Bulk-tank Samples Research Articles

Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples

BackgroundThroughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples.ResultsThe following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%).ConclusionsThe difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

MEASUREMENT OF MICROBIOLOGICAL QUALITY OF RAW GOAT´S MILK BY LASER FLOW CYTOMETRY

The popularity of goat's milk and its products is growing in Slovakia. Among the mandatory health safety features of raw goat's milk is the total bacterial count. It can be determined by the anchor plate-cultivation method, which is, however, tedious, and laborious. Laser flow cytometry is a modern alternative method for measuring the microbiological quality of raw milk. To use the results from laser flow cytometry method for legislative purposes, it is necessary to create a representative conversion of the primarily measured results obtained by this method into the CFU/mL scale. This work describes the creation of such a conversion for raw goat's milk, which will be valid for the Slovak region. After measuring several hundred individual and bulk tank samples of raw goat's milk on a BactoScan FC laser flow cytometer and at the same time using the anchor method according to STN EN ISO 4833-1 and after their subsequent logarithmic transformation, such a conversion was created in the form of log10(CFU/mL) = 1.0174 x log10(IBC/ml) + 2.7483. Subsequently, this was verified by comparing the recalculated and directly measured results in CFU/mL, using additional data set of samples. The influence of the somatic cell count in raw goat's milk, as a possible interfering factor, was also assessed.

Occurrence and antimicrobial susceptibility patterns of Escherichia coli and Escherichia coli O157 isolated from cow milk and milk products, Ethiopia

Escherichia coli is a major foodborne pathogen worldwide. This study was conducted to assess the prevalence, risk factors, and antimicrobial susceptibility of E. coli and E. coli O157 in milk and milk products and hygienic practices in West Shoa, Oromia, Ethiopia. Five hundred fifty-six milk samples comprising 421 udder milk, 57 bulk tank milk, and 78 milk products were investigated. Moreover, a questionnaire was administered to 145 participants to assess hygienic practices. A standard microbiological procedure was used to detect E. coli and E. coli O157. The Kirby Bauer disc diffusion method was used to test the antimicrobial susceptibility of the isolates. Petri film plates were used to enumerate the coliform in raw bulk tank milk. Farm and animal-level E coli prevalence were estimated and association with risk factors was assessed. Escherichia coli was detected in 33.8% (95% confidence interval (CI) 29.9–37.9%) of the samples, of which only one isolate (0.2%) was E. coli O157. Escherichia coli contamination was higher in bulk tank samples (47.4%; 95% CI 34.0–61.0%) than in udder milk (34.7%; CI 30.1–39.4%), cottage cheese (27.0%; 95% CI 14.6–43.9%), and yoghurt 10.5% (95% CI 2.9–24.8%). For the animal-level E. coli contamination, only the study area was identified as a risk factor. Risk factors such as types of milk containers, udder washing practices, hygiene, and management systems were associated with both farm-level and milk products E. coli contamination. Most (59.0%) of the farmers practice udder washing only before milking and the remaining did not practice udder washing at all. The mean coliform count in raw bulk tank milk was higher than the international standard (4.09 log10 CFU/ml). All the 42 E. coli isolates tested were multidrug resistant. The occurrence of E. coli is high, while that of E. coli O157 is too low. The milk-handling practices are poor in the study areas. The high prevalence of E. coli, as well as the high coliform count and higher multi-drug resistance may pose risk to public health and food safety. Therefore, proper hygienic practices throughout the milk chain as well as rational drug use are advised.

Risk factors for Salmonella Dublin on dairy farms in Ontario, Canada

Salmonella Dublin is an emerging pathogen on dairy farms in Canada. In Ontario, Salmonella Dublin has been increasingly isolated from diagnostic laboratory samples. The objective of this observational cross-sectional study was to identify management practices associated with herd positivity for Salmonella Dublin. A convenience sample of 100 dairy farms was visited in Ontario, Canada, from April to August 2022. Farms were visited once to collect blood samples from 20 heifers between 4 and 24 mo old, sample bulk tank milk, and administer an in-person questionnaire on management practices. An additional bulk tank milk sample was collected before the visit by milk transporters. All bulk tank and serum samples underwent ELISA testing to determine Salmonella Dublin positivity (≥35% positivity on ELISA). Of the 1,990 heifers sampled, 44 (2.2%) animals were seropositive for Salmonella Dublin. At least one seropositive heifer was identified on 24% of participating farms. Based on the bulk tank milk samples collected during both sampling periods, 4% of farms were positive for Salmonella Dublin. Overall, of the 100 farms visited, 25% were classified as Salmonella Dublin positive, meaning at least one serum or bulk tank sample was interpreted as positive. A multivariable logistic regression model identified 5 factors associated with herd-level positivity for Salmonella Dublin. Specifically, introducing purchased animals within the last 2 years increased the likelihood that farms were positive for Salmonella Dublin (odds ratio [OR] = 4.6). Farms that had at least one animal leave the premises for a cattle show, embryo collection center, or loan to another farm and return within the last 2 years were also at a higher risk for Salmonella Dublin (OR = 4.9). Farms that removed manure from the surface of bedding in calving pens twice per month or after every calving were at greater risk for Salmonella Dublin than farms that removed manure less frequently (OR = 8.5). Farms that added bedding material to calving areas once or twice weekly were at lower risk for Salmonella Dublin compared with farms that added bedding less than once weekly (OR = 0.1). In addition, farms that kept 3 cows or less per pen in the calving area were at lower risk for Salmonella Dublin. Test positivity for Salmonella Dublin among Ontario dairy farms sampled is high, and dairy producers should consider avoiding management practices that are associated with an increased risk of Salmonella Dublin infection.

Evaluation of Cow-Side Meters to Determine Somatic Cell Count in Individual Cow Quarter and Bulk-Tank Milk Samples.

Intramammary infections, which cause mastitis, can increase treatment and labor costs, decrease milk production, and affect milk quality. Meters that measure quarter somatic cell count (SCC) could be used to make more informed dry cow therapy decisions. The objective of this study was to compare the RT-10 iPhone adapter (RT-10; Dairy Quality Inc., Newmarket, ON, Canada), DeLaval Cell Counter (DSCC; DeLaval, Gurnee, IL, USA), Porta Check Quick Test (PortaCheck, White City, OR, USA), California Mastitis Test (ImmuCell, Portland, ME USA), pH meter (Hanna Instruments, Smithfield, RI, USA), electrical conductivity meter (OHAUS, Parsippany, NJ, USA), and the dual laser infrared temperature thermometer (Klein Tools, Lincolnshire, IL, USA) for measuring SCC in individual Holstein mammary quarters in comparison to a reference standard, the Fourier Transform Spectrometer 600 Combi System (Combi; Bentley Instruments, Chaska, MN, USA). Meters were evaluated using 658 individual cow quarter samples and 100 bulk-tank samples to measure SCC. Individual quarter milk samples from 160 cows from four commercial dairy herds were collected just before dry off and tested within 4 h of collection. To test bulk-tank SCC, 100 bulk-tank milk samples (25 mL) were collected from UC Davis Veterinary Medicine Teaching and Research Milk Quality Lab. Meter SCC values were regressed on observed Combi SCC. Goodness of fit was then evaluated by partitioning the mean square predicted error (MSPE). For individual quarter SCC, RT-10 had the highest coefficient of determination (R2 = 0.86), lowest MSPE, and highest proportion of MSPE due to random variation (96%). Both the RT-10 and DSCC had the highest sensitivity and specificity for identifying quarter SCC above and below 200,000 cells/mL. For bulk-tank SCC, DSCC had the highest coefficient of determination (R2 = 0.45), lowest MSPE, and highest proportion of MSPE due to random variation (80%). The RT-10 and DSCC could be used to measure individual quarter SCC to determine which cows to treat at dry off potentially reducing antibiotic use.

Estimation of genetic parameters for subclinical mastitis using a threshold model in first parity dairy cows under pasture-based systems of Los Ríos Region in Chile

Somatic cell count (SCC) is an indirect measurement to estimate mammary gland health status. This trait provides information regarding the severity of the mammary tissue inflammation in each quarter. Milk samples coming from the farm milk storage vat containing 100,000 to 200,000 cell/mL are considered suspicious, while SCC over 200,000 cell/mL is an indication of subclinical mastitis. Chilean dairy processors penalise farmers monetarily when their bulk tank samples reach levels of 300,000 cell/ mL SCC. The objective of this study was to quantify the additive genetic component of the liability of cows to reach the 300,000 cell/mL threshold. A data set containing the highest SCC test-day record of 10,528 first lactation cows from 15 commercial dairy farms of Los Ríos Region in southern Chile was analysed. The unknown continuous underlying susceptibility of each cow to reach the 300,000 SCC threshold was modelled as a function of a contemporary group formed by the herd, year, and calving season, the regression coefficient of the unknown underlying susceptibility value of a cow on her daily milk yield (MY) and the additive animal genetic effect. Bayesian inference and Gibbs sampling were used to estimate additive and residual variances. The average daily MY and SCC were 17.84±5.25 kg and 125,327±236,297 cell/mL, respectively. The estimated heritability varied from 0.03 to 0.22 and the average was 0.10±0.03. It is concluded that the genetic variability for the susceptibility to reach the 300,000 SCC threshold could be exploited to improve resistance to subclinical mastitis.

Description of the Characteristics of Five Bedding Materials and Association With Bulk Tank Milk Quality on Five New York Dairy Herds.

Environmental mastitis represents a major challenge on dairy farms where contagious pathogens are controlled by improved milking procedures. Therefore, research focused on the environment is important to improve udder health programs. The objectives of this prospective and descriptive study were to (1) describe bedding bacterial counts, pH, and dry matter (DM) of five different bedding types (organic: manure solids, straw, paper fiber; inorganic: sand, recycled sand) and (2) explore the association between bedding bacterial counts with bulk tank milk quality. This study took place within five conveniently selected commercial dairy herds, each with a predominant bedding material in lactating pens. Bedding samples (used n = 237; fresh n = 53) were collected monthly from July 2018 to July 2019 following a standard operating procedure (SOP) to minimize sampling variability. Additionally, a bulk tank (BT) milk sample (n = 40) was collected on the same day unless milk had been picked up prior to arrival. Both BT and bedding samples were submitted to the laboratory for culture and bacterial identification and quantification of Streptococcus spp, coliforms, and non-coliforms as well as detection of several pathogens of mastitis importance. Somatic cell count was evaluated in BT samples. Within bedding type, the correlation between bedding characteristics and bacterial counts in bedding was evaluated using Pearson correlation. Within bedding type, the correlation between bacterial counts in bedding samples and bacterial counts in BT were determined. The Kruskal–Wallis test was used to evaluate the bacterial count by bedding type and to evaluate BT somatic cell count differences based on bedding type. In fresh bedding, bacterial counts were generally higher for manure solids for all bacterial groups compared with other materials. In used samples, organic materials had the highest levels of all bacterial groups. The proportion of samples with detectable organisms of mastitis importance varied within and among herds in both bedding and BT samples throughout the study period. In bedding samples, a higher DM content had the lowest levels of bacterial growth compared with those with lower DM content. Most bedding samples were on the alkaline side within a pH range of 8–11. No relationship between bacterial counts and pH was observed. No associations between BT bacteria counts and bedding bacterial counts were observed. No association between bulk tank somatic cell counts based on bedding type were observed. Despite using an SOP for bedding sampling in an effort to consistently collect samples, we still observed a large amount of variability both within and among bedding samples. This variability may have obscured any potential association between BT milk quality and bedding type.

165 Evaluation of Milk Urea Nitrogen Measurement Precision and Accuracy

Abstract High levels of urea in blood, milk and urine have been linked to poor nitrogen efficiency, increased feed costs, poor reproductive performance and increased environmental impacts of dairy farming. Milk urea nitrogen (MUN) is a commonly used metric to manage herd nitrogen efficiency, with current recommendations for MUN to be between 8–14 mg/dL to maintain milk production and reduce nitrogen losses. However, a previous work suggests commercial analysis of MUN with mid-infrared spectroscopy (MIR) may not be precise enough to determine if a milk sample is within the recommended range. Thus, the objective of this study was to evaluate the precision and accuracy of milk testing lab MUN measurements. Four sets of bulk tank samples were sent to 3 commercial labs and one research lab for analysis by MIR. Samples were sent to commercial labs in duplicate and MUN was also assessed through an enzymatic assay. The Euclidean distance (ED) was calculated as a combined metric of precision and accuracy. The ED was not different between labs and ranged from 0.81–1.27. Repeatability (sr) and reproducibility (sR) were estimated for commercial labs and ranged from (0.297–0.469) and (0.555–0.791) respectively. Differences between individual sample MIR and enzymatic MUN were regressed on the centered enzymatic MUN in a linear mixed model that included a random effect of lab and fixed effects for milk protein and milk fat. Regression results indicate MIR analysis over-predicts MUN at low MUN concentrations and under predicts MUN at high MUN concentrations. Results suggest MIR analysis of MUN is more accurate around milk MUN, protein, and fat concentrations of ~13 mg/dl, 3.4% protein, and 4.2% fat. Further, the combined residual error and random effect of lab suggest the standard error of an MUN MIR measurement is ±1.8.

Genotypic analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin

Nontuberculous mycobacteria (NTM) compose a group of mycobacteria that do not belong to the Mycobacterium tuberculosis complex group. They are frequently isolated from environmental samples such as water, soil, and, to a lesser extent, food samples. Isolates of NTM represent a major health threat to humans worldwide, especially those who have asthma or are immunocompromised. Human disease is acquired from environmental exposures and through consumption of NTM-contaminated food. The most common clinical manifestation of NTM disease in human is lung disease, but lymphatic, skin and soft tissue, and disseminated disease are also important. The main objective of the current study was to profile the farm-level contamination of cow milk with NTM by examining milk filters and bulk tank milk samples. Five different NTM species were isolated in one dairy herd in Wisconsin, with confirmed 16S rRNA genotypes including Mycobacterium fortuitum, Mycobacterium avium ssp. hominissuis, Mycobacterium abscessus, Mycobacterium simiae, and Mycobacterium avium ssp. paratuberculosis (Mycobacterium paratuberculosis). In tank milk samples, M. fortuitum was the predominant species in 48% of the samples, whereas M. chelonae/abscessus and M. fortuitum were the only 2 species obtained from 77 and 23% of the examined filters, respectively. Surprisingly, M. avium ssp. hominissuis, M. paratuberculosis, and M. simiae were isolated from 16.7, 10.4, and 4% of the examined milk samples, respectively, but not from milk filters. Interestingly, NTM isolates from human clinical cases in Wisconsin clustered very closely with those from milk samples. These findings suggest that the problem of NTM contamination is underestimated in dairy herds and could contribute to human infections with NTM. Overall, the study validates the use of bulk tank samples rather than milk filters to assess contamination of milk with NTM. Nontuberculous mycobacteria represent one type of pathogens that extensively contaminate raw milk at the farm level. The significance of our research is in evaluating the existence of NTM at the farm level and identifying a simple approach to examine the potential milk contamination with NTM members using tank milk or milk filters from dairy operations. In addition, we attempted to examine the potential link between NTM isolates found in the farm to those circulating in humans in Wisconsin.

Evaluation of four commercial tests for detecting ceftiofur in waste milk bulk tank samples.

The objective of this study was to identify factors affecting the accuracy of four commercial tests for ceftiofur drug residue in milk samples from bulk tank waste milk (WM). WM samples were collected from 12 California dairy farms which were initially tested using liquid chromatography (LC-MS/MS) to confirm their negative status for drug residues above the FDA established tolerance/safe levels. The milk samples were also tested for fat, protein, lactose, solids non-fat (SNF), somatic cell count (SCC), coliform count, and standard plate count (SPC). Each WM sample was divided into two aliquots, one labeled as negative for drug residues (WMN) and the second spiked with ceftiofur as positive for ceftiofur residues (WMPos). Both types of WM samples were tested to evaluate the performance of 4 commercially available tests: Penzyme® Milk Test, SNAP® β-lactam, BetaStar® Plus and Delvo SP-NT®. Three assays in triplicates for the WMN and WMPos were conducted for each WM sample. Test were evaluated using sensitivity, specificity, positive predictive value, negative predictive value and positive likelihood ratio. Kruskal-Wallis method was used to evaluate the effect of milk quality parameters on true positive (TP) and false negative (FN) test results. All WMPos samples were identified as positive by all four tests, rendering 100% sensitivity for each test. The specificity for Penzyme, BetaStar, Delvo, and SNAP tests were 59.2, 55.5, 44.4, and 29.6, respectively. Overall, all tests correctly identified samples with ceftiofur residues (WMPos), as shown by 100% sensitivity. Greater variability was observed regarding identification of samples free of any drug residue, with Penzyme and BetaStar having the highest risk for correctly identifying TN samples. Our findings indicate that when selecting commercial tests to detect drug residues in WM, milk quality parameters must be considered if the aim is to reduce FP test results.

Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality

The dairy farm environment influences the raw milk microbiota and consequently affects milk processing. Therefore, it is crucial to investigate farm management practices such as the bedding materials. The aim of this study was to evaluate the effect of recycled manure solids (RMS) as bedding material on bulk tank milk and microbiological implications for cheese quality. Bulk tank samples were collected from 84 dairy farms using RMS or straw bedding. The use of RMS did not influence thermophilic and mesophilic aerobic viable counts from spores. However, straw-milk samples gave higher values for mesophilic anaerobic spore-forming bacteria (0.44 log cfu/mL) than RMS-milk samples (0.17 log cfu/mL). The presence of thermoresistant lactic acid bacteria was not increased in milk from farms using RMS. Nevertheless, taxonomic profiles of thermoresistant bacteria isolated were different between the 2 types of milk. More Enterococcus faecalis and Streptococcus spp. were identified in RMS-milk samples. Thermoresistant enterococci and streptococci could easily end up in cheese. Therefore, milk proteolytic activities of these isolates were tested. Neither Streptococcus spp. nor Enterococcus faecium isolates exhibited proteolytic activities, whereas 53% of E. faecalis showed some. Also, only 1 vancomycin-resistant enterococcus was detected. Survival of selected RMS-milk samples isolates (3 E. faecalis and 1 Streptococcus thermophilus) was evaluated during a model Cheddar cheese manufacture. Although those strains survived well, they did not modify the acidification curve of milk. However, they might cause organoleptic defects during cheese maturing.

Bulk tank milk antibody ELISA as a biosecurity tool for detecting dairy herds with past exposure to Mycoplasma bovis

In Australia, one of the biosecurity recommendations to help prevent the introduction of Mycoplasma bovis into a dairy herd is to use a PCR assay on bulk tank milk (BTM) samples to evaluate the M. bovis infection status of potential source herds. An alternative approach is to assess the immunological status of the herd with respect to previous exposure to M. bovis via the use of an ELISA that is commercially available for use on cattle milk and serum. The objectives of this study were to (1) evaluate factors potentially associated with variation in the ELISA BTM optical density coefficient (ODC%) in previously exposed herds, (2) evaluate the association between the proportion of cows that are ELISA positive and the BTM ELISA ODC%, (3) assess agreement between the BTM ELISA and PCR and culture, and (4) compare BTM ELISA ODC% between the "hospital" herd and the main lactating herd on the same farm. Bulk tank milk samples (n = 192) were collected from 19 dairy herds with a history of clinical M. bovis disease and from 6 control herds (herds with no known clinical cases of mycoplasmosis). For 28 of the BTM samples collected, blood was also collected from 50 lactating cows contributing to that bulk tank sample. From 1 herd, concurrent paired BTM samples were collected from the main herd and the hospital herd on 16 occasions. All BTM samples were analyzed by ELISA (Bio-X Bio K 302, Bio-X Diagnostics, Rochefort, Belgium), PCR, and culture. The BTM ELISA ODC% was associated with time since initial M. bovis outbreak and time since the start of the herd's calving period. Following an initial outbreak of M. bovis, the BTM ELISA ODC% was highest in the first 8 mo. In split- and seasonal-calving herds, significantly higher BTM ELISA ODC% results were observed 5 to 8 wk after the commencement of the calving period. A significant association was observed between the within-herd seroprevalence for the lactating herd and BTM ELISA ODC%, but within-herd seroprevalence explained little of the variation in BTM ELISA ODC%. When comparing the BTM ELISA with a multiplex probe PCR and culture followed by 16S to 23S rRNA sequencing, there was virtually no agreement above that expected by chance; prevalence-adjusted bias-adjusted kappa values were 0.22 and 0.25 for ELISA category versus PCR category and culture, respectively. Finally, the hospital herd BTM ELISA ODC% mirrored that for the main herd BTM but was significantly higher. This study demonstrates that this commercially available ELISA used on BTM samples may complement the use of BTM PCR or culture in identifying herds from which purchase of animals may pose a higher biosecurity risk for introduction of M. bovis into noninfected herds.

Spoilage of Microfiltered and Pasteurized Extended Shelf Life Milk Is Mainly Induced by Psychrotolerant Spore-Forming Bacteria that often Originate from Recontamination

Premature spoilage and varying product quality due to microbial contamination still constitute major problems in the production of microfiltered and pasteurized extended shelf life (ESL) milk. Spoilage-associated bacteria may enter the product either as part of the raw milk microbiota or as recontaminants in the dairy plant. To identify spoilage-inducing bacteria and their routes of entry, we analyzed end products for their predominant microbiota as well as the prevalence and biodiversity of psychrotolerant spores in bulk tank milk. Process analyses were performed to determine the removal of psychrotolerant spores at each production step. To detect transmission and recontamination events, strain typing was conducted with isolates obtained from all process stages. Microbial counts in 287 ESL milk packages at the end of shelf life were highly diverse ranging from <1 to 7.9 log cfu/mL. In total, 15% of samples were spoiled. High G+C Gram-positive bacteria were the most abundant taxonomic group, but were responsible for only 31% of spoilage. In contrast, psychrotolerant spores were isolated from 55% of spoiled packages. In 90% of samples with pure cultures of Bacillus cereus sensu lato and Paenibacillus spp., counts exceeded 6 log cfu/mL. In bulk tank milk, the concentration of psychrotolerant spores was low, accounting for merely 0.5 ± 0.8 MPN/mL. Paenibacillus amylolyticus/xylanexedens was by far the most dominant species in bulk tank milk (48% of all isolates), but was never detected in ESL milk, pointing to efficient removal during manufacturing. Six large-scale process analyses confirmed a high removal rate for psychrotolerant spores (reduction by nearly 4 log-units). B. cereus sensu lato, on the contrary, was frequently found in spoiled end products, but was rarely detected in bulk tank milk. Due to low counts in bulk tank samples and efficient spore removal during production, we suggest that shelf life is influenced only to a minor extent by raw-milk-associated factors. In contrast, recontamination with spores, particularly from the B. cereus complex, seems to occur. To enhance milk quality throughout the entire shelf life, improved plant sanitation and disinfection that target the elimination of spores are necessary.

The potential of Fourier transform infrared spectroscopy of milk samples to predict energy intake and efficiency in dairy cows

Knowledge of animal-level and herd-level energy intake, energy balance, and feed efficiency affect day-to-day herd management strategies; information on these traits at an individual animal level is also useful in animal breeding programs. A paucity of data (especially at the individual cow level), of feed intake in particular, hinders the inclusion of such attributes in herd management decision-support tools and breeding programs. Dairy producers have access to an individual cow milk sample at least once daily during lactation, and consequently any low-cost phenotyping strategy should consider exploiting measureable properties in this biological sample, reflecting the physiological status and performance of the cow. Infrared spectroscopy is the study of the interaction of an electromagnetic wave with matter and it is used globally to predict milk quality parameters on routinely acquired individual cow milk samples and bulk tank samples. Thus, exploiting infrared spectroscopy in next-generation phenotyping will ensure potentially rapid application globally with a negligible additional implementation cost as the infrastructure already exists. Fourier-transform infrared spectroscopy (FTIRS) analysis is already used to predict milk fat and protein concentrations, the ratio of which has been proposed as an indicator of energy balance. Milk FTIRS is also able to predict the concentration of various fatty acids in milk, the composition of which is known to change when body tissue is mobilized; that is, when the cow is in negative energy balance. Energy balance is mathematically very similar to residual energy intake (REI), a suggested measure of feed efficiency. Therefore, the prediction of energy intake, energy balance, and feed efficiency (i.e., REI) from milk FTIRS seems logical. In fact, the accuracy of predicting (i.e., correlation between predicted and actual values; root mean square error in parentheses) energy intake, energy balance, and REI from milk FTIRS in dairy cows was 0.88 (20.0MJ), 0.78 (18.6MJ), and 0.63 (22.0MJ), respectively, based on cross-validation. These studies, however, are limited to results from one research group based on data from 2 contrasting production systems in the United Kingdom and Ireland and would need to be replicated, especially in a range of production systems because the prediction equations are not accurate when the variability used in validation is not represented in the calibration data set. Heritable genetic variation exists for all predicted traits. Phenotypic differences in energy intake also exists among animals stratified based on genetic merit for energy intake predicted from milk FTIRS, substantiating the usefulness of such FTIR-predicted phenotypes not only for day-to-day herd management, but also as part of a breeding strategy to improve cow performance.

Methicillin-resistant Staphylococcus aureus (MRSA) in sheep and goat bulk tank milk from Southern Italy

Methicillin-resistant Staphylococcus aureus (MRSA), an important widespread cause of severe infection in both humans and animals, is a pathogen of significant public health concern. In this paper the prevalence of MRSA in sheep and goat bulk tank milk from southern Italy was assessed and nasal swabs from people working on the positive farms were collected. MRSA isolates from milk and nasal swabs were characterized by spa typing, MLST and SCCmec typing and tested for antimicrobial resistance. MRSA was detected in 2 of the 162 (1.23%) bulk tank samples analysed: the first from a sheep farm (Farm 1) and the second from a goat farm (Farm 2). As for farm personnel, no MRSA was detected in the unique nasal swab from Farm 1; while MRSA was detected in all the 3 nasal swabs from Farm 2. The MRSA isolate from Farm 1 was spa type t127, ST1, SCCmec IVa. The MRSA isolates from bulk tank milk and humans in Farm 2 showed an identical genetic profile: spa type t1255, ST398, SCCmec V. In Farm 2 all the isolates had the characteristics of general multidrug resistance and the milk and human MRSA had the same antimicrobial resistance pattern. Although the results showed low prevalence of MRSA in sheep and goat milk from southern Italy, the detection of recognized zoonotic genotypes in milk of both farms and the isolation in people working in one of the positive farm of MRSA strains with a genetic profile identical to that of MRSA from milk, emphasize the public health concern and highlight the need for additional surveillance including the detection of MRSA.

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.

Spore populations among bulk tank raw milk and dairy powders are significantly different

To accommodate stringent spore limits mandated for the export of dairy powders, a more thorough understanding of the spore species present will be necessary to develop prospective strategies to identify and reduce sources (i.e., raw materials or in-plant) of contamination. We characterized 1,523 spore isolates obtained from bulk tank raw milk (n=33 farms) and samples collected from 4 different dairy powder-processing plants producing acid whey, nonfat dry milk, sweet whey, or whey protein concentrate 80. The spores isolated comprised 12 genera, at least 44 species, and 216 rpoB allelic types. Bacillus and Geobacillus represented the most commonly isolated spore genera (approximately 68.9 and 12.1%, respectively, of all spore isolates). Whereas Bacillus licheniformis was isolated from samples collected from all plants and farms, Geobacillus spp. were isolated from samples from 3 out of 4 plants and just 1 out of 33 farms. We found significant differences between the spore population isolated from bulk tank raw milk and those isolated from dairy powder plant samples, except samples from the plant producing acid whey. A comparison of spore species isolated from raw materials and finished powders showed that although certain species, such as B. licheniformis, were found in both raw and finished product samples, other species, such as Geobacillus spp. and Anoxybacillus spp., were more frequently isolated from finished powders. Importantly, we found that 8 out of 12 genera were isolated from at least 2 different spore count methods, suggesting that some spore count methods may provide redundant information if used in parallel. Together, our results suggest that (1) Bacillus and Geobacillus are the predominant spore contaminants in a variety of dairy powders, implying that future research efforts targeted at elucidating approaches to reduce levels of spores in dairy powders should focus on controlling levels of spore isolates from these genera; and (2) the spore populations isolated from bulk tank raw milk and some dairy powder products are significantly different, suggesting that targeting in-plant sources of contamination may be important for achieving low spore counts in the finished product. These data provide important insight regarding the diversity of spore populations isolated from dairy powders and bulk tank raw milk, and demonstrate that several spore genera are detected by multiple spore count methods.

Estimated herd prevalence and sequence types of Coxiella burnetii in bulk tank milk samples from commercial dairies in Indiana.

BackgroundCoxiella burnetii is the etiologic agent of Q fever, a zoonotic disease causing influenza-like illness, pregnancy loss, cardiovascular disease and chronic fatigue syndrome in people. C. burnetii is considered to be enzootic in ruminants, but clinical signs of infection do not always manifest. National studies have documented the presence of C. burnetii in dairy herds in Indiana. This represents an opportunity to better characterize the distribution and prevalence of C. burnetii infection at the state scale, allowing evaluation of the need for surveillance and response planning to occur at this level. A cross-sectional study was conducted to estimate the herd prevalence of C. burnetii in commercial cattle dairies in Indiana and characterize the strains of C. burnetii within these dairies.ResultsBulk tank milk samples were collected between June and August of 2011 by the Indiana State Board of Animal Health (ISBOAH). A total of 316 of these samples were tested for the IS1111 transposon of C. burnetii using quantitative real time polymerase chain reaction (PCR). Single nucleotide polymorphism (SNP) genotyping was used to identify the multispacer sequence genotypes (ST) present in samples where the IS1111 transposon was identified. The geographic distribution of dairies testing positive for C. burnetii DNA and the identified STs were also evaluated. The estimated overall herd prevalence for C. burnetii DNA was 61.1 % (95 % CI 55.6–66.3 %). The highest estimated regional prevalence was 70.2 % in the Central region of Indiana. An ST was identifiable in 74 of the positive 178 samples (41.6 %) and none of the 10 negative samples tested. Of these samples, 71 (95.9 %) were identified as ST20, 2 (2.7 %) as ST8 and a combination of ST20 and ST8 was identified in a single sample.ConclusionsC. burnetii is present in dairy herds throughout Indiana. Indiana follows national trends with ST20 most commonly identified. The presence of multiple STs in a single bulk tank sample indicates that multiple strains of C. burnetii can circulate within a herd. This supports potential transmission of C. burnetii between goats and cattle, presenting the potential for a switch in the dominant genotype found in a given species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0517-3) contains supplementary material, which is available to authorized users.

Identification of dairy farm management practices associated with the presence of psychrotolerant sporeformers in bulk tank milk

Some strains of sporeforming bacteria (e.g., Bacillus spp. and Paenibacillus spp.) can survive pasteurization and subsequently grow at refrigeration temperatures, causing pasteurized fluid milk spoilage. To identify farm management practices associated with different levels of sporeformers in raw milk, a bulk tank sample was obtained from and a management and herd health questionnaire was administered to 99 New York State dairy farms. Milk samples were spore pasteurized [80°C (176°F) for 12 min] and subsequently analyzed for most-probable number and for sporeformer counts on the initial day of spore pasteurization (SP), and after refrigerated storage (6°C) at 7, 14, and 21 d after SP. Management practices were analyzed for association with sporeformer counts and bulk tank somatic cell counts. Sixty-two farms had high sporeformer growth (≥3 log cfu/mL at any day after SP), with an average sporeformer count of 5.20±1.41 mean log10 cfu/mL at 21 d after SP. Thirty-seven farms had low sporeformer numbers (<3 log cfu/mL for all days after SP), with an average sporeformer count of 0.75±0.94 mean log10 cfu/mL at 21 d after SP. Farms with >25% of cows with dirty udders in the milking parlor were 3.15 times more likely to be in the high category than farms with ≤10% of milking cows with dirty udders. Farms with <200 cows were 3.61 times more likely to be in the high category than farms with ≥200 cows. Management practices significantly associated with increased bulk tank somatic cell count were a lack of use of the California mastitis test at freshening and >25% of cows with dirty udders observed in the milking parlor. Changes in management practices associated with cow cleanliness may directly ensure longer shelf life and higher quality of pasteurized fluid milk.

Epidemiological features of BLV natural infection

Dairy farms are heavily infected with Bovine Leukemia Virus (BLV) in Argentina and many other countries, where a control strategy should be design based on the behavior of natural. We conducted a series of studies with the aim to better understand the epidemiology of BLV. Infected new born calves were present in 3 studied farms (8.3%-11%). Proviral load (PVL) was very low in 9/10 new born analyzed calves but rapidly augmented during the first months of age. Cross-sectional studies showed that the rate of high PVL between seroreactors raised together with the prevalence, from 20.2% at 8 months of age to 44.4% in 26 months heifers, with similar levels to adult lactating cows of the same farm. Low fluctuation of blood PVL was observed on animals with naturally-acquired infections. We also observed 10 seroconversions between young heifers with an initial elevation of PVl. The presence of provirus in colostrum was significantly correlated with blood PVL (p≤0.0001). Provirus in milk was detectable in bulk tank (17.2%) and individual samples (40.4%). Colostrum of individual cows showed different provirus/antibodies dual profiles that permit to speculate about different infective/protective potential among infected animals. These findings suggest animals would be exposed to the infective challenge since a very young age. Consequently, it must be control as soon as possible after birth. The main focus should be put on the new-born infected calves that could play the role of main propagators together with the putative oral exposition through colostrum and milk.