Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma

Effects of recombinant bovine interferon (rBoIFN) gamma on mammary gland neutrophil activity during the periparturient period were studied. Bovine mammary gland neutrophils were isolated and incubated in mammary gland secretions obtained from Holstein-Friesian cattle during the last 2 weeks of gestation. Cell functions were evaluated following treatment with 10 U, 100 U, and 1000 U of rBoIFN-gamma. Bacterial phagocytosis, bactericidal activity and chemiluminescence were significantly lower for neutrophils incubated in mammary gland secretions when compared with control neutrophils incubated in Hank's balanced salt solution. Treatment of mammary neutrophils with rBoIFN-gamma reversed the suppressive effects of mammary secretions resulting in higher chemiluminescent activity and significantly more bacterial phagocytosis and bactericidal activity when compared with untreated controls. Results from these preliminary in vitro data suggest that rBoIFN-gamma therapy may modulate mammary gland neutrophil functions in vivo and possibly facilitate the rapid clearance of mastitis-causing pathogens mammary glands during the periparturient period.     

Susceptibility of Pasteurella haemolytica to the bactericidal effects of serum, nasal secretions and bronchoalveolar washings from cattle

Several isolates of logarithmic-phase organisms of Pasteurella haemolytica were shown to be sensitive to an antibody and complement-mediated killing mechanism in adult bovine serum. Data suggested that the classical complement pathway was important in the induction of bactericidal activity of serum. Sera from calves after colostrum feedings (post-colostral sera) killed only 30% of the bacteria in spite of the presence of high levels of antibodies against P. haemolytica. Addition of post-colostral serum to heat-inactivated adult bovine serum decreased the bactericidal capacity of the latter. It was speculated that this inhibition may have been caused by the presence of blocking antibodies (IgA) found in the post-colostral serum. Undiluted nasal secretions collected from adult cattle were not bactericidal to P. haemolytica. The results also suggest that the bronchoalveolar washings (BAW) from vaccinated calves, in spite of having a high antibody titer, were less bactericidal to P. haemolytica than BAW from sham-vaccinated calves (71.12% vs. 83.12%). The bactericidal factor(s) present in BAW from sham-vaccinated calves was heat stable, not complement dependent, and was not related to lysozyme concentration.     

Bactericidal activity of porcine neutrophil secretions

Antimicrobial proteins in neutrophil granules exert their bactericidal activity both within the neutrophil phagolysosome and as components of neutrophil extracellular traps. This study evaluated the bactericidal activity of porcine neutrophil secretions against four bacterial pathogens of swine. Porcine neutrophils were treated with or without phorbol myristate acetate (PMA), then the resulting supernatants were incubated with Escherichia coli K-12, Streptococcus suis, Actinobacillus suis, or Pasteurella multocida, and the surviving colony forming units were enumerated. Supernatants of PMA-activated neutrophils killed an average of 95% of E. coli K-12 cells, relative to supernatants from untreated neutrophils. Inhibition of elastase activity using chloromethylketone (CMK) prior to PMA stimulation significantly reduced the bactericidal activity of the neutrophil supernatants; 57% of the PMA-induced bactericidal activity against E. coli K-12 was estimated to be elastase-dependent. The same neutrophil supernatants had lower bactericidal activity against S. suis, A. suis, and P. multocida, with 30%, 36% and 13% reduction in bacterial numbers, respectively. The cathelicidin porcine myeloid antimicrobial peptide (PMAP)-36 and lactotransferrin were among the proteins identified in the supernatants of PMA-stimulated neutrophils by mass spectrometry. These findings imply that elastase-activated proteins, such as cathelicidins, are partially responsible for the bactericidal effect of porcine neutrophil secretions, but non-elastase-dependent proteins such as lactoferrin may also contribute. Further, the secretions of activated neutrophils were effective in killing the avirulent E. coli K-12 but were less effective against the other bacteria tested, suggesting that these pathogens may have evolved mechanisms to resist neutrophil-mediated killing.     

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.     

The effect of interferon-gamma intramammary administration on mammary phagocyte function

The effect of recombinant bovine interferon-gamma on intramammary phagocyte function of mammary gland was studied in 4 Holstein cows (Study 1) and 7 Holstein cows (Study 2). Recombinant bovine interferon-gamma was intramammarily infused on day 6 of the dry period and phagocytes were collected from lacteal secretions and tested in vitro 24 hours later. Results from Study 1 indicate that phagocytosis of Escherichia coli and Staphylococcus aureus was significantly increased after than before interferon treatment. Similarly, the number of bacteria killed/active phagocyte was enhanced by treatment. Results from Study 2 suggested a trend towards increased production of oxygen dependent bactericidal components and increased killing ability by phagocytes exposed to interferon as compared to control phagocytes. These results from both studies suggest that intramammary infusions of bovine interferon-gamma can stimulate phagocyte function during the early phase of the dry period.     

Interaction of turkey complement with Escherichia coli isolated from turkeys

The role of turkey complement in a serum bactericidal reaction was determined using serum-sensitive and serum-resistant Escherichia coli isolated from turkeys. Inactivation of complement by heating serum (56 C for 40 minutes) or by treating serum with 10 mM EDTA eliminated bactericidal activity. Serum-sensitive E coli organisms were killed by turkey serum treated with 10 mM ethylene glycol-bis-beta-(aminoethyl ether)-N,N,N',N'-tetraacetic acid and 5 mM MgCl2. Exposure of normal turkey serum to serum-sensitive or serum-resistant E coli resulted in equivalent reductions in hemolytic activity of serum. Treatment of serum-resistant E coli with antibody rendered the bacteria sensitive to bactericidal effects of normal turkey serum. Serum-sensitive E coli organisms were readily killed by an alternative complement pathway, serum-sensitive and serum-resistant E coli activated the complement system equally well, and antibody was required for complement-mediated killing of certain serum-resistant E coli organisms from turkeys.     

发情期大熊猫阴道分泌物细菌分析

寄生菌是动物体非特异性免疫的重要组成部分，体表或体内的微生态系统对于牵制或抑制外来有害病原体起到重要作用。生殖道内的寄生菌对于维持生殖道内的稳态起着重要作用。2011年年初在对北京动物园2只健康的雌性大熊猫“萌萌”和“瑛华”进行人工授精过程中，抽取阴道深部（约15cm处）分泌物，于有氧和无氧两种条件下，进行了细菌培养。2只大熊猫的培养结果完全相同。均主要有三株细菌，利用API鉴定试剂条鉴定的结果是L群链球菌、溶血性大肠杆菌和乳杆菌。对数量较多的链球菌和溶血性大肠杆菌进行了药物敏感试验以及16sRNA的测定。药物敏感试验结果显示这两株细菌对大多数抗生素均敏感。16sRNA序列测定结果进一步印证了API菌株鉴定的结果。     

Association of complement sensitivity with virulence of Pasteurella multocida isolated from turkeys

Two strains of Pasteurella multocida, both derivatives of strain P1059, were compared for virulence for 14-week-old turkeys and sensitivity to turkey plasma. Strain P1059-1, a nalidixic-acid-resistant mutant of P1059 with an LD50 of approximately 10(3) colony-forming units (CFU), was more resistant to the bactericidal effects of fresh turkey plasma at 37 C than avirulent strain P1059-1A. P1059-1A, with an LD50 of approximately 10(8) CFU, is an acapsular variant of P1059-1 that spontaneously arose after prolonged passage on artificial medium. The bactericidal effect on P1059-1A was removed when turkey plasma was treated with heat or with zymosan, maneuvers that removed hemolytic complement activity from turkey plasma.     

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.     

In vivo effect of teicoplanin and vancomycin upon haemolytic and bactericidal activity of serum against Staphylococcus aureus

The present study was undertaken to determine whether teicoplanin and vancomycin influence the hemolytic and bactericidal activity of serum obtained from mice (Swiss, aged 15 ± 2 weeks). Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units (CFU/ml) of Staphylococcus aureus that survived after 24 h of incubation in the presence of serum. The results indicate that (1) teicoplanin and vancomycin increase the haemolytic activity of serum; (2) in the serum from animals treated with both teicoplanin or vancomycin, the number of CFU/ml of Staphylococcus aureus declines; and (3) these antibiotics appear to have a similar effect upon the complement system.     

Local intestinal immune response to Escherichia Coli heat-labile (LT) enterotoxin: LT antitoxin levels in healthy, diseased and antigen-fed pigs

With purified LT toxin and IgA, specific anti-LT enterotoxin activity was demonstrated in small intestinal contents of 27 pigs. After 60 days of age, rise in intestinal LT antitoxin titer was observed. Feeding LT containing E. coli antigen increased LT antibody levels in the intestinal secretions, but decreased antibody titers in sera. In post-weaning E. coli diarrhea LT antibody levels in intestinal secretions and sera decreased significantly. This phenomenon can be related to the occurrence of the frequently observed post-weaning E. coli diarrhea.     

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.     

富含亮氨酸和精氨酸的抗菌肽设计

通过构建亮氨酸疏水面和精氨酸亲水面来设计全新的抗菌肽,并评估利用这种螺旋轮投影模型设计出的抗菌肽的生物学活性.检测了设计得到的新型抗菌肽LGR16的最小抑菌浓度(MICs)、对于红细胞和哺乳动物细胞的细胞毒性以及杀菌动力学.结果表明,新型抗菌肽LGR16具有很强的抗菌活性,其对于革兰氏阴性菌和阳性菌的最小抑菌浓度分别为8和4 μg·mL-1.LGR16杀菌速度快,在10 min内能杀死细菌.在最小抑菌浓度该抗菌肽只表现出很弱的溶血活性和对哺乳动物细胞的细胞毒性.综上可见,抗菌肽能够通过在螺旋轮中聚集疏水性或者碱性氨基酸残基来设计,而且本研究得到的抗菌肽LGR16具有替代抗生素的潜质.     

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.     

Research Progress on Pharmacological Action of Cephalonium and Its Application in Prevention and Treatment of Mastitis in Dairy Cow

Cephalonium is a second generation cephalosporin.It is effective to prevent and treat mastitis during dry period.It has a broad antibacterial spectrum,strong bactericidal activity,less allergic reactions and low toxicity,and so on,especially showing a good antibacterial activity to Staphylococcus aureus and Streptococus.In this paper,the physical and chemical properties,pharmacokinetic,pharmacology and toxicology,residue and withdrawal periods,application in the prevention and treatment of mastitis in dairy cows,and the prospect of the development of cephalonium were investigated and reviewed.     

头孢洛宁的药理作用及其在奶牛乳房炎防治中应用的研究进展

头孢洛宁是第2代头孢菌素类抗生素,是防治干乳期奶牛乳腺炎的有效药物,具有抗菌谱广、杀菌力强、过敏反应少、毒性低等优点,尤其对金黄色葡萄球菌、链球菌展现出良好的抗菌活性。文章综述了头孢洛宁的研究现状、理化性质、药理和毒理、药代动力学、残留及弃奶期及其在奶牛乳房炎防治中的应用,并对其应用前景作了展望。     

Lactoperoxidase, thiocyanate, and free cystine in bovine mammary secretions in early dry period and at the start of lactation and their effect on Streptococcus agalactiae growth.

The concentrations of lactoperoxidase (LP) and thiocyanate (SCN-) in the mammary secretions of 4 dairy cows in the early dry period were similar to or higher than concentrations in the milk before drying off. The concentrations of free cystine progressively increased in the secretions beginning 3 to 5 days after the last milking; the mean concentrations were 0.66 mumoles/L before drying off and 6.66 mumoles/L after drying off. The mean concentrations of free cysteine were 0.28 mumoles/L before drying off and 1.4 mumoles/L after drying off. The secretions, when diluted in steamed milk, showed greater stimulation of Streptococcus agalactiae growth as the drying-off period progressed. This increase in stimulatory activity was attributed primarily to the increased concentrations of cystine because cystine counteracts the LP/SCN-/hydrogen peroxide inhibitory system for S agalactiae. This effect on the LP system may account for any increase in susceptibility to S agalactiae under infection during the dry period. In 3 other cows, the mammary secretions on the day of calving had lower mean concentrations of LP, SCN-, and free cystine and cysteine than those obtained 4 to 5 days before, and 7 to 8 days after calving.     

Hemolytic complement levels of neonatal calves delivered from protein-energy malnourished dams and subjected to cold stress

Beef cows were placed on protein-deficient and/or energy deficient rations for the last 150 days of pregnancy. After birth their calves were placed on 1 or 21 C environmental chambers for 3 days, and sera were collected for determination of complement (C) levels. At birth, the mean complement hemolytic (CH50) titer of all calves was 46.0 +/- 1.7 units, but the titer rapidly dropped (P < 0.01) to 31.6 +/- 1.2 by 12 hours after birth. Levels of C activity then began to rise and reached a mean titer of 76.3 +/- 3.0 by 3 days of age. A quadratic curve of predicted CH50 values was constructed from the data. Differences between principal and control groups of calves were not detected. These results suggest that maternal protein-calorie deprivation and limited cold stresses have little effect on levels of C activity in the bovine neonate. Possible explanations for the decrease in CH50 levels after birth are discussed.     

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.     

A sensitive microassay for the determination of hemolytic complement activity in bovine milk

A ⁵¹Cr release microhemolytic complement assay is described to detect hemolytic complement activity in bovine milk. ⁵¹Cr-labeled guinea-pig erythrocytes (GPRBC), which have been sensitized with a subagglutinating amount of rabbit anti-GPRBC, are placed in microtiter plates. Pooled bovine sera as source of complement to achieve about 50% of ⁵¹Cr release were added to each well prior to the addition of the samples on the test. Determination of CH100 titer was obtained by difference of counting between heated and unheated diluted whey samples from a standard linear regression. Comparative hemolytic values throughout lactation were established for the first time and confirmed the improved sensitivity of the assay.