A collection of 102 coagulase-negative staphylococci (cns), isolated from cases of subclinical and clinical bovine mastitis and belonging to 10 different species, were screened by pcr for the presence of genes encoding enterotoxins and enterotoxin-like toxins (sea, seb, sec, sed, see, seg, seh, sei, sej, selk, sell, selm, seln, selo, selp, selq and selu), toxic shock syndrome toxin-1 (tst), and exfoliative toxins A and B (eta and etb). No toxin gene sequences were amplified from any of the isolates, indicating that superantigens encoded by genes detectable by the pcr tests used were not involved in the development of subclinical and clinical mastitis in cattle infected with the cns isolates tested.
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BOVINE mastitis can be caused by several bacterial pathogens, but the coagulase-positive species Staphylococcus aureus is one of the most important causative agents. In addition, many different coagulase-negative staphylococci (cns) have been isolated from the mammary gland of cattle with subclinical and clinical mastitis (Devriese 1979, Jarp 1991, Devriese and others 1994, Taponen and others 2006).
Although cns are generally considered minor pathogens in relation to bovine mastitis, several studies have indicated that they are the most frequently recovered isolates from mastitic samples, especially samples from first-lactation and unbred heifers (Myllys 1995, Zhang and Maddox 2000, Almeida and Oliver 2001). They also cause mastitis in other ruminants such as milking goats (Deinhofer and Pernthaner 1995). The infections caused by cns are usually less severe than those caused by S aureus, but the somatic cell counts of cns-infected cows are generally two to three times higher than those of uninfected cows (Oliver and Jayaro 1995). Mammary tissues infected with cns are more infiltrated by leucocytes and have more connective tissue stroma than uninfected control tissues (Trinidad and others 1990). One study showed that cns infections were responsible for an 8·7 per cent loss in milk production from a 305-day total milk yield (Timms and Schultz 1987).
Although many studies have demonstrated the relative importance of intramammary cns infections, the virulence factors of these bacteria are poorly understood. In S aureus, several virulence factors may contribute to the pathogenesis of bovine mastitis (Zhang and Maddox 2000, Katsuda and others 2005). Many strains of S aureus produce one or more proteins belonging to a group of specific exotoxins, which contains staphylococcal enterotoxins, enterotoxin-like proteins, exfoliative toxins and toxic shock syndrome toxin-1 (tsst-1) (Larsen and others 2000). These proteins are called super antigens. They are structurally and biologically related proteins that have both acute and long-term effects on the immune system of the host. They can stimulate the activation and proliferation of large numbers of T cells, they are pyrogenic, induce cytokines, enhance endotoxic shock and can cause capillary leakage (Larsen and others 2000). In the long term, super-antigens may be involved in modulating the host's immune response and may contribute to the organism's evasion of the host's defences and its persistence (Ferens and Bohach 2000, Fueyo and others 2005). Katsuda and others (2005) showed that approximately 70 per cent of the isolates of S aureus from milk from cows with mastitis possessed superantigenic toxin genes, indicating that these toxins are important in mastitis, and other authors have suggested that the presence of enterotoxins in S aureus may contribute to bovine mastitis (Stephan and others 2001, Srinivasan and others 2006, Vimercati and others 2006, Moon and others 2007).
Several authors have described the presence of staphylococcal enterotoxins in cns species (Orden and others 1992, Cunha and others 2006a, b, 2007). The lesions caused by cns strains and their persistence in the udder might be partly explained by the production of exotoxins. Some isolates of cns from sheep, goats and cows with mastitis have been shown to produce tsst-1 and sec (Orden and others 1992). However, there have been no extensive investigations of the complete battery of enterotoxin and other superantigen genes in cns isolated from the bovine udder. The aim of this study was to screen isolates of Staphylococcus chromogenes, Staphylococcus hyicus, Staphylococcus xylosus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus capitis, Staphylococcus caprae and Staphylococcus auricularis obtained from cattle with subclinical and clinical mastitis for the presence of superantigen genes.
MATERIALS AND METHODS
One hundred and two cns strains, isolated between 2001 and 2002, were included in the study. The strains were selected randomly from pure cultures isolated from samples of milk from clinical and subclinical cases of bovine mastitis submitted for bacteriological examination to the Dutch Animal Health Service. A milk sample was considered to be culture-positive when more than 500 colony-forming units (cfu)/ml of cns were cultured. The cns were differentiated from S aureus by using Slidex Staph Plus (bioMérieux). The Staphylococcus species were further identified by using conventional bacteriological methods, biochemical test kits (api Staph; bioMérieux) and the tdna intergenic spacer pcr described by Baele and others (2000); they belonged to 10 cns species, namely S xylosus (29 strains), S epidermidis (25), S chromogenes (22), S hyicus (12), S simulans (six), S warneri (three), S capitis (two), S sciuri (one), S caprae (one) and S auricularis (one).
To prepare dna, one colony of bacterial cells was suspended in 20 μl lysis buffer (0·25 per cent sodium dodecyl sulphate [sds] and 0·05M sodium hydroxide) and heated at 95°C for five minutes. The samples were centrifuged briefly at 16,000 g at room temperature. After adding 180 μl distilled water and centrifuging for five minutes at 16,000 g, these samples were stored at −20°C and the supernatant was used as the dna extract.
The superantigen genes were detected by pcr tests. For the detection of the enterotoxins, each 30 μl pcr mixture contained 3mM magnesium chloride, 2·5 U Taq dna polymerase, 200μM of dntps, 200 pmol of both primers and 3 μl dna sample. The dna was amplified with a dna thermal cycler (Biometra). Positive control strains for eta, etb, tst, sea-see and seh were kindly provided by H. D. Larsen (Larsen and others 2000). For the detection of seg, sei, selm, seln, selo, selp and sej, S aureus strain a900322 was used, and for selq and sell, S aureus strain ht2005 0018 was used as a positive control strain (Jarraud and others 1999). These strains were provided by M. Bes of the Centre National de Référence des Toxémies Staphylococciques (France). kh454 was used as a positive control for selu and dv70 was used as a positive control for selk (Vancraeynest and others 2006). The primers used in the pcr assays and the expected amplicon sizes and references are shown in Table 1.
After amplification, 5 μl of the amplicon was mixed with 3 μl sample buffer (50 per cent glycerol and 1mM cresol red) and the mixture was electrophoresed. After electrophoresis, the gels were visualised under uv light and photographed. The Gene Ruler 100 bp dna Ladder Plus (mbi Fermentas) was used as a dna size marker.
In the positive control strains, amplicons of the expected size were obtained. However, no genes encoding staphylococcal enterotoxins, enterotoxin-like proteins, exfoliative toxins or tsst-1 were amplified by pcr in any of the isolates of cns.
The following species of cns from human beings and animals have been shown to produce specific exotoxins and tsst-1: S epidermidis, S chromogenes, Staphylococcus haemolyticus, S warneri, S caprae, S xylosus, Staphylococcus saprophyticus and Staphylococcus lentus (Crass and Bergdoll 1986, Valle and others 1991, Orden and others 1992, Cunha and others 2007). Cunha and others (2007) showed that S epidermidis was the predominant species of all the cns that produced toxins. Most of these species were included in the present study.
Several surveys have failed to detect tsst-1 in cns (Kreiswirth and others 1987, Parsonnet and others 1987, Jaulhac and others 1992, Becker and others 2001), but the presence of tsst-1 has been confirmed in S epidermidis from human beings (Crass and Bergdoll 1986) and in S xylosus, S sciuri and S epidermidis from cases of ruminant mastitis (Orden and others 1992). In the present study, tst, the gene encoding tsst-1, could not be demonstrated.
cns have been tested for the presence of genes encoding exfoliative toxins in only two studies (Becker and others 2001, Dakić and others 2005). Both studies dealt with human isolates, and the genes were not detected in any of the isolates. Similarly, in the present study these genes were not detected in any of the isolates of cns.
cns isolated from human beings and from food have also been tested for the presence of enterotoxin-producing genes. Some studies focused on the five classical enterotoxin genes sea-see (Becker and others 2001, Cunha and others 2006a, b, 2007). Others, as in this study, included the more recently described enterotoxin and enterotoxin-like genes (Rosec and Gigaud 2002, Blaiotta and others 2004, Carneiro and others 2004, Dakić and others 2005). Cunha and others (2006a, 2007) found quite a high percentage of isolates from foods positive for one or more of the enterotoxin genes sea-see, but in the other studies, as in the present study, these genes were not detected (Becker and others 2001, Rosec and Gigaud 2002, Blaiotta and others 2004, Carneiro and others 2004, Dakić and others 2005). The studies focusing on food-associated cns may cover cns of both human origin, due to food contamination from the hands or nares of the people processing the food, and animal origin, due to contamination of the meat or milk by cns from the animals. Studies dealing only with enterotoxins in cns from animals are rarer. Valle and others (1990, 1991) detected the production of sea, seb, sec and see in cns strains isolated from goats' milk and suggested that goats may be an important reservoir of enterotoxigenic staphylococci. Orden and others (1992) found that two S xylosus strains out of 40 cns strains isolated from bovine mastitis were enterotoxigenic, producing sec. No other studies on the production of enterotoxins by cns strains from animal mastitis have been published.
The authors who were using pcr (Becker and others 2001, Blaiotta and others 2004, Carneiro and others 2004) performed the reactions using primers that were designed for S aureus genes. The mostly negative results that have been obtained (Becker and others 2001, Blaiotta and others 2004), as in this study, suggest that enterotoxin genes may be absent from cns. However, it is also possible that the genes were not conserved sufficiently for them to be recognised by the primers. On the other hand, enterotoxins have been detected in cns using S aureus primers (Cunha and others 2006a, 2007). Furthermore, the probability that all 17 genes tested in this study were insufficiently conserved is small.
The superantigens encoded by genes that are detectable by the pcr tests used to demonstrate these genes in S aureus appear not to be involved in either the induction of subclinical or clinical mastitis by the cns isolates included in this study, or in the persistence of these isolates in the mammary gland.
The authors thank the Ministry of Science, Research and Technology of Iran for financial support of the scholarship of M. N.
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