Staphylococcus aureus Genotypes of Subclinical Bovine Mastitis Milk in the Middle Western Anatolia

: Background : Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. Subclinical mastitis is characterised by a non-alteration of the milk but can cause food poisoning by production of enterotoxins in milk. Knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments to prevent subclinical mastitis and provide useful data for epidemiological studies. The aim of this study was to characterize the genetic nature of the S . aureus cultured from subclinical bovine mastitis occurring in 16 farms in the middle western Anatolia. Methods : Two hundred sixty eight milk samples positive with California Mastitis Test (CMT) suggesting the subclinical mastitis of lactating cows in 16 different farms in the Middle Western Anatolia were collected and S. aureus were isolated. Identification was carried out by traditional tests and ribotyping confirmed the identification. Staphylococcal Enterotoxins (SE) were detected and typed by Staphylococcal Enterotoxin Test Reversed Passive Latex Agglutination (SET-RPLA) test kit. Genetic characterisation of the isolates was carried out by both ribotyping and pulsed field gel electrophoresis (PFGE). Results : A total of 77 isolates of S. aureus were purified and analysed by both biochemical identification and genotyping. Only 4 isolates (5.19 %) of S. aureus were recorded as enterotoxin positive. Genetic characterisation of the isolates was carried out by ribotyping revealed eight ribotypes while pulsed field gel electrophoresis (PFGE) was more discriminative representing 19 pulsotypes. Conclusion : This study shows no significant association between enterotoxin production, ribogroup and pulsotype profile of the S. aureus isolates collected from the Middle Western Anatolia.


BACKGROUND
Mastitis in dairy cows is a worldwide disease and can be caused by infections with bacteria, yeast and fungi [1][2][3][4]. However, the most causative microorganisms are bacteria with intramammary infection and there are different bacterial species responsible depending on the geographical location and management. Staphylococcus aureus is a major bacterial pathogen in dairy cattle causing clinical and subclinical mastitis [5][6][7] and its prevalence ranges from 5 to 50% in different countries. S. aureus strains can cause acute clinical, and long lasting subclinical mastitis. No alteration is observed in the milk with subclinical mastitis but high somatic cell count is obtained. If the cell count is too high, the milk is inappropriate for the consumers. Halasa et al. [8] reported that this type of mastitis is often chronic and account for up to 30% of all bovine cases which represents an important economic problem for dairy producers with reduction in milk quantity and quality, prolonged costly antibiotic treatments and premature culling. Philpot et al. [9] reported that the reduction in milk production attributed to sub-clinical mastitis may account for 70%-80% of the total losses . Some S. aureus strains have the ability of producing heat stable enterotoxins that cause staphylococcal food poisoning (SFP) [10]. SFP symptoms eg. sickness, abdominal cramps, diarrhoeae and a characteristic projectile emesis [11] appear within a few hours (i.e., 1-6 h) after ingestion of contaminated food, depending on individual susceptibility and toxic dose ingested. The knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments to prevent subclinical mastitis and provide useful data for epidemiological studies.
The occurrence of mastitis in Turkey has been investigated in many studies and although there are some higher percentages in different regions, mastitis prevalence was reported as 30 % in Turkey [12,13]. Many studies focusing on the phenotypic and genotyping characterization of S. aureus isolated from subclinical bovine mastitis in different regions of Turkey were carried out by coagulase gene polymorphisms detected by PCR [14], determination of classical enterotoxigenic characteristics [15], detection of superantigenic toxin genes [16]), comparing the antibiotic resistance profiles. Ünal and İstanbulluoğlu [17] revealed clonal relations of only a small number of S. areus by using Pulsed field gel electrophoresis (PFGE) and a clone of the bacterium was broadly detected in dairy farms in Kırıkkale province. However, there is no study by comparison of different methods about the characterization of strains of S. aureus related to subclinical mastitis occurring in farms in the Middle Western Anatolia.
Therefore, the aim of the present study was to detect staphylococcal enterotoxins and determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from subclinical mastitis in the Middle Western Anatolia by using PFGE and automated ribotyping. To our knowledge, ribotyping is the first study revealing riboprofiles of subclinical mastitis causing S. aureus in Turkey.

Isolates
Two hundred sixty eight milk samples positive with California Mastitis Test (CMT) indicating the subclinical mastitis of lactating cows in 16 different farms in the Middle Western Anatolia were collected for microbial evaluation in 2010-2011. Each milk sample was aseptically collected into sterile bottles just before milking and they were transported in a cool box at 4ºC to the laboratory. The CMT and bacteriological

Ribogroup
Pulsotype analyses were started within 24 h after sampling. For isolation of S. aureus, 10 ml of milk sample were centrifuged at 6000 rpm for ten minutes and after discarding the supernatant, the sediment were inoculated on Mannitol Salt Agar (MSA) with incubation at 37°C for 24 to 48 h for primary isolation. Suspected colonies were selected and subcultured on blood agar medium for obtaining pure culture. S. aureus was identified at the species level using standard biochemical methods including Gram stain, catalase, tube coagulase, oxidase, DNase activities, Growth on Baird Parker medium and fermentation of mannitol [18] and API Staph test (BioMerieux, France). Isolates identified as S. aureus by these classical tests were then evaluated for enterotoxin production and genotyping by both automated ribotyping and PFGE ( Table 1).

Detection of Staphylococcal Enterotoxins (SE)
Strains were grown in 10 mL of tryptone soya broth (CM0219B, Oxoid) for 16-18 h at 37 ˚C by shaking aerobically. After centrifugation, the supernatant was tested for the presence and typing of SEs by Staphylococcal Enterotoxin Test Reversed Passive Latex Agglutination (SET-RPLA) (TD900, Oxoid).

Ribotyping
Strains identified as S. aureus with conventional biochemical tests were then characterized by automated ribotyping using EcoRI in a robotized instrument (Riboprinter™ Microbial Characterization System, Qualicon, DuPont, Wilmington, DE) and the Riboprinter ™ System Data Analysis Program. The procedure used for processing each sample is described in detail by the manufacturer. The identification of each strain was obtained when the corresponding pattern matched one of the pattern of the DuPont Identification library of the Riboprinter® with a similarity %0.86. The characterisation consisted of combining profiles within a similarity range equal or larger than 0.93 to form a dynamic ribogroup that reflected the genetic relatedness of the strains [19]. Each ribogroup was numbered by the system used in the study. A similarity dendogram was generated based on the banding pattern similarities by using SPSS version 21.0.

Pulsed Field Gel Electrophoresis (PFGE) Analyses
The genetic relationships of S. aureus isolates collected from cows with subclinical mastitis were compared by the DNA macrorestriction patterns obtained from PFGE following Sma I digestion. PFGE typing of strains was performed according to the methods of Vanderlinde [20] and Hennekinne et al. [21] with some modifications. The electrophoresis was carried by using a Chef Mapper (Bio-Rad Laboratories, Hercules, CA) and pulse times were ramped from 5 s to 40 s for 19 h. Gels were stained with ethidium bromide, visualized using an ultraviolet transilluminator, and photographed. Strains of S. aureus were placed in groups of identical or related strains by comparing the banding patterns produced, using a combination of photographic visiual inspection and computer analysis (SPSS version 21.0, SPSS Inc., Chicago, IL) was carried out to create a similarity dendogram. A pulsotype (PT) was defined as a unique electrophoretic banding pattern. Strains with identical restriction profiles were assigned as the same type.

Isolation and Identification of S. aureus
A total of 185 isolates were collected from 463 mastitis positive milk samples and identification was carried out by both conventional biochemical tests and ribotyping. However, only 77 isolates collected from 8 out of 16 different farms were identifed as S. aureus by both conventional biochemical tests and ribotyping ( Table 1).

Detection of Staphylococcal Enterotoxins
Using the SET-RPLA test, 73 out of the 77 isolates of the S. aureus (94.8%) showed no enterotoxin activity, only 4 isolates (5.19 %) isolated from mastitis positive milk samples were recorded as positive. Three isolates (numbered 101, 115 and 131) synthesized Staphyloccal enterotoxin B however, only one isolate (122) synthesized Staphyloccal enterotoxin C ( Table  1). No correlation was obtained between enterotoxin profiles and pulsotypes of the isolates ( Table 1).

Ribotyping
In this study, automated riboprinting was applied to the 77 isolates of S. aureus for both confirmation of the classical identification and ribotyping. All the isolates tested were typeable. Restriction of the total DNA with EcoRI yielded about 14 fragments of 1-50 kb in size. A total of eight banding patterns were obtained among the strains (Figure 1). The most prevalent ribotype was Ribotype 5 represented by 20 isolates ( Table 1).
A dendogram based on the similarity was obtained. Ribogroups 7 and 8 showed 98% similarity while all the ribogroups were clustered in a group with a 95% similarity (Figure 2). Automated ribotyping was applied for the first time for evaluation of S. aureus isolates collected from subclinical mastitis positive milk samples in Turkey. All the farms contained different ribogroups of S. aureus except farms E3, B2 and A2. Farms D2 and D3 contained same ribogrous while farms E3, B2 and A2 contained only one ribogroup of S. aureus ( Table 1).
A dendogram that included all patterns was constructed on the basis of the similarity levels. A high degree of similarity was observed between the pulsotypes 4 -7 and 5-6. Secondry level of high degree of similarity was observed between pulsotypes 14-18 and 8-11 (Figure 4).  Table 1.

DISCUSSION
Subclinical mastitis causes elevated somatic cell counts (SCC) but no detectable changes in milk or the udder. The bacteria persist in mammary glands, teat canals, and teat lesions of infected cows and are contagious. Economic damage due to subclinical mastitis has been mainly attributed to the fact that a subclinical cow is a constant source of infection to otber cows and to milk production loss as reported by Swinkels et al. [22]. In this study, only 77 isolates out of 185 were identified as S. aureus as a causative organism of subclinical mastitis suggesting that other bacteria were responsible as indicated in Hegde et al. [23]. S. aureus can access to milk by direct excretion from udders during milking [24] and multiply in milk and produce enterotoxins that cause food poisoning if ingested [25]. Staphylococcal enterotoxins have a remarkable ability to resist heat and denaturation by cooking therefore, even after pasteurization biological activity of toxin remains and can cause food poisoning [26]. A relatively high percentage of classical enterotoxin forming S. aureus strains from bovine subclinical mastitis was recorded in many publications [27][28][29].
Boynukara et al., [15] first investigated classical enterotoxigenic properties of Staphylococcus aureus strains isolated from cows with subclinical mastitis in eastern part of Turkey with 106 S. aureus strains. Twenty seven isolates (25.5%) were found to be enterotoxigenic by reverse passive latex agglutination (RPLA). Of these, 25 (23.6%) were positive for staphylococcal enterotoxin A (SEA), 2 (1.9%) for staphylococcal enterotoxin B. Their study showed that most S. aureus strains isolated from bovine subclinical mastitis produced SEA compared to other SEs. However in this study, the prevelance of SE strains were very low (5.19%) being the SEB (3.89%) and SEC (1.29%) among 77 isolates of S. aureus.

CONLUSION
The strains producing SEs indicates very low potential of poisoning in milk samples collected from subclinical mastitis infected cows in the Middle Western Anatolia. Also, no significant association between enterotoxin production, ribogroup and pulsotype was observed in the current study. Future studies involving biofilm production ability of the isolates as carried out by Khoramrooz et al. [30] may explain a possible correlation between enterotoxin genes with biofilm formation and genotypes.