Growth of Staphylococcus aureus and Streptococcus species in bovine mammary secretions during the nonlactating and peripartum periods following intramammary infusion of lipopolysaccharide at cessation of milking.

The objective was to determine if induced mammary inflammation at cessation of milking influenced growth of gram-positive mastitis pathogens in mammary secretions, particularly during early involution. Growth of all mastitis pathogens evaluated was similar in cell-free fat-free mammary secretions from LPS-infused and control glands. These data indicate that intramammary infusion of LPS at cessation of milking did not alter growth of gram-positive mastitis pathogens in mammary secretion during the nonlactating period. Stage of lactation and the nonlactating period influenced bacterial growth and marked differences between bacteria and among strains of a bacterial species were observed. Staphylococcus aureus grew well in secretions collected during late lactation, but growth decreased during early- and mid-involution and increased again in secretions obtained near parturition. Streptococcus agalactiae and Strep. uberis grew better in mammary secretion obtained during involution than in secretions collected during late or early lactation. Streptococcus dysgalactiae grew well in mammary secretions at all time periods. These data demonstrate the variability of mastitis pathogen growth during physiologic transitions of the bovine udder.     

Growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution of the bovine mammary gland: relation to secretion composition

Mammary secretions from 12 Holstein dairy cows were collected to evaluate growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution and during physiologic transitions of the mammary gland. Mammary secretions obtained during late lactation poorly inhibited growth of E coli and K pneumoniae. However, as involution progressed, mammary secretions increasingly inhibited growth of both coliform mastitis pathogens. Greatest inhibition of E coli and K pneumoniae growth was observed when mammary glands were fully involuted. Growth inhibition remained high until 7 days before parturition, and then it decreased significantly (P less than 0.05) to that observed during late lactation. Inhibition of coliform mastitis pathogen growth was associated with high concentrations of lactoferrin and immunoglobulin G, decreased citrate concentration, and a low citrate to lactoferrin molar ratio. These data suggested that differences in susceptibility or resistance to new intramammary infection with coliform mastitis pathogens during the nonlactating period may be attributable, in part, to marked changes in mammary secretion composition that develop during physiologic transitions of the mammary gland. Resistance of the fully involuted mammary gland to coliform infection may be associated with high concentrations of natural protective factors.     

Hemolytic and bactericidal activities of bovine complement in mammary secretions of cows during the early nonlactating (dry) period

Hemolytic and bactericidal complement-dependent activities were measured in quarter mammary secretions obtained during the first 21 days of the nonlactating (dry) period from 8 Holstein-Friesian cows. We demonstrated an inhibition of hemolytic activity and bactericidal activity against a serum-sensitive Escherichia coli strain. Both hemolytic and inhibitory titers increased markedly during active involution. The bactericidal activity of dry secretions required a minimal threshold of complement and an inhibitory titer lower than the hemolytic titer.     

Bovine leukocytes in mammary secretions during involution: surface protein changes

Changes in surface proteins of leukocytes in bovine mammary secretions were examined during mammary involution. Leukocytes were collected from mammary secretions from involution days (ID) 1 to 36 and were labeled with biotin. Labeled proteins were separated by polyacrylamide-gel electrophoresis, transferred to nitrocellulose by protein blotting, and detected with avidin-peroxidase and color reagent. The population of leukocytes in mammary secretions changed during involution from predominantly polymorphonuclear leukocytes (PMN) at ID 1 to predominantly mononuclear leukocytes by ID 36. The gel profile of surface proteins also changed during involution, and some of those changes may have been caused by shifts in leukocyte populations. Profiles of surface proteins of leukocytes from mammary secretions containing predominantly PMN (ID 1) were different from profiles of PMN from blood. A distinctive change in the pattern of surface proteins on the leukocytes occurred between ID 1 and 3, even though the proportion of PMN did not change appreciably between those days.     

Apoptosis and oxidative stress of infiltrated neutrophils obtained from mammary glands of goats during various stages of lactation

OBJECTIVE: To examine apoptosis in infiltrated neutrophils during involution of mammary glands and compare them with those obtained during late and peak lactation, and to measure oxidative stress and activities of antioxidant enzymes and determine involvement of free radicals in apoptosis of infiltrated neutrophils. SAMPLE POPULATION: Neutrophils from mammary gland secretions of 8 goats at 4 stages (late and peak lactation and 1 and 2 weeks after end of lactation). PROCEDURE: DNA fragmentation was evaluated to characterize apoptosis. Concentration of thiobarbituric acid reactive substances (TBARS) was used to evaluate oxidative stress. Activities of superoxide dismutase and glutathione peroxidase were determined. RESULTS: Neutrophils from secretions obtained after end of lactation of all goats and from late-lactation milk of some goats underwent prominent apoptosis, whereas neutrophils from peak lactation secretions did not. Higher lipid peroxidation and lower antioxidant enzyme activities in neutrophils during involution were observed, compared with those during late and peak lactation. A significant negative correlation existed between TBARS concentrations and antioxidant enzyme activities during the nonlactating period. CONCLUSIONS AND CLINICAL RELEVANCE: Apoptosis is a feature of infiltrated neutrophils during involution of mammary glands in goats. This feature may allow prompt resorption and clearance of infiltrated neutrophils without damaging surrounding tissues. Increased oxidative stress in infiltrated neutrophils from secretions obtained after end of lactation is probably related to a deficiency in antioxidant enzyme activities. Understanding the relationship between apoptosis and oxidative stress will lead to new strategies for manipulating involution and reducing tissue damage.     

A study of bovine T-cell subsets in the blood and mammary gland secretions during the dry period

Blood and mammary secretions were obtained from cows throughout the dry period. Quantitative and qualitative assays were performed to determine the cell types and cell distributions at weekly intervals from day of dry off until parturition. The total cell counts in secretions increased during involution and remained at high levels until a few weeks prepartum. The macrophages were the predominant cell type in mammary secretions whereas the numbers of lymphocytes were always less than neutrophils or macrophages. Enriched mononuclear cell populations derived from blood and mammary secretions were also evaluated using "T-cell rosette" assays. Changes observed in the relative distribution of three T-cell subsets in secretions did not reflect the dynamics of the cells in the peripheral blood. T-cell subsets that predominated in mammary secretions were the EN+ EAET+ and EN-EAET+ phenotypes. Distinct patterns of migration or differentiation of T-cell subsets were suggested by the changes of subsets observed in mammary secretions collected throughout the dry period.     

Persistence of antibiotics in bovine mammary secretions following intramammary infusion at cessation of milking

A study was conducted to determine the persistence of antibiotic preparations for use in nonlactating cows in bovine mammary secretions following intramammary infusion at cessation of milking. Five commercially available antibiotic formulations were evaluated using 311 cows. All quarters of each cow were sampled once only during the nonlactating period and most cows were sampled at or near parturition. Antibiotic residues were detected qualitatively by the Bacillus stearothermophilus disc assay. Great variation between different antibiotics in persistence in mammary secretion was observed. In general, mammary secretions from most mammary glands infused with cloxacillin or penicillin-dihydrostreptomycin were positive at 28–35 days after infusion and some were positive at 42–49 days after infusion. On the other hand, <13% of mammary secretions at 7 days after infusion of novobiocin and 50% of mammary secretions at 14 days after infusion of penicillin-novobiocin were positive for antibiotics. Cephapirin benzathine persisted for about 21 days after infusion. Some samples that were positive for antibiotics after initial testing were negative following heating of samples, suggesting that component(s) of dry secretion can inhibit growth of B. stearothermophilus and influence the interpretation of results. Colostrum samples from all quarters except one were negative for antibiotics. These data suggest that nonlactating-cow antibiotic formulations persist primarily during the early to mid-nonlactating period. Based upon present methods of formulation, it would appear that antibiotic preparations for use in nonlactating cows most likely provide little protection during the periparturient period, at a time when mammary glands are highly susceptible to new intramammary infections.     

Changes in the microscopic anatomy of the bovine teat canal during mammary involution

Changes in the microscopic anatomy of the bovine teat canal were examined during mammary involution. Morphometric analyses revealed a significant (P less than 0.05), temporary dilatation of the teat canal lumen on day 7 of the nonlactating period. Additionally, the teat canal epithelium physiologically atrophied as evidenced by decreased cross-sectional area and thickness during the first 30 days of the nonlactating period, significantly so (P less than 0.05) between days 0 and 7. This physiologic atrophy was due mainly to a reduction in area and thickness of the stratum granulosum and may have resulted from continuing keratinization, a process that led to increased thickness of the keratin layer and formation of a functional plug during later stages of involution. Changes in cells of the stratum granulosum indicated a decrease in the rate of epithelial cell maturation during involution. The mitotic index (percentage of basal cells in mitosis) of the teat canal epithelium decreased significantly (P less than 0.05) between days 0 and 7 of the nonlactating period. Bacteria, observed in histologic sections, appeared to colonize only certain regions of the keratin layer. Seemingly, changes in the teat canal during mammary involution may be important factors in changing susceptibility to new intramammary infection during the early and mid-nonlactating periods.     

Growth inhibition of environmental mastitis pathogens during physiologic transitions of the bovine mammary gland

Ten dairy cows were infused intramammarily near drying off with concanavalin A (conA) or phytohemagglutinin (PHA). Mammary secretions were collected during physiologic transitions of the udder and were used in an in vitro microbiological assay to determine growth inhibition of mastitis pathogens. As mammary involution progressed, in vitro growth inhibition of Klebsiella pneumoniae, Escherichia coli, and Streptococcus uberis increased. Mammary secretions from conA- and PHA-treated glands had significantly increased bacterial growth inhibition. Secretions contained significantly increased concentrations of lactoferrin and a decreased citrate:lactoferrin molar ratio earlier in the dry period than did control mammary secretions. Greatest bacterial growth inhibition was observed in mammary secretions obtained 7 days before parturition. However, differences in secretion composition or bacterial growth inhibition were not found between conA- or PHA-treated and control udder halves during the prepartum period. Bacterial growth inhibition by mammary secretion decreased markedly during early lactation. A highly significant positive correlation was found between bacterial growth inhibition and concentrations of lactoferrin, serum albumin, and immunoglobulin G. A highly significant negative correlation was found in the citrate:lactoferrin molar ratio during early involution and the peripartum period.     

Effect of mammary secretions on functions of polymorphonuclear leukocytes in pigs

OBJECTIVE: To determine the effects of porcine mammary secretions on polymorphonuclear (PMN) leukocyte function and to relate concentrations of estradiol-17beta and cortisol in mammary secretions to PMN cell function. SAMPLE POPULATION: Mammary secretions from 10 healthy sows and blood PMN leukocytes from 27 healthy sows. PROCEDURE: Mammary secretions were collected within 24 hours after parturition (colostrum) and 12 to 13 days later (milk). Chemoattractant properties were assessed by use of a cell migration assay. Phagocytic capacity of PMN cells in colostrum and milk was assessed by recording chemiluminescence following phagocytosis of Escherichia coli or zymosan. Estradiol-17beta and cortisol concentrations were determined by use of radioimmunoassays. RESULTS: Chemoattractant properties of colostrum and milk were significantly greater than that of zymosan-activated serum. However, chemoattractant properties did not differ significantly between the 2 types of secretions. The capacity of PMN cells in colostrum to phagocytose either zymosan or E. coli was less, compared with cells in milk, and the ability of cells in either type of mammary secretion to phagocytose E. coli was greater than the ability to phagocytose zymosan. Concentrations of estradiol-17beta and cortisol were greater in colostrum, compared with milk. No clear relation was evident between PMN cell activity and hormone concentrations in mammary secretions. CONCLUSIONS AND CLINICAL RELEVANCE: Although chemoattractant properties of colostrum and milk did not differ, the phagocytic capacity of PMN cells in colostrum was significantly less than that of cells in milk. This may predispose sows to coliform mastitis during the early postparturient period.     

Frequency of isolation of environmental mastitis-causing pathogens and incidence of new intramammary infection during the nonlactating period

Quarter samples (n = 6,328) of mammary secretions were collected from 160 cows during physiologic transitions of the udder to determine the frequency of isolation of mastitis-causing pathogens and the incidence of new intramammary infections (IMI) during the nonlactating period. None of the cows in the herd was infected with Streptococcus agalactiae, and the prevalence of Staphylococcus aureus was low. Cows were not treated with antibiotics at cessation of milking. A threefold increase in the percentage of quarters infected with major mastitis-causing pathogens developed from late lactation to early involution. Coliforms and streptococci other than Str agalactiae accounted for 94% of major pathogen infections. The number of quarters infected with coagulase-negative staphylococci increased slightly from late lactation to early involution, whereas the number of quarters infected with Corynebacterium bovis decreased markedly. Major pathogens caused 101 of 153 IMI at parturition and greater than 90% were caused by streptococci and coliforms. At parturition, 51 of 52 minor pathogen IMI were caused by coagulase-negative staphylococci. During early lactation, there was a marked decrease in quarters infected with major pathogens; however, the number of quarters with major pathogen IMI during early lactation was 2.3 times higher than the number of quarters infected before cessation of milking. The number of quarters with minor pathogen IMI during early lactation was the same as at parturition, but a marked decrease in quarters infected with coagulase-negative staphylococci and a marked increase in C bovis IMI developed from parturition to early lactation.     

Zum Bau der Milchdrüse des Rindes während der Trockenstehperiode unter besonderer Berücksichtigung der Involutionsprozesse

Light microscopical and histochemical investigations were performed on biopsies, which were obtained from the mammary glands of healthy pregnant cows during different stages of the nonlactating period. The results demonstrate characteristic morphological changes in the different phases of the nonlactating period. They can be interpreted as involution and regeneration processes, which occur in the alveoli and interalveolar tissue of the bovine mammary gland during the nonlactating period.     

Electrolytes and free fatty acids in milk during mammary involution in the cow

Measurements have been made of the concentrations of triglycerides, free fatty acids, sodium, chloride and potassium in milk samples obtained from cows during acute mammary involution and after parturition. Free fatty acid levels in milk increased more than 10-fold during involution. Their appearance did not immediately follow the cessation of milking but followed the increase in permeability of the mammary epithelium, evidenced by changes in the electrolyte content of the milk, that occurs after a delay of a few days. However, the concentration of free fatty acids did not remain high throughout the dry period but had declined to low levels before the change in permeability was reversed at the next parturition. It is concluded that the high level of free fatty acids in milk during mammary involution does not result from equilibration with an extra-mammary pool, but most likely arises from breakdown of triglycerides remaining in the gland which may be accelerated in some manner by the increase of permeability of the mammary epithelium.     

Function of phagocytes obtained from lacteal secretions of lactating and nonlactating cows

Phagocytes, macrophages and neutrophils, were obtained from lacteal secretions of lactating (n = 13) and nonlactating cows (n = 14). Secretions from nonlactating cows were collected at 7 and 14 days after cessation of lactation. Phagocytes were incubated in vitro with Staphylococcus aureus or Escherichia coli, and function was assessed by fluorescent microscopy of cell suspensions stained with acridine orange and crystal violet. A greater percentage of macrophages from nonlactating cow secretions collected on day 14 phagocytized bacteria than did those collected on day 7. A greater percentage of macrophages from nonlactating cow secretions collected on days 7 and 14 phagocytized bacteria than did neutrophils obtained from the same secretions. A similar percentage of phagocytes from nonlactating cow secretions phagocytized bacteria, compared with phagocytes from lactating cow secretions. Results indicated that the intramammary macrophage may be most important in defense of the mammary gland during the early nonlactating period, because it was more phagocytic than the neutrophil and was more active at 14 days than at 7 days into the nonlactating period.     

Changes in the bovine teat canal during the nonlactating period and early lactation, as measured by teat canal impressions.

Changes in dimensions of impressions of the lumen of the teat canals of 13 cows were examined at 17 intervals during the nonlactating period and early lactation. Impressions were made of teats of 2 diagonally opposed quarters of each cow, using dental impression material. Impression length was measured and cross sections of the impressions at the proximal (distal to Furstenburg rosette), distal (proximal to the teat orifice), and middle (midway between the 2), portions of the teat canal were prepared. Cross sections were photographed and enlarged, and circumference and area were determined by use of planimetry. Effects of making repeated impressions during the nonlactating period and early lactation on new infection rates and somatic cell counts were also assessed. Mean length of teat canal impressions decreased between days 0 and 3 of involution and during the prepartum periods. Depending on the level from which they were taken, cross-sectional areas of impressions tended to increase or increased significantly during the period of involution and again in the prepartum period. Significant changes in cross-sectional area were not observed during early lactation. Changes in circumference of proximal, middle, and distal cross sections followed trends similar to area measurements, but were more variable and differences were less statistically significant. On the basis of our findings, we suggest that heightened susceptibility to new infection during mammary involution and the prepartum period may be attributable, in part, to changes in the patency of the teat canal. Making impressions repeatedly throughout the nonlactating period and early lactation did not affect the number of new intramammary infections.     

Protective factors in mammary gland secretions during the periparturient period in the mare

Changes in mare mammary secretion composition were characterized prior to and after foaling. Eighteen mares were used for collection of mammary secretions from 23 days prepartum through 44 days postpartum. Concentrations of lactose, total protein, IgG and lysozyme (activity assay) were determined. Considerable variabilty was observed among mares. Mean lactose concentrations by mare were lower (P<.05) in the prepartum period compared with the postpartum period. Mean total protein and immunoglobulin G concentrations by mare were higher (P<.05) in the prepartum period compared with the postpartum period. Mean lysozyme activities by mare tended to be lower (P<.08) during the prepartum period compared with the postpartum period. Mammary secretion protein profiles, characterized by SDS-PAGE, were consistent with changes in concentrations of specific proteins measured in the secretions. Dynamic changes occurring in mammary secretions during the transition from prepartum accumulation of colostrum to postpartum production of milk include factors like IgG and lysozyme which have protective roles in the neonatal foal.     

Effects of bovine mammary secretion during the early nonlactating period and antibiotics on polymorphonuclear neutrophil function and morphology.

The effect of bovine mammary secretion during the early nonlactating period and of antibiotic preparations on bovine polymorphonuclear neutrophil (PMN) phagocytic function and morphology were evaluated in a series of in vitro multifactorial experiments. Benzathine cloxacillin (CL), benzathine cephapirin (CE), sodium novobiocin (NO), and a combination of dihydrostreptomycin with procaine penicillin G (DP) were prepared in the presence and absence of a peanut oil aluminum monostearate vehicle. The PMN were isolated from bovine blood, and the effect of each antibiotic preparation on PMN function and morphology was evaluated in a buffer, fat, skin, and a combination of fat with skim from bovine mammary secretion during the nonlactating period. The fat and skim were diluted with buffer to approximate their concentration in mammary secretion. Phagocytic functions of PMN were monitored by fluorescent microscopy, which made it possible to estimate both ingestion and intracellular killing of bacteria by PMN. Changes in PMN morphology were monitored by transmission electron microscopy. The ability of PMN to ingest and kill Staphylococcus aureus ATCC 25923 was significantly decreased by fat, skim, CL, CE, NO, and DP. Effects of some antibiotics on ingestion and killing of bacteria by PMN were influenced by the addition of vehicle and by interactions with mammary secretion. Neutrophil morphology was altered by fat, skim, CL, CE, NO, and DP. The detrimental effects of CL, CE, NO, and DP on PMN morphology were influenced (some significantly) by the presence of vehicle and interactions with mammary secretion. There were significant correlations among secretion- and antibiotic-induced changes in PMN ingestion of bacteria, PMN killing of bacteria, and PMN morphology.     

Total and differential cell counts and N-acetyl-β-D-glucosaminidase activity in sow milk during lactation

Colostrum and milk collected from 11 sows throughout lactation were used to estimate total and differential cell counts and N-acetyl--D-glucosaminidase activity (NAGase). The mean log₁₀ cell counts did not change significantly through the four weeks of lactation, ranging between 250 000 and 750 000 cells/ml. Polymorphonuclear neutrophils (PMN) decreased from about 56% of total leukocytes at day 1 (colostrum) to 12–14% at day 14 and day 21. Macrophages were 35% at day 1 and were the predominant cell type throughout the remainder of lactation, peaking at 77–80% at day 14 and 21. The PMN were again increased on day 28 (44% PMN vs 52% macrophage). The mean lymphocyte proportions ranged between 7.0 and 11.3% during the first two weeks of lactation and were decreased to 4.6–5.6% in the second two weeks of lactation. The activity of NAGase declined 9.5 fold (p<0.0001) between day 1 and day 14 with the greatest decline between day 1 and day 3. The activity of NAGase remained constant through the last two weeks of lactation. NAGase activity was significantly correlated with log₁₀ of cell counts in sow milk (r=0.42).     

Effects of intracisternal bead devices on lacteal secretion components, plaque formation, and bacterial infection during the nonlactating period

Effects of placing intracisternal bead devices (ICB) into teat cisterns of 6 dairy cows, from the end of lactation through parturition, were studied. Lacteal secretion samples were collected weekly from each mammary quarter during the nonlactating period to monitor composition changes in ICB-fitted and nonfitted quarters. In quarters remaining uninfected (n = 15), there were significantly higher mean somatic cell counts (P less than 0.05), percentage of neutrophils (P less than 0.019), and cell viability (P less than 0.038), but significantly lower percentage of macrophages (P less than 0.013) in ICB-fitted quarters compared with those in nonfitted quarters. The ICB had no significant effect on mean weekly values for percentage of lymphocytes, pH, lactoferrin, citrate, citrate/lactoferrin molar ratio, serum albumin, alpha-lactalbumin, and N-acetyl-beta-D-glucosaminidase. In infected quarters (n = 9), pH of mammary secretions was significantly (P less than 0.004) higher in ICB-fitted quarters, but concentrations of lactoferrin (P less than 0.004), alpha-lactalbumin (P less than 0.013), and N-acetyl-beta-D-glucosaminidase (P less than 0.028) were significantly lower, compared with those in nonfitted quarters. Coagulase-negative staphylococci comprised approximately 90% of all infections. Over the nonlactating period, 16.4 and 41.5% of samples from nonfitted and ICB-fitted quarters, respectively, contained coagulase-negative staphylococci. Microscopic examination of ICB from uninfected quarters revealed a thin coating of plaque with adhering neutrophils, macrophages, and multinucleated giant cells. Microscopic examination of plaque on devices from ICB-fitted quarters harboring coagulase-negative staphylococci revealed numerous adherent cocci and neutrophils.     

Immunocyte populations in the mammary gland of the rat at different stages of pregnancy and lactation.

Immunocyte populations of apparently normal rat mammary glands were examined at different stages of lactation using immunofluorescence techniques. IgA-and IgG-containing cells were present before parturition; IgA-containing cells were numerous than IgG- or IgM-containing cells during the entire period from pregnancy through parturition and lactation to involution.