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Veterinary Microbiology 106 (2005) 103–107

Detection of the enterotoxins A, B, and C genes in

Staphylococcus aureus from goat and bovine mastitis

in Brazilian dairy herds

Elizabete Rodrigues da Silvaa,1,*, Luiz Simeao do Carmob,2, Nivaldo da Silvaa,1

aLaboratory of Mastitis Control, Veterinary School – Federal University of Minas Gerais/UFMG, Av. Antonio Carlos, 6627,

Campus Pampulha, CEP 30161-970, Belo Horizonte, Minas Gerais, BrazilbFundacao Ezequiel Dias – Laboratory of Staphylococcal enterotoxins, Rua Conde Pereira carneiro,

80, Belo Horizonte, Minas Gerais, Brazil

Received 5 August 2004; received in revised form 30 November 2004; accepted 8 December 2004

Abstract

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 genewas 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.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Goat mastitis; Bovine mastitis; Staphylococcal enterotoxins

1. Introduction

The staphylococcal enterotoxins (SEs) are distin-

guished for their role in the pathogenesis of human and

* Corresponding author. Present address: Embrapa Caprinos,

Estrada Sobral-Groaıras, km 04, Caixa Postal 10, CEP 62011-

970, Sobral, Ceara, Brazil. Tel.: +55 88 677 7000;

fax: +55 88 677 7055.

E-mail address: beteers@hotmail.com (E.R. da Silva).1 Tel.: +55 31 3499 2106.2 Tel.: +55 31 3371 9466.

0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2004.12.005

animal illnesses. These toxins are responsible for food

poisoning outbreaks and toxigenic syndrome in

humans, and may contribute to the persistence of S.

aureus in bovine mammary glands (Ferens et al.,

1998; Llewelyn and Cohen, 2002).

Several reports described the production of SEs by

S. aureus isolated from bovine mastitis with a great

geographical variation in the distribution of enter-

otoxigenic strains (Magalhaes Lopes et al., 1990;

Larsen et al., 2000; Stephan et al., 2001). In addition,

there were differences between S. aureus strains from

.

E.R. da Silva et al. / Veterinary Microbiology 106 (2005) 103–107104

bovine and goat mastitis regarding their ability to

produce enterotoxins (Orden et al., 1992).

Thus, the purpose of this study was to analyze the

distribution of the genes that encode SEA, SEB, and

SEC in S. aureus isolated from bovine and goat

mastitis. Also, the production in vitro of the SEs by

isolates harboring the respective gene was analyzed.

2. Materials and methods

2.1. Sources and strains of S. aureus

One hundred S. aureus strains isolated from goat

(n = 36) and bovine mastitis (n = 64) were studied.

The goats strains originated from Brazilian dairy herds

located in Ceara state and in Rio de Janeiro state, and

were previously isolated and identified (da Silva et al.,

2004). The bovine strains originated from dairy herds

located in Minas Gerais state and were also previously

isolated and identified (Cardoso et al., 1999).

2.2. Production and detection of SEA, SEB, and SEC

All strains were tested for enterotoxins A to C

(SEA-C) by the cellophane-over-agar method for

enterotoxin production and the optimum-sensitivity-

plate (OSP) method for enterotoxin detection and

identification (Robbins et al., 1974). Strains with

doubtful results to OSP were tested with the SET-

RPLA-kit (reversed passive latex agglutination,

Oxoid) following the manufacturer’s instructions.

2.3. Detection of sea, seb, and sec genes

DNA extraction was carried out according to Chen

et al. (2001) and Rosec and Gigaud (2002). The DNA

was suspended in 100 ml of sterile TE pH 7.5,

quantified in spectrophotometer (260 nm) and kept

frozen at �20 8C. The PCR primers were previously

reported by Mehrotra et al. (2000) and are listed in

Table 1. For Multiplex PCR, two sets of primers mixes

were prepared. Set A contained 2 ml of target DNA

(approximately 700 ng), 0.2 ml each of the primers

SEA, SEB, and FemA (10 pmol), 0.4 ml of dNTPs mix

(100 mM each), 0.2 ml of Taq DNA polymerase (1 U)

and 2 ml of PCR 10� buffer. Set B included the same

constituents as in set A except for the primers, which

were SEC and FemA at a volume of 0.4 ml (20 pmol)

and 0.2 ml (10 pmol), respectively. DNA amplification

was carried out in a thermocycler (Mini CyclerTM, MJ

Research) with the following thermal cycling profile:

an initial denaturation at 94 8C for 5 min was followed

by 25 cycles of amplification (denaturation at 94 8C –

2 min, annealing at 57 8C – 1 min, and extension at

72 8C – 1 min), ending with a final extension at 72 8Cfor 4 min. The PCR products were separated at 3.5%

agarose gel, stained with ethidium bromide, and

photographed under UV illumination. The bacter-

iophage DNA of Phi29 digested with HindIII was used

as a molecular marker.

2.4. Specificity testing

The specificity of the primers was tested by

individually analyzing each primer pair and the primer

pairs combined in the Multiplex PCR. The DNAs of

Staphylococcus aureus 89cTP2FUNED (SEAB) and

Staphylococcus aureus FRI 361 (SEC) were used as a

positive control. The specificity of the FemA primers

was confirmed with the DNA of Staphylococcus

aureus ATCC 25923, Staphylococcus epidermidis

ATCC 12228, and Staphylococcus intermedius 08/

96PE-FUNED.

2.5. Reproducibility testing

The reproducibility of the Multiplex PCR products

was tested by inter-assay analysis of 20 isolates (10

from goat mastitis and 10 from bovine mastitis), which

were tested for five consecutive days.

3. Results

3.1. Specificity and reproducibility testing

The reaction with each individual primer pair

resulted in the amplification of single products when

DNA from each reference strain was used as a

template. In the Multiplex, the amplification of three

bands in set A (sea, seb, and FemA) and two bands in

set B (sec and FemA) were successfully obtained

(Fig. 1). The sizes of the products obtained from

control strains in both PCR designs corresponded to

the predicted sizes. Reproducibility was observed in

E.R. da Silva et al. / Veterinary Microbiology 106 (2005) 103–107 105

Table 1

Nucleotide sequences, gene locations, and size of PCR products using specific primers for sea, seb, sec, and FemA genes

Gene Primer Nucleotide sequence (50-30) Location within gene Size of amplified

sea SEA1 GGT TAT CAA TGT GCG GGT GG 349 a 368 102

SEA2 CGG CAC TTT TTT CTC TTC GG 431 a 450

seb SEB1 GTA TGG TGG TGT AAC TGA GC 666 a 685 164

SEB2 CCA AAT AGT GAC GAG TTA GG 810 a 829

sec SEC1 AGA TGA AGT AGT TGA TGT GTA TGG 432 a 455 451

SEC2 CAC ACT TTT AGA ATC AAC CG 863 a 882

FemA Fem1 AAA AAA GCA CAT AAC AAG CG 1444 a 1463 132

Fem2 GAT AAA GAA GAA ACC AGC AG 1556 a 1575

Fig. 1. Detection of the sea, seb, and sec genes by Multiplex PCR in Staphylococcus aureus from goat and bovine mastitis. 3.5% agarose gel—

line 1: molecular marker; lines 2–5: sea, seb, sec, and FemA, respectively; line 6: molecular marker; line 7: Multiplex set A (sea, seb, and FemA);

line 8: Multiplex set B (sec and FemA); line 9: negative control; line 10: molecular marker.

all tested strains. Although there were variations with

band intensity, their presence and sizes were the same.

3.2. Distribution of sea, seb, and sec genes

Among the 100 S. aureus strains analyzed by

Multiplex PCR, 37 (37%) were positive for some of

the SEs genes investigated. Of these 37 strains, 33

(89%) were positive for sec and 4 (11%) were found to

be sea and seb positive.

The gene distribution according to origin of the

isolates is shown in Table 2. Among the 64 strains

from bovine mastitis, 4 (6.3%) co-amplified the sea

and seb genes and 2 (3.1%) were positive for sec gene.

Of the 36 strains from goat mastitis, 31 (86%)

amplified the sec gene.

3.3. Production of SEA, SEB, and SEC

The production of SE was detected in all strains,

which amplified the corresponding gene (Table 3).

The majority of PCR positive strains produced SE in a

quantity detectable by OSP (27/37). On the other

hand, in 10 PCR positive strains the detection of the

SE was possible only by RPLA, and all these were

from goat mastitis. The production of other SEs than

E.R. da Silva et al. / Veterinary Microbiology 106 (2005) 103–107106

Table 3

Detection of the enterotoxins A, B, and C in Staphylococcus aureus

from goat and bovine mastitis

Toxin Number of isolates

PCR OSP RPLA

SEA – – 1

SEB – – –

SEC 33 23 10

SEA+SEB 4 4 NRa

Total 37 27 11

a NR = no realized for this isolates.

Table 2

Distribution of the sea, seb, and sec genes in Staphylococcus aureus

according to origin of the isolates

Gene Origin of isolates Total (%)

Cow (%) Goat (%)

sea – (0.0) – (0.0) – (0.0)

seb – (0.0) – (0.0) – (0.0)

sec 2 (3.1) 31 (86.0) 33 (33.0)

sea+seb 4 (6.3) –(0.0) 4 (4.0)

none 58 (90.6) 5 (14.0) 63 (63.0)

Total 64 (100) 36 (100) 100 (100)

those encoded by the gene detected was also

investigated. Only one strain, which was negative to

PCR, demonstrated positive reaction to RPLA.

4. Discussion

Some S. aureus strains produce one or more

enterotoxigenic toxins including SEA, B, and C.

These SEs represent the main cause of staphylococcal

food poisoning and are potential virulence factors that

contribute toward mastitis pathogenesis (Ferens et al.,

1998). Furthermore, the SEs could be able to indicate

the origin of the S. aureus strains because it was

observed that a higher ratio of isolates from animals

produced SEC and those from humans produced

mainly SEA (Bergdoll, 1990; Orden et al., 1992). Our

results are in agreement because SEC was the most

frequently produced toxin by strains tested in this

study.

The results showed that S. aureus isolated from

goat mastitis has a higher enterotoxigenic potential

than those isolated from bovine mastitis. Similar

results had been described by Orden et al. (1992) who

observed that 67% of the goat mastitis isolates and

only 19% of the bovine mastitis isolates produced SEs.

Valle et al. (1990), who studied the presence of SEs in

Staphylococcus spp. isolated from healthy goats,

concluded that this animal specie is an important

reservoir of enterotoxigenic Staphylococcus. These

findings, along with ours, suggest that SEs may be

more important for S. aureus to establish or persist in

the goat mammary glands than in the bovine’s. The

presence of a higher number of polymorphonuclear

leukocytes in goat mammary glands, particularly

neutrophils, could be a factor that contributes for the

genetic selection of strains that can produce SAgs.

Schuberth et al. (2001) demonstrated the damaging

effect of these toxins on that cellular type.

In this study, only 9.4% of the isolates from bovine

mastitis carried genes and produced some of the SEs

investigated. Previous studies showed intense geo-

graphical variation in the occurrence of enterotoxi-

genic S. aureus isolated from bovine mastitis

(Magalhaes Lopes et al., 1990; Larsen et al., 2000;

Stephan et al., 2001). These results indicate that the

presence of SAgs in S. aureus from bovine mastitis

may be determined by environmental and manage-

ment factors in each geographical area. Moreover, the

results raise doubt about the role of these toxins in the

bovine mastitis pathogenesis. According to Larsen

et al. (2000) a virulence factor is considered important

when the gene encoding it, as well as its expression, is

present in the isolated strains.

The exclusive presence of the sec gene in S. aureus

from goat mastitis, as well as its expression in all

isolates harboring it, suggests that this toxin may play

a role in goat mastitis pathogenesis. Other studies have

also showed that a high percentage of Staphylococcus

spp. isolated from goat mastitis produced SEC (Valle

et al., 1990; Orden et al., 1992). Ferens et al. (1998)

demonstrated that SEC affects the bovine immune

response and may contribute to the persistence of S.

aureus in the mammary gland. At the moment, it is not

known if SEC affects the goat immune system.

However, the presence of the sec gene and its

expression in the majority of the isolates analyzed

in this study suggests that this SAg may be important

for the pathogenicity of S. aureus adaptable to the goat

mammary glands.

E.R. da Silva et al. / Veterinary Microbiology 106 (2005) 103–107 107

A 100% agreement between the gene detection by

Multiplex PCR and detection of the corresponding

toxin by phenotypic test was demonstrated. These

results suggest that the Multiplex design can be used to

characterize and identify enterotoxigenic types of S.

aureus isolated from mastitis. Its capability to

simultaneously detect several genes, as well as its

sensitivity, reproducibility and ease of execution,

makes this technique a useful tool in the field of

veterinary research.

In conclusion, the results indicated that S. aureus

isolated from goat mastitis has a higher enterotoxigenic

potential than those isolated from bovine mastitis.

Additionally, it was demonstrated that S. aureus

causing mastitis in Brazilian goats dairy herds harbored

the sec gene and produced the SEC toxin, suggesting

that it may play a role in goat mastitis pathogenesis.

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