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.

Presence of Mycoplasma bovis in Bulk Tank Milk and Associated Risk Factor Analysis in Serbian Dairy Farms.

Mycoplasma bovis (M. bovis) is a significant pathogen responsible for highly transmissible mastitis in cattle globally. It primarily spreads through colostrum, milk, and semen. Cows with persistent infections act as carriers, intermittently releasing the pathogen, making their milk a pivotal factor in infection transmission. Given the limited seroprevalence surveys in Serbia, this study aimed to detect M. bovis presence in bulk tank milk (BTM), determine route shedding, and evaluate infection risks. BTM samples were collected from 115 dairy farms across Serbia, with M. bovis DNA detected in 11 out of the 115 samples by real-time PCR. Additionally, M. bovis was detected in 1.30% of nasal swabs sampled from apparently healthy animals. A univariate analysis of the risk factors associated with M. bovis presence in the BTM samples revealed correlations with factors such as the breed, farm seropositivity, pre-milking and post-milking disinfection practices, farm type, cow population, milk yield, number of cows in the BTM samples, and parity. Seropositive farms exhibited the highest likelihood of M. bovis presence in milk. Moreover, pre- and post-milking disinfection practices and highly productive cows yielding over 8000 L of milk were identified as risk factors for PCR-positive BTM. In a multivariable mixed regression analysis, a risk factor for the presence of M. bovis infection in the BTM sample was the Holstein breed. These findings underscore a relatively high prevalence of M. bovis in BTM within Serbian dairy farms, suggesting a potential risk for M. bovis spreading through milk and oral route of calves' infection.

The Transmission of Campylobacter Strains in Dairy Herds in Different Housing Systems.

Cattle are frequent carriers of Campylobacter spp.; therefore, these bacteria may be transmitted to humans through meat or milk. Campylobacter spp. in raw milk derives most commonly from secondary fecal contamination during the milking process; however, the udder excretion of Campylobacter may be a cause of milk-borne infection. Studies were carried out on a Campylobacter-positive farm with two different housing systems (with free-stall and tie-stall systems). The sampling process comprised several stages, including samples being taken from animals, such as from raw milk and feces, and from the environment, such as the from floor in the milking parlor and from teat cups. None of the individual raw milk samples or swabs from the floor in the parlor before the milking process were positive for Campylobacter spp. Simultaneously, Campylobacter spp. was isolated from all swabs from the floor after the milking process and in the bulk tank milk samples from the two farms. The incidence of Campylobacter isolated from fecal and teat swab samples ranged from 15.4% to 26.7% and from 8.9% to 25%, respectively. Altogether, 59 recovered Campylobacter isolates were classified, based on sequencing of the flaA short variable region, showing 15 different allele types, and the majority of them were distributed among one farm. Analysis of the virulence and antimicrobial properties showed that genes related to adherence, invasion and cytotoxicity were widely distributed among the Campylobacter recovered strains. In relation to AMR, multidrug resistance was noted in 16.1% of strains.

Extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and risk factors associated with high total bacterial count in bulk tank milk from dairy farms in Colombia.

The objective of the study was to evaluate the frequency and genetic characteristics of ESBL-producing Escherichia coli and Klebsiella spp. and the risk factors associated with a high total bacterial count in bulk tank milk samples of dairy farms in three municipalities of the Antioquia Department, Colombia. Fifteen samples were positive for E. coli and Klebsiella spp. Subsequent analysis of the 16 S rRNA gene sequences confirmed these isolates included E. coli (n = 3), K. oxytoca (n = 11), and K. pneumoniae (n = 1). None of the isolates was positive for ESBL identification by phenotypic methods, but the only the isolate of K. pneumoniae was positive for the blaSHV61 gene by sequence analysis. The antibiotic susceptibility evaluation for all Klebsiella spp. isolates identified resistance to fosfomycin (50%; 6/12) and ampicillin (100%; 12/12). While most of the herds maintain adequate hygienic quality, specific risk factors such as having more than 60 milking cows, frequent changes in milkers, milking in paddocks, and using a chlorinated product for pre-dipping have been identified as associated with a high total bacterial count > 100,000 CFU/mL in bulk tank milk. However, certain variables including the milker being the owner of the animals and the proper washing and disinfection of the milking machine contribute to maintain a high level of hygiene and quality in the raw milk stored in the tanks. In conclusion, the frequency of ESBL producers was relatively low, with only K. pneumoniae testing positive for the blaSHV ESBL type. The presence of these bacteria in milk tanks represents a potential risk to public health for consumers of raw milk and its derivatives.

Detection of Coxiella burnetii in the mammary gland of a dairy goat.

The zoonotic bacterium Coxiella (C.) burnetii can be excreted by infected goats through birth products and milk. The detection of C. burnetii DNA in the mammary gland tissue of infected dairy goats and intermittent milk shedders has been reported, but confirmation of C. burnetii bacteria in the udder remained pending. The pathogen caused abortions in a 152-head dairy goat herd, resulting in the vaccination against C. burnetii of the entire herd with annual boosters. To monitor the C. burnetii shedding at herd level, monthly bulk tank milk (BTM) samples were analyzed using PCR (IS1111). Despite vaccination, C. burnetii DNA was detected in BTM samples within the first 16 months of the study. Therefore, individual milk samples were tested on four different occasions several months apart to identify potential intermittent milk shedders. Only one goat (#67455) tested positive three times. This goat was necropsied to investigate the presence of C. burnetii in the udder and other organs. PCR detected C. burnetii DNA solely in both mammary glands and the left teat cistern. Immunohistological examination identified C. burnetii antigen in mammary gland tissue, confirmed by the detection of C. burnetii bacteria in the mammary epithelial cells using fluorescence in situ hybridization. The removal of goat #67455 led to negative BTM samples until the end of the study. The findings demonstrate the occurrence of C. burnetii in the mammary gland of a naturally infected and vaccinated goat. The presence possibly contributed to intermittent milk shedding of goat #67455, and the mammary gland tissue may serve as a replicative niche for C. burnetii.

Reference Material Production and Milk Protein Concentration as Elements to Improve Bluetongue Serological Diagnosis in Bulk Tank Milk.

The serological surveillance of bluetongue in bulk tank milk is an efficient and cost-effective method for the early detection of bluetongue virus incursions in unvaccinated free areas of the disease. In addition, the availability of standardized and reliable reagents and refined diagnostic procedures with high sensitivity and specificity are essential for surveillance purposes. However, no available reference materials for bluetongue virus serological surveillance in bulk tank milk exist. This study shows the production and characterization of reference material for the implementation of a commercially available bluetongue milk ELISA test in official laboratories, as well as the evaluation of a procedure to increase the sensitivity in samples with low levels of antibodies. This procedure, based on milk protein concentration, allowed us to notably increase the ELISA test's analytical sensitivity, which is useful for milk samples from farms with low within-herd prevalence or pools of bulk tank milk samples. The standardized milk reference material produced here, together with the evaluated procedure to improve analytical sensitivity, could be applied as tools to ensure an accurate diagnosis by official laboratories in bluetongue unvaccinated free areas.

Influência de diferentes tipos de micro-organismos na contagem bacteriana total por citometria de fluxo do leite cru refrigerado / Influence of different types of microorganisms in total bacterial count by flow cytometry of refrigerated raw milk

Contagens bacterianas de 179 amostras de leite cru refrigerado, provenientes de diferentes bacias leiteiras de Minas Gerais, foram realizadas pelos métodos de citometria de fluxo e contagem padrão em placas. O estudo das associações entre a contagem bacteriana total e as contagens bacterianas dos diferentes grupos de micro-organismos foi realizado pela correlação de Pearson. Houve uma forte correlação positiva e significativa entre os valores de contagem de psicrotróficos (r = 0,67) e mesófilos (r = 0,70), determinados pelo método de referência, e os valores da contagem bacteriana total, determinados pela citometria de fluxo. Foi possível estabelecer uma curva de calibração para o citômetro de fluxo utilizando equações de regressão linear. As equações para a contagem bacteriana obtida por citometria de fluxo em função da contagem de mesófilos e psicrotróficos foram: log (CBI) = 0,701 log mesófilos + 1,9091 e log (CBI) = 0,4703 log psicrotróficos + 3,2784, respectivamente. Não foi possível estabelecer curvas de calibrações para termófilos e termodúricos, pois as correlações obtidas entre as contagens desses micro-organismos e a contagem bacteriana total, determinada por citometria de fluxo, foram fracas (r = 0,30 e 0,27, respectivamente).(AU)

Total Bacterial count was performed in 179 raw milk samples from different dairy farms in Minas Gerais State, Brazil, through flow cytometry and standard plate count, that was regarded as the reference method. There was significant strong correlation between the values of psychrotrophic and mesophilic counts determined by the reference method with the values of bacterial count determined by flow cytometry (r = 0.67 and 0.70, respectively), which allowed to establish linear regression equations for bacterial count to calibrate the flow cytometry and estimate the count of mesophilic (log bacterial individual count (BIC) by flow cytometry = 0.701 log mesophilic + 1.9091) and psychrotrophic (log BIC by flow cytometry = 0.4703 log psychrotrophic + 3.2784) bacteria. It was not possible to establish calibration curves for thermophiles and thermodurics due to the weak to moderate correlation coefficients found among these microorganism counts and total bacterial count determined by flow cytometry (r = 0.30 and 0.27, respectively).(AU)

Molecular identification and thermoresistance to boiling of Nocardia farcinica and Nocardia cyriacigeorgica from bovine bulk tank milk

Two strains of Nocardia spp. were isolated from bovine milk of two individual bulk tank. Molecular identification classified the strains as Nocardia farcinica and Nocardia cyriacigeorgica. The thermorresistance to boiling of the isolates was carried out and was observed bacterial growth after boiling. Our findings indicate the potential risk of pathogen transmission to humans through contaminated milk with Nocardia spp.

Association between milking practices and psychrotrophic bacterial counts in bulk tank milk / Asociación entre prácticas de ordeño y recuento de organismos psicrótrofos en leche de tanque de frío

The objective of this work was to determine on-farm risk factors for psychrotrophic bacterial counts in bulk tank milk from dairy farms in Argentina. Raw milk samples from bulk tanks of 27 dairy farms were examined for total psychrotrophic counts (TPC), proteolytic psychrotrophic counts (PPC) and lipolytic psychrotrophic counts (LPC) (dependent or outcome variables). A survey recording infrastructure conditions, milking equipment and milking management (independent variables) was performed. Bivariate association proofs and logistic regression analyses were used to determine association between independent variables and psychrotrophic bacterial counts. Milk cooled in plate heat exchangers or barrel tanks were 16.39 and 10.52 times more likely to yield TPC and PPC above the standard established for high quality milk compared with milk cooled in bulk tanks, respectively. Periodic cleaning of cooling tanks (3 times a week or daily) was associated with lower TPC (approximately 1.5 log CFU/ml) than weekly cleaning frequency and farms where milkers did not wash their hands during milking time were 7.81 times more likely to have higher PPC. No association was found between LPC and any of the independent variables. The only variable associated with TPC and PPC in a logistic regression model was the refrigeration system used on the farm. Dairy farms that possessed bulk milk cooling tanks yielded the lowest bacterial counts. Results of this study highlight the importance of both the type of cooling system used on the farm and its adequate hygienic maintenance for obtaining low pshychrotrophic counts at dairy farm.

El objetivo del trabajo fue determinar los factores de riesgo para altos recuentos de organismos psicrótrofos en leche de tanques de tambos de la Argentina. Se examinaron muestras de leche cruda de tanques de frío de 27 tambos, y se realizó el recuento de organismos psicrótrofos totales (PT), de psicrótrofos proteolíticos (PP) y de psicrótrofos lipolíticos (PL) (variables dependientes). Se realizó una encuesta para registrar las condiciones de infraestructura, el equipo de ordeño y las prácticas de ordeño (variables independientes). Se utilizaron pruebas bivariadas de asociación y regresión logística para determinar la asociación entre las variables independientes y los recuentos de organismos psicrótrofos. La leche enfriada en sistemas de placas de intercambio o tanques tipo cuba tuvo una probabilidad mayor de dar recuentos elevados de PT y PP (16,39 y 10,52) comparada con la enfriada en tanques tipo "panza fría". La limpieza periódica del equipo de frío (3 veces por semana o diariamente) se asoció con bajos recuentos de PT (aproximadamente 1,5 log de UFC/ml). Los tambos cuyos ordeñadores no se higienizaban las manos durante el ordeño tuvieron una probabilidad 7,81 veces mayor de tener recuentos elevados de PP. No se encontró asociación entre el recuento de PL y las variables independientes. La única variable asociada con los recuentos de PT y PP en el modelo de regresión logística fue el sistema de enfriamiento utilizado en el tambo. El tipo de sistema de refrigeración usado y su adecuado mantenimiento higiénico son importantes para la obtención de leche con baja carga de organismos psicrótrofos en el tambo.

Presence of enterotoxigenic Staphylococcus aureus in bulk tank milk from Argentine dairy farms / Presencia de Staphylococcus aureus enterotoxigénico en leche de tanque de frío de tambos de Argentina

Staphylococcus aureus is the most prevalent bovine mastitis pathogen in Argentina. The ability of this organism to produce enterotoxins is linked to staphylococcal food poisoning. Staphylococcal enterotoxins are low molecular weight proteins, highly resistant to heat and proteolytic enzyme activity. The aim of this study was to determine the ability to produce enterotoxins and types of enterotoxins A through E produced among 94 S. aureus isolated from bulk tank milk in Argentina by enzyme-linked immunosorbent assay. Eleven isolates (11.7 %) produced enterotoxins. Seven of them (7.4 %) produced enterotoxin C, two (2.1 %) enterotoxin D, one (1.1 %) enterotoxin B and one (1.1 %) enterotoxins C-D-E. None of the isolates produced enterotoxins A or E alone. Since presence of staphylococcal enterotoxins constitute a potential risk to public health, these findings underscore the need to control S. aureus bovine mastitis and to limit bacterial multiplication in bulk tank milk.

Staphylococcus aureus es el patógeno causante de mastitis más prevalente en Argentina. Las enterotoxinas producidas por este organismo constituyen una de las causas más importantes de intoxicación alimentaria en seres humanos. Las enterotoxinas estafilocócicas son proteínas de bajo peso molecular, termoestables y resistentes a enzimas proteolíticas. El objetivo del presente trabajo fue determinar por enzimoinmunoensayo la presencia de enterotoxinas A-E y establecer su tipo en 94 aislamientos de Staphylococcus aureus obtenidos de leche de tanque de frío de tambos de Argentina. Se identificaron 11 % aislamientos enterotoxigénicos (11,7 %); siete (7,4 %) produjeron enterotoxina C, dos produjeron enterotoxina D (2,1 %), uno produjo enterotoxina B (1,1 %) y uno produjo enterotoxinas C-D-E (1,1 %). No se detectaron aislamientos que produjeran enterotoxinas A o E solamente. Estos hallazgos indican la necesidad de implementar un eficaz control de la mastitis bovina para disminuir la presencia de S. aureus en leche de tanque y evitar riesgos potenciales para la salud pública.

Qualidade higiênica de leite caprino por contagem de coliformes e estafilococos / Goat's milk quality by coliform's and staphylococci counting

O objetivo deste estudo foi investigar os padrões higiênicos do leite de mistura caprino produzido por oito propriedades associadas a uma cooperativa na região do Vale do Taquari - RS. Foram realizadas duas visitas às propriedades durante o período de maior produção leiteira. As propriedades foram avaliadas quanto às instalações, ao manejo de ordenha e ao armazenamento do leite.Foram coletadas amostras de leite e água em cada visita. As amostras de água apresentaram contagem de coliformes dentro dos limites propostos pela legislação vigente, já as de leite apresentaram contagens de coliformes totais que variaram de zero a 1,4 x 10(6)UFC mL-1. Em apenas duas propriedades foram encontrados coliformes fecais. Não foram identificados estafilococos coagulase-positiva nas amostras. Não foi evidenciada relação significativa entre os sistemas de resfriamento e ordenha empregados na propriedade e a presença de coliformes no leite.

The aim of this work was to evaluate the current hygiene-sanitary quality of milk produced by eight dairy goat farms located in Vale do Taquari region, state of Rio Grande do Sul, Brazil. On each farm, two samplings were performed during the highest milk yield period. At each sampling, bulk milk and water samples were taken, and farms were checked for their facilities, milking management and milk storage. Water samples were subjected to fecal coliform enumeration, while milk samples were tested for total and fecal colifoms as well as for coagulase-positive staphylococci. Water samples presented fecal coliform levels according to the national legislation. In milk samples total coliform counts up to 1.4 x 10(6)CFU mL-1 were found. Coagulase-positive staphylococci were not isolated and fecal coliforms were detected in two farms. No correlation was observed between the number of total coliforms and milking management and milk storage.