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The aim of this study was to evaluate the effect of sampling technique and milk fraction on bovine milk microbiota data and to compare the microbiota in milk to microbiota on the teat end and in the teat canal. Representative milk samples are highly important for assessment of bacteriological findings and microbiota in milk. Samples were obtained from 5 healthy lactating dairy cows at udder quarter level during 1 milking. Swab samples from the teat. end and teat canal, and milk samples collected using different techniques and in different milk fractions were included. Milk was collected by hand stripping and through a teat canal cannula before and after machine milking, through a trans-teat wall needle aspirate after milking, and from udder quarter composite milk. The microbiota of the samples was analyzed with sequencing of the V1-V3 region of the 16S rRNA gene. In addition, somatic cell counts and bacterial cultivability were analyzed in the milk samples. Microbiota data were analyzed using multivariate methods, and differences between samples were tested using analysis of similarity (ANOSIM). Differences between samples were further explored via individual studies of the 10 most abundant genera. The microbiota on the teat end, in the teat canal, and in udder quarter composite milk, collected using a milking machine, differed in composition from the microbiota in milk collected directly from the udder quarter. No differences in milk microbiota composition were detected between hand-stripped milk samples, milk samples taken through a teat canal cannula, or milk samples taken as a trans-teat wall needle aspirate before or after milking. We conclude that for aseptic milk samples collected directly from the lactating udder quarter, sampling technique or milk fraction has minor effect on the microbiota composition.

Discoveries of bacterial communities in environments that previously have been described as sterile have in recent years radically challenged the view of these environments. In this study we aimed to use 16S rRNA sequencing to describe the composition and temporal stability of the bacterial microbiota in bovine milk from healthy udder quarters, an environment previously believed to be sterile. Sequencing of the 16S rRNA gene is a technique commonly used to describe bacterial composition and diversity in various environments. With the increased use of 16S rRNA gene sequencing, awareness of methodological pitfalls such as biases and contamination has increased although not in equal amount. Evaluation of the composition and temporal stability of the microbiota in 288 milk samples was largely hampered by background contamination, despite careful and aseptic sample processing. Sequencing of no template control samples, positive control samples, with defined levels of bacteria, and 288 milk samples with various levels of bacterial growth, revealed that the data was influenced by contaminating taxa, primarily Methylobacterium. We observed an increasing impact of contamination with decreasing microbial biomass where the contaminating taxa became dominant in samples with less than 104 bacterial cells per mL. By applying a contamination filtration on the sequence data, the amount of sequences was substantially reduced but only a minor impact on number of identified taxa and by culture known endogenous taxa was observed. This suggests that data filtration can be useful for identifying biologically relevant associations in milk microbiota data.

This study investigated milk somatic cell count (SCC), polymorphonuclear leukocytes (PMNs) and milk composition in dairy cows, which were kept in tie stalls, during the first days after pasture turnout. Thirty-five cows of the Swedish Red Breed, free of clinical signs of mastitis and with a geometric mean of SCC 67 × 103 cells/mL, were turned out to pasture after the morning milking on day 0 and then monitored for the next five days on pasture. Samples of cow composite milk were taken at every milking and analysed for SCC, PMN percentage of the total SCC and milk composition. There was a marked increase in both PMN proportion and SCC with the highest SCC value during the study recorded at the evening milking on day 0. The highest value in morning milk was observed on day 1. Milk SCC values in evening and morning milk declined after day 0 and day 1, respectively, but remained on a higher level compared with before turnout to pasture. Milk composition was only slightly altered. Since the changes in ...

A technical stop in automatic milking systems may result in a severely prolonged milking interval (PMI) with subsequent impact on milk somatic cell count (SCC). This study investigated the inflammatory reaction, milk composition and yield during SCC peak observed in composite milk after exposing cows to a single PMI of 24h. At the first milking after the PMI, a sharply increased proportion of milk polymorphonuclear leukocytes (PMN) but marginally increased SCC were observed. The peak in SCC was not seen until morning milking day 2 after the PMI, notably, concomitantly with a decreased PMN proportion. An increase in blood lactose, milk bovine serum albumin and serum amyloid A (SAA) and a drop in milk alpha lactalbumin (ALA) were seen concomitantly with the peak in PMN. All parameters mentioned, had returned to base line after day 2. The changes in SCC and SAA had the longest duration. Lactate dehydrogenase in afternoon milk was decreased during the whole study as was also afternoon milk yield. Interleukin-1β could not be detected in milk at any time. SAA and ALA, respectively, may influence chemotaxis and the changed concentrations observed after the PMI might have contributed to the increased migration of PMN to milk.

Dairy production is not traditional in Vietnam. The farmers have little practical knowledge and udder health control is generally lacking. In order to give the farmers appropriate advice, knowledge about the distribution of udder pathogens is crucial. The aim of the study was to investigate the prevalence of sub-clinical mastitis and to identify udder pathogens isolated from smallholder dairy herds in Southern Vietnam. Twenty farms with a herd somatic cell count (SCC) ranging from low (≤ 400 × 10(3)cells/mL) to high (400 × 10(3)cells/mL) were randomly selected. Milk samples were collected from 458 quarters of 115 clinically healthy cows. SCC was analyzed on farm by a portable cell counter. Bacteriological samples were taken using Mastistrip(©) cassettes and sent to Sweden for examination. For all herds the mean herd SCC was 632 × 10(3)/mL milk. The prevalence of subclinical mastitis at quarter SCC basis was 63.2 % and at cow basis 88.6 %. Only 40 % of all cows were bacteriologically negative in all quarters. Streptococcus agalactiae was the most commonly found bacteria species, isolated from 96 of the 458 quarter samples, in 13 of the 20 farms. The results indicate pronounced subclinical mastitis problems among the dairy cows in this region mainly due to infections with S. agalactiae. The high prevalence of this highly contagious pathogen is probably attributable to the generally poor milking hygiene and low awareness of proper measures to prevent occurrence and spread of udder infections. A strict, targeted action program for the herds in this area is required in order to lower the prevalence of subclinical mastitis.

Milking-related release of oxytocin, prolactin, and cortisol was studied following three pre-milking treatments. Six Murrah buffaloes were treated with direct application of milking cluster (O), a 1-min pre-stimulation (M), and combined feeding and pre-stimulation (MF). Machine milk yield, stripping yield and milk composition were recorded. Milk ejection occurred significantly earlier with MF than M and O (P3–5 ng/l in oxytocin concentration. Increase in oxytocin concentration over a threshold level and milk ejection occurred simultaneously and were closely correlated (r=0·83, Pr=0·86, PP

Cytokine kinetics were examined in milk and in afferent and efferent lymph of the supramammary lymph node after intramammary infusion of endotoxin from Escherichia coli. Cows were sampled 0, 2 and 4h after infusion (p.i.). Neutrophils appeared in afferent lymph 2h p.i., and in efferent lymph and milk 4h p.i. The milk contained high concentrations of interleukin-8 (IL-8) at 2 and 4h p.i. IL-8 was also found in lymph, but at lower concentrations. The tumor necrosis factor-alpha (TNF-alpha) concentration tended to increase in afferent lymph at 2h p.i., and increased in milk at 4h p.i. The level of IL-1beta increased at 4h p.i. in milk, but was not detected in lymph. Interferon-gamma was not detected in any sample, at any time. The results indicate a primary role for IL-8 in the recruitment of neutrophils into the gland, and suggest that IL-1beta and TNF-alpha are not necessary for IL-8 production and release in response to endotoxin.

A total of 414 coagulase-positive staphylococcal strains obtained at the mastitis laboratory, National Veterinary Institute, Uppsala, Sweden, were studied. One hundred and seventy seven strains were used for a frequency study. Ninety-seven per cent were identified as Staphylococcus aureus, 2% as Staphylococcus intermedius and 1% as Staphylococcus hyicus. Two hundred and thirty seven strains with atypical hemolysis reactions on bovine blood agar were randomly selected, with the aim to increase the number of S. intermedius and S. hyicus strains available for testing. Eight different characteristics, including physiological, enzymatical and biochemical properties, were used to identify the coagulase-positive Staphylococcus species. The results of this study suggest that the following tests should be included for correct identification of the 3 different species of coagulase-positive staphylococci: P agar supplemented with acriflavin, beta-galactosidase and hemolytic reaction on chocolate agar. These 3 tests are simple and quick to perform and enable accurate for easy differentiation of the 3 coagulase-positive Staphylococcus species.

Background The first hours after antigen stimulation, interactions occur influencing the outcome of the immunological reaction. Immunoglobulins originate in blood and/or are locally synthesized. The transfer of Ig isotypes (Igs) in the udder has been studied previously but without the possibility to distinguish between the endothelium and the epithelium. The purpose of this study was to map the Ig transfer through each barrier, separately, and Ig transfer in the local lymph nodes of the bovine udder during the initial innate immune response. Methods The content of IgG1, IgG2, IgM, IgA and albumin (BSA) was examined in peripheral/afferent mammary lymph and lymph leaving the supramammary lymph nodes, and in blood and milk before (0 h) and during 4 hours after intramammary challenge with Esherichia coli endotoxin in 5 cows. Results Igs increased most rapidly in afferent lymph resulting in higher concentrations than in efferent lymph at postinfusion hour (PIH) 2, contrary to before challenge. Ig concentrations in milk were lower than in lymph; except for IgA at 0 h; and they increased more slowly. Afferent lymph:serum and efferent lymph:serum concentration ratios (CR) of Igs were similar to those of BSA but slightly lower. Milk:afferent lymph (M:A) CRs of each Ig, except for IgG2, showed strikingly different pattern than those of BSA. The M:A CR of IgG1, IgM and IgA were higher than that of BSA before challenge and the CR of IgA and IgG1 remained higher also thereafter. At PIH 2 there was a drop in Ig CRs, except for IgG2, in contrast to the BSA CR which gradually increased. The M:A CR of IgM and Ig A decreased from 0 h to PIH 4, in spite of increasing permeability. Conclusion The transfer of Igs through the endothelium appeared to be merely a result of diffusion although their large molecular size may hamper the diffusion. The transfer through the epithelium and the Ig concentrations in milk seemed more influenced by selective mechanisms and local sources, respectively. Our observations indicate a selective mechanism in the transfer of IgG1 through the epithelium also in lactating glands, not previously shown; a local synthesis of IgA and possibly of IgM, released primarily into milk, not into tissue fluid; that IgG2 transfer through both barriers is a result of passive diffusion only and that the content of efferent lymph is strongly influenced by IgG1, IgM and IgA in the mammary tissue, brought to the lymph node by afferent lymph.

Background A single prolonged milking interval (PMI) e.g. after a technical stop in an automated milking system is of concern for the producer since it is associated with a short-lasting increase in milk somatic cell count (SCC), which is a major quality criterion used at the dairy plants. The content of polymorphonuclear leukocytes (PMN) and how the milk quality is influenced has not been much investigated. The SCC peak occurs without any obvious antigen challenge, possibly indicating a different leukocyte attraction mechanism after a PMI than we see during mastitis. Methods Composite cow milk samples were taken at the milkings twice daily during 7 days before and 5 days after a PMI of 24 h. Milk was analyzed for SCC, PMN, fat, protein and lactose, and at some occasions also casein and free fatty acids (FFA). Results During the PMI the proportion of milk PMN increased sharply in spite of marginally increased SCC. The peak SCC was not observed until the second milking after the PMI, in the afternoon day 1. However, the peak SCC value in morning milk did not occur until one day later, concomitantly with a decrease in the proportion of PMN. After declining, SCC still remained elevated while PMN proportion was decreased throughout the study as was also the milk yield, after the first accumulation of milk during the PMI. Milk composition was changed the day after the PMI, (increased fat and protein content; decreased lactose, whey protein and FFA content) but the changes in the following days were not consistent except for lactose that remained decreased the rest of the study. Conclusion The PMI resulted in increased SCC and proportion of PMN. Additionally, it gave rise to minor alterations in the milk composition in the following milkings but no adverse effect on milk quality was observed. The recruitment of PMN, which was further enhanced the first day after the PMI, appeared to be independent of milk volume or accumulation of milk per se. Hence, we suggest that there is a special immunophysiological/chemoattractant background to the increased migration of leukocytes into the milk compartment observed during and after the PMI.

Cell con-centrations and cell populations were studied in fraction-collected milk by use of cytofluorometric methods. The results indicate differences in the condition of udders producing milk with more and less than 100 000 cells per ml, respectively

Quarter samples of foremilk and residual milk were taken approximately every second week from 2 days post partum (pp) throughout lactation month 9, from 5 dairy cows in their second lactation period. Bacteriologically positive milk samples were excluded. The aim was to study the variation in total and differential leukocyte counts, N-acetyl-beta-D-glucosaminidase (NAGase), antitrypsin (ATR) and serum albumin (BSA) in milk during the lactation period and different stages of oestrous cycle. Also the between milkings variation was studied from lactation month 4 to 9. At 2 days pp, each fraction of milk contained significantly higher numbers of leukocytes and had a higher activity of NAGase and ATR than later in the lactation period. In foremilk the highest content of BSA was also recorded at 2 days pp. From lactation month 2 to 9, stage of lactation had, in general, a slight effect on the variation in the variables measured. The total leukocyte count in residual milk tended to increase as lactation proceeded. The proportion of monocyte-macrophages in foremilk was significantly decreased during the last 4 months. NAGase and BSA in both fractions and ATR in residual milk increased significantly towards the end of the lactation period. From lactation month 4 to 9 the highest recorded ranges of variation between milkings, within quarter and stage of lactation, in the total leukocyte count, proportions of neutrophils, lymphocytes, monocyte-macrophages, NAGase, ATR and BSA in foremilk were 215 x 10(3)/ml, 42%, 34%, 54%, 6.68 units, 0.36 units and 0.14 mg/ml respectively. The corresponding figures in residual milk were higher except for the variation in BSA which was slightly lower in residual milk than in foremilk. In residual milk there was a positive correlation between the proportion of neutrophils and the total leukocyte count, when calculated on data from all cows and the entire experimental period. During the oestrous periods, the proportion of neutrophils in residual milk was higher than during the dioestrous periods. Foremilk and residual milk differed in the total as well as the differential leukocyte counts in all the various stages of lactation, whereas the contents of NAGase, ATR and BSA were equal in both fractions. The exception was 2 days pp when the proportions of lymphocytes were equal in both fractions and BSA-significantly higher in foremilk than in residual milk.