PRODUCTION OF ENTEROTOXINS OF STAPHYLOCOCCUS SPP . ISOLATED FROM SAMPLES OF SHEEP MILK

In our study was followed occurrence of mastitis in herd of 430 sheep of breed zoslachtena valaska with hand milking technology examined two times during one lactation season. Individual examination consisted from clinical examination of udder and microbiological examination of milk samples. By PCR was determined presence of genes coding production of enterotoxins, and by ELISA methods production individual types of enterotoxins. From individual forms of mastitis were frequently detected subacute (6.7%), subclinical (5.7%) and acute (2.9%). The coagulase-negative staphylococci (CNS) were identified in 102 (65.4%) from all 156 positive isolates. The CNS and S. aureus caused subacute (5.1%), subclinical (3.9%) and acute (2.4%) forms of mastitis. The most frequently isolated were S. epidermidis, followed by S. chromogenes and S. xylosus from ewes with subacute and subclinical mastitis. From acute and chronical forms of mastitis were predominantly isolated S. aureus, S. uberis and S. epidermidis. The production of staphylococcal enterotoxins (SE) SEA, SEB, SEC, SED and the presence of genes sec (3), sea (2), seb (2) and sed (2) were determined in S. aureus, S. epidermidis, S. schleiferi and S. chromogenes, respectively. The results suggested on the high occurrence (12.4%) of subacute and subclinical forms. Confirmed production of enterotoxins and presence of genes coding their production present a risk for human health and decreased a quality of milk and products from sheep ́s milk.


INTRODUCTION
Mastitis has been considered an economically important disease in the production of sheep.The occurrence of mastitis in sheep is in interval from 4.0 to 50.0%.Staphylococci are the main aetiological agents of small ruminant intramammary infections (IMI), and Staphylococcus aureus is the most frequent isolate from clinical mastitis cases and coagulase-negative species are the most frequent in subclinical IMI.The annual incidence of clinical IMI in dairy sheep is generally lower than 5%, but in a small percentage of herds the incidence may exceed 30-50% of the animals, causing mortality (gangrenous mastitis) or culling of up to 70% of the herd (Fthenakis, 1994, 1995; Vautor et al., 2009).
Antibiotic treatment of mastitis leads to significant increase in milk quantity and quality, lower somatic cell count and is likely associated with reduction in prevalence of clinical mastitis among herds, which is economically beneficial (Contreras et al., 2007).
The important factor of virulence of Staphylococcus spp.besides to antibiotic resistance is production of enterotoxins, which showed high health risk for human.Milk and other dairy products are reported to be frequently associated with SE food poisoning.It was supposed that milk of infected animals constitute the main source of staphylococcal enterotoxigenity of animal origin (Omoe et al., 2002;Scherrer et al., 2004).
During the many years the production of enterotoxins was connected only with S. aureus.Many authors report, that other species of CNS (S. intermedius, S. hyicus) may producing of enterotoxins (Becker et al., 2001, Beatriz et al., 2006).
The aim of our study was the observed the occurrence of mastitis in herd of sheep with hand milking and determined of enterotoxigenic bacteria of Staphylococcus spp.isolated from milk samples during one milking season.

Animals and milking
The experiment was carried out in herd of 430 sheep of breed zoslachtena valaska, which during winter season were stabled in bricked shed with deep bedding.During milking season (from April to September) was hand milking carrying out in cover strung cage with three fixation places two times per day.

Examination of health status of sheep and milk samples
Complex examination of health status of udder in sheep was carried out at the start (April) and at the end of milking (September).The clinical examination was carried out according to Hariharan et al. ( 2004) and milk from individual halves was evaluated by NK-test (Bioveta a.s., Inovice na Hané, Czech Republic) according to Fthenakis (1994).

Laboratory analyses
From the every individual milk sample were inoculated 0.05 ml, onto blood agar (Oxoid LTD, Hampshire, UK) and cultivated at 37°C for 24h.Based on the colony morphology, bacteria Staphylococcus spp. was selected for the tube coagulase test (Staphylo PK, ImunaPharm, SR).Suspect colonies Staphylococcus spp., Streptococcus spp.and Enterobacteriacae spp.were isolated on blood agar, cultivated at 37°C for 24h and identified biochemical using the STAPHYtest, STREPTOtest, ENTEROtest (Erba-Lachema, Brno, Czech Republic) and identification by software TNW Pro 7.0 (Erba-Lachema, Brno, Czech Republic).
Identification of genes coding staphylococcal enterotoxins (sea, seb, sec, sed, see) was carried out by PCR method according to Becker et al. (1998).DNA was isolated by QiAMP tissue kit (Oiagem, Hilden, Germany).For control were used reference strains for types of SE: SEA, SEB SEC, SED, SEE (Bergdoll; CNCTC, Brno, Czech Republic).For the detection of genes was used oligonucleotid primers sea -see (Becker et al., 1998).
Validation of production of SE in vitro in every strain was confirmed by ELISA method, by set Ridascreen ® Set A, B, C, D, E (R-Biopharm AG, Darmstad, Germany).

RESULTS
From total number of sheep (n = 820) in Figure 1, on the base of complete examination were determined 19.5% occurrence of mastitis (n = 160) during one milking season.The most frequently were found subacute (6.7%), subclinical (5.7%) and acute (2.9%) forms of mastitis in 126 sheep (15.4%).
The presence of genes coding production of enterotoxins in bacteria Staphylococcus spp.(n = 115) described Table 2. Four strains of S. epidermidis showed presence of gene sea (1), seb (2) and sed (1), but by ELISA method was confirmed only production SE types A (1) and B (1) in two strains of staphylococci.In S. chromogenes and S. schleferi was determined presence of one gene sec, and gene sed in S. schleferi was conjugated with production correspondent type of SE.Three strains of S. aureus was characterized by presence of genes sea (1) and sec (2), but the production of SEC was determined only in one strain.

DISCUSSION
How common mastitis is in sheep is extremely variable.Studies of cull ewes at slaughter in Britain show a very high prevalence ranging from 13 to 50%, indicating that clinical mastitis is likely an important cause of culling of ewes in the UK (Conington et al., 2008).
Hariharan et al. ( 2004) showed 6.7% positive samples of milk from 492 examinated sheep in which were isolated CNS.The most frequently isolated were S. equorum, S. xylosus and S. simulans from ewes with subclinical mastitis during lactation.
Burriel (1997) reported that S. simulans, S. xylosus, S. hyicus were the predominant species of CNS in the milk from meat ewes, S. epidermidis and S. schleiferi was the predominant species in the milk of dairy ewes, what was also confirmed in our study.In addition to the high incidence of subclinical and subacute mastitis, which were caused by CNS and S. aureus, in some strains were determined production of SE.
The staphylococcal enterotoxins are recognised as being agents of intoxication such as staphylococcal food poisoning syndrome in man and they may be involved in other types of infections (Zschöck et al., 2005).1990) tested 342 Staphylococcus spp.bacteria for their ability to produce enterotoxins, which were isolated from various parts of the body of small ruminants.Staphylococcal enterotoxins were produced by 74.3% of 70 coagulase-positive bacteria and 22% of coagulase-negative bacteria.Most enterotoxigenic bacteria were isolated from the teat skin and milk.These bacteria most frequently produced staphylococcal enterotoxin of type C, namely either alone (67.9%) or in combination with other type of SE.From our results it follows that within 115 Staphylococcus spp.bacteria the production of SEC and SEA was recorded frequently, than SEB and SED, all the same as a frequently presence of sec gene.
The production of enterotoxins SEA, SEC and SED by S. aureus field strains isolated from mastitis animals has been investigated in several studies (Matsunaga et al., 1993; Zschöck et al., 2000.In our study presence of genes coding SE in species of CNS (S. epidermidis, S. chromogenes, S. schleiferi) was also certified.

CONCLUSION
By complex examination in herd of sheep we determined 12.4% occurrence of subacute and acute forms of mastitis caused predominantly by CNS, S. aureus and S. uberis.Very important is the early diagnosis of mastitis in sheep during the milking season.At the start of the treatment of subclinical mastitis showing the subclinical forms of mastitis can significantly eliminate clinical stage of subacute and acute forms of mastitis.In bacteria S. aureus, S. epidermidis, S. schleiferi and S. chromogenes were by PCR detected the presence of enterotoxins genes sec (3), sea (2), seb (2) and sed (2), as well as production of SE -SEA (2), SEC (2), SEB (1) and SED (1) by ELISA method.Because of the importance of these toxins for public health and food safety, an efficient screening for the prevalence of enterotoxic strains in mastitis is required.

Table 2
Staphylococcal enterotoxins and genes coding production of SE in bacteria Staphylococcus spp.(n=15) in herd of sheep