Longitudinal study on the effects of intramammary infection with non-aureus staphylococci on udder health and milk production in dairy heifers.

We conducted a longitudinal study to evaluate the effect of non-aureus staphylococci (NAS) causing subclinical intramammary infections (IMI) on quarter milk somatic cell count (qSCC) and quarter milk yield (qMY). In total, 324 quarters of 82 Holstein Friesian heifers were followed from calving to 130 d in milk (DIM) and were sampled 10 times each at 14-d intervals. The IMI status of each quarter was determined based on bacterial culture results at the current and previous or next sampling day, or both. The qSCC was determined on each sampling day and the average qMY on sampling day was available through stored daily milk weight data in the management program of the automatic milking system. A transient IMI (tIMI) was defined as a case where a specific pathogen was isolated from a quarter on only one sampling day and not on the previous or next sampling day. When the same bacterial strain, as defined by random amplification of polymorphic DNA-PCR, was isolated from the same quarter on multiple sampling days, it was defined as a persistent IMI (pIMI) status on those sampling days; a pIMI episode was defined as the combination of multiple consecutive pIMI statuses with the same bacterial strain on different sampling days. During this study, 142 subclinical IMI with NAS occurred in 116 different quarters from 64 animals, yielding in total 304 NAS isolates belonging to 17 different species. The prevalence of NAS was highest in the first 4 DIM. Overall, the predominant species was Staphylococcus chromogenes (52% of the isolates), followed by S. epidermidis (9.2%), S. xylosus (8.2%), and S. equorum (5.9%). Staphylococcus chromogenes was the only species for which an effect on qSCC and qMY could be analyzed separately; the other NAS species were considered as a group because of their low prevalence. Eighteen out of 40 IMI (45%) caused by S. chromogenes persisted over at least 2 sampling days, whereas only 10 of 102 (9.8%) IMI caused by other NAS species persisted for at least 2 sampling days. The average duration of pIMI episodes was 110.4 d for S. chromogenes and 70 d for the other NAS species. Remarkably, 17 of the 18 pIMI episodes with S. chromogenes started within the first 18 DIM. The qSCC was highest in quarters having a pIMI with a major pathogen, followed by quarters having a pIMI with S. chromogenes, and a pIMI with other NAS. Transient IMI with other NAS or with a major pathogen caused a small but significantly higher qSCC, whereas the qSCC in quarters having a tIMI with S. chromogenes was not statistically different compared with noninfected quarters. No significant differences in qMY were observed between quarters having a pIMI or tIMI with S. chromogenes or with the other NAS species compared with noninfected quarters, despite the higher qSCC. Quarters having a pIMI with major pathogens showed significantly lower daily milk production. Surprisingly, quarters that cured from an IMI with S. chromogenes had a significantly lower qMY than noninfected quarters.

Incidence of milk leakage after dry-off in European dairy herds, related risk factors, and its role in new intramammary infections.

The incidence of milk leakage (ML) after dry-off (DO) and related risk factors was studied in 1,175 dairy cows from 41 commercial herds in 8 European countries: Belgium, Czech Republic, Denmark, France, Germany, Italy, the Netherlands, and Spain. Milk leakage was assessed twice for 30 s each during 3 visits at 20 to 24 h, 30 to 34 h, and 48 to 52 h after DO. Information related to dry-cow management and udder health was collected at herd and cow level, including individual somatic cell count (ISCC) from test-day controls and occurrence of clinical mastitis cases from DO until 30 d in lactation. Mixed-effect logistic regression analyses were used to identify possible risk factors for ML and to study the association between ML and new intramammary infections. Intramammary infections were defined as clinical mastitis cases during the dry period and in the first 30 d in lactation or a rise in ISCC from before to after the dry period (threshold: 200,000 cells/mL) or both. Milk leakage was observed in 24.5% of the cows between 20 and 52 h after DO, where the herd incidence varied between 0.0 and 77.8%. The reduction in number of milkings in the weeks before DO had statistically significant effect on the ML incidence. When the milking frequency was reduced from 3 times/d to 2 or maintained at twice a day, cows had 11 (95% CI = 3.43-35.46) or 9 (95% CI = 1.85-48.22) times higher odds of leaking milk, respectively, compared with cows where the milking frequency was reduced from twice to once a day. Also, the milk production 24 h before DO was associated with ML incidence. Hence, cows with a milk production between 13 and 21 L or above 21 L had 2.3 (95% CI = 1.48-3.53) and 3.1 (95% CI = 1.79-5.3) times higher odds of leaking milk, respectively, compared with cows with a milk production below 13 L. A higher ML incidence was present in the group of cows with an average ISCC in the last 3 mo before DO ≥200,000 cells/mL (odds ratio = 1.7; 95% CI = 1.13-2.41) compared with cows with an average ISCC <100,000 cells/mL. Quarters with ML tended to have 2.0 times higher odds of developing clinical mastitis compared with quarters not leaking milk. Cows with ML tended to have 1.5 times higher odds of intramammary infections (i.e., an increase of ISCC or clinical mastitis) compared with cows without ML.

Effect of bismuth subnitrate on in vitro growth of major mastitis pathogens.

The mode of action of bismuth subnitrate in teat sealant formulations as a preventative for intramammary infections during the dry period is unknown. Although previous studies proposed an action mechanism-creating a physical barrier in the teat canal to prevent bacterial invasion-it has not been proven experimentally. We hypothesized that bismuth subnitrate has an inhibitory effect on bacterial growth, in addition to its barrier effect. The objective of this study was to assess the effect of bismuth subnitrate on bacterial growth of major mastitis-causing agents. A strain of Streptococcus uberis (SR115), 2 strains of Staphylococcus aureus (SA3971/59 and SA1), and a strain of Escherichia coli (P17.14291) were tested in vitro for their ability to grow in the presence or absence of bismuth subnitrate. Disk diffusion testing, impedance measurement, and evaluation of bacterial growth in shaking conditions were the methods used to test this hypothesis. A reduction of growth in the presence of bismuth subnitrate occurred for all the strains tested. However, we observed strain and species variations in the extent of growth inhibition. These results suggest that an inhibitory effect on bacterial growth by bismuth subnitrate could partially explain the efficacy of bismuth-based formulations for preventing intramammary infections over the dry period. Further research is required to test the effect of teat sealant formulations on bacterial growth.

Molecular epidemiology of Streptococcus uberis intramammary infections: Persistent and transient patterns of infection in a dairy herd.

A longitudinal observational study was carried out to explore transmission dynamics and duration of infection of Streptococcus uberis. Quarter milk samples were collected aseptically for bacterial culture from all lactating cows once a month over a 10-mo period. Molecular typing of S. uberis mastitis was performed using pulsed-field gel electrophoresis (PFGE). Molecular typing was used to determine episodes of S. uberis intramammary infection (IMI). Comparisons of spontaneous cure among PFGE types were performed using Fisher's exact chi-squared tests. Differences of duration among PFGE types and between periods of lactation were tested with Kaplan-Meier survival curves and Cox's proportional hazard model. Among a total of 851 quarter samples, 145 milk samples were detected with S. uberis presence. Based on results of PFGE, 66 episodes of S. uberis IMI were determined. From the 8 main PFGE types (A-H), PFGE type D, E, F1, F2, G, and H had only one episode indicating no evidence for transmission, subsequently defined as environmental S. uberis strains. In contrast, PFGE types A1, A2, B, C1, and C2 had at least 2 infection episodes caused by the same strain in different quarters or cows, indicating that these strains would be able to transmit to other quarters or cows. These strains were defined as contagious strains. The majority of IMI were attributable to PFGE type A1 (55%), B (17%), and A2 (11%). Spontaneous cures were observed in 35 IMI episodes. Of these 35 IMI cures, 91.4% were in IMI with duration of infection of 1 mo, n = 25, and 2 mo, n = 6. The remaining 8.6% was in IMI with duration of infection >2 mo, n = 4. Based on results from Cox's proportional hazard model, environmental S. uberis episodes were likely to have spontaneous cure with shorter duration compared with contagious S. uberis with PFGE type B (hazard ratio = 8.4). Quarters infected with S. uberis strain PFGE type A in early lactation were more likely to persist compared with those infected in late lactation (hazard ratio = 7.57). In conclusion, the majority of S. uberis IMI in this herd were transient and showed spontaneous cure. In addition to environmental S. uberis IMI, at least 3 types of contagious IMI S. uberis can be defined as (1) short duration of IMI and likely to have spontaneous cure, (2) long duration and unlikely to have spontaneous cure, and (3) wide range of duration of IMI either transient or persistent where spontaneous cure may occur depending on host defense capacity.

Efficacy of cabergoline in a double-blind randomized clinical trial on milk leakage reduction at drying-off and new intramammary infections across the dry period and postcalving.

The abrupt cessation of milking at dry-off may induce milk leakage, which may increase the risk of new intramammary infections (IMI). This study assessed the efficacy of 1 i.m. injection of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France) at drying-off on milk leakage after dry-off and new IMI across the dry period and postcalving compared with a placebo (negative control) and an intramammary antibiotic treatment (positive control) under field conditions. The study was a double-blind, randomized, 3-arm, multicenter, clinical trial performed under Good Clinical Practice conditions. Data from 900 dairy cows of various breeds from 63 farms in France, Germany, and Hungary were analyzed. Only quarters with no bacterial growth at drying-off and a cow somatic cell count ≤200,000 cells/mL were included. Quarters infected with major or minor pathogens or cows with high somatic cell count at time of inclusion were excluded. Cows that qualified for the study were visited 7 times in total before and after drying-off and after calving. Presence (yes/no) of milk leakage was recorded on the day after dry-off. A new infected quarter (new IMI) was defined as one with a major pathogen present in any one of the 2 postcalving samples. Two mixed logistic regression models were fitted to the data to evaluate the efficacy of cabergoline in the reduction of milk leakage and new IMI. One i.m. injection of cabergoline at drying-off significantly reduced the incidence of milk leakage the day after dry-off compared with both placebo and antibiotic treatment. Cabergoline-treated cows significantly reduced the risk of new IMI by major pathogens across the dry period and postcalving by 21% when compared with placebo cows (20.5 vs. 26.0%, respectively). However, when milk leakage was added to the model, the significance of cabergoline was reduced. We interpreted this to show that milk leakage is an intervening variable between treatment with cabergoline and lower risk of new IMI. The antibiotic treatment significantly decreased the odds of new IMI compared with both cabergoline and placebo. However, because several countries are currently disallowing the preventive use of antibiotics at dry-off in noninfected quarters, the dry-off facilitator cabergoline may therefore be of particular value to reduce the risk of new IMI across the dry period.

Quantifying calf mortality on dairy farms: Challenges and solutions.

In the Netherlands, the mortality rate of ear-tagged calves <1 yr is one of the indicators that is continuously monitored in census data and is defined as the number of deceased calves relative to the number of calf-days-at-risk. In 2017, yearly calf mortality rates were published in the lay press and resulted in discussions about the calculation of this parameter among stakeholders because the same parameter appeared to be calculated in many different ways by different organizations. These diverse definitions of calf mortality answered different aims such as early detection of deviations, monitoring trends, or providing insight into herd-specific results, but were difficult to understand by stakeholders. The aim of this study was to evaluate several definitions of calf mortality for scientific validity, usefulness for policymakers, and comprehensibility by farmers. Based on expert consultations, 10 definitions for calf mortality were evaluated that assessed different age categories, time periods, and denominators. Differences in definitions appeared to have a large effect on the magnitude of mortality. For example, with the original mortality parameter, the mortality rate was 16.5% per year. When the first year of life was subdivided into 3 age categories, the mortality rate was 3.3, 4.5, and 3.1% for postnatal calves (≤14 d), preweaned calves (15-55 d), and weaned calves (56 d-1 yr), respectively. Although it was logical that these mortality rates were lower than the original, the sum of the 3 separate mortality rates was also lower than the original mortality rate. The reason was that the number of calves present in a herd and the risk of mortality are not randomly distributed over a calf's first year of life and the conditional nature of mortality rates when calculated for different age categories. Ultimately, 4 parameters to monitor calf mortality in Dutch dairy herds were chosen based on scientific value, usefulness for monitoring of trends, and comprehensibility by farmers: perinatal calf mortality risk (i.e., mortality before, during, or shortly after the moment of birth up to the moment of ear-tagging), postnatal calf mortality risk (≤14 d), preweaned calf mortality rate (15-55 d), and weaned calf mortality rate (56 d-1 yr). Slight differences in definitions of parameters can have a major effect on results, and many factors have to be taken into account when defining an important health indicator such as mortality. Our evaluation resulted in a more thorough understanding of the definitions of the selected parameters and agreement by the stakeholders to use these key indicators to monitor calf mortality.

Does clinical mastitis in the first 100 days of lactation 1 predict increased mastitis occurrence and shorter herd life in dairy cows?

The objectives of this study were to estimate the direct effects of clinical mastitis (CM) occurring in early productive life (defined as the first 100 d of the first lactation) of Holstein dairy cows on the future rate of CM occurrence and on the length of total productive lifetime. Information on CM cases and other data occurring in 55,144 lactations in 24,831 cows in 5 New York State Holstein herds was collected from January 2004 until February 2014. For the first objective, a generalized linear mixed model with a Poisson distribution was used to study the effects of CM cases occurring in the first 100 d of a cow's first lactation, as well as farm indicator and number of days in the cow's lifetime, on the future lifetime rate of CM. Only cows that had completed their productive life [i.e., all had been culled (or sold) or had died; n = 14,440 cows] were included in this analysis. For the second objective, a Cox proportional hazards model was used to study the effects of CM cases occurring in the first 100 d of a cow's first lactation on the length of total productive lifetime. The model was stratified by farm. All 24,831 cows were included in this analysis with right censoring. Cows experienced between 0 and 4 CM cases in the first 100 d of lactation 1. Over their lifetime, cows experienced between 0 and 25 CM cases. During the study period, 10% of all cows died and nearly half of all cows were culled. The average length of productive life, including censored observations, was 2.0 yr after first calving. Compared with cows having no CM cases in the first 100 d of lactation 1, cows with 1 CM case in that time period had a 1.5 times higher rate of total number of CM cases over their lifetime. Cows with 2 (or 3 or more) CM cases in the first 100 d of lactation 1 had a 1.7 times (or 2.6 times) higher rate of total number of CM cases over their lifetime. For each additional CM case occurring in the first 100 d of lactation 1, the hazard rate of culling increased by 34%. Given economic conditions for preferentially culling mastitic cows, the study findings may help farmers make optimal decisions with regard to culling of such cows.

Relationship between intramammary infection prevalence and somatic cell score in commercial dairy herds.

We examined consistency of the relationship between intramammary infection (IMI) and somatic cell score (SCS) across several classes of cow, herd, and sampling time variables. Microbial cultures of composite milk samples were performed by New York Quality Milk Production Services from 1992 to 2004. SCS was from the most recent Dairy Herd Improvement test before IMI sampling. Records were analyzed from 79,308 cows in 1,124 commercial dairy herds representing a broad range of production systems. Three binary dependent variables were presence or absence of contagious IMI, environmental IMI, and all IMI. Independent variables in the initial models were SCS, SCS2, lactation number, days in milk, sample day milk yield, use of coliform mastitis vaccine, participant type (required by regulation or voluntary), production system (type of housing, milking system, and herd size), season of sampling, year of sampling, and herd; also the initial models included interactions of SCS and SCS2 with other independent variables, except herd and milk yield. Interaction terms characterize differences in the IMI-SCS relationship across classes of the independent variables. Models were derived using the Glimmix macro in SAS (SAS Institute Inc., Cary, NC) with a logistic link function and employing backward elimination. The final model for each dependent variable included all significant independent variables and interactions. Simplified models omitted SCS2 and all interactions with SCS. Interactions of SCS with days in milk, use of coliform mastitis vaccine, participant type, season, and year were not significant in any of the models. Interaction of SCS with production system was significant for the all IMI model, whereas interaction of SCS with lactation number was significant for the environmental and all IMI models. Each 1-point increase in SCS (or doubling of somatic cell count) was associated with a 2.3, 5.5, and 9.1% increase in prevalence of contagious, environmental, and all IMI, respectively. Empirical receiver operator characteristic curves and areas under the curve were derived for final and simplified models. The areas under the curve for simplified and final models within each type of IMI differed by 0.009 or less. We concluded that the relationship of IMI with SCS was generally stable over time and consistent across seasons, production systems, and cow factors.

Longitudinal assessment of dairy farm management practices associated with the presence of psychrotolerant Bacillales spores in bulk tank milk on 10 New York State dairy farms.

The ability of certain spore-forming bacteria in the order Bacillales (e.g., Bacillus spp., Paenibacillus spp.) to survive pasteurization in spore form and grow at refrigeration temperatures results in product spoilage and limits the shelf life of high temperature, short time (HTST)-pasteurized fluid milk. To facilitate development of strategies to minimize contamination of raw milk with psychrotolerant Bacillales spores, we conducted a longitudinal study of 10 New York State dairy farms, which included yearlong monthly assessments of the frequency and levels of bulk tank raw milk psychrotolerant spore contamination, along with administration of questionnaires to identify farm management practices associated with psychrotolerant spore presence over time. Milk samples were first spore pasteurized (80°C for 12 min) and then analyzed for sporeformer counts on the initial day of spore pasteurization (SP), and after refrigerated storage (6°C) for 7, 14, and 21 d after SP. Overall, 41% of samples showed sporeformer counts of >20,000 cfu/mL at d 21, with Bacillus and Paenibacillus spp. being predominant causes of high sporeformer counts. Statistical analyses identified 3 management factors (more frequent cleaning of the bulk tank area, the use of a skid steer to scrape the housing area, and segregating problem cows during milking) that were all associated with lower probabilities of d-21 Bacillales spore detection in SP-treated bulk tank raw milk. Our data emphasize that appropriate on-farm measures to improve overall cleanliness and cow hygiene will reduce the probability of psychrotolerant Bacillales spore contamination of bulk tank raw milk, allowing for consistent production of raw milk with reduced psychrotolerant spore counts, which will facilitate production of HTST-pasteurized milk with extended refrigerated shelf life.

Use of an Individual-based Model to Control Transmission Pathways of Mycobacterium avium Subsp. paratuberculosis Infection in Cattle Herds.

Johne's disease (JD) is a chronic enteric disease in cattle caused by Mycobacterium avian subsp. paratuberculosis (MAP). Eradicating JD is a difficult task due to the long incubation period of MAP, inefficient diagnostic tests, and delayed clinical signs. Effective control strategies can help farmers to reduce prevalence, but those most acceptable to farmers combine specific information about lactation performance and testing results, which existing models do not provide. This paper presents an individual-based model of MAP infection dynamics and assesses the relative performance of the applied alternative control strategies. The base dairy herd model included the daily life events of a dairy cow and reflects several current dairy management processes. We then integrated MAP infection dynamics into the model. The model adopted four different test-based control strategies based on risk-based culling decisions and three hygiene scenarios. The model tracked the source of each infection and quantified the efficacy of each control strategy in reducing the risks of different transmission routes. The results suggest that risk-based culling can reduce prevalence compared with no control, but cannot eliminate the infection. Overall, this work provides not only a valuable tool to investigate MAP transmission dynamics but also offers adaptability to model similar infectious diseases.

The effect of Mycobacterium avium ssp. paratuberculosis infection on clinical mastitis occurrence in dairy cows.

Endemic diseases can be counted among the most serious sources of losses for livestock production. In dairy farms in particular, one of the most common diseases is Johne's disease, caused by Mycobacterium avium ssp. paratuberculosis (MAP). Infection with MAP causes direct costs because it affects milk production, but it has also been suspected to increase the risk of clinical mastitis (CM) among infected animals. This might contribute to further costs for farmers. We asked whether MAP infection represents a risk factor for CM and, in particular, whether CM occurrences were more common in MAP-infected animals. Our results, obtained by survival analysis, suggest that MAP-infected cows had an increased probability of experiencing CM during lactation. These results highlight the need to account for the interplay of infectious diseases and other health conditions in economic and epidemiological modeling. In this case, accounting for MAP-infected cows having an increased CM occurrence might have nonnegligible effects on the estimated benefit of MAP control.

Longitudinal relationship between fecal culture, fecal quantitative PCR, and milk ELISA in Mycobacterium avium ssp. paratuberculosis-infected cows from low-prevalence dairy herds.

Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of ruminant Johne's disease, presents a particular challenge with regard to infection mitigation on dairy farms. Diagnostic testing strategies to identify and quantify MAP and associated antibodies are imperfect, and certain facets of the relationship between diagnostic tests remain to be explored. Additional repeated-measures data from known infected animals are needed to complement the body of cross-sectional research on Johne's disease-testing methods. Statistical models that accurately account for multiple diagnostic results while adjusting for the effects of individual animals and herds over time can provide a more detailed understanding of the interplay between diagnostic outcomes. Further, test results may be considered as continuous wherever possible so as to avoid the information loss associated with dichotomization. To achieve a broader understanding of the relationship between diagnostic tests, we collected a large number of repeated fecal and milk samples from 14 infected cows, in addition to bulk milk samples, from 2 low-prevalence dairy herds in the northeast United States. Predominately through the use of mixed linear modeling, we identified strong associations between milk ELISA optical density, fecal quantitative PCR, and fecal culture in individual animals while concurrently adjusting for variables that could alter these relationships. Notably, we uncovered subtleties in the predictive abilities of fecal shedding level on milk ELISA results, with animals categorized as disease progressors reaching higher ELISA optical density levels. Moreover, we observed that spikes in fecal shedding could predict subsequent high ELISA values up to 2 mo later. We also investigated the presence of MAP in individual milk samples via PCR and noted an association between poor udder hygiene and MAP positivity in milk, suggesting some level of environmental contamination. The paucity of positive milk samples and the complete absence of detectable MAP in the bulk tank throughout the study period indicate that contamination of milk with MAP may not be of chief concern in low-prevalence herds. An enhanced understanding of the interrelationships between diagnostic tests can only benefit the development of testing strategies and objectives, which in turn may lessen MAP infection prevalence in dairy herds.

Comparative risk assessment for new cow-level Mycobacterium avium ssp. paratuberculosis infections between 3 dairy production types: Organic, conventional, and conventional-grazing systems.

Johne's disease, a granulomatous enteritis of ruminant animals, is a hidden threat on dairy farms, adversely affecting animal welfare as well as herd productivity. Control programs in the United States advocate for specific management practices to temper the spread of the causal organism (Mycobacterium avium ssp. paratuberculosis, MAP), such as improving calving area hygiene and limiting introduction of replacement stock with unknown infection status. A need remains for direct exploration of Johne's disease prevention strategies in the United States with respect to production type. Alongside the growing demand for organic products, the safety of organic dairy practices with respect to MAP control is warranted. Further, conventional herds for which organic practices such as pasture grazing are used should be situated within the risk spectrum. We developed a risk assessment model using the US Voluntary Bovine Johne's Disease Control Program as a framework, with the goal of evaluating the risk of new cow-level MAP infections. A total of 292 organic and conventional farms in 3 states were surveyed on management practices, and an overall analysis was conducted in which each farm was first scored on individual practices using a range of "no risk" to "high risk," according to the literature. The sum of all risk factors was then analyzed to quantify and compare the risk burden for each production type. Organic herds received higher overall risk scores compared with both conventional grazing and nongrazing subtypes. To identify which factors contributed to the overall increased risk for organic herds, the management practices were categorized and evaluated by logistic regression. We determined that the increased risk incurred by organic herds was predominantly due to decisions made in the calving area and preweaned calf group. However, although certain individual risk factors related to calf management are commonly involved in prevention strategies (e.g., cow/calf separation) and were thus included in the overall risk assessment, empirical evidence linking them to the spread of MAP is lacking. Instead, these factors are problematic when executed with other management decisions, leading to a hypothesized synergism of transmission risk. To this end, we developed a set of compound risk factors, which were also evaluated as outcomes in logistic regression models, with production type serving as the predictor of interest. Organic farms in our study were more susceptible to risks associated with the synergism of study variables. Notably, organic producers were most likely to allow calves to spend extended time with the dam, while also lacking a dedicated calving area. Additionally, calves in organic herds were more often permitted to nurse even with poor udder hygiene on farm. A heightened vigilance toward calving area hygiene is therefore indicated for these herds.

Intramammary immunization with ultraviolet-killed Escherichia coli shows partial protection against late gestation intramammary challenge with a homologous strain.

The objective of this study was to evaluate the efficacy of intramammary immunization with UV-killed Escherichia coli ECC-Z on prevention of intramammary colonization after a challenge with a dose of the homologous E. coli ECC-Z live bacteria. A total of 10 cows were included in a study to evaluate the efficacy of intramammary immunization. All 10 cows received an intramammary immunization of 100 cfu of UV-killed E. coli ECC-Z bacteria into one hind quarter at the time of dry off. Approximately 2wk before the anticipated calving date, both hind quarters of all cows were challenged with 100 cfu of live E. coli ECC-Z bacteria. Five of the cows were vaccinated parenterally with a commercial J5 bacterin, and 5 cows served as controls with no parenteral vaccination. The cows were then followed over time and infection risk, clinical scores, somatic cell count, and milk production were observed over time. The results of these 10 cows showed partial protection of intramammary immunization on the outcome of a subsequent homologous intramammary challenge. Immunization resulted in a lower probability of infection, a lower bacteria count, lower somatic cell counts and milk conductivity, a lower clinical mastitis score, and increased milk production compared with unimmunized control quarters. Once the analysis was corrected for immunization, parenteral J5 vaccination had no significant effect on any of the measured parameters. These results provide the first evidence that intramammary immunization may improve the outcome of an intramammary E. coli infection in late gestation and onset of mastitis immediately following parturition. Unlike systemic vaccination, which generally does not reduce the intramammary infection risk, the intramammary immunization did show a 5-times reduced odds of an established intramammary infection after challenge. Cytokine profiles indicated a local return of proinflammatory response after challenge as the data showed a more pronounced increase in in IFN-γ with a subsequent negative feedback due to a spike in the level of IL-10 in immunized quarters relative to nonimmunized quarters. Although these results are preliminary and obtained on only 10 cows, the results provide insight into the biological benefits of triggering mucosal immunity in the mammary gland.

Implications of PCR and ELISA results on the routes of bulk-tank contamination with Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (MAP), the etiologic agent of Johne's disease in dairy cattle, may enter the bulk tank via environmental contamination or direct excretion into milk. Traditionally, diagnostics to identify MAP in milk target either MAP antibodies (by ELISA) or the organism itself (by culture or PCR). High ELISA titers may be directly associated with excretion of MAP into milk but only indirectly linked to environmental contamination of the bulk tank. Patterns of bulk-milk ELISA and bulk-milk PCR results could therefore provide insight into the routes of contamination and level of infection or environmental burden. Coupled with questionnaire responses pertaining to management, the results of these diagnostic tests could reveal correlations with herd characteristics or on-farm practices that distinguish herds with high and low environmental bulk-tank MAP contamination. A questionnaire on hygiene, management, and Johne's specific parameters was administered to 292 dairy farms in New York, Oregon, and Wisconsin. Bulk-tank samples were collected from each farm for evaluation by real-time PCR and ELISA. Before DNA extraction and testing of the unknown samples, bulk-milk template preparation was optimized with respect to parameters such as MAP fractionation patterns and lysis. Two regression models were developed to explore the relationships among bulk-tank PCR, ELISA, environmental predictors, and herd characteristics. First, ELISA optical density (OD) was designated as the outcome in a linear regression model. Second, the log odds of being PCR positive in the bulk tank were modeled using binary logistic regression with penalized maximum likelihood. The proportion of PCR-positive bulk tanks was highest for New York and for organic farms, providing a clue as to the geographical patterns of MAP-positive bulk-tank samples and relationship to production type. Bulk-milk PCR positivity was also higher for large relative to small herds. The models revealed that bulk-milk PCR result could predict ELISA OD, with PCR-positive results corresponding to high bulk-milk ELISA titers. Similarly, ELISA was a predictor of PCR result, although the association was stronger for organic farms. Despite agreement between high bulk-milk ELISA titers and positive PCR results, a large proportion of high ELISA farms had PCR-negative bulk tanks, suggesting that farms are able to maintain satisfactory hygiene and management despite a presence of MAP in these herds.

The effects of progressing and nonprogressing Mycobacterium avium ssp. paratuberculosis infection on milk production in dairy cows.

Longitudinal data from 3 commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Mycobacterium avium ssp. paratuberculosis (MAP) infection status and progression path on milk production. Disease status, as indicated by MAP test results, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues and feces at slaughter. Milk production data were collected from the Dairy Herd Information Association. Animals with positive MAP test results were categorized, based on test results over the full course of the study, as high path (at least one high-positive culture) or low path (at least one positive culture or ELISA). The cumulative numbers of positive ELISA and culture results were recorded. The effects of both MAP infection path, status, and number of positive tests on milk production were analyzed using a mixed linear model with an autocorrelation random effect structure. Low- and high-path animals produced more milk before their first positive test than always-negative animals, especially high-path animals. Although mean production decreased after a first positive test, low-path animals were shown to recover some productivity. High-path animals continued to exhibit a decrease in milk production, especially after their first high-positive fecal culture. These results show that not all animals that test positive for MAP will have long-term production losses. Milk production decreased significantly with each additional positive test. Ultimately, production loss appeared to be a function of MAP infection progression.

Postpartum uterine diseases and their impacts on conception and days open in dairy herds in Italy.

The objective of this study was to describe the incidence and the impact of postpartum uterine diseases in postpartum cows on future uterine status and reproductive performance in large Italian dairy herds. This study provides an important quantitative estimate of uterine and postpartum diseases incidence that afflict high-producing Italian dairy cows. The total number of cows included in the study was 1498 on three farms; all cows were followed from the dry period until 300 days postpartum. All farms used high-quality data collection systems and standard operating procedures: weekly herd health visits, monthly Dairy Herd Improvement Association visits, and, due to cheese-making milk quality requirements, a supplementary milk sample collected at 7 ± 3 days postpartum evaluated for milk components. Clinical metritis in primiparous cows did not change the time to the first artificial insemination (AI) or days open; conversely, clinical metritis in multiparous cows had impact on the time to first AI (hazard ratio: 0.66, P < 0.01) and resulted in a lower conception rate at first insemination and a increase in days open (odds ratio: 0.64, P < 0.05). Clinical endometritis had a strong deleterious effect on first AI conception rate (odds ratio: 0.34, P < 0.05) and days open across all lactations (hazard ratio: 0.68, P < 0.05). Persistent metritis, defined as the presence of both clinical metritis and clinical endometritis in the same animal in the same lactation, caused low conception rate both in the first-lactation and in older cows and had a strong negative effect on the proportion of pregnant cows at 300 days (P < 0.05). In conclusion, the impact of endometritis on fertility was true across lactation groups. A good management and precocious diagnosis of the pathologies is not resolutive to restore good fertility parameters, and understanding the immune response in first-lactation cows may be of value for developing alternative intervention protocols for older-lactation cows.

Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms.

Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies.

Effects of pathogen-specific clinical mastitis on probability of conception in Holstein dairy cows.

The objective of this study was to estimate the effects of pathogen-specific clinical mastitis (CM), occurring in different weekly intervals before or after artificial insemination (AI), on the probability of conception in Holstein cows. Clinical mastitis occurring in weekly intervals from 6 wk before until 6 wk after AI was modeled. The first 4 AI in a cow's lactation were included. The following categories of pathogens were studied: Streptococcus spp. (comprising Streptococcus dysgalactiae, Streptococcus uberis, and other Streptococcus spp.); Staphylococcus aureus; coagulase-negative staphylococci (CNS); Escherichia coli; Klebsiella spp.; cases with CM signs but no bacterial growth (above the level that can be detected from our microbiological procedures) observed in the culture sample and cases with contamination (≥ 3 pathogens in the sample); and other pathogens [including Citrobacter, yeasts, Trueperella pyogenes, gram-negative bacilli (i.e., gram-negative organisms other than E. coli, Klebsiella spp., Enterobacter, and Citrobacter), Corynebacterium bovis, Corynebacterium spp., Pasteurella, Enterococcus, Pseudomonas, Mycoplasma, Prototheca, and others]. Other factors included in the model were parity (1, 2, 3, 4 and higher), season of AI (winter, spring, summer, autumn), day in lactation of first AI, farm, and other non-CM diseases (retained placenta, metritis, ketosis, displaced abomasum). Data from 90,271 AI in 39,361 lactations in 20,328 cows collected from 2003/2004 to 2011 from 5 New York State dairy farms were analyzed in a generalized linear mixed model with a Poisson distribution. The largest reductions in probability of conception were associated with CM occurring in the week before AI or in the 2 wk following AI. Escherichia coli and Klebsiella spp. had the greatest adverse effects on probability of conception. The probability of conception for a cow with any combination of characteristics may be calculated based on the parameter estimates. These findings may be helpful to farmers in assessing reproduction in their dairy cows for more effective cow management.

Efficacy of vaccination on Staphylococcus aureus and coagulase-negative staphylococci intramammary infection dynamics in 2 dairy herds.

The aim of this study was to evaluate vaccine efficacy of a commercial vaccine (Startvac, Hipra Spain) aimed at reducing intramammary infections (IMI) with Staphylococcus aureus and coagulase-negative staphylococci under field conditions. During the 21-mo duration of the study, 1,156 lactations from 809 cows were enrolled in 2 herds. During the first phase of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (at ~6mo). At that point, when 50% vaccination coverage was reached, cows that were due to calve were randomly assigned to be vaccinated or left as negative controls. Cure rate, rate of new infection, prevalence, and duration of infections were analyzed. Vaccination resulted in a moderate reduction in incidence of new staphylococcal IMI and a more pronounced reduction in duration of IMI associated with reduction of the basic reproduction ratio of Staph. aureus by approximately 45% and of coagulase-negative staphylococci by approximately 35%. The utilization of vaccine in combination with other infection-control procedures, such as excellent milking procedures, treatment, segregation, and culling of known infected cattle, will result in an important reduction in incidence and duration of intramammary staphylococcal infections.