Characterization of immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution

The aim of this study was to characterize the immune response in Staphylococcus aureus chronically infected bovine mammary glands during active involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic naturally acquired S. aureus intramammary infections (IMI) were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical analysis were taken. Protein expression of toll-like receptor 2 (TLR2) and TLR4 was significantly higher in S. aureus-infected quarters than in uninfected controls at the three involution stages studied. Protein expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α and IL-17 was significantly affected by IMI; being higher in S. aureus-infected than uninfected quarters during all evaluated stages. In S. aureus-infected and uninfected quarters protein expression of lactoferrin increased from day 7–14 of involution, decreasing significantly to day 21 in mammary quarters with chronic infections. The number of monocytes-macrophages was significantly higher in S. aureus-infected than in uninfected control quarters at 7 and 21 d of involution. The number of T lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters at 7 and 14 d of involution while the number of B lymphocytes was significantly higher in S. aureus-infected than in uninfected quarters during all evaluated stages, showing a progressive increase as involution advanced. These results demonstrated a sustained and exacerbated innate and adaptive immune response during chronic S. aureus IMI, playing a critical role in the infection control during active involution.     

Somatic cell and innate immune responses in mammary glands of lactating cows to intramammary infusion of Bifidobacterium breve at pre-drying off period

Intramammary infusion of Bifidobacterium breve (B. breve)-induced somatic cell (SC) counts, chemiluminescent response (CL), lactoferrin (LF) concentrations and mastitis-causing pathogens from quarters with subclinical mastitis were measured to evaluate innate immune response of mammary glands in dairy cows at 3 to 4 weeks before drying off. SC counts in 7 quarters of 7 control cows and 5 quarters of 6 cows with mastitis increased markedly on day 1 and SC values in control cows were significantly (P<0.05) increased and returned to pre-infusion levels on day 5 after B. breve-infusion. CL values in both groups increased markedly on day 1 and then decreased after B. breve-infusion; however, CL values in cows with mastitis did not return to normal levels on day 5 and at postpartum. The CL values were highly correlated with their SC counts in milk from both groups. LF concentrations increased toward day 3 after B. breve-infusion and were higher in cows with mastitis. B. breve-infusion eliminated 16.6% (1/6) of pathogens from 6 quarters with chronic subclinical mastitis. B. breve-induced SC responses in quarters from 3 cows with mastitis showed characteristic patterns of recovery, persistent and new infections. B. breve-induced SC counts in quarters from the cows in the pre-drying off were lower (25.7–70.6%) than those of the cows in mid-lactation. The intrinsic innate immune response in cows on pre-drying off may be decreased and appears to be insufficient to eliminate pathogens from mammary gland in the pre-drying off.     

An Evaluation of a Teat Dip with Dodecyl Benzene Sulfonic Acid in Preventing Bovine Mammary Gland Infection from Experimental Exposure to Streptococcus agalactiae and Staphylococcus aureus

The effectiveness of a teat dip with dodecyl benzene sulfonic acid (1.94%) for the prevention of intramammary infections was determined in cows experimentally challenged with Streptococcus agalactiae and Staphylococcus aureus. The infection rates with Streptococcus agalactiae and Staphylococcus aureus were 62.5% and 75% in undipped quarters, 12.5% and 21.5% in dipped quarters with a reduction rate of 80% and 71% respectively. The significance of some findings in relation to mastitis control are discussed.     

Association of CXCR1 polymorphisms with apoptosis,necrosis and concentration of milk neutrophils in early lactating dairy heifers

Associations between polymorphisms in the candidate gene CXCR1, encoding the chemokine (C-X-C motif) receptor 1, and udder health have been identified before. In the present study, associations between the CXCR1 genotype (whole coding region) and apoptosis, necrosis, and concentration of milk polymorphonuclear neutrophilic leukocyte (PMNL) of 292 quarters belonging to 73 early lactating dairy heifers were studied. In uninfected quarters, % milk PMNL apoptosis was higher in c.980GG heifers [least squares means (LSM) 27%] compared to c.980AG heifers (LSM 16%), whereas in infected quarters, % milk PMNL apoptosis was higher in c.642GG heifers (LSM 29%) compared to c.642AG heifers (LSM 18%). Differences in milk PMNL concentration between infected and uninfected quarters were smaller in c.980AG heifers than in c.980GG heifers. An association between the CXCR1 genotype and necrosis of milk PMNL could not be demonstrated. Results indicate that CXCR1 polymorphisms influence viability and concentration of milk PMNL and provide a foundation for future research.     

Staphylococcus aureus chronic intramammary infection modifies protein expression of transforming growth factor beta (TGF-β) subfamily components during active involution

The objectives of this study were to determine whether Staphylococcus aureus chronic intramammary infection (IMI) influences protein expression of TGF-β subfamily components and collagen I and to examine the histomorphometric changes that occur in mammary stroma and parenchyma during active mammary gland involution. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic natural S. aureus IMI were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical and morphometric analysis were taken. Protein expression of TGF-β1, TGF-β2 and TGF-β3 was significantly higher in chronically infected quarters than in uninfected controls at the three involution stages studied. Immunostaining of TGF-βR1 and TGF-βR3 and collagen I was significantly higher in S. aureus-infected quarters than in uninfected controls at every involution time evaluated. The percentages of tissue area composed of parenchyma and intralobular stroma were significantly higher in S. aureus-infected than in uninfected quarters. Chronic S. aureus mastitis modifies protein expression of the three TGF-β isoforms and type 1 and 3 receptors, which was associated with changes directed to limit the scope of inflammation and injury to the host.     

Innate immune response of mammary gland induced by intramammary infusion of Bifidobacterium breve in lactating dairy cows

This study aimed to evaluate innate immune responses of mammary glands induced by intramammary infusion of Bifidobacterium breve in dairy cows. Somatic cell counts in quarters of cows showed a marked increase following B. breve infusion on days 1 and 2. Opsonized-stimulated chemiluminescence response in quarter milk was significantly (P<0.05) increased by B. breve infusion on days 1 to 3 compared to that of pre-infusion. Lactoferrin concentrations in B. breve-infused quarter milk increased significantly (P<0.05) on days 2 to 4 and 6 compared to those of pre-infusion. IgG and IgA concentrations in B. breve-infused quarters significantly (P<0.05) increased on days 2 to 4 for IgG and days 3, 4, 6 and 8 for IgA compared to those of pre-infusion. Interleukin (IL)-1β and IL-8 mRNA levels in somatic cells from B. breve-infused quarters were significantly (P<0.05) upregulated on day 1 compared to those on days 0 and 14. Conversely, IL-6 mRNA levels in somatic cells from B. breve-infused quarters on days 0, 1 and 14 and NF-κB mRNA levels on day 0 were significantly (P<0.05) down-regulated compared to those of control. IL-1β, tumor necrosis factor (TNF)-α and IL-6 concentrations increased on days 1, 3 and 7 after B. breve infusion in quarters. Intramammary infusion of B. breve (3 × 10⁹ cfu) induces a massive influx of leukocytes and enhances innate immune response in mammary glands. This event may contribute to the enhancing host defense in the mammary gland.     

Subclinical mastitis causes alterations in nitric oxide, total oxidant and antioxidant capacity in cow milk

The aim of this study was to investigate total antioxidant (TAC), and oxidant capacity (TOC) and nitric oxide (NO) levels in milk of cows with subclinical mastitis. Brown Swiss and Holstein breed cows were screened with California Mastitis Test (CMT) to determine mammary glands with subclinical mastitis. Moreover, somatic cell counts (SCC) were determined electronically in all milk samples. Mammary quarters were classified as healthy (n = 25) or subclinical mastitis (n = 35) based on CMT scores and somatic cell count (SCC: ?200,000/ml or >200,000/ml) in milk. Nitric oxide, TOC and SCC levels were significantly higher (p < 0.001, p < 0.005 and p < 0.001, respectively) in milk from mammary quarters with subclinical mastitis compared to those from healthy mammary quarters. In conclusion, subclinical mastitis results in higher NO concentrations, TOC and SCC, and NO and TOC were positively correlated with SCC. Moreover, alterations in NO levels and TOC in milk could be used as an alternative diagnostic tool to screen for subclinical mastitis.     

Bovine mycoplasma mastitis from intramammary inoculation of small numbers of Mycoplasma bovis. I. Microbiology and pathology

Intrammary inoculation of 70 colony forming units (cfu) of Mycoplasma bovis into one quarter of four previously non-infected cows resulted in severe mastitis in inoculated and uninoculated quarters. Hematogenous spread of the infection was most likely, as mycoplasma was isolated from the peripheral blood of three of four cows and the milking machine was designed to prevent quarter to quarter communication of milk and air. The presence of large numbers of mycoplasma exceeding 10⁶ cfu/ml of milk preceded the onset of overt sero-purulent mastitis by 1–3 days. In general, the severity, duration of the infection and within cow spread of mastitis to adjacent quarters after the inoculation of 70 cfu was indistinguishable from naturally occurring mycoplasma mastitis.The pathology of the chronically infected quarters consisted of alveolar involution and moderate to severe mononuclear infiltration and an increase in interalveolar and loose connective tissue. The quarters of one cow resolving the infections at the time of slaughter were not as severely affected and contained numerous milk-producing alveoli and many alveoli with hyperplasia of the alveolar cells.     

Quarter,cow, and farm risk factors for intramammary infections with major pathogens relative to minor pathogens in Thai dairy cows

A cross-sectional study was carried out from May to September 2011 on 35 smallholder dairy farms in Chiang Mai, Thailand, to identify the quarter, cow, and farm factors that relate to intramammary infections (IMI) from major specified pathogens, compared to infections from minor pathogens. Data on general farm management, milking management, and dry cow management were recorded for each herd. Quarter milk samples were collected from either clinical or subclinical mastitis quarters. Dependent variables were binary data defining the specified major pathogens, including Streptococcus agalactiae (7.1 %), Streptococcus uberis (9.4 %), Streptococcus dysgalactiae (4.0 %), and other streptococci (16.7 %), as a case, and all minor pathogens as a control, in each dependent variable. The occurrence of S. agalactiae IMI was lower in first-parity cows and cows with short milking time. Cows with body condition score (BCS) <2.5 had higher occurrence of S. agalactiae IMI. The occurrence of S. uberis IMI was higher in quarters with California mastitis test (CMT) score 2, score 3, and having clinical mastitis and in farms with increasing age of vacuum system. Quarters with CMT score 3, having clinical mastitis, cow with manual milking after detaching milking cluster, and farms with high bulk milk somatic cell counts (BMSCC >500,000 cells/ml) had higher occurrence of S. dysgalactiae IMI. For other streptococci, quarters having clinical mastitis, BCS <2.5, and pulling down of milking cluster while milking increased occurrence of other streptococci IMI relative to minor pathogen IMI. These results highlight the importance of individual cow factors, milking characteristics, and BMSCC in determining the risk of IMI from major pathogens.     

Effect of precalving intramammary treatment with pirlimycin in nulliparous Holstein heifers

A clinical trial was conducted to determine whether prepartum intramammary pirlimycin reduces the proportion of nulliparous heifers with intramammary infection (IMI) during early lactation and improves milk production. Quarter milk samples were collected from 428 heifers, systematically allocated to treatment and control groups, 6 to 12 d before the expected calving date and 2 to 8 d after calving. At the prepartum visit, heifers in the treatment group (n = 219) received an infusion of pirlimycin hydrochloride in all 4 quarters; the control heifers (n = 209) received no infusions. Intramammary infection was detected in 69% of the heifers and 33% of the quarters before calving. After calving, the proportion of treated heifers with IMI was significantly lower than the proportion of control heifers (31% versus 45%). Staphylococcus aureus was isolated from 10% of the heifers and 3% of the quarters before calving. After calving, the proportion of IMIs due to S. aureus was significantly lower in the treated heifers than in the control heifers (5.6% versus 10%). Antibiotic treatment increased the percentage of cures and prevented new IMIs caused by gram-positive bacteria after calving. The incidence of new IMIs caused by gram-negative bacteria and yeast was higher among treated heifers than among control heifers. There was no overall effect of treatment on milk production, but there was a significant interaction effect of treatment and the interval between treatment and calving. An increase of 302 kg of milk was observed when antibiotic treatment was applied more than 1 wk before calving. Treatment did not affect the milk somatic cell count on the 1st 3 test days after calving.     

The Use of Induced Mammary Infections for Evaluating Dry Cow Treatment Products II. Trial of a Proposed Method to Compare Three Levels of Novobiocin

Infections were induced at the end of lactation in all udder quarters of 19 cows by the infusion of 0.2 mL of a 10^-4 dilution in milk of a six hour milk culture of Staphylococcus aureus, strain 305 (A.T.C.C No. 29740). Two right or two left udder quarters were infected at 15 days and the opposite two five days before the last milking of lactation. Following the last milking all four udder quarters of eight cows were treated with 400 mg novobiocin in 10 mL of 2% aluminum monosterate in peanut oil, gelled. All udder quarters of eight other cows were treated with 50 mg novobiocin in the same vehicle and the udder quarters of three cows were treated with the vehicle only.

At calving, eight of 32 quarters treated with 400 mg novobiocin were still infected, as were 18 of 32 treated with 50 mg of novobiocin and all those quarters treated with vehicle only. Results were identical from udder quarters infected 15 and five days before drying off.

No significant differences were found between quarters in milk yield on the last day of lactation, nor the length of the dry period. An increasing number of udder quarters were infected at calving with increase in lactation age of the cow, although the small number of cows would not allow a firm conclusion. A significant difference in results was found between front and hind udder quarters, only five of 32 front quarters were infected at calving as compared to 21 to 32 hind quarters.

The method proposed was found to give essentially the same results as those from a large field trial using the same antibiotic. It should therefore be useful in evaluation trials of new antibiotic products for dry cow treatment.

     

Effect of management practices and animal age on incidence of mastitis in Nili Ravi buffaloes

Buffalo is an economically important dairy animal in South Asia but mostly ignored in research priorities. In this retrospective study, the effect of management practices and age of animal on the incidence of mastitis in Nili Ravi buffaloes was investigated. A total of 1,560 quarters of buffaloes (n?=?390) were screened by visual examination of the udder and milk (clinical mastitis) and California mastitis test (subclinical mastitis). Household data was collected on a predesigned questionnaire and analyzed. The prevalence of subclinical mastitis, clinical mastitis, and blind quarters was 41.8, 13.6, and 9.7 %, respectively. The highest prevalence was noted in the hind quarters and left side as compared to that in the forequarters and right side. This data significantly (p?p?p?R ²?=?0.772) with mastitis. This study concluded that some factors alone or in combination with other factors influence significantly the occurrence of mastitis, and to minimize the infection, these factors should be considered. The outcome of the study will be valuable for policy-making for positive management practices and implementation of preventive measures.     

Relations Between Udder Infection and Somatic Cells in Camel (Camelus dromedarius) Milk

Quarter milk samples (n = 391) from 101 camels were examined to study the occurrence and causes of mastitis in traditionally managed camels in eastern Sudan and to evaluate the value of the California Mastitis Test (CMT), somatic cell count (SCC) and adenosine triphosphate (ATP) in the detection of subclinical mastitis in the camel.One hundred and seventy (43.5%) of the quarter milk samples yielded pathogenic bacteria. Streptococcus agalactiae, other Streptococcus spp., Staphylococcus aureus, coag–ulase–negative staphylococci, and Escherichia coli were isolated from milk. Thirty–two (8.2%) quarter milk samples yielded mixed cultures, and 189 (48.3%) yielded no growth.Mean values for CMT, SCC and ATP were higher for quarters infected with major pathogens. However, a significant number of quarter milk samples had elevated values in these tests but were from quarters from which no bacteria were isolated. The ability of the tests to predict a positive bacteriology increased slightly when 2 or 3 tests were combined. kw|Keywords|k]inflammation; k]diagnostic tests; k]Mastitis; k]CMT; k]ATP; k]bacteriology; k]Sudan     

Effects of intramammary infection and parity on calf weaning weight and milk quality in beef cows

The objectives of this study were to determine 1) the effect of intramammary infection on calf weaning weight, milk somatic cell count, and milk composition, and 2) the effect of parity on percentages of infected cows, infected quarters, and blind quarters. The number of infected quarters, milk somatic cell counts, milk components, and intramammary infection were studied at weaning in 164 beef cows. The percentage of infected cows ranged from 61.9% at first parity to 66.7% at fifth to ninth parities. Cows with three or four infected quarters had higher (P < .01) milk somatic cell counts than cows with zero, one, or two infected quarters. Among bacterial isolates, Staphylococcus aureus-infected quarters had the highest (P < .01) milk somatic cell count. Percentages of butterfat and lactose were lower (P < .01) in milk from infected quarters than from uninfected quarters. Infections by S. aureus and coagulase-negative staphylococci were the most common and accounted for 67 to 78% of the infections. Percentages of infected quarters and infections caused by S. aureus increased with parity (P < .01). Intramammary infections did not affect (P > .10) calf weaning weight. In conclusion, intramammary infection had no effect on calf weaning weight but increased milk somatic cell count and decreased the percentage of protein, lactose, solids-not-fat, and butterfat. The number of infected and blind mammary quarters increased with parity.     

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.     

An Outbreak of Pseudomonas Mastitis in Dairy Cows

An outbreak of mastitis in a dairy herd is described in which the causative organism was Pseudomonas aeruginosa. Cases occurred either in dry cows or in animals which had very recently calved. The fact that all four quarters were involved is a very strong indication that the bacteria had been introduced in the dry cow therapy.     

Activity of cyclooxygenase-2 and nitric oxide in milk leucocytes following intramammary inoculation of a bio-response modifier during bovine Staphylococcus aureus subclinical mastitis

The objective of the study was to evaluate the effects of intramammary infusion of a bio-response modifier (BRM) prepared from Nocardia globerula on certain inflammatory markers and percentage of neutrophil/lymphocyte in mammary secretions during bovine Staphylococcus aureus subclinical mastitis (SCM). The somatic cell count (SCC), total bacterial count (TBC) in milk, cyclooxygenase-2 (COX-2) activity, production of nitrite and nitrate (NOx) in milk leukocytes and neutrophil % and lymphocyte % in milk were evaluated before and after intramammary infusion of BRM in healthy and quarters inflicted with S. aureus SCM. Intramammary infusion of BRM significantly enhanced the SCC in earlier phase with subsequent reduction on day 7 after initiation of treatment. Whereas, the reduction of TBC was observed from day 3 onwards. The COX-2 activity and NOx production in milk cell increased initially on day 3 of post treatment but reduced on day 5 in SCM infected quarters following BRM infusion. The neutrophil % and lymphocyte % in milk also enhanced significantly on day 3 but reduced on day 5 in SCM infected quarters in response to BRM infusion. Initial influx of SCC, increased neutrophil%, lymphocyte % and enhanced COX-2 and NOx activity indicate the immunomodulatory potential of BRM in S. aureus SCM. Reduction of TBC could be due to increased leukocytosis or direct microbicidal activity of the activated milk cells. The beneficial effect of the BRM could be used as alternative therapy in the control of S. aureus SCM in cows, either alone or in conjunction with antibiotic therapy.     

Relationships between the phagocytic ability of milk macrophages and polymorphonuclear cells and somatic cell counts in uninfected cows

Phagocyte numbers and activities were compared in milk from 2 groups of uninfected mammary-gland quarters from 3 cows each: 6 quarters with a high (> or = 200 000/mL) somatic cell concentration (SCC), analyzed as 4 individual quarters and 1 pooled sample; and 12 quarters with a low SCC (< 200 000/mL), analyzed as 6 paired samples. The concentrations and ability of macrophages and polymorphonuclear (PMN) cells to phagocytize fluorescent microspheres were determined by flow cytometry after exposure of the cells to the microspheres. The macrophages and PMNs contained 2 major subpopulations, characterized by low phagocytic (LP) or high phagocytic (HP) ability. The quarters with high SCCs had significantly lower percentages of HP cells than did the quarters with low SCCs (P < 0.01). Whether mammary-gland quarters or cows were the unit of analysis, the HP/LP ratio was negatively related to the SCC (P < 0.04), which explained more than 50% of the SCC variability. Thus, poor bovine mammary-gland phagocytic ability may be associated with high SCC. Longitudinal studies are suggested to further explore and characterize these relationships.     

The prophylactic effect of a dry-cow antibiotic against Streptococcus uberis

The prophylactic use of a dry-cow antibiotic for reducing the incidence of mastitis due to Streptococcus uberis was studied in four seasonally calving dairy herds involving 378 cows. The treatment was a long-acting dry-cow antibiotic preparation administered immediately after the last milking of lactation. New intramammary infections were identified by comparing the bacteriological status of quarters at drying off with that after calving, or through manual udder palpation during the dry period. The administration of dry-cow antibiotic to uninfected quarters at drying off reduced the overall incidence of new infections with Streptococcus uberis from 12.3% for untreated quarters to 1.2% of quarters (p<0.01). The reduction was significant (p<0.01) for both dry-period and post-calving infections. The susceptibility of uninfected quarters to new infection by Streptococcus uberis appeared to be unrelated to the infection status of a cow at drying off. Clinical infections during the dry period were most prevalent (97%) in quarters identified as having open teat canals. Fewer open teat canals (p<0.05) were observed among antibiotic treated quarters over the first 4 weeks of the dry period. Treated quarters had a lower (p<0.05) incidence of new clinical infection during the ensuing lactation and lower somatic cell counts. This did not affect production levels of milk, milk fat or protein. The results clearly indicated a prophylactic benefit for the dry cow antibiotic treatment against new Streptococcus uberis infections during the dry period.     

Bacterial cure and somatic cell count response of dairy cows with a positive California Mastitis Test at calving to therapy with cephapirin sodium

Cows without signs of clinical mastitis were evaluated by the California Mastitis Test at calving (Day 0). Milk samples from 117 of 184 quarters (64 cows) were positive by this test for mastitis and were submitted for bacterial culture and determination of somatic cell counts. Cows with infected quarters were randomly allocated to treatment with cephapirin sodium by intramammary infusion or to be untreated as controls. Two and 4 weeks following calving, milk was again sampled from the infected quarters and tested. By the 4-week evaluation, the quarters treated with cephapirin sodium had significantly (P < or = .05) fewer positive bacterial cultures and somatic cell counts were significantly (P < or =.05) reduced compared with untreated control quarters.