Microbiota of bovine milk, teat skin, and teat canal: Similarity and variation due to sampling technique and milk fraction.

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.

Quarter milking for improved detection of increased SCC.

The aim of the present study was to investigate whether milk composition and milk yield are changed in relation to a moderate increase in milk somatic cell count (SCC) in separate udder quarters. During a period of 13 weeks, 4158 bulk quarter milk samples from 68 cows were collected and analysed for milk SCC and milk composition. The sampling was done twice weekly. The cows were in different stages of lactation and in different lactation numbers. For calculations, three groups of cows were formed according to their SCC value. Group 1 cows, where all quarters had an SCC <100,000 cells/ml at all sampling occasions, were considered to be non-affected. Group 2 cows had one udder quarter with an increased SCC >100,000 cells/ml and 1.5-fold higher than the opposite quarter at one sampling occasion. For group 3 cows, the increase in SCC remained for several consecutive sampling occasions. Data from group 1 cows revealed that front and rear quarters were similar when compared with each other. For group 3 cows, the lactose content in milk decreased significantly, simultaneously with the increase in SCC and remained decreased for two sampling occasions after the initial increase in SCC. It was concluded that deviations in lactose content within front and rear quarters, respectively, may be a useful tool for detection of moderately increased SCC in separate udder quarters.

Total and differential leucocyte counts and lymphocyte subpopulations in lymph, afferent and efferent to the supramammary lymph node, during endotoxin-induced bovine mastitis.

Leucocyte trafficking in afferent and efferent mammary lymph and the supramammary lymph node in cows was examined during 4 h after intramammary infusion of endotoxin from Escherichia coli. Total and differential leucocyte counts were measured in milk, blood and lymph. The proportions of CD4(+), CD8(+), major histocompatibility complex (MHC) class II(+) and IgM(+) lymphocytes were examined in the lymph and lymph node. At post-infusion hour (PIH) 4, the flow rates of both lymph fluids had increased approximately eightfold. Total leucocyte concentration increased in afferent lymph, but decreased in efferent lymph. Neutrophils increased in afferent lymph at PIH 2 and in efferent lymph and milk at PIH 4. The predominant cell type in afferent lymph shifted from lymphocyte to neutrophil while lymphocyte was still at PIH 4 the predominant type in efferent lymph. Among the lymphocytes, B cells were predominant in afferent lymph and lymph node at PIH 4 while T cells, mainly CD4(+) cells, were predominant in efferent lymph both at PIH 0 and PIH 4. The CD4 : CD8 ratio was higher in efferent lymph and the challenged lymph node than in afferent lymph and the control node, respectively. There was a significant difference in proportions of each lymphocyte subpopulation except for IgM(+) cells, between afferent and efferent lymph after infusion. According to the results, there was already during the first hours of the immune response, a non-random trafficking of neutrophils and lymphocyte subpopulations resulting in a changed distribution of cells in afferent and efferent lymph and a difference in lymphocyte reactivity between the two lymph fluids.

Cytokines in mammary lymph and milk during endotoxin-induced bovine mastitis.

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.

Identification of coagulase-positive staphylococci isolated from bovine milk.

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.

Flow and composition of afferent mammary gland lymph.

Afferent mammary lymphatic flow was characterized in conscious lactating cows during milking and prior to, during, and after intramammary infusion of endotoxins. Lymph flow (13 to 45 ml/h) was pulsatile with monophasic and multiphasic episodes. Flow resulted from 62 to 67 episodes per h. Episodes varied from 1 to 53 s in duration. Maximum instantaneous flow ranged from 163 to 245 microliters/s. Flow did not increase consistently during milking. Lymph flow increased (5.5- to 8-fold) during endotoxin-induced mastitis. Flow rates were elevated for up to 48 h after infusion of endotoxin. Compositional comparisons between afferent mammary lymph and blood plasma showed distinct differences. Lymph contained 7, 6, and 10 times less protein, albumin, and globulin, respectively, than did plasma. Glucose concentrations were equivalent. Lymph had 17 times less cholesterol and about one-half as much triglyceride and calcium as did plasma. gamma-Glutamyl-transaminase and aspartate transaminase were substantially higher in plasma than in lymph. Afferent mammary lymph has unique compositional characteristics. The lymph ducts contained an intrinsic mechanism for lymph movement. Moreover, this mechanism was altered by inflammation. The techniques herein provide a better understanding of the mammary lymphatic system.

Total and differential leukocyte counts, N-acetyl-beta-D-glucosaminidase activity, and serum albumin content in foremilk and residual milk during endotoxin-induced mastitis in cows.

Foremilk, residual milk, and blood samples were studied for 10 days during acute mastitis episodes induced by endotoxin infused via the teat canal. Quarter milk and blood samples were collected frequently for 3 days after the infusion and thereafter once or twice daily. Leukocyte concentration in milk and blood was determined by flow cytometry. Within 2 hours after infusion of the endotoxin, clinical mastitis was observed. Total leukocyte concentration and proportion of neutrophils increased significantly (P < 0.05) by postinfusion hour (PIH) 2 in foremilk and by PIH 4 in residual milk. From PIH 2, serum albumin content and N-acetyl-beta-D-glucosaminidase activity were significantly increased in both fractions. Neutrophils were the predominant leukocyte population in both fractions until PIH 59. From PIH 72, lymphocytes were the predominant cell population until PIH 175 in foremilk and until PIH 223 in residual milk. Serum albumin content and N-acetyl-beta-D-glucosaminidase activity in residual milk was significantly lower than in foremilk from PIH 4 to 24 and from PIH 24 to 59, respectively. Regarding total and differential leukocyte counts, values for the 2 fractions followed the same pattern throughout the course of inflammation, probably owing to frequent sample collection. Total and differential cell counts tended to differ between the fractions during some periods, although differences were not statistically significant. When samples were taken less frequently, the total leukocyte concentration in residual milk was higher than that in foremilk. Although sample collections were frequent, clustering of immature neutrophils was not observed in the cytofluorogram of blood leukocytes in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Variations during lactation in total and differential leukocyte counts, N-acetyl-beta-D-glucosaminidase, antitrypsin and serum albumin in foremilk and residual milk from non-infected quarters in the bovine.

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.

Flow cytofluorometric studies on the alteration of leukocyte populations in blood and milk during endotoxin-induced mastitis in cows.

Alterations in the various leukocyte populations in milk, blood, and mammary lymph were studied by use of the flow cytometric method during acute mastitis episodes induced by endotoxin infusion (50 micrograms of lipopolysaccharide of Salmonella typhimurium SH 4809) via the teat canal. Lymph samples were collected via a semipermanent catheter from an afferent duct to the supramammary lymph node. Milk somatic cell count increased at 4 hours after infusion of endotoxin. Neutrophils were the predominant cell population for up to 59 hours after infusion. Numbers of lymphocytes and monocytes-macrophages in milk also increased after the endotoxin infusion. The total cell count in milk started to decrease during the third postinfusion day and returned to preinfusion values during the fourth day. Lymphocyte numbers remained high for about 1 week after the infusion, and lymphocytes were the predominant cell population between postinfusion days 4 and 8. Total blood leukocyte count decreased during the first 6 hours after infusion, followed by an increase until postinfusion hour 31. The proportion of neutrophils in blood increased during the first day, whereas that of lymphocytes decreased. Lymph flow rate and leukocyte numbers in lymph increased after endotoxin infusion. The proportion of neutrophils in the lymph increased during the first 6 hours, whereas that of lymphocytes decreased. After postinfusion hour 6, the inverse course of events was seen.

Sampling of lymph from lymph vessels afferent to the supramammary lymph gland in the cow.

A technique for catheterization of a lymph vessel afferent to the supramammary lymph gland in the cow is described. The operation was made with the cow under general anaesthesia. The lymph vessel was catheterized with a 30 cm long heparinized polyethylene catheter with an external diameter of 2.5 to 3 mm. Generally at least 5 ml of lymph could be collected within a period of 3 to 5 minutes. In 11 out of 15 cows lymph could be collected for 7 days or longer.

Concentrations of penicillin, streptomycin, and spiramycin in bovine udder tissue liquids.

Concentrations of benzylpenicillin and spiramycin adipate were determined in bovine plasma and milk and in lymph draining the udder tissue after IM or IV administration. Combined benzylpenicillin and dihydrostreptomycin sulfate concentrations were also determined in the same fluids after intramammary injection. A superficial parenchymal lymph vessel, afferent to the supramammary lymph gland of the left quarters, was cannulated with a polythene catheter from which the lymph was allowed to drain freely. After injections of 9.5 mg of benzylpenicillin/kg of body weight IM, a mean peak concentration (PC) in lymph (3.7 micrograms/ml), constituting 77% of the PC in plasma (4.8 micrograms/ml), was obtained 0.5 to 1 hour after PC in the plasma. The benzylpenicillin lymph concentration was close to that in plasma for about 7 hours after injection. Thereafter, the benzylpenicillin lymph concentration continued to exceed that in plasma, but not that in milk. After IV administration of spiramycin adipate, the lymph concentration was almost identical to that in plasma. After intramammary injection of procaine benzylpenicillin (400 mg), in combination with the same amount of dihydrostreptomycin sulfate, into 2 udder quarters each, mean PC in the lymph of 3.5 micrograms/ml and 8.4 micrograms/ml, respectively, were obtained 6 hours after injection. In plasma, the mean PC of benzylpenicillin (0.07 micrograms/ml) and of dihydrostreptomycin sulfate (0.85 micrograms/ml) were obtained after 4 and 6 hours, respectively. In milk from the nontreated quarters, a mean concentration of 5 ng of benzylpenicillin/ml was obtained, whereas dihydrostreptomycin sulfate (greater than or equal to 0.3 microgram/ml) was not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteroides fragilis, Streptococcus intermedius and group B streptococci in ascending infection of pregnancy. An animal experimental study.

By means of bacteriological, histological and immunofluorescence methods, the ascending spread of endocervically inoculated bacteria was studied in 14 pregnant sheep. 8 ewes near term were exposed to group B streptococci, Bacteroides fragilis or Streptococcus intermedius for 2-4 days. 6 other animals in mid pregnancy were exposed to one of the bacteria mentioned for 30 days. Before inoculation, aerobic and anaerobic culture specimens from cervix and amniotic fluid were obtained. At delivery by caesarean section, in addition to culture specimens, membrane and placental tissues were collected. Bacterial detection by means of indirect immunofluorescence showed significant invasion of the membranes and the placentas by the microorganisms inoculated. In one case, inoculated with S. intermedius, the ascending infection could be shown to pass the intact membranes reaching the amniotic fluid. It is concluded that endocervical bacteria in the indigenous microflora might cause pregnancy complications by establishing ascending infection. Furthermore, as the bacteria tested have been implicated in premature rupture of the membranes, an aetiological role of ascending infection in this pregnancy complication is postulated.

A case of partial cerebellar hypoplasia in a cat.

A case of spontaneous ataxia accompanied by partial cerebellar hypoplasia in a two month old siamese kitten is described. The cause of cerebellar hypoplasia is unknown but the feline panleucopenia virus is number one to be suspected.