乳房炎奶牛金黄色葡萄球菌毒素基因的检测及PFGE分型研究

作者针对临床及亚临床乳房炎奶牛乳汁中金黄色葡萄球菌分离株的毒素基因进行检测和脉冲场凝胶电泳(PFGE)基因分型,比较2种类型乳房炎金黄色葡萄球菌分离株的差异.无菌法采集奶样,采用国际标准方法从中分离金黄色葡萄球菌,用多重PCR方法扩增nuc基因和mecA基因以确证金黄色葡萄球菌(SA)和耐甲氧西林金黄色葡萄球菌(MRSA).进一步用PCR方法检测SA的各种毒素基因(SEs、ETs、TSST 1和PVL基因等).利用限制性内切酶Sma Ⅰ对SA基因组DNA进行酶切和PFGE分析,最后利用BioNumerics软件进行聚类分析.结果:19.3％(23/119)的临床乳房炎奶样和14.8％(26/176)的亚临床乳房炎奶样确定为金黄色葡萄球菌阳性样品,分别从中分离鉴定出43株和26株金黄色葡萄球菌,其中临床乳房炎分离株中有5株为mecA基因阳性.临床乳房炎奶牛奶样中检测到SA的SEA、SEB、SED、SEJ和PVL毒素基因,检出率分别为3.8％(1株)、11.5％(3株)、19.2％(5株)、7.7％(2株)和31.2％(10株);亚临床乳房炎奶牛乳样中仅检测到SA的SEA和PVL毒力基因,检出率分别为7.0％(3株)和84.1％(37株).表明临床与亚临床乳房炎奶牛乳汁中SA菌株携带的毒素基因不一样,SEs可能是临床乳房炎菌株的重要致病基因,PVL可能是亚临床乳房炎菌株的重要致病基因.69株SA使用Sma Ⅰ酶切分型后,可分为7个大簇、50个基因型,来源相同的SA分型后大部分位于同一簇内.临床乳房炎奶牛乳汁中检测到MRSA菌株,PVL基因在亚临床乳房炎中的检出率为临床乳房炎的2.7倍.PFGE方法能较好的区分临床乳房炎和亚临床乳房炎的SA分离菌株.     

金黄色葡萄球菌性奶牛乳房炎多价疫苗的研制和免疫效果评价

为研制用于预防金黄色葡萄球菌性奶牛乳房炎的免疫疫苗,使用4株血清型为外膜多糖336型(336PS)、荚膜多糖5型(CP5)和荚膜多糖8型(CP8)强毒力金黄色葡萄球菌试制出金黄色葡萄球菌多价疫苗,并通过抗体水平监测、攻毒保护试验以及两个牧场的田间试验对免疫效果进行了评价。抗体水平监测显示,首免后30 d即可产生较高的抗体水平,二次免疫后有效抗体水平可持续到产后210 d;人工攻毒保护试验显示,疫苗对金黄色葡萄球菌引起的临床型乳房炎的保护率为89%;田间试验结果表明,该疫苗对奶牛临床型乳房炎的保护率在75.2%～83.3%之间,对隐性乳房炎的保护率在49.9%～52.2%之间,免疫组日产奶量较对照组平均提高1.26 kg以上。上述结果表明,所试制的疫苗可以显著降低泌乳牛金黄色葡萄球菌引起的临床型和隐性乳房炎发病率并提高产奶量。     

Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe

This study was carried out to determine the prevalence of coagulase-negative staphylococci in clinical and subclinical mastitis in commercial and small-scale farms in Zimbabwe. Thirty five quarter milk samples from clinical mastitis cases and 371 quarter milk samples from cows with subclinical mastitis were cultured for bacterial pathogens. The most frequent pathogens isolated in clinical mastitis were the enteric bacteria (31.4%), followed by coagulase negative staphylococci (22.9%) and then Staphylococcus aureus (17.1%), whereas in subclinical mastitis S. aureus (34.2%) and coagulase-negative staphylococci were (33.2%) the most common. Bacillus species were only isolated in milk samples from subclinical mastitis. Coagulase-negative staphylococci were observed in mixed infections with other bacteria in only 2.2 of the 406 milk samples from clinical and subclinical mastitis where they were isolated together with Bacillus species in 6 of the 9 mixed infection cases. About 95% of the milk samples from which 131 coagulase-negative staphylococci were isolated had correspondingly high somatic cell counts. The coagulase-negative staphylococci isolated most frequently were S. chromogenes (7.9%), S. epidermidis (7.4%) and S. hominis (5.9%). They were all associated with high somatic cell counts. All the coagulase-negative staphylococci isolates were susceptible to cloxacillin and erythromycin, and more than 90% of the isolates were susceptible to neomycin, penicillin and streptomycin. The highest resistance was to tetracycline (17.6%), followed by lincomycin (13.7%). About 8% of the isolates were resistant to both penicillin and streptomycin.     

Use of enzyme-linked immunosorbent assay to measure bovine milk and serum antibodies to alpha toxin, beta toxin, and capsular antigens of Staphylococcus aureus

An enzyme-linked immunosorbent assay (ELISA) procedure was used to quantitate milk and serum antibodies (IgG) to Staphylococcus aureus alpha and beta toxins, and S. aureus 2-8 and Smith diffuse strain capsular antigens. Milk samples were collected on two occasions. A comparison was made between levels of milk antibodies specific for the two toxins and capsular antigens for 41 cows that were infected with S. aureus on both sampling dates, and 18 cows not S. aureus-infected on either date. Staphylococcus aureus-infected cows were grouped according to somatic cell counts. All groups of infected cows, regardless of somatic cell counts, had significantly higher milk antibody levels to alpha and beta toxins than did the non-infected cows (P less than .002). Serum samples taken for 13 infected and 4 non-infected cows also indicated that significant elevations in anti-alpha toxin and anti-beta toxin IgG were present in S. aureus-infected cows, compared to non-infected cows. A similar immune response was not seen to capsular antigens, however. No significant differences were present between the two groups of cows for either milk or serum antibodies to Smith diffuse strain capsular antigens. Milk antibodies to 2-8 capsule were significantly elevated only in infected cows with somatic cell counts greater than 10(6)/ml, compared to non-infected cows; no differences were present for serum antibodies to 2-8 capsule between infected and non-infected cows. These results indicate that significant increases in milk (and possibly serum) antibodies to alpha and beta toxins are present in cows with chronic staphylococcal mastitis, apparently resulting from a systemic immune response to these toxins. There does not appear to be a similar immune response to capsular antigens.     

Dynamics of Staphylococcus aureus infections during vaccination with an autogenous bacterin in dairy cattle

The effect of an autogenous vaccine against Staphylococcus aureus on S. aureus prevalence and mastitis, as well as on somatic cell count (SCC), was studied in a dairy herd with a high prevalence of S. aureus. The vaccination group (n = 35; 22 cows and 13 heifers) and the control group (n = 36; 23 cows and 13 heifers) received the vaccine or a placebo, respectively, according to the following protocol: all animals: basic immunization (twice, 3 weeks apart); cows: booster dose at the time of drying off, 5 and 2 weeks before calculated calving date; heifers: booster dose 2 and 5 weeks before calculated calving date. The vaccine or the placebo was administered subcutaneously in the area of the supramammary lymph nodes. Quarter milk samples were collected monthly and subjected to SCC and bacteriological evaluation. At this time, the animals were also checked for signs of clinical mastitis. Non-clinical S. aureus mastitis diagnoses were based on udder quarter SCC and a positive S. aureus culture. In order to compare the SCC in individual whole milk samples, records from the monthly milk quality testing were evaluated. Cow and udder quarter prevalence of S. aureus intramammary infections calculated for the experimental animals and quarters, respectively, did not differ between groups. However, during the lactation period following the boostcr dose, the prevalence of S. aureus increased in both groups (P < 0.05). The cumulative incidence of various mastitis diagnoses (clinical, subclinical, latent infection) due to S. aureus on an animal basis did not differ between groups. On an udder quarter basis, the cumulative incidence of subclinical mastitis was higher in vaccinated animals than in control animals (33.8 versus 26.0%; P < 0.05). This was mainly due to a higher cumulative incidence of subclinical mastitis in vaccinated than control heifers. The SCC in composite milk samples did not differ between groups, but increased as lactation progressed. The herd prevalence of S. aureus differed considerably throughout the study period, but declined consistently to below 10% at the end of the study period. Recent herd checks revealed a prevalence of S aureus infections of < 5%. It is concluded that the autogenous bacterin tested in this study did not have the desired effect on the prevalence of S. aureus infections and mastitis or SCC. The decline in S. aureus prevalence was very probably due to other factors than specific immunization against S. aureus.     

Field trial of a staphylococcal mastitis vaccine in dairy herds: clinical, subclinical and microbiological assessments

Objective To assess the efficacy of a new staphylococcal mastitis vaccine under commercial dairying conditions.
Design A field trial involving 1819 cows and heifers conducted on seven dairy herds in Victoria. The trial was done 'blind'; approximately half the animals were vaccinated and the remainder were untreated controls.
Procedure The vaccine was given twice during the last 10 weeks of pregnancy. Effects of vaccination were assessed, during the ensuing lactation, on the basis of clinical and sub-clinical mastitis and microbiological investigations of the milk.
Results A total of 273 cases of clinical mastitis were recorded. Staphylococcus aureus was isolated from 112 of these, 45 cases in vaccinates and 67 cases in controls; the difference was not statistically significant. One herd was notable in having a high incidence of clinical staphylococcal mastitis. This herd accounted for 15.8% of the animals in the field trial but 54.5% of cases of clinical staphylococcal mastitis. For this herd, vaccinated animals had significantly lower incidence of clinical staphylococcal mastitis and prevalence of subclinical mastitis, relative to controls. An unexpected feature of the trial as a whole was the low incidence of clinical mastitis from which S aureus was isolated in pure culture (26.3% of cases) and the high incidence of clinical Streptococcus uberis mastitis (22.7% of cases).
Conclusions The trial showed that the vaccine was efficacious in reducing the incidence of clinical mastitis and prevalence of subclinical mastitis in a herd that had a serious staphylococcal mastitis problem.     

Efficacy of a herd-specific vaccine against Staphylococcus aureus to prevent post-partum mastitis in dairy heifers

A placebo-controlled field study was performed to evaluate the effect of a herd-specific vaccine against Staphylococcus aureus on intramammary infection (IMI), somatic cell count (SCC) and clinical mastitis. Three hundred and twenty-one heifers were assigned to two groups. Heifers in the vaccination group (n = 164) were vaccinated twice, i.e. 5 and 2 weeks before their expected calving date. Animals of the control group (n = 157) received the same treatment with a placebo containing no bacterial antigen. Quarter milk samples were collected immediately after parturition prior to the first machine milking, 3-4 weeks after calving and before the onset of treatment in animals with signs of clinical mastitis during the first 3 months after calving. For comparison of SCC the data from the monthly milk test records were evaluated. The prevalence of S. aureus in quarter milk samples taken at calving and 3-4 weeks post-partum did not differ significantly between the vaccine and control group. Incidence of clinical mastitis during the first 3 months after calving and the prevalence of S. aureus in quarter milk samples taken before the onset of treatment did not differ significantly between the groups. The SCC was lower in vaccinated than in control heifers. However, the difference was only significant on the third milk test day. Regarding prevalence of IMI with S. aureus and incidence of clinical mastitis the use of a herd-specific vaccine against S. aureus did not prove to be efficient on this farm.     

Mastitis severity induced by two Streptococcus uberis strains is reflected by the mammary immune response in vitro

Streptococcus uberis is the most common environmental mastitis pathogen causing udder inflammations of different severities in dairy cows. The aim of the study was to investigate if the different clinical outcome of mastitis induced by different strains of S. uberis can be reflected in the mammary immune response. Mammary epithelial cells and somatic milk cells were treated with heat inactivated and living S. uberis of strain A and strain B in vitro. Strain A was repeatedly isolated from a chronically infected quarter during 8 months, and persisted in the quarter despite antibiotic treatment. Strain B caused an acute clinical mastitis and was not further isolated after a single antibiotic treatment. Treatment with Strain B induced a more pronounced increase of mRNA-expression of various immune factors (interleukin-8, interleukin-1beta, RANTES, and lactoferrin) in mammary epithelial cells than strain A. In contrast to mammary epithelial cells the response of removed somatic milk cells showed no differences between the stimulation with two S. uberis strains. Tumor necrosis factor-alpha mRNA expression was not differently induced by the two strains. In conclusion, the characteristics of different severities of mastitis that are induced by different S. uberis strains in vivo can also be reflected at the level of the immune response of the mammary gland in vitro.     

Vaccination against experimental staphylococcal mastitis in ewes

Experiments were carried out in ewes using a new vaccine developed for the prevention of mastitis caused by Staphylococcus aureus. The vaccine comprised three major components: (i) killed S aureus cells which had been cultured to induce synthesis of pseudocapsule; (ii) toxoided staphylococcal beta haemolysin and (iii) the adjuvant dextran sulphate. Ewes systemically vaccinated twice during pregnancy developed significantly elevated circulating levels of IgG1 and IgG2 anti-pseudocapsule antibody, as well as increased serum titres of anti-beta haemolysin. Five different strains of S aureus were used to challenge both vaccinated and control ewes by the intramammary route during the ensuing lactation. The incidence of acute gangrenous mastitis and nonacute, clinical mastitis was significantly lower in vaccinated than in control groups after challenge with each strain. Vaccinated ewes produced significantly more milk than control ewes after challenge with four of the five strains of S aureus.     

奶牛乳腺炎的细菌学研究

在对山东7个地区14个奶牛场临床型和隐性乳腺炎调查的基础上采集234头临床乳腺炎病牛乳样、241个隐性乳腺炎乳样并分别做了细菌学检查,结果表明:泌乳期临床型乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌、链球菌、酵母菌和棒状杆菌为主;干奶期临床型乳腺炎病原菌以大肠杆菌、链球菌、金黄色葡萄球菌、凝固酶阴性葡萄球菌和酵母菌为主;隐性乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌链球菌、酵母菌、假单胞菌和棒状杆菌为主;厌氧菌在隐性乳腺炎、干奶期乳腺炎和干奶期乳腺炎乳样的捡出率分别为 5．82％,4．17%,10．16％;隐性乳腺炎、干奶期乳腺炎细菌的共感染率较高,与泌乳期乳腺炎病原菌的差异极显著(P<0．01),隐性乳腺炎与干奶期乳腺炎病原菌共感染率差异不显著(P >0．05)。     

凉州区奶牛乳房炎主要病原分离鉴定及药敏试验

乳房炎是奶牛最常见的疾病,随着奶牛产业的蓬勃发展,乳房炎已经成为制约该产业的瓶颈之一。从甘肃省武威市凉州区5个奶牛养殖场采集了1 000头荷斯坦奶牛共计3 991份奶样,对临床型和非临床型(隐性)乳房炎分别通过临床症状和兰州隐性乳房炎诊断液(LMT)进行诊断。结果显示:该地区奶牛临床型乳房炎发病率18.20%,亚临床型乳房炎发病率36.50%,总阳性率54.70%,总阳性乳区率54.72%。从阳性奶样中分离纯化得到的主要病原菌为链球菌属、金黄色葡萄球菌和大肠杆菌,且在不同类型乳房炎中的分布情况不同,临床型中链球菌属最多(54.37%),亚临床型中金黄色葡萄球菌最多(48.56%)。对主要病原菌进行药敏试验,结果表明治疗凉州区奶牛乳房炎的最佳药物应为喹诺酮类和庆大霉素类。     

Staphylococcus aureus lipoteichoic acid triggers inflammation in the lactating bovine mammary gland

The response of the bovine mammary gland to lipoteichoic acid (LTA), which is a major pathogen-associated molecular pattern of Gram-positive bacteria, was investigated by infusing purified Staphylococcus aureus LTA in the lumen of the gland. LTA was able to induce clinical mastitis at the dose of 100 microg/quarter, and a subclinical inflammatory response at 10 microg/quarter. The induced inflammation was characterized by a prompt and massive influx of neutrophils in milk. LTA proved to induce strongly the secretion of the chemokines CXCL1, CXCL2, CXCL3 and CXCL8, which target mainly neutrophils. The complement-derived chemoattractant C5a was generated in milk only with the highest dose of LTA (100 microg). The pro-inflammatory cytokine IL-1beta was induced in milk, but there was very little if any TNF-alpha and no IFN-gamma. The re-assessment of CXCL8 concentrations in milk whey of quarters previously challenged with S. aureus, by using an ELISA designed for bovine CXCL8, showed that this chemokine was induced in milk, contradicting previous reports. Overall, S. aureus LTA elicited mammary inflammatory responses that shared several attributes with S. aureus mastitis. Purified LTA looks promising as a convenient tool to investigate the inflammatory and immune responses of the mammary gland to S. aureus.     

Experimental trial in heifers vaccinated with Staphylococcus aureus avirulent mutant against bovine mastitis

Staphylococcus aureus, is the most frequently isolated pathogen from cases of bovine mastitis. Vaccination against S. aureus seems to be a rational approach for the control of staphylococcal mastitis. In the present work we evaluate the response of heifers vaccinated with a S. aureus avirulent mutant to the intramammary challenge with a S. aureus virulent strain. Clinical signs, production of milk, shedding of S. aureus cells, somatic cell count (SCC) and antigen-specific IgG in blood and milk, were determined. Two subcutaneous doses of a culture of the mutant, used as vaccine, was administered to four pregnant heifers 30 and 10 days before calving. The vaccinated heifers and four non-vaccinated were challenged 10 days after calving with the homologous virulent S. aureus strain, which was inoculated by intramammary route into two quarters of each animal. No local tissue damage was observed due to the administration of the vaccine. A significantly increase of specific IgG to S. aureus RC122 was detected in blood and milk of vaccinate heifers as well as a slight increase in daily milk yield during the trial. No significant difference on shedding of bacteria in milk and SCC were found among groups. In conclusion, vaccination of heifers before calving by an avirulent mutant vaccine of S. aureus, induced specific and significant antibody responses and provide better post-challenge conditions in vaccinated heifers.     

Coagulase-negative staphylococci as cause of bovine mastitis- not so different from Staphylococcus aureus?

In this review of the literature, mastitis-causing coagulase-negative staphylococci (CNS) and Staphylococcus aureus are compared. Staphylococci are the bacteria most commonly isolated from bovine mastitis, and CNS are now predominant over S. aureus in most countries. CNS include various species, but only a few prevail in bovine mastitis. S. aureus can cause clinical mastitis, but often causes subclinical mastitis, which remains persistent and increases milk somatic cell count. CNS, traditionally regarded as minor pathogens, seem to lack the ability to cause severe mastitis. CNS can, however, persist in the mammary gland and moderately increase milk somatic cell count. Resistance to various antimicrobials is more common in CNS than in S. aureus, but CNS mastitis responds much better to antimicrobial treatment than S. aureus mastitis.     

Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

ABSTRACT: Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.     

Changes in blood and milk lymphocyte sub-populations during acute and chronic phases of Staphylococcus aureus induced bovine mastitis

Staphylococcus aureus (S. aureus) often causes long-lasting chronic sub-clinical udder infections in dairy cows. To investigate if this can be due to a negative impact of S. aureus on lymphocytes important for the immune defence, alterations in proportions and expression intensity of CD4+, CD8+, WC1+, B and IL-2R+ lymphocytes was studied in blood and milk, as S. aureus mastitis developed from acute clinical to chronic sub-clinical form. Six healthy dairy cows were inoculated with S. aureus in one udder quarter per cow, and one quarter per cow acted as an uninfected control. Blood samples, and milk samples from infected and non-infected quarters were collected before infection and for five weeks after infection. All infected quarters developed acute clinical mastitis, of which five turned into chronic sub-clinical mastitis. In infected quarters, the proportions of all lymphocyte sub-sets, except WC1+ cells, differed in acute phase compared to pre-infection, while the dominant finding in the chronic phase was increased expression intensities per cell. An impact on blood lymphocytes and milk lymphocytes in non-infected quarters also occurred, mainly during the chronic phase. The most prominent finding was the increased proportion and expression of B-lymphocytes in blood, infected and non-infected quarters during chronic sub-clinical mastitis. As S. aureus can invade and survive intracellularly, a preferential stimulation of B-cells, suggesting development of a humoral response, may not be sufficient to eliminate intracellular bacteria, which could explain the persistence of the infection.     

奶牛隐性乳房炎奶样中埃希氏大肠杆菌共同性抗原初探

用LMT的方法对新疆8个奶牛场采集的1020份奶样进行隐性乳房炎的检测,并对阳性样品进行了大肠杆菌的分离与鉴定。从不同牛场分离菌株中挑选8株用家兔制备免疫血清,用试管凝集对从乳房炎奶样分离出的其它21株大肠杆菌进行共同性抗原的筛选。结果显示,免疫血清效价达1∶2560～1∶5120,85.7%的待检菌株与其中3种血清凝集反应呈阳性。对制备血清的8株菌进行SDS-PAGE电泳分析,菌体蛋白图谱显示在20~84 ku处存在大量相同或相似的蛋白条带;用制备的免疫血清进行免疫印迹分析,与8株菌的菌体蛋白在70 ku处呈现明显的抗原抗体反应。说明这些菌株存在共同性抗原。     

Adjuvant effect of ginseng extracts on the immune responses to immunisation against Staphylococcus aureus in dairy cattle

A crude ginseng extract (GS) and the purified ginsenoside R(b1) (R(b1)) were evaluated for their adjuvant effects in dairy cattle at immunisation with ovalbumin (OVA) and/or a Staphylococcus aureus bacterin used for prevention of bovine mastitis. To evaluate a suitable dose of GS as an adjuvant, 36 lactating cows were randomly divided into six groups. The cows were inoculated twice intramuscularly with a 2-week interval, with saline solution, OVA in saline, or OVA in combination with 4, 16 or 64 mg GS, or Al(OH)(3). The level of specific antibodies to OVA in serum and milk whey was measured before immunisations and 1-5 weeks after the second immunisation. The antibody response in serum was significantly higher in animals immunised with OVA and GS than in animals immunised with OVA alone. A significant increase in milk antibody titres compared with OVA only was only found 2 weeks after the second immunisation in the group immunised with OVA and 4 mg GS. In the second part of the study, 18 heifers were randomly divided into three groups and were immunised twice intramuscularly with a two week interval, with the S. aureus bacterin (control), or with the bacterin in combination with 4 mg GS or 1mg R(b1). The specific antibody response to S. aureus and the lymphocyte proliferation after stimulation with PWM, concanavalin A (Con A) or a specific S. aureus antigen was evaluated in blood samples taken before and after immunisations as specified above. Addition of R(b1) resulted both in significantly higher antibody production and lymphocyte proliferation in response to PWM, Con A and S. aureus antigens than in the control group. Addition of GS induced a significantly higher lymphocyte proliferation in response to PWM and Con A than the control, but had no additional effect on the antibody production. In conclusion, both GS and R(b1) were safe adjuvants, and R(b1) had the strongest adjuvant effects, when used for immunisation against S. aureus in dairy cattle. Field trials are warranted to test the ability of GS and R(b1) to enhance the efficacy of mastitis vaccines in protection against intramammary infections.     

Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully.     

Altered leukocyte responsiveness in dairy cows with naturally occurring chronic Staphylococcus aureus mastitis

Changes in inflammatory parameters, leukocyte surface markers, functional responses and cytokine mRNA expression of leukocytes of dairy cows with naturally occurring chronic Staphylococcus aureus (S. aureus) mastitis and healthy cows were determined to elucidate the leukocyte responses to S. aureus infection of the mammary gland. Increased values in inflammatory parameters and matrix metalloproteinase activities in milk revealed the characteristics of cows with chronic mastitis. Expression of L-selectin and CD18 molecules on neutrophils and proportion of CD8 cells in milk from cows with S. aureus mastitis were significantly (P<0.05) increased compared with those found in healthy cows. The FcR-stimulated CL response of blood neutrophils was significantly (P<0.05) decreased in cows with S. aureus mastitis. Significantly (P<0.05) decreased mitogenic responses of lymphocytes were found in cows with S. aureus mastitis; however, the values were not restored to those of healthy cows when stimulated with both mitogens and the cytokine IL-1β. The mRNA expression of TNF-α, IL-1β and IL-8 on milk leukocytes from cows with S. aureus was found to be increased compared with that of healthy cows. The changes of immune responses found in cows with S. aureus mastitis appear to be influenced by the severity and duration of inflammation in infected quarters. The down-regulation of the leukocyte functions found in cows with S. aureus mastitis appears to be associated with the progress of the chronic stage of S. aureus mastitis.