Longitudinal study of the distribution of Mycobacterium avium subsp. paratuberculosis in the environment of dairy herds in the Michigan Johne’s disease control demonstration herd project

Abstract

The objective of this study was to describe the distribution of Mycobacterium avium subsp. paratuberculosis (MAP) in the environment of infected dairy farms over time. Johne’s disease (JD) prevalence was monitored annually in 7 Michigan dairy herds. Environmental samples were collected bi-annually and cultured for MAP. Of 731 environmental samples that were cultured, 81 (11%) were positive. The lactating cow floor and manure storage areas were the areas most commonly contaminated, representing 30% and 33% of positive samples, respectively. When herd prevalence was > 2%, MAP was cultured from the lactating cow floor and/or manure storage area 75% of the time. When herd prevalence was ≤ 2%, MAP was never cultured from samples collected. For every 1 unit increase in number of positive environmental samples, within herd JD prevalence increased 1.62%. Environmental contamination with MAP is consistent over time on infected dairy farms, and management practices to reduce environmental contamination are warranted.

Introduction

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease (JD), is prevalent worldwide. The National Animal Health Monitoring and Surveillance (NAHMS) Dairy 2007 study estimated that 68.1% of the dairy herds in the United States were infected with MAP (1). This is up from 21.6% reported in the NAHMS Dairy 1996 study (2). Based on data from the 1996 NAHMS study, annual economic losses for the dairy industry due to JD were estimated to range from $200–250 million (3). Cattle generally become infected with MAP as young calves, but do not exhibit signs of the disease until years later (4). Due to the chronic nature of the disease, and its long incubation period, testing and culling infected animals as a method of control has been relatively ineffective by itself (5–7). Instead, strategies for controlling JD have focused on minimizing the exposure of calves, the animals most susceptible to becoming infected, to MAP, thereby preventing new infections.

While calves can become infected with MAP in utero (8–9), or through ingestion of colostrum or milk from infected cows, this generally only occurs when the dam is in the late stages of the disease (10–11). Most post-natal infections occur through the ingestion of the bacterium from a contaminated environment (4,12). Thus, factors playing a role in transmission include the amount of MAP being shed into the environment, the location contaminated, and the length of time the bacteria survive in that environment.

As an obligate intracellular pathogen, MAP does not replicate outside the host (12), but it can survive for over a year in the environment (13). Wildlife, birds (14–16), even invertebrates such as flies and worms (17–19) commonly found around dairy farms can become infected with MAP, and occasionally shed the bacterium into the environment. While the amount of MAP shed by these nontraditional hosts is negligible compared with that shed by cattle (20), these hosts represent a way by which the bacterium can persist and multiply outside of the primary host. The presence of “dormancy-related genes” in the MAP genome suggests that, in the absence of essential nutrients, MAP may enter a state of dormancy and then return to a viable, infectious state when conditions become favorable (13). Under field conditions in Australia, the sheep strain of MAP was cultured from pasture 12 mo after removal of livestock (21).

Studies have been conducted to determine the extent of MAP contamination on infected dairy farms (22–24). The bacterium has been found in numerous locations on dairy farms including calving pens and weaned calf pens (24), both of which are high-risk areas for transmitting the disease to herd replacements. The areas most commonly culture positive for MAP are those where manure from adult cattle accumulates, and include manure storage areas (lagoons, manure spreaders) and high-traffic, common cow areas (feed alleys, holding pens, return alleys) (22–24). Targeted culturing of these areas can be used to identify MAP-infected herds. In the most recent USDA’s Johne’s Program Standards (25), targeted environmental culturing was approved as an entry-level screening test for dairy herds desiring to participate in the Voluntary Johne’s Disease Control Program. Also, the number of positive environmental cultures and the amount of MAP in those samples are positively correlated with the within herd prevalence (22,24,26).

To date, studies investigating MAP contamination on dairy farms have been cross-sectional, with the environment being sampled at only 1 point in time (22,24,26). The temporal relationship between MAP environmental contamination and within herd JD prevalence remains undefined. There is limited information on how MAP contamination in the environment changes as within herd JD prevalence changes. Therefore, the objective of this study was to characterize the distribution of MAP in the environment of infected dairy farms, and describe if, or how, that distribution changes as within herd prevalence changes. The intention was to identify areas on infected farms that consistently culture positive for MAP. By understanding what areas on infected farms are consistently contaminated with MAP, even in the face of changing herd prevalence, more focused and economical herd screening programs can be developed.

Materials and methods

Farms

A total of 7 Michigan dairy herds participated in this study, which was part of the larger Michigan Johne’s Disease Control Demonstration Project. Herds were selected based on the following criteria: 1) herds were known to be infected with JD upon enrollment; 2) the producer was willing to participate in a longitudinal study for at least 5 y; and 3) the herd was representative of a typical Michigan dairy farm in terms of herd size and housing management. Upon enrollment, and annually thereafter, a JD risk assessment was performed for each herd. Based on the risk assessment and the individual herd’s goals and management capabilities, a JD control program was implemented on each herd and updated as necessary throughout the study. Study herd size ranged from 94 to 513 adult cows. Only 1 herd expanded significantly (231 to 445 cows) during the course of this study. Herd size for the other 6 herds remained consistent throughout the study period. Housing management practices consisted of total confinement (4 herds), combination of confinement and grazing (2 herds), and 1 rotational grazing herd which was confined during the winter months. Confinement housing consisted of free stalls (6 farms) or a combination of tie stalls and free stalls (1 farm).

Determination of herd prevalence

Fecal culture was performed on all adult cows in each herd annually. Prevalence was calculated as the number of cows with positive fecal cultures, divided by the total number of cows tested that year.

Environmental sampling

Environmental samples were collected from each farm every 6 mo. At each visit, 1 sample was collected from the feeding area, primary water source, and floor from each of the following areas: pre-weaned calf, weaned heifer, maternity, and lactating cow. A sample from the primary manure storage area (generally a lagoon or manure spreader) was also collected. Thus, a total of 13 environmental samples were collected at each herd visit. In addition, samples of pasture, pasture water sources, deer feces, and recycled sand bedding were collected and cultured when appropriate.

An attempt was made to acquire as representative a sample from each designated area as possible. For feed and flooring samples respectively, a clean, gloved hand was used to collect 10 random “grab” samples from various locations in the designated area. The samples were mixed together thoroughly and placed in 720 mL sterile Whirl-Pak bags. A composite sample from all sources (buckets, water tanks, automatic waterers, ponds) providing drinking water to cattle in a given area was collected in a sterile, 1-L bottle. The water sample was thoroughly agitated before filling a 120-mL plastic specimen cup and submitting for culture. For manure lagoons, samples were collected 15 cm below the surface from 4 to 6 locations and pooled to fill a 120 mL specimen cup. For manure spreaders, a 120 mL sample was collected from the beaters (box spreaders) or dispensing area (liquid spreaders). For recycled sand bedding and pastures respectively, random “grab” samples were collected; 5 from the surface and 5 from the underlying surface at depths varying from 6–24 cm. All samples from each area were mixed together in a clean bucket and a pooled sample placed in a 720-mL sterile Whirl-Pak bag for culture. During each farm visit, the farmstead, particularly around feed storage areas, pastures, fields, and any adjacent woods where deer sightings were reported, were walked and samples of deer feces collected when found. Environmental samples were collected from December 2002 through November 2006.

Bacterial culture

All fecal and environmental samples were submitted for MAP culture to the Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, Michigan, USA. Prior to June 2004, all samples were cultured by standard solid culture on Herrold’s Egg Yolk (HEY) medium. Thereafter, samples were cultured using the ESP^® culture system II (ESP II; TREK Diagnostics Systems, Cleveland, Ohio, USA). Processing and decontamination of samples prior to inoculation of culture media remained the same throughout the study, and consisted of a modification of the Cornell method described previously (27). Briefly, 2 g of each sample were added to 35 mL of sterile distilled water. The sample was vigorously shaken for 15 s, and then allowed to set at room temperature for 30 min. A 5-mL sample from the center of the supernatant was pipetted into a centrifuge tube containing 25 mL 1/2XBHI-HPC (half strength brain heart infusion broth with 0.9% 1-Hexadecylpyridinium) and gently mixed. Tubes were incubated at 35°C to 37°C overnight. Samples were then centrifuged at 3000 × g for 20 min at 22°C. The supernatant was decanted. A 1-mL volume of antibiotic mixture (50 μg amphotericin B, 100 μg vancomycin, and 100 μg naladixic acid in 1/2 XBHI) was added to the sample and vortexed to resuspend the pellet for final decontamination. Samples were incubated at 35°C to 37°C overnight before inoculating onto culture media.

Culture positive samples were confirmed as having MAP using Kinyoun’s acid-fast stain and real-time polymerase chain reaction (RT-PCR) for the IS900 insertion sequence. Real-time PCR was performed after 42 d on all signal negative ESP II samples. Samples were only reported as negative if they were signal negative on ESP II and negative on PCR.

Descriptive data analysis

Culture results were recorded in, and descriptive statistics generated, using a commercial computer spreadsheet (Microsoft Office Excel; Microsoft Corporation, Redmond, Washington, USA).

Statistical data analysis

The number of environmental samples collected at each collection date varied across and within herds depending on the housing management (pasture versus confinement), season (pastures were not sampled during winter months when cows were confined and/or access was restricted due to snow cover), and availability (deer feces were not consistently found on all farms). The association between the within herd JD prevalence and the number of culture positive environmental samples over time was therefore restricted to those samples that were consistently collected on all farms (feed, flooring, and water from the pre-weaned calf, weaned heifer, lactating cow, and maternity areas, and manure storage area). Environmental samples were collected every 6 mo, while herd prevalence was only calculated once every 12 mo. Thus, for every year, 2 samples were collected from each area on the farm. For ease of analysis, environmental culture results were aggregated by calendar year and animal location (pre-weaned calf, weaned heifer, lactating cow, maternity, and manure storage areas). Using within herd JD prevalence as the outcome of interest, its association with time (study year) and the number of positive environmental samples was assessed using linear regression, controlling for repeated measures within herds using generalized estimating equations (GEE) using an exchangeable correlation structure. The regression model was built starting with univariable analysis for study year and the total number of positive environmental samples each year. To determine if within herd prevalence was associated with MAP contamination in specific areas on the farm, similar univariable linear regression models were assessed using the number of positive environmental samples in the pre-weaned calf, weaned heifer, lactating cow, maternity, and manure storage areas as the independent variables, respectively. Area-specific variables with a P-value of > 0.15 on univariable analysis were then considered in a multivariable linear regression model using step-wise backward selection. The final multivariable model consisted of only those variables with a P-value of < 0.05.

Model fit for all respective regression models was assessed using an extension of cumulative residuals as discussed in Lin et al (28). The cumulative sums of the residuals for each independent variable in the respective regression models were plotted, along with the residuals of 10 000 simulated realizations from a zero-mean Gaussian distribution. The Kolmogorov-type supremum test was calculated along with its associated P-value. This process was repeated with alternative functional forms of the variable based on the initial pattern of the cumulative sums of residuals in an attempt to improve model fit when warranted. The greater the Kolmogorov-type test statisitic and its P-value, the better the model fits the data, and P-values < 0.05 were considered indicative of poor, or insufficient, model fit.

All statistical analyses were performed using commercially available software (Proc Genmod, SAS 9.1, SAS Institute, Cary, North Carolina, USA).

Results

Herd prevalence

Initial apparent JD prevalence based on whole herd fecal culture in the study herds ranged from 2% to 11%. Over the 4-year course of this study, apparent JD prevalence within these herds ranged from 0% to 42%. In 1 herd, the prevalence increased dramatically, from 7% to 42% in the 2nd year of the study then gradually declined to 12% by year 4. This occurred despite the herd having been closed for over 20 y and herd size remaining constant. Apparent prevalence within the herd that purchased cattle to double herd size increased slightly (9% to 11%) over the study period. Johne’s disease prevalence in the other 5 herds tended to decrease or plateau between years 3 and 4 of this study.

Environmental cultures

A total of 731 environmental samples were collected with 81 (11%) culturing positive for MAP. Culture results by location are summarized in Table 1. Over the 4-year course of the study, positive environmental samples were identified on 6 of the 7 farms. The 1 farm with no positive environmental samples had extremely low fecal culture prevalence, ranging from 0% to 2%.

Table 1.

Distribution of Mycobacterium avium subsp. paratuberculosis (MAP) in the environment of 7 Michigan dairy farms

Location	Number of samples	Number positive	Location (%)	Total (%)
Calf feed	51	2	3.9	2.5
Calf floor	57	4	7.0	4.9
Calf water	49	0	0.0	0.0
Heifer feed	50	0	0.0	0.0
Heifer floor	53	3	5.7	3.7
Heifer water	52	0	0.0	0.0
Maternity feed	52	0	0.0	0.0
Maternity floor	56	8	14.3	10.0
Maternity water	54	5	9.3	6.2
Lactating cow feed	52	2	3.8	2.5
Lactating cow floor	54	24	44.4	30.0
Lactating cow water	53	2	3.8	2.5
Lagoon/manure spreader	53	27	50.9	33.3
Recycled sand	5	4	80.0	4.9
Other	40	0	0.0	0
Total	731	81	11.1	100.0

The areas most commonly contaminated were the lactating cow floor and the manure storage areas, representing 30% and 33% of the positive samples, respectively. One or both of these areas was positive on 75% of the environmental collection dates. Both of these areas were positive at least once in the 6 herds with positive environmental samples, and often multiple times, on different sampling dates.

Ten percent of the positive environmental samples came from the maternity floor and 6% from maternity water samples. The maternity area was positive for MAP at least once in 4 of the 6 herds. Fecal culture prevalence in those herds at the time the maternity area was positive ranged from 5.4% to 42%.

The pre-weaned calf area was contaminated in 3 of the 6 herds. Apparent prevalence of MAP shedding in those herds at the time ranged from 8.6% to 17%. On one of the farms, the calves were housed in a group pen across an alley from a contaminated maternity pen, with the potential for cross-contamination. On the other 2 farms, the calves were housed in separate barns, well away from any possible contamination or run-off from adult cattle.

Recycled sand bedding represented 5% of the positive environmental samples; however, these samples came from only 1 farm with fecal culture prevalence ranging from 12% to 42% at the time the samples were collected.

Most of the environmental samples contaminated with MAP originated from flooring or manure storage (n = 70) as compared to feed (n = 4), or water (n = 7). Two of the positive feed samples came from the calf area adjacent to a contaminated maternity pen on a farm when within herd JD prevalence was 14%. The other 2 positive feed samples came from fence-line feed alleys in free stall barns housing lactating cows. All of the MAP positive water samples originated from adult cow areas, with 5 occurring in the maternity area and 2 in the lactating cow area.

When compiled, the number of positive environmental samples decreased as herd prevalence decreased (Figure 1). Once herd prevalence fell to < 2%, MAP was never cultured in the environment of any area sampled. When herd prevalence was > 2%, MAP was cultured from the lactating cow floor and/or manure storage areas 75% of the time. All the positive samples in the 2% to 5% herd prevalence category originated from either the lactating cow floor or manure storage areas. When herd prevalence exceeded 5%, MAP began to be isolated from areas in addition to the lactating cow floor or manure storage areas, with the most common area being the maternity floor. Within individual herds, the trend for decreasing MAP environmental contamination (based on the percent of culture positive environmental samples) with decreasing within herd JD prevalence was not always as obvious (Table 2).

Figure 1.

Percentage Mycobacterium avium subsp. paratuberculosis (MAP) positive environmental samples by within herd Johne’s disease prevalence.

Table 2.

Percent of MAP culture positive cows and environmental samples by herd over time (includes all environmental samples collected)

	2003a		2004		2005		2006
Herd	Cows (%)	Environment (%)	Cows (%)	Environment (%)	Cows (%)	Environment (%)	Cows (%)	Environment (%)
1	10.3	0	14a	24.1a	20.3	18.4	4.4	6.5
2	10.2	6.7	4.1	3.6	2.9	7.4	1.9	11.5
3	8.6	14.3	5.4	17.9	10.6	21.4	11	11.5
4	10.6	0	6.4a	14.8a	2	0	4	7.1
5	NT	NT	5.3	19.2	5	7.7	6.3	19.2
6	1.8	0	0.6	0	0.6	0	2	0
7	7	6.7	42.1	17.2	16.9	28.6	12.1	23.8

^aIndicates samples cultured on Herrold’s Egg Yolk (HEY).

NT — Herd not tested.

Over the course of this study, the JD prevalence within each herd changed, and the herds moved up and down across the prevalence categories outlined in Figure 1. For example, the < 2% category represents data from 3 herds; the 2% to 5% category, 5 herds; the 6% to 15% category, 6 herds; and the > 15% category, 3 herds.

Statistical data analysis

The results of univariable linear regression models to assess the association between herd prevalence over time and the number of positive environmental samples overall and in each area are summarized in Table 3. The results of the final multivariable linear regression model assessing the association of within herd JD prevalence with MAP environmental contamination in specific areas of the farm are shown in Table 4.

Table 3.

Univariable linear regression analysis using within herd Johne’s disease prevalence as outcome

		95% confidence limit
Model	Estimate	Lower	Upper	P-value	Kolmogorov-type supremum test P-value
Study year	−0.54	−1.43	0.35	0.2374	0.38
Number positive environmental samples — total	1.62	0.82	2.42	< 0.0001	0.53
Number positive environmental samples — pre-weaned calf area	3.01	1.41	4.60	0.0002	0.30
Number positive environmental samples — weaned heifer area	6.01	1.16	10.85	0.0152	0.32
Number positive environmental samples — lactating cow area	1.03	−0.82	2.89	0.2737	0.11
Number positive environmental samples — maternity area	6.24	0.77	11.72	0.0254	0.29
Number positive environmental samples — manure storage area	1.41	−0.0012	2.83	0.0502	0.21

Table 4.

Final multivariable linear regression model using within herd Johne’s disease prevalence as the outcome

		95% confidence limits
Variable	Estimate	Lower	Upper	P-value	Kolmogorov-type supremum test P-value
Number positive environmental samples — pre-weaned calf area	5.12	3.58	6.65	< 0.0001	0.48
Number positive environmental samples — weaned heifer area	6.19	2.52	9.87	0.0010	0.42
Number positive environmental samples — maternity area	5.68	2.10	9.25	0.0018	0.20

The regression estimate for the association between within herd JD prevalence and study year was negative, suggesting that the prevalence in these herds declined over time, even though that decline was not statistically significant. Regardless, there was a significant association between decreasing JD herd prevalence and number of positive environmental samples. For every 1 unit increase in the number of annual positive environmental samples, the within herd JD prevalence increased 1.62%. (P < 0.0001). When contamination within specific areas of the farm were assessed, for every 1 unit increase in the number of positive environmental samples in the pre-weaned calf, weaned calf, and maternity areas, within herd JD prevalence increased by 5.12%, 6.19%, and 5.68%, respectively. Environmental contamination in the lactating cow and manure storage areas was not statistically associated with increasing JD prevalence because these were the areas that were consistently contaminated on the farms, even when within herd prevalence was very low.

Discussion

The strength of this study is its longitudinal nature, such that changes in the distribution of environmental MAP contamination could be monitored as within herd JD prevalence changed on infected dairy farms following the implementation of on-farm JD control programs. Mycobacterium avium subsp. paratuberculosis was cultured consistently (75% of the time) over time in the manure storage area and/or the lactating cow floor when within herd culture prevalence was > 2%; indicating a consistent reservoir of MAP contamination, even when relatively few cows in the herd were actively shedding. However, once the number of cows shedding the bacterium in the herd fell to < 2%, MAP was not cultured from any location sampled. Logically, the fewer cows shedding MAP, the less contamination there is in the environment; and the less likely it is for an environmental sample to contain MAP at a level detectable by current culture methods. It is also possible manure management and sanitation practices implemented by the herds for JD control purposes resulted in less manure accumulation, thereby decreasing the potential for MAP environmental contamination. The fact MAP was never cultured from environmental samples of 1 herd that consistently had low within herd JD prevalence (< 2%) does not mean that the environment for this herd was not contaminated with MAP. More likely the level of MAP contamination was minimal and below the detection threshold of the sampling protocol.

Herd prevalence had to increase only slightly to 5% before MAP was cultured in areas in addition to the lactating cow floor and manure storage areas, with the most common area being the maternity floor. This is not surprising, as this is an area populated with adult cows. From a JD control standpoint, it is concerning because calves, the animals most susceptible to becoming infected with MAP, are being born in those areas. This emphasizes the importance of maternity pen management in any JD control program.

A surprising finding in this study was the positive environmental samples in the pre-weaned calf area on 3 farms. While it is possible to explain cross-contamination from a contaminated maternity pen across an alley in the same barn on one farm; the other 2 farms with positive calf areas had separate calf barns, located well away from adult cattle. It is possible these areas became contaminated through farm personnel or feeding/cleaning equipment traveling between cow and calf barns. The other possibility is that some of the calves on these farms were shedding MAP. While it has been traditionally thought newly infected cattle do not start shedding MAP for several months, or until adulthood; recent reports suggest calves may indeed shed MAP, albeit transiently, and typically, at low levels (28–29). Regardless, the finding of MAP in the pre-weaned calf area should be considered a risk for infection to the calves housed there, and appropriate precautions should be taken.

Isolating MAP in 4 of 5 samples of recycled sand bedding (although originating from only 1 farm) was also an interesting finding, and raises the issue of where that bedding should be used. If the traditional JD paradigm that cattle become less susceptible to infection with age is accepted, using this sand to bed the adult herd likely represents minimal risk for spreading the infection. However, care should be taken to ensure it is not used in calf, young heifer, or maternity pen areas.

Our findings were similar to those reported in previous studies (22–24), in that the areas most commonly contaminated with MAP on infected dairy farms were those with the greatest concentration of manure (lactating cow floor and manure storage areas) from adult cows, the animals at greatest risk of shedding the bacterium. Also, as in previous studies (22,24,26), there was an overall tendency for the amount of MAP in the environment to increase as within herd JD prevalence increased (Tables 3 and 4, Figure 1). The difference between this study and those referenced was that this study was longitudinal while the others were cross-sectional. The significance is that these findings were consistent over time in the face of increasing and decreasing JD prevalence within the same herds. This adds strength to the importance of environmental contamination as a source of MAP transmission to susceptible cattle.

At the individual herd level, there was not an obvious consistent downward trend in MAP environmental contamination as within herd JD prevalence decreased in all herds (Table 2). Factors to consider are: the potential presence of 1 or 2 “super-shedders” in the herd, the relatively long time MAP survives in the environment and the diligence each herd gave to sanitation. The “super-shedder” phenomenon in regards to MAP infection is a recently introduced theory in which 1 infected cow sheds billions of bacteria into the environment each day (30). Thus, 1 or 2 “super-shedders” in a herd could disproportionately contaminate the environment, resulting in a high environmental load of MAP when compared with the absolute number of shedders in the herd. The prolonged survivability of MAP in the environment may result in a lag period following the removal of cows actively shedding MAP during which the environmental load of the bacteria in the herd’s environment is maintained. The environmental load does not decrease to undetectable levels until the MAP finally dies or is physically removed. As in the general population, cleaning and manure removal were done more effectively in some herds than in others. Subjectively, the herds that did not follow the expected pattern of decreasing environmental MAP contamination in conjunction with decreasing within herd JD prevalence, were the ones that were less diligent in cleaning. Regardless, as long as MAP is detectable in the environment, susceptible cattle in the herd are at risk of becoming infected.

In contrast, linear regression analysis demonstrated a statistically significant association between within herd JD prevalence and the number of positive environmental samples. As the number of contaminated environmental samples increased, so did within herd prevalence. Also, the culturing of MAP from areas other than the lactating cow and manure storage areas was likewise associated with increasing herd prevalence. The reason a similar association was not found in the lactating cow and manure storage areas is likely due to the fact that these were the areas most consistently contaminated over time, even when prevalence was relatively low.

Potential factors influencing the results of this study were sample collection and culture system. An attempt was made to collect representative environmental samples from the same areas on each farm throughout the course of the study. All the study herds were infected with MAP and it is possible any given area may have been contaminated with MAP, but the sample collected either did not contain the bacteria, or did not contain enough of it to be detectable by the culture methods used. Therefore, due to sampling error, the MAP contamination on these dairy herds is likely to be more extensive than reported.

During the course of this study, the laboratory switched from solid culture on HEY medium to the ESP II liquid culture system. Subsequently, the number of positive environmental cultures increased, while JD prevalence on most of the study herds was on a downward trend. The most likely explanation for this is that the ESP II culture system is more sensitive than HEY and was able to detect MAP at lower levels (31).

Quantitative analysis of the level of MAP contamination in environmental samples over time, in conjunction with changing within herd JD prevalence would have strengthened this study. A high volume of contamination in the environment is associated with increased infection pressure, and subsequently higher within herd prevalence (7). In a cross-sectional study, Raizman et al (22), reported a positive correlation between the volume of MAP isolated from environmental samples and herd prevalence. Unfortunately, the laboratory reporting of quantitative culture results was inconsistent over the course of this study, precluding such analysis. The lack of quantitative analysis of MAP environmental contamination on these herds over time is a limitation of this study, and something that remains to be pursued in the future.

In this study, when herd fecal culture prevalence was > 2%, MAP was isolated from the lactating cow floor and/or manure storage area 75% of the time. If samples cultured using only the ESP II culture system are considered, the lactating cow floor and/or the manure storage area was positive 81% of the time. Thus, culturing these 2 areas was a sensitive method for determining the presence of, and, to a lesser degree, the extent of MAP in a dairy herd. This protocol could be adapted to monitor the progress of JD control programs at the individual herd or regional level. Periodically culturing these areas on an individual farm could provide some indication whether or not the herd’s JD control program is working. As within herd JD prevalence declines, eventually the lactating cow area and manure storage should consistently culture negative for MAP. At the state, regional, or national level, culturing the lactating cow and manure storage areas could provide an economical and efficient method for determining the number of dairy herds infected with JD, which in turn, could help direct the allocation of JD control resources.

In conclusion, MAP was widely distributed in the environment of the Michigan dairy farms participating in this study. The lactating cow floor and manure storage areas (areas with the greatest concentration of manure from the greatest number of adult cows), were the locations that most commonly cultured positive for MAP. Periodic targeted sampling of these areas may provide an efficient and economic tool for monitoring the progress of JD control programs at the individual herd and regional levels. An increasing number of MAP positive environmental cultures was associated with increasing within herd JD prevalence. It was not uncommon for MAP to be cultured from the maternity and pre-weaned calf areas, areas where there is a high risk for transmitting JD to the next generation of herd replacements. This underscores the need to emphasize cleanliness in these areas when recommending JD control programs. Finally, as long as MAP is present in the herd environment, susceptible cattle are at risk of becoming infected with JD. Thus, when using testing as part of a JD control program, targeted testing of the environment may be as important as testing individual cattle.