8.

    Sockett DCCarr DJCollins MT. Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle. Can J Vet Res 1992; 56:148153.

    • Search Google Scholar
    • Export Citation
  • 9.

    Collins MT. Diagnosis of paratuberculosis. Vet Clin North Am Food Anim Pract 1996; 12:357371.

  • 10.

    Whitlock RHWells SJSweeney RW, et al. ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method. Vet Microbiol 2000; 77:387398.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Dargatz DAByrum BABarber LK, et al. Evaluation of a commercial ELISA for diagnosis of paratuberculosis in cattle. J Am Vet Med Assoc 2001; 218:11631166.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Nielsen SSGrohn YTEnevoldsen C. Variation of the milk antibody response to paratuberculosis in naturally infected dairy cows. J Dairy Sci 2002; 85:27952802.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Nielsen SSGrønbæk CAgger JF, et al. Maximum-likelihood estimation of sensitivity and specificity of ELISAs and faecal culture for diagnosis of paratuberculosis. Prev Vet Med 2002; 53:191204.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Hendrick SHDuffield TEKelton DE, et al. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows. J Am Vet Med Assoc 2005; 226:424428.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Nielsen SS. Transitions in diagnostic tests used for detection of Mycobacterium avium subsp. paratuberculosis infections in cattle. Vet Microbiol 2008; 132:274282.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Nielsen SSToft N. Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-gamma assay and faecal culture techniques. Vet Microbiol 2008; 129:217235.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Wells SJ. The prospects for herd level control of paratuberculosis—a scientists view, in Proceedings. 8th Int Colloq Paratuberculosis 2005;174181.

    • Search Google Scholar
    • Export Citation
  • 18.

    Windsor PAWhittington RJ. Evidence for age susceptibility of cattle to Johne's disease. Vet J 2010; 184:3744.

  • 19.

    Caldow GL. The prospects for herd level control of paratuberculosis in cattle: a consultant's view, in Proceedings. 8th Int Colloq Paratuberculosis 2005;165173.

    • Search Google Scholar
    • Export Citation
  • 20.

    van Roermund HJWde Jong MCM. Horizontal transmission experiment of paratuberculosis, in Proceedings. 8th Int Colloq Paratuberculosis 2005;672678.

    • Search Google Scholar
    • Export Citation
  • 21.

    van Roermund HJBakker DWillemsen PT, et al. Horizontal transmission ofMycobacterium avium subsp. paratuberculosis in cattle in an experimental setting: calves can transmit the infection to other calves. Vet Microbiol 2007; 122:270279.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Sweeney RWWhitlock RHRosenberger AE. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J Clin Microbiol 1992; 30:166171.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Streeter RNHoffsis GFBech-Nielsen S, et al. Isolation ofMycobacterium paratuberculosis from colostrum and milk of subclinically infected cows. Am J Vet Res 1995; 56:13221324.

    • Search Google Scholar
    • Export Citation
  • 24.

    Giese SBAhrens P. Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture. Vet Microbiol 2000; 77:291297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Pithua PGodden SMWells SJ, et al. Efficacy of feeding plasma-derived commercial colostrum replacer for the prevention of transmission of Mycobacterium avium subsp paratuberculosis in Holstein calves. J Am Vet Med Assoc 2009; 234:11671176.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Nielsen SSBjerre HToft N. Colostrum and milk as risk factors for infection with Mycobacterium avium subspecies paratuberculosis in dairy cattle. J Dairy Sci 2008; 91:46104615.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Dieguez FJArnaiz ISanjuan ML, et al. Management practices associated with Mycobacterium avium subspecies paratuberculosis infection and the effects of the infection on dairy herds. Vet Rec 2008; 162:614617.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Merkal RS. Paratuberculosis: prevalence, diagnosis, prevention, and treatment, in Proceedings. 17th Annu Conv Am Assoc Bovine Pract 1985;6466.

    • Search Google Scholar
    • Export Citation
  • 29.

    Pithua PWells SJGodden SM, et al. Clinical trial on type of calving pen and the risk of disease in Holstein calves during the first 90 d of life. Prev Vet Med 2009; 89:815.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Wells SJWagner BA. 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. J Am Vet Med Assoc 2000; 216:14501457.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Pithua PWells SJGodden SM, et al. Experimental validation of a nested polymerase chain reaction targeting the genetic element ISMAP02 for detection ofMycobacterium avium subsp. paratuberculosis in bovine colostrum. J Vet Diagn Invest 2010; 22:253256.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Hosmer DWLameshow S. Applied logistic regression. 2nd ed. New York: Wiley and Sons, 2000.

  • 33.

    Dohoo IMartin WStryhn H. Veterinary epidemiologic research. Charlottetown, PE, Canada: AVC Inc, 2003.

  • 34.

    Kirkwood BRStern JAC. Essential medical statistics. 2nd ed. Malden, Mass: Blackwell Science, 2003.

  • 35.

    Rockhill BNewman BWeinberg C. Use and misuse of population attributable fractions. Am J Public Health 1998; 88:1519.

  • 36.

    Natarajan SLipsitz SRRimm E. A simple method of determining confidence intervals for population attributable risk from complex surveys. Stat Med 2007; 26:32293239.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    National Agricultural Statistics Services. Farms, land in farms, and livestock operations: 2003–2004. Available at: usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1259. Accessed Dec 22, 2009.

  • 38.

    McDonald JSAnderson AJ. Total and differential somatic cell counts in secretions from noninfected bovine mammary glands: the early nonlactating period. Am J Vet Res 1981; 42:13601365.

    • Search Google Scholar
    • Export Citation
  • 39.

    McDonald JSAnderson AJ. Total and differential somatic cell counts in secretions from noninfected bovine mammary glands: the peripartum period. Am J Vet Res 1981; 42:13661368.

    • Search Google Scholar
    • Export Citation
  • 40.

    Zurbrick BGCzuprynski CJ. Ingestion and intracellular growth of Mycobacterium paratuberculosis within bovine blood monocytes and monocyte-derived macrophages. Infect Immun 1987; 55:15881593.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Coussens PM. Mycobacterium paratuberculosis and the bovine immune system. Anim Health Res Rev 2001; 2:141161.

  • 42.

    Raizman EAWells SJGodden SM, et al. The distribution ofMycobacterium avium ssp. paratuberculosis in the environment surrounding Minnesota dairy farms. J Dairy Sci 2004; 87:29592966.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Stewart SGodden SBey R, et al. Preventing bacterial contamination and proliferation during the harvest, storage, and feeding of fresh bovine colostrum. J Dairy Sci 2005; 88:25712578.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Godden S. Colostrum management for dairy calves. Vet Clin North Am Food Anim Pract 2008; 24:1939.

  • 1.

    Sweeney RW. Transmission of paratuberculosis. Vet Clin North Am Food Anim Pract 1996; 12:305312.

  • 2.

    Benedictus GDijkhuizen AAStelwagen J. Economic losses due to paratuberculosis in dairy cattle. Vet Rec 1987; 121:142146.

  • 3.

    Johnson-Ifearulundu YJKaneene JB. Epidemiology and economic impact of subclinical Johne's disease: a review. Vet Bull 1997; 67:437447.

    • Search Google Scholar
    • Export Citation
  • 4.

    Ott SLWells SJWagner BA. Herd-level economic losses associated with Johne's disease on US dairy operations. Prev Vet Med 1999; 40:179192.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    National Animal Health Monitoring System. Johne's disease on US Dairies, 1991–2007. Available at: nahms.aphis.usda.gov/dairy/index.htm#dairy2007. Accessed Dec 22, 2009.

  • 6.

    St Jean G. Treatment of clinical paratuberculosis in cattle. Vet Clin North Am Food Anim Pract 1996; 12:417430.

  • 7.

    Whitlock RH, Hutchinson LT, Glickman LT, et al. Paratuberculosis (Johne's disease) update. Bovine Pract 1986;21:2430.

  • 8.

    Sockett DCCarr DJCollins MT. Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle. Can J Vet Res 1992; 56:148153.

    • Search Google Scholar
    • Export Citation
  • 9.

    Collins MT. Diagnosis of paratuberculosis. Vet Clin North Am Food Anim Pract 1996; 12:357371.

  • 10.

    Whitlock RHWells SJSweeney RW, et al. ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method. Vet Microbiol 2000; 77:387398.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Dargatz DAByrum BABarber LK, et al. Evaluation of a commercial ELISA for diagnosis of paratuberculosis in cattle. J Am Vet Med Assoc 2001; 218:11631166.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Nielsen SSGrohn YTEnevoldsen C. Variation of the milk antibody response to paratuberculosis in naturally infected dairy cows. J Dairy Sci 2002; 85:27952802.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Nielsen SSGrønbæk CAgger JF, et al. Maximum-likelihood estimation of sensitivity and specificity of ELISAs and faecal culture for diagnosis of paratuberculosis. Prev Vet Med 2002; 53:191204.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Hendrick SHDuffield TEKelton DE, et al. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows. J Am Vet Med Assoc 2005; 226:424428.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Nielsen SS. Transitions in diagnostic tests used for detection of Mycobacterium avium subsp. paratuberculosis infections in cattle. Vet Microbiol 2008; 132:274282.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Nielsen SSToft N. Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-gamma assay and faecal culture techniques. Vet Microbiol 2008; 129:217235.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Wells SJ. The prospects for herd level control of paratuberculosis—a scientists view, in Proceedings. 8th Int Colloq Paratuberculosis 2005;174181.

    • Search Google Scholar
    • Export Citation
  • 18.

    Windsor PAWhittington RJ. Evidence for age susceptibility of cattle to Johne's disease. Vet J 2010; 184:3744.

  • 19.

    Caldow GL. The prospects for herd level control of paratuberculosis in cattle: a consultant's view, in Proceedings. 8th Int Colloq Paratuberculosis 2005;165173.

    • Search Google Scholar
    • Export Citation
  • 20.

    van Roermund HJWde Jong MCM. Horizontal transmission experiment of paratuberculosis, in Proceedings. 8th Int Colloq Paratuberculosis 2005;672678.

    • Search Google Scholar
    • Export Citation
  • 21.

    van Roermund HJBakker DWillemsen PT, et al. Horizontal transmission ofMycobacterium avium subsp. paratuberculosis in cattle in an experimental setting: calves can transmit the infection to other calves. Vet Microbiol 2007; 122:270279.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Sweeney RWWhitlock RHRosenberger AE. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J Clin Microbiol 1992; 30:166171.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Streeter RNHoffsis GFBech-Nielsen S, et al. Isolation ofMycobacterium paratuberculosis from colostrum and milk of subclinically infected cows. Am J Vet Res 1995; 56:13221324.

    • Search Google Scholar
    • Export Citation
  • 24.

    Giese SBAhrens P. Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture. Vet Microbiol 2000; 77:291297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Pithua PGodden SMWells SJ, et al. Efficacy of feeding plasma-derived commercial colostrum replacer for the prevention of transmission of Mycobacterium avium subsp paratuberculosis in Holstein calves. J Am Vet Med Assoc 2009; 234:11671176.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Nielsen SSBjerre HToft N. Colostrum and milk as risk factors for infection with Mycobacterium avium subspecies paratuberculosis in dairy cattle. J Dairy Sci 2008; 91:46104615.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Dieguez FJArnaiz ISanjuan ML, et al. Management practices associated with Mycobacterium avium subspecies paratuberculosis infection and the effects of the infection on dairy herds. Vet Rec 2008; 162:614617.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Merkal RS. Paratuberculosis: prevalence, diagnosis, prevention, and treatment, in Proceedings. 17th Annu Conv Am Assoc Bovine Pract 1985;6466.

    • Search Google Scholar
    • Export Citation
  • 29.

    Pithua PWells SJGodden SM, et al. Clinical trial on type of calving pen and the risk of disease in Holstein calves during the first 90 d of life. Prev Vet Med 2009; 89:815.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Wells SJWagner BA. 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. J Am Vet Med Assoc 2000; 216:14501457.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Pithua PWells SJGodden SM, et al. Experimental validation of a nested polymerase chain reaction targeting the genetic element ISMAP02 for detection ofMycobacterium avium subsp. paratuberculosis in bovine colostrum. J Vet Diagn Invest 2010; 22:253256.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Hosmer DWLameshow S. Applied logistic regression. 2nd ed. New York: Wiley and Sons, 2000.

  • 33.

    Dohoo IMartin WStryhn H. Veterinary epidemiologic research. Charlottetown, PE, Canada: AVC Inc, 2003.

  • 34.

    Kirkwood BRStern JAC. Essential medical statistics. 2nd ed. Malden, Mass: Blackwell Science, 2003.

  • 35.

    Rockhill BNewman BWeinberg C. Use and misuse of population attributable fractions. Am J Public Health 1998; 88:1519.

  • 36.

    Natarajan SLipsitz SRRimm E. A simple method of determining confidence intervals for population attributable risk from complex surveys. Stat Med 2007; 26:32293239.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    National Agricultural Statistics Services. Farms, land in farms, and livestock operations: 2003–2004. Available at: usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1259. Accessed Dec 22, 2009.

  • 38.

    McDonald JSAnderson AJ. Total and differential somatic cell counts in secretions from noninfected bovine mammary glands: the early nonlactating period. Am J Vet Res 1981; 42:13601365.

    • Search Google Scholar
    • Export Citation
  • 39.

    McDonald JSAnderson AJ. Total and differential somatic cell counts in secretions from noninfected bovine mammary glands: the peripartum period. Am J Vet Res 1981; 42:13661368.

    • Search Google Scholar
    • Export Citation
  • 40.

    Zurbrick BGCzuprynski CJ. Ingestion and intracellular growth of Mycobacterium paratuberculosis within bovine blood monocytes and monocyte-derived macrophages. Infect Immun 1987; 55:15881593.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Coussens PM. Mycobacterium paratuberculosis and the bovine immune system. Anim Health Res Rev 2001; 2:141161.

  • 42.

    Raizman EAWells SJGodden SM, et al. The distribution ofMycobacterium avium ssp. paratuberculosis in the environment surrounding Minnesota dairy farms. J Dairy Sci 2004; 87:29592966.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Stewart SGodden SBey R, et al. Preventing bacterial contamination and proliferation during the harvest, storage, and feeding of fresh bovine colostrum. J Dairy Sci 2005; 88:25712578.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Godden S. Colostrum management for dairy calves. Vet Clin North Am Food Anim Pract 2008; 24:1939.

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