On-farm environmental sampling is an effective method for herd-level diagnosis of Mycobacterium avium ssp. paratuberculosis (MAP) infection and between-herd prevalence estimation. So far, no prevalence study enrolling important livestock-farming regions has been conducted. As the structure of dairy farming differs between main livestock-farming regions in Germany, our objective was to assess the between-herd prevalence of paratuberculosis for these regions in a standardized approach. Methods: In total, 457 randomly selected dairy farms from three regions of Germany (North: 183, East: 170, South: 104) were sampled between 2017 and 2019. Environmental samples (boot-swabs, aggregate feces and/or liquid manure samples) were cultured and analyzed using an IS900-qPCR for MAP determination. Of the 457 selected farms, 94 had at least one MAP-positive environmental sample with significant differences between regions regarding the apparent (North: 12.0%, East: 40.6%, South: 2.9%) or corrected true (North: 14.8%, East: 50.1%, South: 3.6%) between-herd prevalence. In conclusion, regional differences of between-herd prevalence of paratuberculosis are substantial in Germany, indicating the need for control approaches with different aims. Taking into account regional MAP prevalence, MAP-control programs should focus on on-farm prevalence reduction or on mitigating the risk of between-herd transmission, depending on region.

Herd-level diagnosis of paratuberculosis using a poolmilk ELISA (pool size: n ≤ 50) is a novel, economical, and convenient method to identify blood serological Mycobacterium avium ssp. paratuberculosis (MAP) antibody-positive herds. To date, the diagnostic performance of the pool-milk ELISA has been described only under laboratory conditions where herd prevalence was simulated by the preparation of milk pools consisting of milk samples of cows with a known MAP status determined by fecal culture. In our observational study, test performance under field conditions was studied using pooled milk and individual blood samples. A total of 486 herds within the MAP prevalence reduction program of Lower Saxony, from which pooled milk and individual blood ELISA results were available, were assigned to this study. Data were analyzed for the period between January 1 and December 31, 2018, the first year after herd testing became obligatory in this federal state of Germany. To evaluate whether pooled milk samples reliably distinguish between herds with a MAP-apparent blood serological within-herd prevalence (MAP-Ab-WHP app ) ≥5% and herds with a MAP-Ab-WHP app <5%, the distribution of the MAP-Ab-WHP app was compared between pool-positive and pool-negative herds. The MAP-Ab-WHP app was 3.4% (median; 95% confidence interval = 0-11.4%) in pool-positive herds and 1.2% (median; 95% confidence interval = 0-6.4%) in pool-negative herds. Only 10.8% (n = 12) of the pool sample-negative herds had a MAP-Ab-WHP app ≥5% and were therefore false negatives, given the aims of the MAP prevalence reduction program. Hence, the pool-milk sampling strategy seems well suited to dis-tinguish between herds with a MAP-Ab-WHP app ≥ 5% and herds with a MAP-Ab-WHP app <5% since only 10% of serum MAP-ELISA positive herds were missed. Employing a logistic regression model, we estimated that the minimum blood serological MAP-Ab-WHP app to detect a pool-positive herd with a probability of 95% was 8%, which fits well with the aim of the MAP prevalence reduction program to focus on herds with a MAP-Ab-WHP app of ≥5%. Despite the limitations of the control approach, which include milk pool sample collection and a low sensitivity of the ELISA used in milk pools and serum samples, the aims of the MAP prevalence reduction program can be achieved. The results of these field data support that pool-milk sample ELISA is a useful, economical, and low labor-intensive tool to identify herds seropositive for MAP in a MAP prevalence reduction program.

In a cross-sectional study, it was identified that three regions in Germany differed with respect to their herd-level prevalence for paratuberculosis in dairy cattle. In the study presented here, the same farms were analyzed to identify those components of biosecurity and farm management with the highest impact on Mycobacterium avium ssp. paratuberculosis (MAP) introduction and establishment in a farm. Hence, the data analyzes included 183, 170 and 104 herds from the study regions north, east and south, respectively. A herd was considered MAP-positive if at least one fecal environmental sample was positive. Twenty-six different possible risk factors from five different components of biosecurity and farm management were analyzed. We show that the average management of calf feeding increased the odds for a MAP-positive farm by 5.22 times (95% confidence interval (CI) = 1.25–21.83). With every 100-cow increase in farm size, the risk for a farm to test MAP-positive increased by 1.94 times (CI = 1.15–3.27), 1.14 times (CI = 1.02–1.27) and 5.53 times (CI = 0.44–68.97) in the north, east and south study regions, respectively. Furthermore, the purchase of cattle with an unknown MAP status increased the risk for a farm testing MAP-positive by 2.86-fold (CI = 1.45–5.67). Our results demonstrate that herd size, unknown MAP status of the purchased cattle and different aspects of calf feeding play an important role in the MAP status of a farm and should be in focus in regions with different MAP between-herd prevalence. Additionally, farm individual risk patterns should be identified during (veterinary) biosecurity consultancy.