Effect of intramammary injection of RbIL-8 on milk levels of somatic cell count, chemiluminescence activity and shedding patterns of total bacteria and S. aureus in Holstein cows with naturally infected-subclinical mastitis

Summary The effect of intramammary injection of recombinant bovine interleukin-8 (rbIL-8, 1 mg/10 ml of saline) on quarter milk levels of somatic cell count (SCC), chemiluminescence (CL) activity and counts of total bacteria and Staphylococcus aureus (S. aureus) was investigated, using 10 Holstein cows with an early stage or a late stage of subclinical mastitis naturally infected with S. aureus. In the late-stage group, milk SCC and CL activity had significant rises with maximum levels at 6 h, following maintained high levels thereafter post-cytokine injection. The counts in milk total bacteria and S. aureus were insignificantly decreased, being increased back on day 7 post-cytokine injection. Thus, the cytokine was inefficient for the late-stage subclinical mastitis. However, in the early-stage group milk SCC and CL activity declined to under pre-injection levels on day 7 after marked and significant rises at 6 h and day 1 post-cytokine injection. The milk total bacterial count decreased significantly on days 0.25 and 2. Furthermore, the milk S. aureus count was decreased significantly on days 1, 2, 3 and 7 by the cytokine injection. These results suggest that the rbIL-8 has a potential as a therapeutic agent of the subclinical mastitis of dairy cows, if the cytokine is applied at an initial stage of infection.     

应用体细胞计数监测奶牛隐性乳房炎

奶牛乳汁中体细胞数的变化受多方面因素的影响。本文主要论述用体细胞计数检测奶牛隐性乳房炎技术、以及将体细胞数作为标记性状、选育具有乳房炎抗性奶牛的研究。     

Effect of subcutaneous injection of ginseng on cows with subclinical Staphylococcus aureus mastitis

Cows with subclinical mastitis caused by Staphylococcus aureus were subjected to subcutaneous injections with either an extract from the root of Panax ginseng CA Meyer at a dose of 8 mg/kg body weight per day for 6 days, or with saline as a control. The injection areas were checked for adverse reactions. The daily milk production was measured before and after treatment. Blood was collected for total and differential leucocyte counts, identification of lymphocyte subpopulations using flow cytometry, lymphocyte proliferation test, and neutrophil phagocytosis and oxidative burst assay. Quarter milk samples were collected for bacteriological analysis and somatic cell counts (SCC). After the end of treatment, the numbers of S. aureus-infected quarters and milk SCC tended to decrease in ginseng-treated cows. Phagocytosis and oxidative burst activity of blood neutrophils were significantly increased 1 week after ginseng treatment, but the proliferative response of blood lymphocytes did not change significantly. The number of monocytes in ginseng-injected cows was significantly higher 1 week post-treatment than pre-treatment, and the number of lymphocytes was significantly higher than pre-infusion at 2 and 3 weeks after ginseng treatment. Similar changes were not observed in the control group. The present findings indicate that ginseng treatment can activate the innate immunity of cows and may contribute to the cow's recovery from mastitis. It is therefore suggested that ginseng has a potential as a stimulator of the immune system of dairy cows.     

Somatic cell count in milk of selenium-supplemented dairy cows after an intramammary challenge with Staphylococcus aureus

The objective of this study was to evaluate the effect of selenium (Se) supplementation on milk somatic cell count (SCC) in dairy cows. Twelve multiparous Holstein-Friesian cows were fed a diet containing a suboptimal Se concentration (<0.05 ppm, dry basis) starting 2 months before calving. Supplemented cows (n=6) received a single s.c. injection of barium selenate (1 ml/50 kg BW) 45 days prior to calving, whereas control group was kept unsupplemented. Twenty weeks after calving, two mammary quarters (right side) of each cow were challenged with 205,000 cfu/ml of Staphylococcus aureus (strain Newbould 305). Blood was collected bi-weekly until day 150 of lactation for the analysis of blood glutathione peroxidase (GPx1; EC 1.11.1.9) activity. To re-isolate the challenging pathogen and to evaluate SCC, aseptic milk samples were collected daily starting on the day of challenge, and finishing 7 days after inoculation. Unsupplemented cows had a lower activity of GPx1 through the experiment (P<0.001). Natural log SCC (lnSCC) was higher in unsupplemented than Se-supplemented cows (P=0.04), showing evidence of significance after 5 days. Selenium supplementation of dairy cows fed a diet containing a suboptimal Se concentration, resulted in higher blood activity of GPx1, and lower mean lnSCC after an intramammary challenge with Staph. aureus.     

Effect of intramammary infusion of recombinant bovine GM-CSF and IL-8 on CMT score,somatic cell count,and milk mononuclear cell populations in Holstein cows with <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> subclinical mastitis

The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400 μg/5 mL/quarter, n = 9), rbIL-8 (1 mg/5 mL/quarter, n = 9), and phosphate-buffered saline (5 mL/quarter, n = 7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.     

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.     

Effect of vitamin B2 on somatic cell counts in milk of clinical Staphylococcus aureus mastitis.

Effects of intravenous injection of Vitamin B2 (VB2) on the nitroblue tetrazolium (NBT) reductivity of peripheral blood neutrophils and the somatic cell counts (SCC) in quarter milk of Staphylococcus aureus mastitis were investigated. The NBT reductivities of neutrophils were enhanced at 2 days after single injection of VB2 (5.0 and 2.5 mg/kg), and were also enhanced at 4 days after initial injection of continuous 3 days of VB2 (2.5 mg/kg). The SCC in quarter milk were significantly decreased at 3, 7 and 14 days after initial injection of continuous 3 days of VB2 (2.5 mg/kg), however, S. aureus in the infected quarter was not cured bacteriologically by VB2 injection.     

Effects of autogenous toxoid-bacterin in lactating cows with Staphylococcus aureus subclinical mastitis

To evaluate clinical effects of autogenous toxoid-bacterin treatment for Staphylococcus aureus subclinical mastitis in lactating cows, 22 cows which had at least one S. aureus infected quarter were selected from among cows at a S. aureus prevalent dairy farm. Eleven cows were injected with their own autogenous toxoid-bacterin and the others were maintained as non-injected control. In the toxoid-bacterin injected group, 27% of infected quarters were cured during the 12-week trial, compared to 5% in the control group. New intramammary infections with S. aureus were only detected in 3 quarters of the control group. Mean IgG antibody titer against S. aureus somatic antigens and alpha-toxin in serum and milk were significantly increased in the toxoid-bacterin injected group (p<0.05) and remained higher than those of the control group which showed no significant changes (p<0.05). In contrast to the control group, from 3 weeks after the second injection of the toxoid-bacterin injected group, mean S. aureus cfu/ml in milk samples from injected quarters with S. aureus was significantly decreased until the end of the study (p<0.05). In the toxoid-bacterin injected group, significant decreases of mean SCC were detected from milk samples from infected quarters with S. aureus from week 7 to week 10 (p<0.05). These data show that autogenous toxoid-bacterin treatment against S. aureus subclinical mastitis in lactating cows may increase the cure rate of the infections, reduce the severity of the infections and also prevent occurrence of the new infections.     

Effect of somatic cell count on milk and protein yields and female fertility in Tunisian Holstein dairy cows

The effects of test-day somatic cell scores (SCS) variations on milk and protein yields, and calving to first service and calving to conception intervals were studied in Tunisian Holsteins. There were 34,129, 25,700, and 18,077 test-day production records collected on first, second, and third parity cows, respectively. Records were of cows calving between 1996 and 2004 in 160 herds. Somatic cell scores and milk and protein yields were analysed using a linear model that included herd-test-day date and herd–year interactions, calving season, calving age, and calving to conception interval. Reproductive trait model included herd–year interaction, calving season, calving age, and month of insemination. Effects of SCS on milk and protein yields were studied by regressing current test-day yields on corresponding and preceding test-day SCS, while effects of SCS on fertility traits were investigated by separately regressing calving to first service and calving to conception intervals corrected for environmental and management factors on SCS corrected for actual milk yield. A cow produced around 19.0 kg (SD = 8.0 kg) and 0.6 kg (SD = 0.3 kg) milk and protein yields on a daily basis and had an average of 3.8 (SD = 2.1) SCS in the first three lactations. SCS varied consistently (p < 0.05) with herd-test-day date and herd–year interactions in all lactations. Days in milk, calving age, and calving to conception interval were all together important sources of variation (p < 0.05) for SCS mainly in the first and second parities. Test-day milk and protein yields were unfavourably affected by high SCS recorded in the same test-day and with a lesser degree by SCS observed in the nearest preceding test-day. Reduction in milk and protein productions from increased SCS varied from 0.23 to 1.76 kg and from 6 to 75 g, respectively. Likewise, increased test-day SCS lengthened both calving to first service (mean interval = 94.9 days; SD = 49.1 days) and calving to conception (mean interval = 161 days; SD = 69.6 days) intervals by 1.3 to 2.0 days for each unit increase in SCS. Using SCS in addition to milk traits as a criterion to select semen and improving veterinary care should result in increased milk and protein yields and in satisfactory fertility measures.     

Effects of intramammary infusion of Bifidobacterium breve on mastitis pathogens and somatic cell response in quarters from dairy cows with chronic subclinical mastitis

The present study assessed the effects of intramammary infusion of Bifidobacterium breve (B. breve) on mastitis‐causing pathogens and on the somatic cell counts (SCC) in lactating cows with chronic subclinical mastitis. The bacteriological cure rates of 42 quarters from 42 cows infected with Staphylococcus aureus, Corynebacterium bovis, coagulase‐negative staphylococci, and environmental streptococci were 18.2% (2/11), 14.3% (1/7), 58.8% (10/17), and 28.6% (2/7), respectively, on day 14 after B. breve infusion. In a second trial, B. breve was infused into 18 quarters from 18 cows with chronic subclinical mastitis from which pathogens had not been isolated; the rates of quarters showing SCC > 50 × 10⁴ cells/ml prior to B. breve infusion that decreased to < 30 × 10⁴ cells/ml after infusion were significantly (p < .01) increased to 61.1% (11/18) on day 14 compared to that prior to infusion (0/18). The intramammary infusion of B. breve appears to be a non‐antibiotic approach for elimination of minor pathogens and decreasing SCC in quarters with chronic subclinical mastitis in dairy cows.     

Normal somatic cell count and subclinical mastitis in Murrah buffaloes

This study was conducted to investigate the normal somatic cell count (SCC) and to define subclinical mastitis in Murrah buffaloes. Data were collected from 60 clinically normal buffaloes stationed at five farms of Chitwan Nepal and Buffalo Research Center, Hissar, India. Somatic cell count was measured using the Newman-Lampert staining technique. The upper limit of SCC was determined >or=200 000/ml of milk based on the mean +/- 2SD of a total SCC. Abnormal data of the SCC was repeatedly removed, which lie beyond the values of more than mean + 2SD until all the data come to lie within (mean + 2SD). Averages of SCC of right front and right hind quarters were significantly higher than left front and left hind quarters. Nearly 94% of California mastitis test (CMT) negative quarters were having somatic cells >or=200 000/ml. The mean SCC of CMT positive quarter was significantly higher (P < 0.01) than CMT negative quarters. Subclinical mastitis was diagnosed on the basis of samples with SCCs >or=200 000/ml with positive bacterial cultures. Subclinical mastitis was found in 21.7% buffaloes and 8% of the quarter foremilk samples. Neutrophil counts were significantly higher in subclinical mastitis milk.     

Differentially expressed genes associated with Staphylococcus aureus mastitis of Canadian Holstein cows

To study pathway specific gene expression within the immune-endocrine axis of dairy cows with Staphylococcus aureus mastitis, mRNA was collected from blood mononuclear cells (BMCs) and milk somatic cells (MSCs) of cows (n = 7) identified as culture positive for S. aureus and their matched negative control cows (n = 7) with no evidence of S. aureus mastitis. Labeled cDNA probes derived from BMCs and MSCs of infected and healthy cows were applied to a bovine immune-endocrine cDNA array containing 167 genes. Genes with a log₂ ratio ≥ 0.5 were considered to be up-regulated and genes with a log₂ ratio ≤ −0.5 to be down-regulated. In total, 22 genes were differentially displayed in BMCs and 16 genes in MSCs of case versus controls. Expression of selected genes in BMCs and MSCs were confirmed by real-time PCR. The RT-PCR results were highly correlated with microarray measurements. Some of these genes, such as interleukin (IL)-8 have been previously implicated in other bacterial diseases, and are known to regulate immune responses; whereas, others may reflect novel pathways or genes involved in progressive mammary gland disease. For example, IL-18 was up-regulated in BMCs but not MSCs of mastitic quarters, while IL-17 was more highly expressed in MSCs compared to BMCs. This study identified a number of differentially expressed genes associated with bovine S. aureus mastitis and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.     

A comparative randomized field trial on intramammary and intramuscular dry cow antibiotic treatment of subclinical Staphylococcus aureus mastitis in dairy cows

The efficacy of two dry cow treatment (DCT) regimens for subclinical Staphylococcus aureus mastitis was evaluated in naturally infected dairy cows. At dry-off, cows were assigned to two treatment groups by randomized blocks on the basis of parity and somatic cell count (SCC). Two antibiotic DCT regimens were used, namely: (1) a single intramammary infusion containing sodium nafcillin, procaine benzylpenicillin and dihydrostreptomycin; and (2) systemic cefquinome administered intramuscularly, twice at a 24-h interval. In the intramammary (IMM) treatment group, the S. aureus intramammary infection (IMI) rate was reduced from 40% (56/140 quarters) before dry-off to 20% (28/140) after calving. Seventy per cent (39/56) of the S. aureus-positive quarters were negative after calving, and 13% (11/84) of the negative quarters were positive after calving. In the systemic treatment group, the S. aureus IMI rate increased from 39% (29/74 quarters) before dry-off to 55% (41/74) after calving. Twenty-eight per cent (8/29) of the S. aureus-positive quarters were negative after calving and 45% (20/45) of the negative quarters were positive after calving. The odds ratio of an S. aureus-positive quarter being negative after calving in the IMM group relative to the systemic therapy group was 44.6 (95% confidence interval = 2.1-909.1, P < 0.01). Parity, quarter, milk SCC and N-acetyl-beta-D-glucosaminidase were tested in the model, and were found to have no significant effect on S. aureus cure rates or new IMI rates. The IMM treatment resulted in a higher cure rate compared with that observed in previous studies. The very low cure rate after systemic cefquinome treatment was comparable to the spontaneous cure rate observed in untreated controls in previous studies. The unfavourable results of the cefquinome systemic DCT might reflect inadequate pharmacokinetic properties of the drug regarding poor udder penetration in subclinical mastitis and short antimicrobial effect compared with the IMM treatment.     

Effect of puerperal mastitis in primiparous cows on milk production, cell count and culling

A retrospective cohort study was performed to investigate the effect of clinical mastitis in heifers during the first week post partum (p.p.) on subsequent health and productivity. Primiparous cows that had calved between August 15th, 1996 and August 14th, 1997 (n = 1389) were included in the study. Milk samples were collected from each quarter post partum prior to first milking. The predominant group of bacteria found were Staphylococcus spp. (69%) followed by Streptococcus spp.(12%) and E. coli (5%). Intramammary infections at calving increased the risk of clinical mastitis within the first week p.p. The cow incidence of mastitis until day 7 p. p. was 38%. Cows were classified into three groups. Group 1: Animals with clinical mastitis prior to calving (n = 35); Group 2: Animals with clinical mastitis between calving and 7 days p. p. (n = 503); Group 3: Animals without mastitis until 7 days p. p. (n = 851). Mastitis prior to parturition and mastitis within the first week p.p. increased the risk of further cases of mastitis and culling during the first 45 days of lactation. Milk yield of Group 2 was lower and somatic cell counts were higher than in Group 3.     

The effect of polyethylene intramammary devices on the somatic cell count and milk production of dairy cows

SUMMARY Polyethylene intramammary devices (IMO) were inserted into all 4 quarters of 15 multiparous dairy cows. Fifteen cows, matched for parity and production, were controls. The insertion of IMD's was easily achieved and produced no adverse effects. Throughout the 150-day test period, the mean somatic cell count of cows with IMD's in situ was 216,000 cells per ml, compared with 119,000 cells per ml in controls (P<0.01). Total production of milk, butterfat and protein was not significantly different between the 2 groups. The new infection rate was too low to allow assessment of the value of the IMD for mastitis prevention.     

Antibiotic therapy for subclinical mastitis in early lactation; effects on infection, somatic cell count and milk production

Cows with consistently high somatic cell counts (SCC) and bacterial infection, but no clinical signs of mastitis, were identified early in lactation. Some of these quarters were treated with Cloxacillin, while other similar quarters were left untreated. Prior to treatment, 74% of infected quarters had SCC higher than 300,000 cells/ml while 85% of uninfected quarters has SCC lower than 300,000 cells/ml. Infection was eliminated by treatment in five out of the eight quarters treated with Cloxacillin, and in these quarters SCC decreased from 4,200,000 cells/ml before treatment to 160,000 cells/ml after treatment. Milk production and composition were not affected significantly although production was about 14% higher in treated quarters than untreated quarters. The possible role of antibiotic therapy for subclinically infected quarters early in lactation was discussed in relation to a reduced rate of new infection in a herd.     

Pharmacokinetics of azithromycin in lactating dairy cows with subclinical mastitis caused by Staphylococcus aureus

Lucas, M. F., Errecalde, J. O., Mestorino, N. Pharmacokinetics of azithromycin in lactating dairy cows with subclinical mastitis caused by Staphylococcus aureus. J. vet. Pharmacol. Therap. 33 , 132–140. Azithromycin is a time‐dependent antimicrobial with long persistence. The main characteristics of azithromycin suggest that it could be useful for treating bovine mastitis caused by Staphylococcus aureus. To investigate this possibility, its pharmacokinetic (PK) behavior was studied. Six Holstein lactating cows with subclinical mastitis were administered two 10 mg/kg intramuscular (i.m.) doses of azithromycin, with a 48‐h interval. Milk and plasma concentrations were measured by microbiological assay. The MIC₉₀ was determined in 51 S. aureus isolations to calculate pharmacokinetic/pharmacodynamic (PK/PD) parameters. Milk maximal concentration (C_max) was 7.76 ± 1.76 μg/mL (16.67 h post‐first administration) and 7.82 ± 2.18 μg/mL (14 h post‐2^nd administration). In plasma C_max was 0.18 ± 0.03 μg/mL (2 h post‐1^rst administration) and 0.11 ± 0.03 μg/mL (14 h post‐2^nd administration). Azithromycin was eliminated from the milk with a half‐life (T½λ) of 158.26 ± 137.7 h after 2^nd administration, meanwhile plasma T½λ resulted shorter(13.97 ± 11.1 h). The mean area under the concentration vs. time curve from 0 to 24 h (AUC_0‐24h) was 153.82 ± 34.66 μg·h/mL in milk secretion and 2.61 ± 0.59 μg·h/mL in plasma. Infection presence in the quarters had a significant effect (P < 0.05) on the area under the concentration vs. time curve from 0 to infinity (AUC_0‐∞) and clearance from the mammary gland (Cl_mam/F). Moreover, it had influence on milk bioavailability (F_milk), T½λ, AUC_0‐∞ and mean residence time (MRT) in milk, which values resulted increased in mastitic quarters. In this study, it was determined that the production level and the mammary health status have an influence on PK parameters of azithromycin treatments in bovine mastitis.     

Effect on outcome of intramammary challenge exposure with Staphylococcus aureus of somatic cell concentration and presence of an intramammary device

Results of experimental Staphylococcus aureus intramammary challenge of all quarters of 6 cows, each fitted with an intramammary device (IMD) in 2 quarters, and of 10 quarters of 3 cows not fitted with an IMD were reported. Infection was established in all 34 quarters, regardless of presence or absence of an IMD. Neither the course nor severity of early S aureus intramammary infection were influenced by the presence of an IMD or by differences in milk somatic cell (MSC) concentration in the gland at the time of bacterial challenge infusion, up to a MSC concentration of nearly 1 million/ml. Cumulative success of experimental infection in this and a previous study from our laboratory was nearly 100% in glands in which the MSC concentration was less than 1 million/ml and about 17% when the MSC concentration exceeded 1 million/ml.