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.     

Characteristics of Staphylococcus aureus from subclinical bovine mastitis in Brazil

A total of 127 Staphylococcus aureus strains isolated from milk samples of cows with subclinical bovine mastitis was examined for biotype, phage pattern, in-vitro antibiotic susceptibilities and ability to produce enterotoxins. The majority of the strains showed features consistent with bovine rather than human origin. All strains were sensitive to the antibiotics tested, except penicillin and streptomycin. Enterotoxigenicity was observed in 6 (4.7%) strains and only enterotoxins A and C were produced.     

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.     

Chloramphenicol resistance plasmids in Staphylococcus aureus isolated from bovine subclinical mastitis.

Chloramphenicol resistance (CmR) could be detected in 11 of 217 Staphylococcus aureus isolates from bovine subclinical mastitis. All isolates were assigned to biotypes A or C. The CmR-determinants were found to be located exclusively on small plasmids of approximately 4.6 kb as revealed by protoplast transformation. The 11 CmR-plasmids could be differentiated on the basis of restriction endonuclease analyses. The restriction maps of these CmR-plasmids identified two separate groups. One group demonstrated homology to the plasmid pC 221, the other to the plasmid pC 223. Both prototype plasmids, pC 221 and pC 223, had been isolated from S. aureus of human origin.     

S.aureus诱导的小鼠亚临床型乳房炎乳腺病理变化试验

乳房炎是奶牛最常见和危害最大的传染病之一,乳房炎动物模型和系统性的乳房炎病理变化研究是深入研究乳房炎的基础。本研究以分娩后7~12 d的C57BL/6小鼠为模型动物,将从隐性乳房炎的奶牛乳汁分离的金黄色葡萄球菌(S.aureus)50μL(1×107CFU)或等量的无菌生理盐水通过乳导管将缓慢注入小鼠第4、5对乳腺,分别于感染后2、4、8、12、18、24、36、48、60 h和72 h处死小鼠,对乳腺的病理变化进行了系统性的观察。结果显示,对照小鼠乳腺结构完整,连接紧密,腺泡腔充满乳汁,而试验组小鼠2 h即有免疫细胞出现,而且随着时间的推移乳腺内的免疫细胞数量逐渐增加,60 h时小鼠乳腺组织开始脂肪化退变。本研究结果,对奶牛乳房炎乳腺病理变化的研究具有参考意义,也为进一步进行乳腺免疫机制研究奠定了模型基础。     

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.     

Detection of superantigenic toxin genes in Staphylococcus aureus strains from subclinical bovine mastitis

The aim of this study was to determine the presence of genes encoding enterotoxins (sea-sej) and toxic shock syndrome toxin-1 (tst) of Staphylococcus aureus strains (n = 130) isolated from subclinical bovine mastitis in Turkey by polymerase chain reaction. Sixty-one (46.9%) isolates were found to contain one or more toxin genes. The most frequently found enterotoxin genes were seg (16.2%) and sei (16.2%), followed by sec (15.4%), sed (10.8%), and sej (10.8%), respectively. The tst gene was detected in seven (5.4%) isolates. None of S. aureus strains harbored sea, seb, see, and seh genes. Since these toxins are recognized agents of staphylococcal food poisoning, it must be considered that the consuming raw milk and raw milk products would pose public health risk as high prevalence of toxigenic S. aureus was found in this study.     

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.     

The epidemiology of Staphylococcus aureus infections from subclinical mastitis in dairy cows during a control programme

The objective was to investigate the spread of S. aureus in seven dairy herds. Milk samples were taken to determine mastitis pathogens and somatic cell count. An approved hygiene programme was established to control the spread of S. aureus from quarter to quarter. S. aureus isolates were differentiated by geno- and phenotyping to trace their spread within a herd.In two herds S. aureus showed a high prevalence, but were eliminated from the herds by the control programme. In these herds and a third herd typing results identified a particular type that was found much more frequently than all other types. The frequent types were repeatedly detectable during the study. In three other herds with a lower S. aureus prevalence and also in one herd with a long history of S. aureus and high prevalence subclinical mastitis was caused by several distinguishable S. aureus types. These types occurred simultaneously in the particular herd and showed little or no tendency to spread from quarter to quarter. They seemed to circumvent the control procedures resulting in a relatively high rate of new infected animals and therefore were hardly eliminated. The typing results and the clinical observations indicated that strains differed in their tendency to spread and their ability to infect udder quarters. In three herds typing methods identified a predominant type with the common epidemiological features of a contagious mastitis pathogen, while in the other herds the S. aureus type patterns were similar to that of environmental pathogens.     

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.     

In vitro susceptibility of Staphylococcus aureus strains isolated from cows with subclinical mastitis to different antimicrobial agents

Sensitivity to commercial teat dips (nonoxinol-9 iodine complex and chlorhexidine digluconate) of 56 Staphylococcus (S.) aureus strains isolated from quarter milk samples of various German dairy herds treated with different teat dipping schemes was investigated in this study. The minimum inhibitory concentration was determined using a broth macrodilution method according to the German Veterinary Association guidelines. The main objective of the current study was to induce in vitro resistance induction of S. aureus to chemical disinfectants. Ten different strains were repeatedly passed ten times in growth media with sub-lethal concentrations of disinfectants. Nine strains showed a significant reduction in susceptibility to the nonoxinol-9 iodine complex but only one strain developed resistance to chlorhexidine digluconate. Stability of the acquired resistance was observed in all S. aureus strains adapted to the nonoxinol-9 iodine complex and chlorhexidine digluconate. In contrast, simultaneous resistance to different antibiotics was not observed in any of the ten investigated S. aureus strains. However, the isolates exhibited a high degree of resistance to penicillin G. Based on these results, resistance of S. aureus to chemical disinfectants may be more likely to develop if the chemicals are used at concentrations lower than that required for an optimal biocidal effect.     

Enhanced virulence of Staphylococcus aureus from bovine mastitis induced by growth in milk whey

The virulence towards mice of Staphylococcus aureus strains from bovine mastitis was enhanced upon growth in milk whey compared to homologous organisms grown in tryptic soy broth (TSB). In the mouse mastitis model, S. aureus grown in milk whey caused more severe lesions than homologous strains grown in TSB. Staphylococcus aureus strain F1440 grown in milk whey induced 75% mortality and local necrotic reaction in subcutaneously inoculated mice, whereas the homologous strain grown in TSB caused only 5% mortality and slight skin reaction. Extracellular capsule on milk whey-grown, S. aureus could not be demonstrated. However, diffuse type colony morphology could be correlated with an increased virulence of S. aureus towards mice.     

Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey

The genetic relatedness of coagulase (coa) positive Staphylococcus aureus isolated from cows with subclinical mastitis in Turkey was investigated by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Among 700 milk samples positive in the California Mastitis Test (CMT), species specific PCR identified 200 (28.6%) isolates as S. aureus and 161 (80.5%) of these isolates were positive for the 3' end of the coa gene by PCR. Most isolates (n=135, 83.9%) produced a single band on coa PCR, with molecular sizes ranging from 500 to 1400bp, whereas a small number of isolates (n=26, 16.1%) yielded two amplification products. Coa RFLP analysis using AluI and Hin6I revealed 23 and 22 band patterns, respectively. The detection of double bands by coa PCR, previously reported in human isolates, suggests that milking personnel can play a role in the transmission of S. aureus.     

Epidemiological investigation of subclinical bovine mastitis in Algeria and molecular characterization of biofilm-forming Staphylococcus aureus

Tropical Animal Health and Production - Thirty dairy farms were selected for this study; the first objective of our study was to investigate the prevalence of subclinical bovine mastitis (SCM) in...     

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.     

原料乳和临床乳房炎金黄色葡萄球菌毒力基因检测及药敏分析

对临床乳房炎（57株）和原料乳（44株）金黄色葡萄球菌菌株,用PCR方法检测mecA基因、PVL基因、ETs基因、SEs基因和TSST-1基因;采用CLSI指导说明执行琼脂稀释法药敏性试验。结果显示原料乳菌株中,84.09%携带有毒素基因,其中PVL的检出率为84.09%,肠毒素的检出率为52.27%,主要流行的肠毒素基因为sea（56.82%）,均未检测到携带mecA、ETs、TSST-1、sei和sej基因的菌株;同时得到10种毒素基因型,其主要流行的毒素基因型为PVL＋sea（29.55%）和PVL（27.27%）。临床菌株中,78.95%携带有毒素基因,其中PVL的检出率为28.07%,肠毒素的检出率为77.19%,主要流行的肠毒素基因为sea（47.37%）,没有检测到携带ETs、TSST-1和seh基因菌株;同时得到25种毒素基因型,其主要流行的毒素基因型为sea（19.30%）,其次是seb（7.02%）,sea＋sed＋sej（3.51%）和PVL＋sea＋seb＋sec＋seg＋sei（3.51%）。6株（10.53%）携带有mecA基因菌株均含有较多毒素基因。原料乳分离株对甲氧苄啶和头孢西丁的耐药率较高,分别为100%和86.36%,其次对氯霉素、红霉素、苯唑西林、头孢哌酮和庆大霉素的耐药率分别为11.36%、4.55%、2.27%、2.27%和6.82%,所有原料乳菌株均对环丙沙星敏感,同时得到8种耐药谱,多重耐药率达22%;临床乳房炎菌株对红霉素和甲氧苄啶的耐药率较高,分别为100%和71.93%,其次对氯霉素、庆大霉素、环丙沙星、头孢西丁和苯唑西林的耐药率分别为28.07%、26.07%、24.56%、19.30%和7.02%,临床乳房炎菌株对头孢哌酮和四环素的敏感率为100%,同时得到13种耐药谱,多重耐药率达77.19%。所有原料乳和临床乳房炎菌株均对万古霉素和阿米卡星敏感。临床乳房炎菌株携带的毒素基因和多重耐药率比原料奶菌株高,同时在临床乳房炎乳中检测到MRSA菌株,提示我们应加强乳及其乳制品的管理,并对奶牛乳房炎加以重视。     

Effect of intramammary injection of rboGM-CSF on milk levels of chemiluminescence activity, somatic cell count, and Staphylococcus aureus count in Holstein cows with S. aureus subclinical mastitis

The effect of intramammary injection of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF, 400 microg/10 mL) on quarter milk levels of chemiluminescence (CL) activity, and somatic cell count (SCC) and shedding pattern of Staphylococcus aureus was investigated. Ten Holstein cows, naturally infected with S. aureus were used, with either early-stage or late-stage subclinical mastitis. Injection of rboGM-CSF caused a remarkable increase in milk CL activity with a peak at 6 h after the cytokine injection in the early- and late-stage groups. In the early-stage group, milk SCC stayed around preinjection level at 6 h, rose significantly on days 1 and 2, and was followed by a smooth and significant decline to an under preinjection level (below 200 000 cells/mL) on day 7 postinjection. Alternatively, in the late-stage group, milk SCC rose significantly at 6 h after the cytokine injection and maintained high levels thereafter. The milk S. aureus count decreased drastically by the cytokine injection in the early-stage group. The bacterial count was moderately decreased in the late-stage group, but increased back to preinoculation levels on day 7 after the cytokine injection. The results suggest that the rboGM-CSF has a potential as a therapeutic agent for S. aureus infection causing subclinical mastitis of dairy cows, if the cytokine is applied at the initial stage of infection.     

Detection of virulence-associated genes in Staphylococcus aureus isolated from bovine clinical mastitis milk samples in Guangxi

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals and is one of the most common etiological agents of clinical and subclinical bovine mastitis. The purpose of this study was to determine the presence of genes encoding clfA, fnbA, fnbB, cap5, cap8, hla, hlb, nuc, sea, and tst of S. aureus strains (n?=?39) isolated from bovine clinical mastitis in Guangxi by polymerase chain reaction amplification. The results of the present study indicated that all isolates were found to contain one or more virulence-associated genes. The most frequently encountered genes were fnbA (97?%) and nuc (90?%), followed by hla (85?%) and hlb (82?%), respectively. None of the investigated S. aureus strains harbored fnbB and sea genes. The data in the present study showed a relatively wide distribution of the genes fnbA and nuc among the investigated isolates, indicating that they play an important role on bovine mastitis pathogenesis. The study provides a valuable insight into the virulence-associated genes of this important pathogen.     

Characterization of leukocytotoxic and superantigen-like factors produced by Staphylococcus aureus isolates from milk of cows with mastitis.

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays.Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described.The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41%; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2%; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.