Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea

One hundred and sixty-six Staphylococcus aureus isolates from mastitic milk samples from different cows on 26 farms were investigated for staphylococcal enterotoxins(SEs) and toxic shock syndrome toxin-1(TSST-1) by polymerase chain reaction(PCR) and reverse passive latex agglutination assay(RPLA). SEs and the TSST-1 gene were detected in thirty-seven isolates based on a multiplex PCR; SEA was detected in 32 isolates, SEB in 3 isolates, SEC in 1 isolate, and SEA and the TSST-1 gene in 1 isolate. Of the 37 enterotoxigenic isolates, thirty-three isolates were enterotoxigenic according to RPLA, where 29 isolates produced SEA, 3 isolates produced SEB, and 1 isolate produced SEC. The enterotoxin-producing S. aureus isolates were further characterized by pulsed-field gel electrophoresis(PFGE). A macrorestriction analysis revealed 11 PFGE patterns. Among the 33 enterotoxigenic S. aureus isolates, 45.4% exhibited the same PFGE pattern I. Accordingly, although the enterotoxin-producing S. aureus isolates from bovine mastitis were genetically diverse, 1 common genotype prevailed on the farms, indicating that PFGE pattern I isolates may be the most disseminated in Korea.     

Characterization of Staphylococcus aureus strains isolated from bovine mastitis in Japan

Fifty-three strains of Staphylococcus aureus isolated from cows affected with mastitis from 21 prefectures in Japan were characterized by phenotypic and genotypic methods. Thirty-three (62.3%) strains showed biotype K-beta+CV:A, coagulase type VI, and sensitivity to bovine phages of group III or IV. These 33 strains could be subdivided into two groups on the basis of the production of staphylococcal enterotoxins (SEs) and on toxic shock syndrome toxin-1 (TSST-1). By pulsed-field gel electrophoresis, the 16 SEC- and TSST-1-producing strains showed similar patterns that differed by only a few fragments, suggesting that they were genetically closely related. Fifteen of 17 non SEC-producing strains which did not produce any other SEs and TSST-1 were genetically different from the SEC-producing strains and showed genetic diversity.     

Mastitis in the lactating mink female (Mustela vison S.) and the development of "greasy kits"

"Greasy kits" is the result of a multifactorial disease complex with few known definitive aetiological factors. Mastitis has been hypothesized as a triggering factor although classical clinical signs of mastitis (rubor, tumor, dolor, calor) are rarely seen in lactating Danish mink females. In this study we sacrificed 2 groups of lactating mink females with a total of 78 mammary glands at day 19-30 after giving birth. The first group had raised normal mink kits while the other group had suffered severe attacks of greasy kits. We found no clinical or histopathological evidence of mastitis but isolated streptococci and staphylococci from 2 mammary glands in females raising greasy kits. These glands showed no clinical or histological signs of inflammation attributable to bacteria and we conclude that mastitis is not necessary for the generation of greasy kits.     

Experimental staphylococcal mastitis in the mouse: the persistence of chronic infection from one lactation to the next.

Chronic staphylococcal mastitis was established in 50 mammary glands of 25 mice. The mice were mated and in the subsequent lactation there was alveolar mastitis in one of 50 glands. A further five glands had abscesses that contained staphylococci but there was no alveolar mastitis. The results are discussed in relation to the large number of infections that persist from one lactation to the next in cows not subjected to mastitis control measures.     

Production of staphylococcal enterotoxins and TSST-1 by coagulase negative staphylococci isolated from ruminant mastitis.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 by 40 coagulase negative staphylococci isolated from sheep, goat and cow mastitis was studied. Both ELISA double sandwich and Western blot were used to detect the production of these toxins. Only two strains of S. xylosus were enterotoxigenic, producing SEC. TSST-1 was seen to be produced by 5 strains of S. xylosus, 1 S. sciuri and 2 S. epidermidis. Results obtained by ELISA and by Western blot agreed in all cases except in one strain of S. epidermidis which was only positive using ELISA.     

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.     

Anti-inflammatory effects of intramammary infusions of glycyrrhizin in lactating cows with mastitis caused by coagulase-negative staphylococci

OBJECTIVE: To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS). ANIMALS: 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis. PROCEDURE: Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of alpha-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL. RESULTS: In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of alpha-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.     

Inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.     

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.     

Dynamics of leukocytes and cytokines during experimentally induced Streptococcus uberis mastitis

Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.     

Effects of Staphylococcus aureus mastitis on bovine mammary gland plasma cell populations and immunoglobin concentrations in milk

Bovine mammary tissue and milk samples were examined to determine effects of chronic Staphylococcus aureus mastitis on the humoral immune response. Parenchymal and teat end tissues from lactating bovine mammary glands were stained immunohistochemically to determine distribution of immunoglobulin (Ig) G1-, IgG2-, IgA-, and IgM-producing plasma cells. Numbers of all Ig-producing plasma cells tended to be higher in tissues from S. aureus infected quarters compared with controls, but most differences were not statistically different. Numbers of IgG1-producing plasma cells at the Furstenberg's rosette area of infected quarters were significantly (P less than 0.05) higher than uninfected quarters. There were no significant differences in concentrations of Ig isotypes in milk from S. aureus infected and uninfected quarters. Data suggest that the antigenic effect of chronic S. aureus infection on the humoral immune response of the bovine mammary gland is minimal. Persistency of S. aureus infection may result, in part, from suboptimal stimulation or immunosuppression of the mammary immune system.     

Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds

To determine the distribution of genes that encode enterotoxins A, B and C, 36 strains of Staphylococcus aureus isolated from goat mastitis and 64 isolated from bovine mastitis were analyzed by Multiplex PCR. Of the total strains studied, 37 (37%) were detected to have some of the SEs genes. From the bovine mastitis strains, 4 (6.3%) co-amplified the sea and seb genes and 2 (3.1%) were positive for the sec gene. From the goat mastitis strains, 31 (86%) tested positive to the Multiplex, and the sec gene was detected in all of them. The production of SE was detected in all strains harboring the corresponding gene. The results demonstrated that S. aureus isolated from goat mastitis had a higher enterotoxigenic potential than those isolated from bovine mastitis. Additionally, the presence of the sec gene in the majority of goat mastitis strains suggests a possible involvement of SEC in goat mastitis pathogenesis.     

Microbiological colonisation of bovine teat canal--significance and influencing factors

The teat canal of lactating dairy cattle is of particular importance for the defence of facultative pathogenic and pathogenic microorganisms (e.g., staphylococci) invading the bovine udder. Furthermore the canal is usually colonized with microorganisms, too. In addition to microorganisms inhibiting mastitis pathogens the teat canal is colonized by staphylococci. The microbial colonization can be influenced by the environment of the animals, the care and disinfection of the teat skin and indirectly by the effects of forces being associated with machine milking. Because of vacuum fluctuations occurring under the teat tip microorganisms, which colonize the teat canal, can invade the bovine mammary gland and cause infections there. This paper gives a review of the microbial colonization of the bovine teat duct and of influencing factors on the microbial populations as well as of the significance of the teat canal colonization for the development of mastitis.     

Mastitis and other diseases of the goat's udder.

Udder problems of modern dairy goats are similar to those seen in dairy cows. Anomalies of the goat's udder and teats are common, and many may be hereditary. Skin diseases of the udder include viral infections, mange, sunburn, wounds, and staphylococcal dermatitis. There are numerous known causes of caprine mastitis. These include streptococci, hemolytic and nonhemolytic staphylococci, corynebacteria, and mycoplasmas. Diagnosis of mastitis in goats is often difficult, as the udder secretion may remain grossly normal and somatic cell counts in nonmastitic goats are higher than the recognized normal range for cows. The importance of nonhemolytic staphylococcal cultures remains uncertain. Nonhemolytic staphylococci were isolated at the New York State Mastitis Laboratory from 30% of normal halves and from 22% of halves of udders from goats with assorted clinical problems. Treatment and prophylaxis of caprine mastitis closely parallel the standard technique recommended for bovine mastitis.     

Intramuscular treatment of subclinical staphylococcal mastitis in lactating cows with penicillin G, methicillin and their esters

The relationship between antibiotic milk concentrations and bacteriological efficacy was investigated in groups of lactating cows with subclinical mastitis due to either penicillin G-sensitive or penicillin G-resistant Staphylococcus aureus. Treatments consisted of the intramuscular injection of procaine penicillin G, or its weak base ester penethamate hydriodide, and sodium methicillin, or its weak base ester tamethicillin. Antibiotics were administered once daily for 2 or 4 days at accepted dosages. After four daily, treatments with procaine penicillin G and penethamate hydriodide, infections were eliminated from 56.5% and 68.8%, respectively, of quarters infected with penicillin G-sensitive staphylococci, and from 14.3% and 7.7%, respectively, of quarters infected with penicillin G-resistant staphylococci. After four daily treatments with sodium methicillin and tamethicillin, infections were eliminated from 32.4% and 48.6%, respectively, of quarters infected with penicillin G-resistant staphylococci. The better efficacy of penethamate hydriodide and tamethicillin was considered to be linked to the higher milk drug concentrations obtained with these drugs as opposed to the lower concentrations measured in the milk after treatment with the parent drugs. Cure rates were generally higher after treatment for 4 days than after the 2-day course of therapy. Treatment efficacy decreased progressively with increasing age of the cows. Intramuscular treatment of subclinical staphylococcal mastitis in lactating cows can serve as a useful model for screening existing and new antibacterial agents and drug products intended for the parenteral treatment of clinical staphylococcal mastitis.     

Viral infections and bovine mastitis: a review

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.     

Antibody response to toxic shock syndrome toxin-1 of Staphylococcus aureus in dairy cows

Antibody response to toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus in dairy cows was examined by enzyme linked immunosorbent assay (ELISA). Serum antibody to TSST-1 was not detected in 39 (76.5%) of 51 calves, which were 1-6 months of age. In contrast, TSST-1 antibody was demonstrated in 1728 (72.6%) of 2380 lactating cows housed on 36 dairy farms. The ELISA values of antibody ranged from 0.2 to 3.0 OD and presented a distribution with the peak at 1.6 OD. The mean ELISA value differed between farms, and it increased slightly along with parturient history. Somatic cell counts of milk from 174 lactating cows was compared with TSST-1 antibody and tst1,000,000 cells per ml. The mean ELISA values in milk were lower than those of sera, but they rose as somatic cells increased. The tst gene of S. aureus detected in 76.0-86.2% of the milk samples containing somatic cells > 500,000 cells per ml, a level which indicates mastitis. The data suggests that many lactating cows may be infected by TSST-1- producing S. aureus.     

Binding of a surface protein of Staphylococcus aureus to cultured ovine mammary gland epithelial cells.

Staphylococcus aureus is the most persistent pathogen causing ovine mastitis. This study investigated S. aureus binding to cultured epithelial cells obtained from the mammary gland. A staphylococcal 145kDa cell wall adhesin, originally isolated from a bovine mastitis strain, was detected in lysostaphin-solubilized ovine mastitis strains and in the encapsulated strain A. This adhesin was able to bind to cultured ovine mammary gland epithelial cells (MGEC) and to a rat intestinal epithelial cell line (RIE-1), exhibiting different electrophoretic mobilities that could be attributable to protein polymorphism. Inhibition assays using antibodies against 145kDa adhesin and against whole bacteria showed the specificity of the binding to cells. The role of this protein in adherence was assessed by adherence inhibition tests carried out in vitro with radiolabeled bacteria and cultured epithelial cells. Preincubation of bacteria with antibodies against adhesin 145kDa or against strain c195 resulted in a statistically significant decrease of adherence. These experiments suggest that adherence of S. aureus to MGEC may be critical for colonization.     

Invasive potential of bacterial isolates associated with subclinical bovine mastitis

This work describes differences in the invasive ability of bacterial isolates associated with mastitis. Invasion ability was determined by the uptake and survival in a primary culture of bovine mammary epithelial cells (BMEC). BMEC were isolated from a healthy lactating cow and characterized by their morphology, immunostaining for cytokeratin and the detection of beta- and kappa-casein mRNAs. Ten bacterial isolates comprising the staphylococcal species Staphylococcus aureus (3), Staphylococcus epidermidis (1), Staphylococcus haemolyticus (1), Staphylococcus equorum (2), Staphylococcus xylosus (1) and Brevibacterium stationis (2) obtained from raw milk of cows with mastitis from backyard farms were assayed for their ability to invade BMEC. Only two S. aureus and one S. epidermidis isolates were able to invade BMEC, at similar levels to the S. aureus control strain ATCC 27543. In conclusion, using the in vitro model of infection used in this study, differences in bacterial invasion capability may be detected.     

Immunology of experimental staphylococcal mastitis in mice

Anti-staphylococcal serum, inoculated intramammarily, significantly reduced the number of bacteria recovered from mammary glands in which staphylococcal mastitis had been induced. In addition, there was an alleviation of some of the pathological clinical signs following the immune-serum treatment. Intramammary inoculated normal serum and systemically inoculated immune serum failed to elicit any protection.

The role of cell-mediated immunity in relation to staphylococcal mastitis was not clearly established.