Absence of changes in the milk microbiota during Escherichia coli endotoxin induced experimental bovine mastitis.

Changes in the milk microbiota during the course of mastitis are due to the nature of a sporadic occurring disease difficult to study. In this study we experimentally induced mastitis by infusion of Escherichia coli endotoxins in one udder quarter each of nine healthy lactating dairy cows and assessed the bacteriological dynamics and the milk microbiota at four time points before and eight time points after infusion. As control, saline was infused in one udder quarter each of additionally nine healthy cows that followed the same sampling protocol. The milk microbiota was assessed by sequencing of the 16 S rRNA gene and a range of positive and negative controls were included for methodological evaluation. Two different data filtration models were used to identify and cure data from contaminating taxa. Endotoxin infused quarters responded with transient clinical signs of inflammation and increased SCC while no response was observed in the control cows. In the milk microbiota data no response to inflammation was identified. The data analysis of the milk microbiota was largely hampered by laboratory and reagent contamination. Application of the filtration models caused a marked reduction in data but did not reveal any associations with the inflammatory reaction. Our results indicate that the microbiota in milk from healthy cows is unaffected by inflammation.

Microbiota data from low biomass milk samples is markedly affected by laboratory and reagent contamination.

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.

The Effect of Lipopolysaccharide-Induced Experimental Bovine Mastitis on Clinical Parameters, Inflammatory Markers, and the Metabolome: A Kinetic Approach.

Mastitis is an inflammatory condition of the mammary tissue and represents a major problem for the dairy industry worldwide. The present study was undertaken to study how experimentally induced acute bovine mastitis affects inflammatory parameters and changes in the metabolome. To this end, we induced experimental mastitis in nine cows by intramammary infusion of 100 µg purified Escherichia coli lipopolysaccharide (LPS) followed by kinetic assessments of cytokine responses (by enzyme-linked immunosorbent assay), changes in the metabolome (assessed by nuclear magnetic resonance), clinical parameters (heat, local pain perception, redness, swelling, rectal temperature, clot formation, and color changes in the milk), and milk somatic cell counts, at several time points post LPS infusion. Intramammary LPS infusion induced clinical signs of mastitis, which started from 2 h post infusion and had returned to normal levels within 24-72 h. Milk changes were seen with a delay compared with the clinical signs and persisted for a longer time. In parallel, induction of IL-6 and TNF-α were seen in milk, and there was also a transient elevation of plasma IL-6 whereas plasma TNF-α was not significantly elevated. In addition, a robust increase in CCL2 was seen in the milk of LPS-infused cows, whereas G-CSF, CXCL1, and histamine in milk were unaffected. By using a metabolomics approach, a transient increase of plasma lactose was seen in LPS-induced cows. In plasma, significant reductions in ketone bodies (3-hydroxybutyrate and acetoacetate) and decreased levels of short-chain fatty acids, known to be major products released from the gut microbiota, were observed after LPS infusion; a profound reduction of plasma citrate was also seen. Intramammary LPS infusion also caused major changes in the milk metabolome, although with a delay in comparison with plasma, including a reduction of lactose. We conclude that the LPS-induced acute mastitis rapidly affects the plasma metabolome and cytokine induction with similar kinetics as the development of the clinical signs, whereas the corresponding effects in milk occurred with a delay.

Prevalence of subclinical mastitis and isolated udder pathogens in dairy cows in Southern Vietnam.

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.

The effect of a single prolonged milking interval on inflammatory parameters, milk composition and yield in dairy cows.

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-1ß 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.

Relationship between somatic cell count, polymorphonuclear leucocyte count and quality parameters in bovine bulk tank milk.

The somatic cell count (SCC) in bovine bulk tank milk is presently used as an indicator of raw milk quality, reflecting the udder health status of the herd. During mastitis, SCC increases, mostly owing to an influx of polymorphonuclear leucocytes (PMN) from blood into milk, with a concomitant change in milk composition. Bulk tank milk samples were categorized according to their SCC, as well as polymorphonuclear leucocyte count (PMNC), to study relationships between SCC, PMNC and various raw milk quality traits, i.e. contents of total protein, whey protein, casein, fat and lactose, casein number, proteolysis and rheological properties. The proportion of PMN, obtained by direct microscopy, was significantly higher in samples with high SCC compared with low SCC samples. SCC and PMNC were strongly correlated, yielding a correlation coefficient of 0.85. High SCC samples had lower lactose and casein contents, lower casein number and more proteolysis than low SCC samples. Samples with high PMNC had a lower casein number than low PMNC samples. Samples with high and low SCC or PMNC did not differ in respect to rheological properties. Our results do not indicate that PMNC is a better biomarker than SCC for raw bulk tank milk quality, as previously proposed.

Is there a special mechanism behind the changes in somatic cell and polymorphonuclear leukocyte counts, and composition of milk after a single prolonged milking interval in cows?

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.

Transfer of immunoglobulins through the mammary endothelium and epithelium and in the local lymph node of cows during the initial response after intramammary challenge with E. coli endotoxin.

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.

Effect of different milking routines on milking-related release of the hormones oxytocin, prolactin and cortisol, and on milk yield and milking performance in Murrah buffaloes.

Milking-related release of oxytocin, prolactin, and cortisol was studied following three premilking 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 (P<0.05; 2.50, 5.10 and 6.33 min, respectively). In all treatments, milk ejection occurred with small increases >3-5 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, P<0.05). There was a positive correlation between total time oxytocin concentration remained elevated over threshold levels and machine yield (r=0.86, P<0.05). For treatment O, milk ejection was inhibited during machine milking, while a marked increase in oxytocin occurred during hand stripping (6 and 16 ng/l, respectively). For treatment M, mean oxytocin concentrations remained unchanged during prestimulation but increased during subsequent machine milking and hand stripping (6.38, 18.06 and 12.36 ng/l, respectively). For treatment MF, although there was a 3.6-fold increase during pre-stimulation, oxytocin increased by 10-fold and 3-fold during machine milking and hand stripping, and was significant for machine milking (P<0.05, 17.32, 47.86, 18.13 ng/l, respectively). Milk-ejection-related cortisol release was visible only in treatment MF. For treatments O and M, prolactin concentration increased prior to the increase in oxytocin. The stripping yield was higher, and fat content in the stripping yield significantly lower, for treatment O indicating incomplete milking. Thus buffaloes are easily disturbed even by small changes in milking routines.