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.     

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.     

Accelerated bovine mammary involution induced by infusion of concanavalin A or phytohemagglutinin

Right mammary quarters of dairy cows were infused with concanavalin A (conA) or phytohemagglutinin (PHA) near drying off. Significant changes in milk yield and composition were observed by 24 hours after infusion. In general, conA or PHA treatment resulted in significantly greater compositional changes earlier in the dry period compared with control quarters. As involution progressed, lactoferrin, serum albumin, immunoglobulin G, pH, and numbers of somatic cells increased, whereas citrate and the molar ratio of citrate:lactoferrin decreased. Mammary secretion accumulation at 7 days of involution was significantly lower in treated compared with control quarters. The data indicated that intramammary infusion of conA or PHA near drying off accelerates bovine mammary involution, resulting in elevated levels of natural protective factors.     

Studies on the modulation of leucocyte subpopulations and immunoglobulins following intramammary infusion of beta 1,3-glucan into the bovine udder during the dry period

Inflammatory and immunological reactions after intramammary infusion of beta 1,3-glucan were studied during the steady dry period and involution phase of the bovine udder. The effects of a single intramammary infusion of two different doses (100 and 200 mg) of beta 1,3-glucan were evaluated during the steady dry period. In a second study, the effects of beta 1,3-glucan at drying off were studied by using two treatment regimens; a single infusion at drying off, compared with two infusions of the compound, at drying off and again 2 weeks later. Total and differential leucocyte counts were measured in both blood and udder secretions. Additionally, the expression of receptors for CD14 and MHC class II on leucocytes, and the expression of receptors for CD4, CD8, WC1, IL2R and B-cells on lymphocytes was measured in mammary secretions by flow cytometric analyses. The concentrations of immunoglobulins in udder secretions were measured by radial immunodiffusion. The results showed that a single intramammary infusion of beta 1,3-glucan during the steady dry period causes transient enhancement of some aspects of the inflammatory and immune responses. The increases in somatic cell counts, numbers of monocytes/macrophages, and in proportions of CD14+ and MHC class II+ leucocytes in udder secretions were dose-dependent. Infusion of beta 1,3-glucan also slightly increased the proportion of CD4+ lymphocytes and the concentrations of IgG1 and IgG2 in dry secretions. Infusion of beta 1,3-glucan at drying off seemed to accelerate the involution process through an increase in somatic cells, particularly in the numbers of macrophages, in mammary secretions. The numbers of lymphocytes and polymorphonuclear leucocytes, the proportions of IL2R+ lymphocytes, the proportions of CD14+ or MHC class II+ leucocytes and the concentrations of IgG1 and IgG2 also increased in comparison with untreated controls. Moreover, a second infusion of beta 1,3-glucan tended to prolong this response, indicating that this might be an effective means of enhancing the mammary defence against udder infections closer to calving. In conclusion, the results indicate the intramammary infusion of beta 1,3-glucan could be used to enhance the defence mechanisms of the bovine udder against infections, especially during early involution.     

Studies on the Modulation of Leucocyte Subpopulations and Immunoglobulins following Intramammary Infusion of β1,3‐glucan into the Bovine Udder during the Dry Period

Inflammatory and immunological reactions after intramammary infusion of β1,3‐glucan were studied during the steady dry period and involution phase of the bovine udder. The effects of a single intramammary infusion of two different doses (100 and 200 mg) of β1,3‐glucan were evaluated during the steady dry period. In a second study, the effects of β1,3‐glucan at drying off were studied by using two treatment regimens; a single infusion at drying off, compared with two infusions of the compound, at drying off and again 2 weeks later. Total and differential leucocyte counts were measured in both blood and udder secretions. Additionally, the expression of receptors for CD14 and MHC class II on leucocytes, and the expression of receptors for CD4, CD8, WC1, IL2R and B‐cells on lymphocytes was measured in mammary secretions by flow cytometric analyses. The concentrations of immunoglobulins in udder secretions were measured by radial immunodiffusion. The results showed that a single intramammary infusion of β1,3‐glucan during the steady dry period causes transient enhancement of some aspects of the inflammatory and immune responses. The increases in somatic cell counts, numbers of monocytes/macrophages, and in proportions of CD14 + and MHC class II + leucocytes in udder secretions were dose‐dependent. Infusion of β1,3‐glucan also slightly increased the proportion of CD4 + lymphocytes and the concentrations of IgG₁ and IgG₂ in dry secretions. Infusion of β1,3‐glucan at drying off seemed to accelerate the involution process through an increase in somatic cells, particularly in the numbers of macrophages, in mammary secretions. The numbers of lymphocytes and polymorphonuclear leucocytes, the proportions of IL2R + lymphocytes, the proportions of CD14 + or MHC class II + leucocytes and the concentrations of IgG₁ and IgG₂ also increased in comparison with untreated controls. Moreover, a second infusion of β1,3‐glucan tended to prolong this response, indicating that this might be an effective means of enhancing the mammary defence against udder infections closer to calving. In conclusion, the results indicate that intramammary infusion of β1,3‐glucan could be used to enhance the defence mechanisms of the bovine udder against infections, especially during early involution.     

Course of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in mammary secretions of the goat during end-pregnancy and early lactation

The epidermal growth factor (EGF) plays a crucial role in mammogenesis in many species. In ruminants, studies are limited, as EGF does not occur in peripheral plasma and specific analytical systems do not exist. Therefore a heterologous radioimmunoassay based on rhEGF was set up to monitor EGF in mammary gland secretions from goats during end-pregnancy and early lactation. IGF-I was measured with an established radioimmunoassay. Samples were collected from 13 goats for 25 days ante-partum and 25 days post-partum. Mammary gland secretions were obtained ante-partum by removing a small amount of the udder secretions (control half) or milking (stimulated half). Post-partum normal milk samples were collected. Blood samples were drawn by jugular venipuncture for the same period. EGF was found to occur in different molecular weight forms in the mammary glands. For routine measurements these proteins were extracted with acetone and not further separated. IGF-I and EGF concentrations in mammary secretions and similarly IGF-I in blood were high ante-partum and decreased slightly towards birth. IGF-I but not EGF is found in the peripheral plasma. Whereas IGF-I concentrations in blood were quite constant post-partum, IGF-I and EGF dropped in mammary secretions close to the detection limits. The decrease was more pronounced in the stimulated half than in the control half. The data support a synergistic role for EGF and IGF-I for mammogenesis. Both factors are further influenced by the milking stimulus and thus the functional state of the udder.     

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.     

Lactoferrin and citrate concentrations at drying-off and during early mammary involution of dairy cows

Quarter milk samples were taken from 48 clinically healthy, pregnant Finnish Ayrshire and Friesian dairy cows at the last milking before drying-off, and 2 and 6 days later to determine the lactoferrin and citrate concentrations in the udder secretion. The mean lactoferrin concentration in the milk increased from 5.29 mg ml(-1) on the last day of drying-off process to 8.09 and 11.26 mg ml(-1), 2 and 6 days later, respectively. Citrate concentration decreased from 1.85 mg ml(-1) to 1.54 and 1.09 mg ml(-1), respectively. Median molar ratio (citrate to native-Lf) decreased from 153 to 86 and further to 44. Lf and citrate concentrations in milk varied greatly among cows. Sampling time had a statistically significant effect on lactoferrin and citrate concentrations in milk while the breed and the parity of the cows had no effect on either of these variables.     

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.     

Effects of intramammary infusions of interleukin-8 on milk protein composition and induction of acute-phase protein in cows during mammary involution

The effects of interleukin-8 (IL-8) on bovine mammary functions such as milk protein secretion and the blood-milk barrier during mammary involution were evaluated. Following the final milking, recombinant bovine (rb) IL-8 (5 or 25 microg) and a saline placebo were individually infused into the left- and right-front teat cisterns of 6 cows, respectively. Three cows without treatment at the final milking were also used as controls. Mammary secretions and blood were collected at -24, 0, 10, 24, 72, 168, 336, and 720 h after infusion. In the mammary glands infused with 25 microg of rbIL-8, the increases in somatic cell counts and in the concentrations of serum albumin, IgG1 and IgG2, and the decreases in the concentrations of alpha- and beta-casein and beta-lactoglobulin were greater than in the control glands. In the mammary glands infused with 5 microg of rbIL-8, compared to the glands infused with 25 microg of rbIL-8, these changes were moderate. These results indicate that rbIL-8 impairs the integrity of the blood-milk barrier and suppresses milk-specific protein secretions. In the cows infused with 25 microg of rbIL-8, the rectal temperature and serum haptoglobin level were transiently elevated after the infusion, showing that intramammary infusion of rbIL-8 could elicit systemic inflammation.     

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.     

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.     

Immunoglobulins, lysozyme and lactoferrin in the teat and udder of the dry cow during endotoxin-induced inflammation.

Immunoglobulins (Ig) and antibacterial proteins like lysozyme and lactoferrin are components of the humoral defence against infections. Changes in Ig, lysozyme and lactoferrin concentrations during endotoxin-induced inflammation in the test cistern and udder quarter of the dry cow were studied. Surgical closure of the passage between teat and udder cisterns enabled studies of reactions in the teat cistern without interference of the mammary gland. After endotoxin infusion, IgG1, IgG2, lysozyme, and to some extent IgM, increased in the teats and udder quarters, and were positively correlated with changes in somatic cell counts. No significant changes were observed in IgA or lactoferrin. The origin and significance of Ig, lysozyme and lactoferrin in the bovine teat and udder are discussed. Ig probably originated both from serum and from local plasma cells, while leukocytes appeared to be the source of lysozyme during inflammation. Secretory epithelium appeared to be the source of lactoferrin. Support for this theory was the almost total absence of lactoferrin in teat cistern samples.     

The effect of experimental infectious mastitis on leukocyte subpopulations and cytokine production in non-lactating ewes.

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichia coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin + lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1 beta, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.     

Partial purification and characterization of proteins with growth promoting activities from ovine mammary gland secretions.

Developmental regulation of growth promoting activities in mammary secretions of pregnant Awassi ewes was defined, and growth factors contained in these secretions were partially purified and characterised. Mammary secretions from pregnant ewes enhanced fibroblast cell (AKR-2B) and mammary cell (CID-9 cell strain) proliferation to levels comparable to that induced by 10% Foetal calf serum. Major milk proteins in mammary secretions collected from pregnant ewes one month prior to lambing up to one week after lambing, were resolved by SDS-PAGE, while gelatinases were resolved by zymography. Gelatinase activity was noted prior to P134 and decreased thereafter to reach a minimum during lactation. This decrease was concomitant with the onset of casein production. It is during this critical developmental period that highest growth promoting activity in mammary secretions was detected.Secretions with highest growth promoting activity were fractionated by ion exchange and gel filtration chromatography. Two heat-resistant, trypsin/chymotrypsin sensitive, growth-promoting activities were characterised. The first, designated ovine mammary derived growth factor-1 (oMDGF-1), had around a 30 kDa molecular weight and eluted at 0.65 M NaCl gradient on cation ion exchange chromatography. The second, oMDGF-2, eluted under gel filtration conditions at a molecular weight of 50 kDa and 150 kDa. oMDGF-1 induced changes in Connexin 43, but not in beta-casein mRNA expression by CID-9 mammary cells.In conclusion, growth factor activities in ewe mammary secretions peak during gestation at a period that overlaps maximal gelatinase expression and precedes milk protein synthesis. The factors modulate mammary cell function and may play a role in mammary gland development.     

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.     

Changes in oestrogen,progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs

Changes in the concentrations and affinities of receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) were studied in mammary glands of healthy bitches with regard to age, the location in the mammary chain and the stage of the oestrous cycle. Uterus was used as the reference tissue for the evaluation of steroid receptors. Mammary and uterine samples from 7 healthy bitches were taken at five stages of the oestrous cycle in such a way that all the locations in the mammary chain were represented at each stage of the cycle (10 samples/dog). ER, PR and EGF-R were detected by biochemical assays using increasing concentrations of tritiated (steroids) or iodinated (EGF) ligands. A significant direct correlation was found between the ER and PR concentrations for mammary and uterine samples. No significant correlation was found between the steroid receptors and EGF-R concentrations. Mammary ER concentrations were significantly higher in bitches of 5 years of age or older than in younger ones; in posterior glands (4th and 5th pairs) than in anterior glands; and in the mid-luteal phase. Mammary PR did not vary significantly with age or location but was significantly lower in the early luteal phase than in other phases. A similar decrease in PR concentrations was observed in the uterus during the early luteal phase and uterine ER and PR concentrations were very low in the mid-luteal phase. Mammary EGF-R were not significantly higher in the early or mid-luteal phase than in pro-oestrus or anoestrus.The differences observed between the uterine and mammary steroid receptor concentrations during the oestrous cycle could be due to different mechanisms for regulating steroid receptor expression in the two tissues. Mammary EGF-R concentrations may be linked, as in other species, to cellular proliferation and/or to the serum progesterone concentrations.Abbreviations BSA bovine serum albumin - EGF epidermal growth factor - EGF-R receptor for epidermal growth factor - ER oestrogen receptor - K _d dissociation constant - LH luteinizing hormone - p probability of error - PR progesterone receptor     

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.     

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.     

The Effect of Experimental Infectious Mastitis on Leukocyte Subpopulations and Cytokine Production in Non-lactating Ewes

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichta coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin+ lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1β+ (IL-1β), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1β, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.