Microbial communities in feed, bedding material, and bulk milk - experiences from a feeding trial.

There is an increasing interest in the microbiota of the dairy value chain, from field to fork. Studies to understand the effects of environmental, feed and management factors on the raw milk microbiota have been performed to elucidate the origin of the bacteria and find ways to control the presence or absence of specific bacteria. In this study, we explored the microbiota in feedstuff, bedding material and milk on a Swedish dairy farm to investigate the effects of feeding different silages on the bacterial compositions throughout the dairy value chain. Three ensiling treatments were evaluated: without additive, with acid treatment, and with inoculation of starter culture. The silage treatments were fed as partial mixed rations to 67 dairy cows for 3 weeks each, with one treatment fed twice to evaluate if a potential change in milk microbiota could be repeated. The highest average total bacteria counts were found in the used bedding material (9.6 log10 cfu/g), while milk showed the lowest (3.5 log10 cfu/g). Principal coordinate analysis of the weighted UniFrac distance matrix showed clear separation between 3 clusters of materials: 1) herbage, 2) silage and partial mixed ration, and 3) used bedding material and milk. Surprisingly, the expected effect of the ensiling treatments on silage microbiota was not clear. Transfer of major bacteria from the silages and resulting partial mixed rations to the used bedding material was observed, but rarely to milk. The milk microbiota showed most resemblance to that of the used bedding material. Lactobacillus was a major genus in both feed and milk, but investigations at amplicon sequence variant level showed that in most cases the sequences differed between materials. However, low total bacteria count in the milk in combination with a high diversity suggests that results may be biased due to environmental contamination of the milk samples. Considering that the study was performed on a research farm, strict hygienic measures during the feeding experiment may have contributed to the low transfer of bacteria from feed to milk.

The microbiota of ensiled forages and of bulk tank milk on dairy cattle farms in northern Sweden - a case study.

Factors contributing to variations in the quality and microbiota of ensiled forages and in bulk tank microbiota in milk from cows fed different forages were investigated. Nutritional quality, fermentation parameters and hygiene quality of forage samples and corresponding bulk tank milk samples collected in 3 periods from 18 commercial farms located in northern Sweden were compared. Principal coordinates analysis revealed that the microbiota in forage and bulk milk, analyzed using 16S rRNA gene-based amplicon sequencing, were significantly different. The genera Lactobacillus, Weissella and Leuconostoc dominated in forage samples, whereas Pseudomonas, Staphylococcus and Streptococcus dominated in bulk milk samples. Forage quality and forage-associated microbiota were affected by ensiling method and by use of silage additive. Forages stored in bunker and tower silos (confounded with use of additive) were associated with higher levels of acetic and lactic acid and Lactobacillus. Forage ensiled as bales (confounded with no use of additive) was associated with higher dry matter content, water-soluble carbohydrate content, pH, yeast count and the genera Weissella, Leuconostoc and Enterococcus. For bulk tank milk samples, milking system was identified as the major factor affecting the microbiota and type of forage preservation had little impact. Analysis of common amplicon sequence variants (ASVs) suggested that forage was not the major source of Lactobacillus found in bulk tank milk.

Prevalence and spatial distribution of infectious diseases of dairy cattle in Ontario, Canada.

We investigated the prevalence and spatial distribution of selected pathogens associated with infectious diseases of dairy cattle in Ontario, Canada. The cross-sectional study surveyed bulk tank milk for antibodies against bovine leukemia virus (BLV), Mycobacterium avium ssp. paratuberculosis (MAP), and Salmonella Dublin, and for the presence of mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis). Between October 2021 and June 2022, bulk tank milk samples were obtained from every commercial dairy farm in Ontario (n = 3,286). Samples underwent ELISA testing for the presence of BLV, MAP, and S. Dublin antibodies, and quantitative PCR testing for the detection of specific antigens of pathogens associated with mastitis. Bayesian models were used to estimate prevalence, and spatial analysis was carried out to identify regional clusters of high pathogen prevalence. Prevalence varied for different pathogens, and BLV was widespread across dairy farms in Ontario, with an estimated prevalence of 88.3%. The prevalence of MAP, Staph. aureus and S. Dublin in Ontario dairy herds was 39.8%, 31.5%, and 5.1%, respectively. The vast majority of dairy herds in Ontario were free of intramammary infections caused by Strep. agalactiae and M. bovis. Clusters of increased positive test rates were detected for S. Dublin, MAP, and Staph. aureus, indicating potential geographic risk factors for pathogen transmission. For S. Dublin, an area of increased test positivity rates was detected in southwestern Ontario, close to the Canada-United States border where most of the dairy herds in Ontario are located. Conversely, a localized cluster of positive test outcomes involving 14 subdivisions located in the southeastern region of Ontario was detected for Staph. aureus. Findings from our survey highlight the importance of the testing of aggregated samples and conducting spatial analysis as part of disease surveillance programs, and for implementing risk-based trading approaches among dairy producers.

Prevalence and associated risk factors of anti-Coxiella burnetii antibodies in dairy cattle herds using bulk tank milk analysis in Kabylia area, north Algeria.

Coxiella burnetii, or Q fever agent, has notable implications for human and livestock health. Infections in cattle primarily manifest through reproductive issues where infected animals shed the bacterium in birth fluids, placental tissues, and milk, serving as potential sources of transmission. Bovine herds become reservoirs, contributing to the environmental contamination of farming areas. Comprehensive studies on the prevalence, transmission routes, and associated risk factors among cattle contribute to the development of effective control strategies, ultimately safeguarding both livestock and public health.Here we determine the prevalence of Coxiella burnetii antibodies against in dairy cattle farms from Kabylia (northern Algeria) and identify the associated risk factors. Bulk tank milk samples from 184 farms were analyzed by indirect ELISA technique, 49 of them were tested positive which corresponds to a prevalence rate of 26.63% (95% CI 20.25-33.01%). Multivariate analysis by logistic regression showed that the risk factors associated with detection of anti-Coxiella burnetii antibodies are: cohabitation of cattle with small ruminants(OR = 3.74 95% CI [1.41-8.92]), exposure to prevailing winds (OR = 5.12 95% CI [2.11-13.45]), and the veterinarian visits frequency(OR = 5.67 95% CI [2.55-13.60]). These findings underscore the susceptibility of dairy cattle to Q fever in the Kabylia region, highlighting practices that pose risks. We recommend the implementation of hygienic measures and adherence to proper farming conditions to mitigate the transmission of Q fever and reduce the associated zoonotic risk.

Serological testing of an equal-volume milk sample - a new method to estimate the seroprevalence of small ruminant lentivirus infection?

BACKGROUND: In cattle attempts to evaluate within-herd prevalence of various infectious and parasitic diseases by bulk-tank milk (BTM) testing with ELISA have been made with moderate success. The fact that BTM is composed of variable and unknown volumes of milk from individual lactating animals weakens the relationship between numerical result of the ELISA and the within-herd prevalence. We carried out a laboratory experimental study to evaluate if a pooled milk sample created by mixing an equal volume of individual milk samples from seropositive and seronegative goats, henceforth referred to as an equal-volume milk sample (EVMS), would allow for accurate estimation of within-herd seroprevalence of caprine arthritis-encephalitis (CAE) using 3 different commercial ELISAs. By mixing randomly selected milk samples from seronegative and seropositive goats, 193 EVMS were created - 93 made of seronegative samples and 100 with the proportion of seropositive individual milk samples (EVMS%POS) ranging from 1 to 100%. EVMS%POS could be considered as a proxy for the within-herd seroprevalence. Then, OD of EVMS (ODEVMS) of the 193 EVMS was measured using 3 commercial ELISAs for CAE - 2 indirect and 1 competitive. RESULTS: The cut-off values of ODEVMS indicating SRLV infection were determined. The regression functions were developed to link ODEVMS with EVMS%POS. A significant monotonic relationship between ODEVMS measured with 2 commercial indirect ELISAs and EVMS%POS was identified. Two regression models developed on this basis described approximately 90% of variability and allowed to estimate EVMS%POS, when it was below 50%. High ODEVMS indicated EVMS%POS of > 50%. CONCLUSION: Our study introduces the concept of serological testing of EVMS as a method of detecting SRLV-infected herds and estimating the proportion of strongly seropositive goats. Further field studies are warranted to assess practical benefits of EVMS serological testing.

Management practices associated with Johne's bulk tank milk ELISA positivity.

Johne's disease (JD) control is often based on the culling of positive animals and the adoption of management practices that minimize exposure of young stock to the pathogen (Mycobacterium avium ssp. paratuberculosis). Throughout 2010 to 2013, the province of Ontario, Canada, instituted a voluntary Johne's control program consisting of whole-herd testing and risk assessment. The JD risk assessment evaluated 5 management areas to characterize herd JD risk. Using a modified milk ELISA technique with an optical density cut-off of 0.089, province-wide bulk tank milk (BTM) testing was used to assess the prevalence of JD high-risk herds at the end of the control program and again 4 yr after its completion. Approximately 71% of Ontario bulk tanks were classified as positive in 2017 compared with roughly 46% in 2013. In 2019, the same JD risk assessment used in the original program was readministered on 180 Ontario dairy farms. Using this cross-sectional approach, logistic regression models were built using data from the original program risk assessment and follow-up risk assessment as well as the BTM ELISA results to determine management factors associated with the control of JD. We demonstrated that management of the maternity area is an important factor in the control of Johne's disease. Although it is believed that the highest risk group for JD infection is calves under 6 mo, the cleanliness scores of older heifers and their exposure to mature cow manure was significantly associated with JD control; farms with highly contaminated weaned and bred heifers and those that had exposure to mature cow manure were more likely to be unsuccessful in their JD control efforts. Careful management of young calves appears to be important for JD control, and this management should continue even after calves have left the maternity area.

Regression trees to identify combinations of farming practices that achieve the best overall intrinsic quality of milk.

Many studies over the last 30 years have shown the effects of farming practices on milk compounds. Combinations of practices may have antagonistic or synergistic effects on milk compounds, but these combination effects remain underinvestigated. Research needs to focus on overall intrinsic milk quality (including sensory, technological, health, and nutritional dimensions) and identify the combinations that can optimize it. The aim of this study was to identify which combinations of farming practices achieved the best scores for sensory, technological, health, and nutritional dimensions and for overall intrinsic milk quality. Ninety-nine private farms were visited once each to sample their bulk tank milk and survey their farming practices. The surveyed practices concerned herd characteristics, feeding management, housing conditions, and milking and milk storage conditions on the day of test. Analyses of bulk tank milk were designed to evaluate the overall intrinsic quality of the milk for 2 target products: raw milk cheese and semi-skimmed UHT milk. Regression trees were then used to identify the combinations of farming practices that achieved the best scores on each dimension and on overall intrinsic quality of the milk. Breed and diet (type of forage) were the most influential factors for sensory and health dimensions and for technological and nutritional dimension scores, respectively, in the cheese assessment. Overall cheese quality was highly positively correlated with these 4 dimension scores. Therefore, breed and diet emerged as the most influential practices in the regression tree for overall cheese quality. However, the combinations of practices that resulted in the best quality scores differed according to dimension studied and product targeted. This suggests that advice on farming practices to improve intrinsic milk quality needs to be adapted according to the end-purpose of the collected milk. This innovative approach combining on-farm data and regression trees provides farm managers with a valuable and practical tool to prioritize practices in terms of their role in shaping milk quality, and to identify the combinations of practices that promote good milk quality and practice thresholds or modalities needed to achieve it.

Comparison of non-aureus staphylococcal and mammaliicoccal species found in both composite milk and bulk-tank milk samples of dairy cows collected in tandem.

Non-aureus staphylococci and the closely related mammaliicoccal species (NASM) are the most common causes of bovine subclinical mastitis on modern dairy farms and are highly prevalent in bulk-tank milk. The purpose of this study was to determine the distribution of NASM in both composite cow milk (CCM) and bulk-tank milk (BTM) samples collected in tandem in commercial Flemish dairy herds and to estimate the origin of the different (subgroups of) NASM species present in BTM by applying strain typing (random amplification of polymorphic DNA or random amplified DNA [RAPD]). A single cross-sectional sampling was performed over 5 herds that volunteered to participate in the study. Composite cow milk samples (n = 356) were collected from all lactating cows (except those with clinical mastitis) during a milking in tandem with 6 BTM samples per herd sequentially collected immediately post that milking (n = 30). In total, 421 and 80 NASM isolates were recovered and identified by MALDI-TOF mass spectrometry from the CCM and BTM samples, respectively and a total of 21 and 12 different NASM species were identified from CCM and BTM samples, respectively. Staphylococcus cohnii was the most prevalent NASM species found in BTM followed by Staphylococcus haemolyticus, Staphylococcus epidermidis, Mammaliicoccus lentus, and Staphylococcus equorum, whereas from CCM samples the most common species were S. hemolyticus, S. cohnii, S. equorum, S. epidermidis, and Staphylococcus chromogenes. The prevalent NASM species in both CCM and BTM samples was distinct for each herd, corroborating other studies observing a herd-specific NASM microbiota. Random amplified DNA analysis was performed on 9 NASM species (S. chromogenes, S. epidermidis, S. haemolyticus, S. equorum, Mammaliicoccus sciuri, Staphylococcus xylosus, S. cohnii, Staphylococcus debuckii, and M. lentus) because these species were isolated from both sample types in a herd. The same RAPD types were found in both sample types for all NASM species selected for strain typing in varying degrees. When assessing the distribution of NASM species, differences within NASM species should be examined meaning a closer look should be taken at the strain level rather than at the species level only.

Herd-level prevalence of Mycoplasma bovis in Swedish dairy herds determined by antibody ELISA and PCR on bulk tank milk and herd characteristics associated with seropositivity.

Mycoplasma bovis is an important pathogen causing pneumonia, mastitis, and arthritis in cattle, leading to reduced animal welfare and economic losses worldwide. In this cross-sectional study, we investigated the prevalence of M. bovis in bulk tank milk (BTM) and herd characteristics associated with a positive antibody test result in Swedish dairy herds. Bulk tank milk samples from all Swedish dairy herds (n = 3,144) were collected and analyzed with ID Screen antibody ELISA and PCR. Information on herd characteristics was collected from the national Dairy Herd Improvement database. To identify herd characteristics associated with the presence of antibodies in BTM, logistic regression was used in 4 different models. The apparent herd-level prevalence of M. bovis infection based on antibodies in BTM was 4.8%, with large regional differences ranging from 0 to 20%. None of the BTM samples was positive by PCR. All the antibody-positive herds were situated in the south of Sweden. The logistic regression model showed that larger herds had higher odds of detectable antibodies in BTM (herd size >120 cows, odds ratio = 8.8). An association was also found between antibodies in BTM and both a higher late calf mortality (2-6 mo) and a higher young stock mortality (6-15 mo). This study showed a clear regional difference in the apparent prevalence of M. bovis infection based on antibodies. The relatively low prevalence of M. bovis in Sweden is a strong motivator for the cattle industry to take steps to prevent further spread of the infection. It is essential that the M. bovis status of free herds be known, and the regional differences shown in this study suggest that testing is highly recommended when live cattle from high-prevalence areas are being introduced into herds. We do not recommend using PCR on BTM to detect infected herds, owing to the low detection frequency in this study.

Analysis of Prototheca and yeast species isolated from bulk tank milk collected in Tokachi District, Japan.

Bovine mastitis, a major infectious disease affecting milking cows, leads to reduced milk yield and quality, reduced animal welfare, and an increased need for culling. Although its major causative agents are bacteria, yeast species and achlorophyllous algae of the Prototheca genus are well known as causative agents of bovine refractory mastitis. Nevertheless, few studies have analyzed specific yeasts and Prototheca in this context. Herein, we present survey data of yeast species and Prototheca species isolated from bulk tank milk in the Tokachi district of Japan from April 2020 through March 2021. The species of 276 isolates were determined. Yeast species accounted for 184 isolates, of which Pichia kudriavzevii was the most prevalent species. Regarding Prototheca species, only Prototheca bovis was isolated (92 isolates). Prototheca bovis and Pichia kudriavzevii were detected throughout the year and were detected repeatedly on the same farm. Kluyveromyces marxianus was the second most frequently isolated yeast species after Pichia kudriavzevii. Candida parapsilosis, the fourth most frequently isolated yeast species, was found discontinuously. Analysis of monthly data indicated that Kluyveromyces marxianus and Candida parapsilosis were mainly found during the winter and summer months, respectively. Candida akabanensis and Pichia cactophila were the third and fifth most frequently isolated yeast species, respectively. They were detected repeatedly in bulk tank milk samples from the same farms. Results obtained from bulk tank milk underscore the prevalence of these species. These study results are expected to contribute to the elucidation of problematic yeast and Prototheca species.

Milking system and premilking routines have a strong effect on the microbial community in bulk tank milk.

In this study, we investigated the variation in the microbial community present in bulk tank milk samples and the potential effect of different farm management factors. Bulk tank milk samples were collected repeatedly over one year from 42 farms located in northern Sweden. Total and thermoresistant bacteria counts and 16S rRNA gene-based amplicon sequencing were used to characterize microbial community composition. The microbial community was in general heterogeneous both within and between different farms and the community composition in the bulk tank milk was commonly dominated by Pseudomonas, Acinetobacter, Streptococcus, unclassified Peptostreptococcaceae, and Staphylococcus. Principal component analysis including farm factor variables and microbial taxa data revealed that the microbial community in milk was affected by type of milking system. Milk from farms using an automatic (robot) milking system (AMS) and loose housing showed different microbial community composition compared with milk from tiestall farms. A discriminant analysis model revealed that this difference was dependent on several microbial taxa. Among farms using an automatic milking system, there were further differences in the microbial community composition depending on the brand of the milking robot used. On tiestall farms, routines for teat preparation and cleaning of the milking equipment affected the microbial community composition in milk. Total bacteria count (TBC) in milk differed between the farm types, and TBC were higher on AMS than tiestall farms (log 4.05 vs. log 3.79 TBC/mL for AMS and tiestalls, respectively). Among tiestall farms, milk from farms using a chemical agent in connection to teat preparation and a more frequent use of acid to clean the milking equipment had lower TBC in milk, than milk from farms using water for teat preparation and a less frequent use of acid to clean the milking equipment (log 3.68 vs. 4.02 TBC/mL). There were no significant differences in the number of thermoresistant bacteria between farm types. The evaluated factors explained only a small proportion of total variation in the microbiota data, however, despite this, the study highlights the effect of routines associated with teat preparation and cleaning of the milking equipment on raw milk microbiota, irrespective of type of milking system used.

Comparison Between Full-Length 16S rRNA Metabarcoding and Whole Metagenome Sequencing Suggests the Use of Either Is Suitable for Large-Scale Microbiome Studies.

Since the number of studies of the microbial communities related to food and food-associated matrices almost completely reliant on next-generation sequencing techniques is rising, evaluations of these high-throughput methods are critical. Currently, the two most used sequencing methods to profile the microbiota of complex samples, including food and food-related matrices, are the 16S ribosomal RNA (rRNA) metabarcoding and the whole metagenome sequencing (WMS), both of which are powerful tools for the monitoring of foodborne pathogens and the investigation of the microbiome. Herein, the microbial profiles of 20 bulk tank milk filters from different dairy farms were investigated using both the full-length 16S (FL-16S) rRNA metabarcoding, a third-generation sequencing method whose application in food and food-related matrices is yet in its infancy, and the WMS, to evaluate the correlation and the reliability of these two methods to explore the microbiome of food-related matrices. Metabarcoding and metagenomic data were generated on a MinION platform (Oxford Nanopore Technologies) and on a Illumina NovaSeq 6000 platform, respectively. Our findings support the greater resolution of WMS in terms of both increased detection of bacterial taxa and enhanced detection of diversity; in contrast, FL-16S rRNA metabarcoding has proven to be a promising, less expensive, and more practical tool to profile most abundant taxa. The significant correlation of the two technologies both in terms of taxa diversity and richness, together with the similar profiles defined for both highly abundant taxa and core microbiomes, including Acinetobacter, Bacillus, and Escherichia genera, highlights the possible application of both methods for different purposes. This study allowed the first comparison of FL-16S rRNA sequencing and WMS to investigate the microbial composition of a food-related matrix, pointing out the advantageous use of FL-16S rRNA to identify dominant microorganisms and the superior power of WMS for the taxonomic detection of low abundant microorganisms and to perform functional analysis of the microbial communities.

An observational study on the relationship between zinc concentrations in bulk tank milk and in serum and farmer-reported zinc supplementation of dairy cattle for facial eczema prophylaxis.

AIMS: To describe the concentration of Zn in bulk tank milk (BTM) in a sample of New Zealand dairy farms, investigate the association between the method of Zn administration for facial eczema prophylaxis and Zn concentrations in BTM and investigate the relationship between the concentration of Zn in serum and that in BTM. METHODS: Multiple BTM samples (n = 3,330) collected during milk pick-up by the milk tanker driver were stored and tested for 121 farms, in Northland (n = 50), Waikato (n = 51) and Southland (n = 20) from February to May 2017. Enrolled farms provided retrospective information on the type of Zn supplementation (if any) used for the prevention of facial eczema and the timeframe over which supplementation occurred. In addition, the concentration of Zn in serum was measured in blood samples collected from ≥15 cattle per farm for 22 farms from Northland (n = 11) and Waikato (n = 11), and compared against the concentrations of Zn in BTM on the day of blood sampling. A linear mixed model was used to model log Zn concentrations in BTM using method of Zn supplementation, region, milk fat and protein percentage, volume of milk, and frequency of milk pick-up as risk factors. A mixed logistic regression model was used to assess the relationship between Zn concentrations in BTM and the presence of cows with a concentration of Zn in serum of ≥20 µmol/L. RESULTS: The median Zn concentration in BTM was 67.9 (min 38.9, max 146.6) µmol/L. The median range of Zn concentrations for repeated samples of BTM within farm was 22.6 µmol/L. In comparison to farms that did not use any form of Zn supplementation, farms that supplemented Zn through a slow-release capsule, oral drench, in feed or a combination of in-feed and water were associated with increased concentrations of Zn in BTM (p < 0.001). There was no difference in Zn concentrations in BTM between farms that administered Zn through the water only and farms that did not administer Zn (p = 0.22). Every 15.3 µmol/L increase in Zn concentration in BTM was associated with 2.2 times (95% CI=1.7-2.9) the odds of a cow having Zn concentration in serum ≥20 µmol/L. CONCLUSION AND CLINICAL RELEVANCE: Zn concentration in BTM is highly variable between farms, days and Zn administration method. Zn concentration in BTM content has modest potential as a way to signal whether a herd has achieved the high Zn status considered to be protective against FE.

Characterization of the microbiota and resistome of bulk tank milk samples from Prince Edward Island dairy farms.

Bulk tank milk (BTM) is regularly used for surveillance on dairy farms for disease conditions such as mastitis and Johne's disease. In this study, we used 16S rRNA sequencing and bait-capture enrichment to characterize the microbiota and resistome of BTM, and investigate potential differences between the cream or pellet fractions. A total of 12 BTM samples were taken from 12 Prince Edward Island dairy farms, in Atlantic Canada, in duplicates. The DNA was analyzed by high-throughput sequencing of the 16S rRNA gene and a suite of antimicrobial resistance (AMR) genes. Target-capture enrichment of AMR genes was conducted before shotgun sequencing. The bioinformatics pipelines QIIME 2 and AMR++ were used for microbiota and resistome analysis, respectively. Differences between microbiotae were evaluated qualitatively with nonmetric multidimensional scaling and quantitatively with permutational ANOVA of UniFrac distances. A total of 47 phyla were present across the BTM samples. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the most abundant phyla. At the genus level, Corynebacterium, Acinetobacter, Lactobacillus, and Turicibacter were the most abundant. There was no significant difference in the Faith's phylogenetic diversity between the cream and pellet fraction. Faith's phylogenetic diversity differed marginally by stall type. There were 10,217 hits across 80 unique AMR genes, with tetracycline resistance being the most common class.

Temporal trends in bulk tank milk antibody ELISA and PCR test results for bovine viral diarrhoea in New Zealand pastoral dairy herds.

Aims: To describe temporal trends in bulk milk antibody ELISA and PCR testing for bovine viral diarrhoea (BVD) in New Zealand pastoral dairy herds and to assess the use of historical accession data to predict herd-level BVD incursions. Methods: Data on all diagnostic testing of bulk milk for BVD performed by the Livestock Improvement Corporation (Hamilton, NZ) over eight lactation seasons from 1 June 2010 to 31 May 2018 were analysed. This included anonymised herd identification, geographic location, herd size, sample collection date, sample to positive (S/P) ratio for antibody ELISA results, and cycle threshold values for PCR detecting viral RNA. Multivariable logistic regression was used to explore the relationship between historical accession data and the risk of herds having at least one positive bulk milk PCR test result in the 2017 season. Results: There were 156,034 bulk milk BVD diagnostic testing accessions for 10,495 uniquely identified dairy herds over the 8-season period. The prevalence of tested herds with at least one positive bulk milk PCR test result decreased from 14.6% (407/2,786) in the 2010 season to 5.6% (355/6,309) in the 2017 season with similarly marked declines in S/P ratios. In the 2017 season, 2,961/6,309 (46.9%) herds had S/P ratios greater than the 0.75 cut-off value indicating recent or active BVD virus transmission within the herd while 1,422/6,309 (22.5%) herds were classified as having negative or low S/P ratios. Herds that cleared BVD from the milking herd experienced a mean decline in S/P ratio of 0.11 units per year (min 0.05; max 0.18). In the multivariable analysis, the overall incidence risk of herds experiencing a BVD incursion in the 2017 season was 3.8% (146/3,848) and there were three significant predictors in the final model: herd size, PCR status in the 2014 season, and change in S/P ratio between the 2014 and 2015 seasons. The area under the receiver operating curve for the final model was 0.695 indicating poor discrimination. Conclusions and clinical relevance: The prevalence of dairy herds in New Zealand with positive bulk milk PCR test results and high S/P ratios has decreased over time, suggesting fewer herds are actively infected with BVD and that herd immunity may also be declining. Although monitoring trends in bulk milk test results provides useful information on changes in individual herd status, it is difficult to accurately predict when new incursions will occur and farmers should continue to maintain good biosecurity.

Large genetic diversity of Arcobacter butzleri isolated from raw milk in Southern Italy.

Arcobacter butzleri is a zoonotic foodborne pathogen able to cause enteric and extraintestinal diseases. Its occurrence in foodstuff is well recognized worldwide but data on its presence in foods from Southern Italy are scarce. In this study the results on the occurrence and genotyping of Arcobacter spp. in bulk milk samples collected in Southern Italy are reported. Out of 484 samples, 64 (13.2%) resulted positive for the presence of Arcobacter spp. Using Real Time PCR but as few as 31.2% of these samples turned out as positive by using the cultural method, showing an overall prevalence of 4.1%. All isolates were identified as A. cryaerophilus using the biochemical identification whilst the sequencing of the atpA gene revealed that all the isolates were A. butzleri. Among the confirmed isolates, 16 different Sequence Types (ST) were identified using the Multi Locus Sequence Typing (MLST), 14 (87.5%) of which were previously unreported. Our survey reveals the presence of A. butzleri in bulk tank milk from Southern Italy and highlights the discrepancy between the two approaches used both for the detection (i.e., real time PCR vs cultural method) and the identification (i.e., biochemical test vs aptA sequencing) of Arcobacter spp In addition, a large genetic diversity among the isolates was detected and this makes the identification of source of the infections very challenging in outbreaks investigation.

Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy).

Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates.

Development and implementation of a risk assessment and management program for enzootic bovine leukosis in Atlantic Canada.

Over the past 30 yr, the prevalence of bovine leukemia virus (BLV) infection has increased in North America, including Atlantic Canada, at both the herd and individual cow levels. This has occurred despite increased awareness of the disease and its deleterious effects and despite implementation of management practices aimed at reducing disease transmission. Our objectives were to identify risk factors associated with the within-herd prevalence of BLV-infected cows by using a risk assessment and management program workbook, as well as to determine the current level of BLV prevalence in the Atlantic Canada region. We hypothesized that previously established risk factors, including management practices associated with calf rearing and fly control, would affect within-herd BLV prevalence. Bulk tank milk (BTM) samples were collected in January and April of 2016 and again during the same months in 2017 and 2018 from all dairy farms shipping milk in the region. Samples were tested with ELISA for levels of anti-BLV antibodies to estimate within-herd prevalence. Regional BLV prevalence at the herd level was 88.39% of dairy herds infected in 2016 and 89.30% in 2018. All dairy farms shipping milk and who had BTM samples collected in 2017 (n = 605) were eligible to participate in the risk assessment and management program questionnaire (RAMP), which was developed and distributed to all bovine veterinarians in Atlantic Canada. One hundred and six RAMP were returned, with representation from all 4 provinces. The RAMP results were combined with the mean BTM ELISA results, and univariable logistic regression followed by multivariable logistic regression was performed to investigate the association between RAMP risk factors and the estimated within-herd BLV prevalence. Factors in the multivariable model significantly associated with the odds of a herd being classified as >25% estimated within-herd prevalence included history of diagnosis of clinical BLV and calves receiving colostrum from cows with unknown BLV status. Differences in within-herd prevalence were not associated with hypodermic needle and injection practices, rectal sleeve practices, or using bulls for natural breeding, based on these 106 dairy farms.

On-Farm Surfaces in Contact with Milk: The Role of Staphylococcus aureus-Containing Biofilms for Udder Health and Milk Quality.

Staphylococcus aureus is a Gram-positive bacterium that causes intramammary infections and bulk tank milk (BTM) contamination in dairy operations around the world in spite of on-farm application of preventive measures. The study was conducted on a 30-cow dairy farm in the Ñuble Region of Chile. For BTM culture and somatic cell count (SCC) analysis, three consecutive BTM samples were collected. Samples for bacterial culture (n = 16) were collected from macroscopic adherence on previously washed, sanitized, and dry milking equipment surfaces in direct contact with milk during milking or cooling. A total of 48 S. aureus isolates from BTM, milking equipment, and cows' quarters with intramammary infections were analyzed by pulsed-field gel electrophoresis (PFGE). Selected milking equipment pieces were removed for biofilm visualization using scanning electron microscopy (SEM). S. aureus was isolated from all three BTM samples; the average SCC for the three BTM samples was 1,429,333 cells/mL. Fourteen of the 16 samples of milking equipment (87.5%) were culture positive for S. aureus. Biofilms were visualized by SEM in all four removed milking equipment pieces. Microorganisms observed by SEM in those biofilms were mainly coccus-shaped bacteria, and microbiological culture of these biofilms yielded viable S. aureus isolates in all samples. All pulsotypes observed among S. aureus isolates from BTM were indistinguishable from those in milking equipment surfaces. All PFGE pulsotypes observed among S. aureus isolates from biofilms on rubber liners were indistinguishable from isolates from intramammary infections in cows. Our findings suggest that milking equipment films may act as source of S. aureus contamination for BTM and cows during milking, thus compromising the microbiological quality of milk used for manufacturing dairy products.

Prevalence, economic assessment, and risk factors of gastrointestinal nematodes infecting herds in tropical, dry and temperate climate regions in Mexico.

This study investigated exposure to gastrointestinal (GI) nematodes on dairy cattle farms by antibody level determination in bulk tank milk (BTM) samples and its influence on production to detect the risk factors for infection in different climate regions in three states of Mexico. From January to April 2017, BTM samples were collected from 1058 dairy cattle herds and used to establish three Köppen climate classes (tropical, dry and temperate) and states of Mexico. A questionnaire on farm management was applied. The overall herd prevalence of parasites was 67.20%. The highest percentage of positive herds was detected in Veracruz state (78.45%). In addition, the highest prevalence among the climate regions was found in the tropical climate (78.59%). In general, production losses were approximately 1.37-1.78 kg of milk/cow per day. The annual costs of milk production losses per farm were estimated for three different climate regions, ranging between $5541.49 and 6982.50 US$, and those in the three states varied between 5974.10 and 8660.06 US$. The costs for anthelmintic treatments for young stock and adult cows ranged between 57.51 and 192.75 US$, respectively, among the three climate regions and between 46.02 and 189.49 US$, respectively, among the three states. The overall annual costs of milk yield loss per cow were estimated to be 150.74 US$ for the climate regions and 190.54 US$ for the three states of Mexico, followed by the treatment costs for young stock (4.02 US$) and adult cows (3.99 US$). The results suggest that the economic losses due to GI nematodes in Mexican dairy herds are approximately 248 million US$ per annum. Four final models were built based on multivariate logistic regression for potential statistical association from the ELISA results using climatic/environmental and management factors so that each model used different risk factors that were significantly associated with helminth infections in dairy herds.