ORIGINAL ARTICLE

Detection of classical enterotoxins of Staphylococcus aureusin raw sheep, goat, camel, and water buffalo milkby ELISA method

Ebrahim Rahimi

Received: 9 November 2011 /Accepted: 1 December 2011 /Published online: 13 December 2011# Springer-Verlag London Limited 2011

Abstract Milk is considered a nutritious food because itcontains several important nutrients including proteins andvitamins. Conversely, it can be a vehicle for several patho-genic bacteria such as Staphylococcus aureus. The aim ofthe present study was to investigate the presence of classicalenterotoxins of S. aureus in raw milk in Iran. For thispurpose, from January 2011 to October 2011, a total of 88raw milk samples from sheep (n028), goat (n027), camel(n014), and water buffalo (n019) were collected from 64randomly selected herds in Isfahan, Chaharmahal vaBakhtiari, and Khuzestan provinces, Iran, and tested for staph-ylococcal enterotoxins (SEs) presence by enzyme-linkedimmunosorbent assay method. Out of 88 samples studied, 11samples (12.5%) were positive for at least one SE. Theprevalence rate of SEs contamination in raw sheep, goat, andwater buffalo milk samples were 10.7%, 18.5%, and 15.8%.No classical SEs was founded from raw camel milk samples.Of these, four were positive for staphylococcal enterotoxin A(SEA), four for SEC, two SEA and SEC, and one for SEA andSED. None of the samples was positive for SEB andSEE. Statistical evaluation showed that there were nosignificant differences (P>0.05) between the presenceof SEs raw sheep, goat, and water buffalo milk samplestested. The quality of Iranian milk is still low, and effortsfrom the government and the entire productive chain arerequired to attain consumer safety.

Keywords Staphylococcal aureus . Entrotoxins . Rawmilk .

Water buffalo . Sheep . Goat . Camel

Introduction

Staphylococcus aureus is one of the most common agents inbacterial food poisoning outbreaks. It is also a major causativepathogen of clinical or subclinical mastitis of dairy domesticruminants. Poultry, meat, and egg products as well as milk andmilk products have been reported as common foods that maycause staphylococcal food poisoning (Le Loir et al. 2003). S.aureus strains produce a spectrum of protein toxins and viru-lence factors thought to contribute to the pathogenicity of thisorganism. The staphylococcal enterotoxins (SEs) have beenclassified into many different types. These enterotoxins areheat-stable and resistant to the action of digestive enzymes(Balaban and Rasooly 2000). The most common types ofthese enterotoxins are SEA to SEE. Isolates carrying toxingenes SEA to SEE are responsible for 95% of staphylococcalfood poisoning outbreaks (Hwang et al. 2007; Pelisser et al.2009). The remaining staphylococcal food-borne disease out-breaksmay therefore be associatedwith other newly identifiedSEs (Omoe et al. 2002; Rosec and Gigaud 2002; Rall et al.2008). Therefore, the presence of S. aureus in food can beconsidered a potential health risk.

As milk is a major nutrient for infants, children, con-valescents, and old people, the presence of classical enter-otoxins in milk and dairy products is a matter of concern.Milkis a good substrate for S. aureus growth and enterotoxinproduction. In addition, enterotoxins retain their biologicalactivity even after pasteurization (Asao et al. 2003). Accord-ing to Anderson et al. (1996), staphylococcal enterotoxins arevery resistant to heat; staphylococcal enterotoxin A (SEA), forexample, retains some biological activity after 28 min at121°C. Thus, because of the importance of these toxins inpublic health and food sectors, an efficient screening to detectthe prevalence of enterotoxic strains in foods is required. Sur-veys to detect classical enterotoxins and identify enterotoxin

E. Rahimi (*)Department of Food Hygiene, School of Veterinary Medicine,Islamic Azad University,Shahrekord Branch, P.O.Box 166, Shahrekord, Irane-mail: ebrahimrahimi55@yahoo.com

Comp Clin Pathol (2013) 22:181–184DOI 10.1007/s00580-011-1383-4

genes in S. aureus from milk and milk products have beenconducted in many countries, e.g., Norway (Jorgensen et al.2005), Italy (Morandi et al. 2007), Brazil (Rall et al. 2008),Turkey (Boynukara et al. 2008; Ertas et al. 2010), Sudan(Shuiep et al. 2009), and Vietnam (Huong et al. 2010). Butthere is limited information regarding the presence of SEs invarious food stuff in Iran. Although, governmental regulationof milk pasteurization and sanitation in dairy processing plantshas been in existence in Iran for many years, direct saleof unpasteurized milk and dairy products from producersto the consumer is not uncommon in many regions.Therefore, this study was conducted to investigate thepresence of classical enterotoxins of S. aureus in rawsheep, goat, camel, and water buffalo milk in Isfahan,Chaharmahal va Bakhtiari, and Khuzestan provinces,Iran, by enzyme-linked immunosorbent assay (ELISA).

Materias and methods

Sample collection

From January 2011 to October 2011, a total of 28 sheep bulkmilk samples were collected from 21 sheep breeding farms, 27goat bulk milk samples were collected from 23 goat breedingfarms, 14 camel bulk milk samples were collected from 10camel breeding farms, and 19 buffalo bulk milk samples werecollected from 14 buffalo breeding farms in Isfahan,Chaharmahal va Bakhtiari, and Khuzestan provinces, Iran.The animals whose milk samples collected for this study wereclinically healthy and themilk samples showed physical (color,pH, and density) consistency. The samples were immediatelytransported to the laboratory in a cooler with ice packs andwere processed within an hour of collection.

Preparation of samples

Milk samples were centrifuged (10 min/3,500×g/10°C), andremoved of upper cream layer by absorbing in generously.The defatted milk diluted 1:20 with distilled water and wasfiltered with disposable syringe filter holder.

Determination of SEs by ELISA

All reagents were of analytical quality and purchased fromMerck. The presence of classical enterotoxins of S. aureus inmilk samples was detected with ELISA (RIDASCREEN®SET A, B, C, D, E Art. No: R4101, R-Biopharm AG,Germany). The assay was performed according to themanufacturer’s recommendation and as described elsewhere(Rahimi and Ghasemian Safai 2010). The mean lowerdetection limit of the assay was 0.1 ng/L. All experimentswere performed in duplicate.

Microtiter strips were inserted in to the microwell holder(a strip has eight wells from A–H). One hundred millilitersof the prepared sample were added to wells from A to G ofone microtiter strip and 100 μL of the positive control wasadded to well H, mixed gently by rocking the plate manually,and incubated for 60 min at room temperature. The liquid wascompletely poured off the wells. The wells were filled with250 μL of washing buffer and the liquid poured out again (thewashing procedure repeated two times). One hundred milli-liters of the diluted enzyme conjugate was added to the wells,gently mixed by rocking the plate manually, and incubated for60 min at room temperature. The walls were washed threetimes with 250 μL of washing buffer. Fifty microliters ofsubstrate and 50 μL of chromogen were added to the wells.After incubation for 30 min in the dark at room temperature,100 μL stop solution reagent was added to each well. Thewells were mixed gently by rocking the plate manually and theabsorbance was measured at 450 nm using an ELISA reader(Start Fax 2100, UK) with 30 min of the addition of the stopsolution. The cut-off value was obtained with adding, 0.15 tothe mean value of the negative control.

Statistical analysis

Data were transferred to Microsoft Excel spreadsheet (Micro-soft Corp., Redmond, WA, USA) for analysis. Using SPSS18.0 statistical software (SPSS Inc., Chicago, IL, USA),chi-square test and fisher’s exact two-tailed test analysiswas performed and differences were considered significant atvalues of P<0.05.

Results

In this study, 88 raw sheep, goat, camel, and water buffalomilk samples were analyzed for classical SEs (SEA, SEB,SEC, SED, and SEE) with ELISA. The prevalence rate of SEscontamination in raw sheep, goat, and water buffalo milksamples were 10.7%, 18.5%, and 15.8%. No classical SEswas founded from raw camel milk samples. In other words, of

Table 1 Distribution of classical Staphylococcus enterotoxins insheep, goat, camel, and water buffalo raw milk in Iran

Raw milksamples

Sampletested

Positivesamples

Types of enterotoxin

A C A and C A and D

Sheep 28 3 (10.87%) 1 1 1 0

Goat 27 5 (18.5%) 1 3 1 0

Camel 14 0 (0.0%) 0 0 0 0

Water buffalo 19 3 (15.8%) 2 0 0 1

Total 88 11 (12.5%) 4 4 2 1

182 Comp Clin Pathol (2013) 22:181–184

88 raw milk samples, 11 (12.5%) were contaminated with oneor more SEs (Table 1). Eight samples (9.1%) were contami-nated only with one enterotoxin and three (3.4%) carried twoenterotoxin. With this type of determination, the SEA entero-toxin was found to be the one most present (seven positivesamples), followed by SEC (six positive samples) and SED(one positive samples). No SEB and SEE were identified inraw milk samples. Table 1 shows distribution of classical SEssheep, goat, camel, and water buffalo milk samples collectedin Isfahan, Chaharmahal va Bakhtiari, and Khuzestanprovinces, Iran. Type A enterotoxin was found in one sheepmilk, one goat milk, and one water buffalo milk samples. TypeC was found in one sheep milk and three goat milk samples.Type A and C enterotoxin was detected in one sheep milk andthree goat milk samples. Type A and D enterotoxin was foundin one water buffalo milk sample (Table 1).

Discussion

S. aureus is one of the leading causes of food-borne disease;for example, De Buyser et al. (2001) estimated that 85.5%of outbreaks associated with consumption of milk and dairyproducts, especially raw milk and raw milk products, werecaused by S. aureus. Many sources of milk contaminationexist, including infected cows, animal and human skin, foodhandlers, milking machinery, other equipment, and air.Although Iranian law does not establish limits for the amountof S. aureus in milk, it is known that the amount of enterotoxinsproduced by enterotoxigenic strains achieve levels that aresufficient enough to bring about symptoms of food-bornedisease when S. aureus concentration exceeds 105 CFU/mL(Tranter 1996). SEA is the most common enterotoxinrecovered from food poisoning outbreaks followed by SEDand SEC, and it is known that 95% of staphylococcal foodpoisoning outbreaks are caused by SEA to SEE (Balaban andRasooly 2000).

In this study, 11 (12.5%) out of 88 raw milk samples werepositive for at least one enterotoxin. According to detectionlimit (0.1 ng/L) of ELISA method in our study, concentra-tion levels of SEs positive samples were higher than of0.1 ng/L. The most frequently observed SE, was SEA,observed in seven (8.0%) samples, followed by SEC (sixsamples, 6.8%) and SED (one sample, 1.1%). These findingsare in agreement with Serraino et al. (2004), Normano et al.(2005), Manfereda et al. (2005), Morandi et al. (2007), Rall etal. (2008), Rahimi and Ghasemian Safai (2010), and Rahimiet al. (2011), who reported frequent enterotoxin A in manydairy products.

Our data show that presence of SEs in different raw milksamples are in close correlation with sample origin. Forexample, type A enterotoxin was found in all of positivewater buffalo milk samples and only in two of eight positive

sheep and goat milk samples, while SEC mainly wasdetected from sheep and goat milk samples. These resultsare in agreement with the data reported from other countries(Akineden et al. 2008; Jorgensen et al. 2005; Loncarevic etal. 2005; Morandi et al. 2007; Nørrung and Buncic 2008;Rall et al. 2008; Scherrer et al. 2004; Tang et al. 2011).However, other studies have shown that SEC is the mostfrequent enterotoxin produced by S. aureus isolated fromcow’s milk (Aragon-Alegro et al. 2007; Jablonsky andBohach 1997; Jorgensen et al. 2005; Normanno et al.2005, 2007 ), and SEAwas usually typical of human isolates(Balaban and Rasooly 2000). Variation in the resultsreported in other studies may be a result of different samplingtechniques employed, seasonal effects, and/or laboratorymethodologies employed in different studies.

More studies on the occurrence of enterotoxigenic S. aureusare needed to establish microbiological criteria of foods in otherpart of the country. Also, future epidemiological studies areneeded to enterotoxigenic S. aureus isolated or their toxins infood, and investigations should also be performed to find therelationship between the presence of this pathogen or SEs infood and its ability to cause disease in humans. To our knowl-edge, this is the first survey to estimate the presence of classicalenterotoxins of S. aureus in raw sheep, goat, camel, and waterbuffalo rawmilk used for human consumption in Iran. It can beconcluded that raw milk is contaminated by this pathogen inthis area as well as in other countries and might constitute a riskfor S. aureus enterotoxin food poisoning.

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