Epidemiological Evidence of Lesser Role of Thermostable Direct Hemolysin (TDH)–Related Hemolysin (TRH) Than TDH on Vibrio parahaemolyticus Pathogenicity

Abstract

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007–2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007–2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

Introduction

V ibrio parahaemolyticus is recognized as one of the major foodborne pathogens worldwide. Thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH)–producing V. parahaemolyticus are both considered virulent strains (Yeung and Boor, 2004), and they have similar biological, physiological, genetic, and immunological characteristics (Honda et al., 1988, 1989; Nishibuchi et al., 1989). TRH-producing strains as well as TDH-producing strains induce fluid accumulation in a rabbit ileal loop assay to detect diarrhea-inducing substrates (Honda et al., 1990). Increased chloride secretion followed by elevated intracellular calcium also occurs in intestinal epithelial cells (Takahashi et al., 2000). In some reports, the tdh-negative and trh-positive V. parahaemolyticus were isolated from 1.1% to 7.8% of overseas travelers with diarrhea in Japan during 1983–1995 (Suzuki et al., 1994; Obata et al., 1996; Suzuki et al., 1997). Shirai et al. (1990) detected the trh gene in >35% of 214 clinical strains isolated from overseas travelers with diarrhea in a Japanese quarantine in which 24% lacked the tdh gene (Shirai et al., 1990). There are few reports of foodborne infections caused by trh-positive strains, but infections caused by tdh-positive V. parahaemolyticus are common. Therefore, information regarding seafood contaminated with trh-positive strains is limited in the world (Johnson et al., 2010; Nakaguchi, 2013; Ottaviani et al., 2013).

In addition, the role of TRH as a pathogenic factor is still under discussion, though the pathogenicity of TRH has been investigated. In this study, the prevalence of trh-positive V. parahaemolyticus in bivalves was investigated and compared with the prevalence of outbreaks associated with trh-positive V. parahaemolyticus in V. parahaemolyticus outbreaks evaluated from the available data in foodborne infections in Japan. Based on the results, the lesser significance of TRH in V. parahaemolyticus infections is discussed.

Materials and Methods

Bivalve samples

A total of 74 samples including 2 species of domestic bivalves—47 hen clams (Mactra sulcatari) and 27 short-neck clams (Tapes japonica)—was purchased from seafood markets and retail shops in various regions (Table 1) in Japan from June to October (2007–2008). A total of 177 samples including 4 species of domestic bivalves—29 bloody clams (Anadara broughtonii), 45 hen clams, 60 short-neck clams, and 43 rock oysters (Crassostrea nippona)—was also purchased from seafood markets and retail shops (Table 2) in Japan from June to September (2010). The bivalve shells were removed aseptically, and the flesh was used as a test sample. A 25-g portion of each test sample was mixed with 225 mL of alkaline peptone water (APW) (Nissui Co., Tokyo, Japan) in a stomacher bag, gently homogenized by hand, and incubated at 35–37°C for 18 h.

Table 1.

Detection of Pathogenic Vibrio parahaemolyticus in Bivalves in Japan, 2007–2008

			Polymerase chain reaction detection
Seafood	Region	No. of samples tested	No. of tdh positive and trh negative sample (%)	No. of tdh negative and trh positive sample (%)	No. of tdh and trh positive sample (%)
Hen clam	North	44	3 (6.8%)	0	1 (2.3%)
	Central	2	0	1 (50%)	0
	Western	1	0	1 (100%)	0
	South	0	0	0	0
	Subtotal	47	3 (6.4%)	2 (4.3%)	1 (2.1%)
Short-neck clam	North	4	0	3 (75%)	0
	Central	5	0	1 (20%)	2 (40%)
	Western	11	1 (9.1%)	0	0
	South	7	0	3	0
	Subtotal	27	1 (3.7%)	7 (25.9%)	2 (7.4%)
Total		74	4 (5.4%)	9 (12.2%)	3 (4.1%)

Table 2.

Detection of Pathogenic Vibrio parahaemolyticus in Bivalve in Japan, 2010

			Polymer chain reaction detection			Isolation
Seafood	Region	No. of samples tested	No. of tdh positive and trh negative sample (%)	No. of tdh negative and trh positive sample (%)	No. of tdh and trh positive sample (%)	No. of samples with V. parahaemolyticus (%)	No. of samples with tdh positive and trh negative V. parahaemolyticus (%)	No. of samples with tdh-negative and trh2-positive V. parahaemolyticus (%)	No. of samples with tdh and trh1-positive V. parahaemolyticus (%)
Bloody clam	North	19	0	2 (10.5)	0	19 (100)	0	2 (10.5)	0
	Central	0	0	0	0	0	0	0	0
	Western	8	0	2 (25.0)	0	8 (100)	0	2 (25.0)	0
	South	2	0	0	0	2 (100)	0	0	0
	Subtotal	29	0	4 (13.8)	0	28 (96.6)	0	4 (13.8)	0
Hen clam	North	31	1 (3.2)	1 (3.2)	0	27 (87.1)	1 (3.2)	1 (3.2)	0
	Central	14	0	4 (28.6)	1 (7.1)	14 (100)	1 (7.1)	4 (28.6)	0
	Western	0	0	0	0	0	0	0	0
	South	0	0	0	0	0	0	0	0
	Subtotal	45	1 (2.2)	5 (11.1)	1 (2.2)	41 (91.1)	2 (4.4)	5 (11.1)	0
Short-neck clam	North	6	0	1 (16.7)	0	4 (66.7)	0	0	0
	Central	28	3 (10.7)	15 (53.6)	4 (14.3)	28 (100)	3 (10.7)	5 (17.9)	1 (3.6)
	Western	12	4 (33.3)	2 (16.7)	2 (16.7)	12 (100)	1 (8.3)	1 (8.3)	0
	South	14	1 (7.1)	4 (28.6)	3 (21.4)	14 (100)	1 (7.1)	4 (28.6)	0
	Subtotal	60	8 (13.3)	22 (36.7)	9 (15.0)	58 (96.7)	5 (8.3)	10 (16.7)	1 (1.7)
Rock oyster	North	23	0	1 (4.3)	0	20 (87.0)	0	1 (4.3)	0
	Central	11	0	0	0	11 (100)	0	0	0
	Western	6	0	1 (16.7)	0	6 (100)	0	0	0
	South	3	0	0	0	3 (100)	0	0	0
	Subtotal	43	0	2 (4.7)	0	39 (90.7)	0	1 (2.3)	0
Total		177	9 (5.1)	33 (18.6)	10 (5.6)	168 (94.9)	7 (4.0)	20 (11.3)	1 (0.6)

Detection of tdh and trh from enrichment culture

The enrichment cultures (0.1 mL) of samples were centrifuged at 10,000×g for 10 min. After the supernatant was removed, the pellet was resuspended in 100 μL of 50 mM NaOH in sterilized distilled water and heated at 100°C for 10 min. The solution was cooled and 16 μL of 1 M Tris-HCl (pH 7.0) was added to neutralize the solution. After centrifugation at 10,000×g for 10 min, the supernatant was transferred to a new tube for polymerase chain reaction (PCR) and used as template DNA for PCR assays targeting tdh and trh (both trh1 and trh2) (Tada et al., 1992). The PCR mixture (50 μL) consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01 mM EDTA, 0.1 mM dithiothreitol, 0.05% Tween 20, 0.05% Nonidet P-40, 5% glycerol, 0.2 mM of each of the four deoxynucleoside triphosphates (dNTP mixture) (Takara, Ohtsu, Japan), and 0.5 U of Taq polymerase (Takara Ex Taq; Takara). The amplification conditions were set at 1 cycle of 96°C for 5 min, followed by 35 cycles of amplification consisting of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, followed by 7 min at 72°C.

Isolation of V. parahaemolyticus

The APW enrichment culture (0.01 mL) of samples in 2010 was streaked onto CHROMagar Vibrio agar medium (CV agar) (CHROMagar, Paris, France) for detection of V. parahaemolyticus. At the same time, 0.1 mL each of the enrichment culture and the 10-fold dilutions of 10⁻¹ to 10⁻⁶ in phosphate-buffered saline (PBS) containing 2% NaCl was also plated onto CV agar for growing of many colonies, and incubated at 37°C for 18 h. Since serotype O3:K6 is a major serotype of TDH-producing V. parahaemolyticus, the anti-K6 antigen immunomagnetic separation (IMS) method was performed to improve isolation of TDH-producing organisms (Hara-Kudo et al., 2001). The enrichment culture (1 mL) was added to 25 μL of immunomagnetic beads coated with antibody against the V. parahaemolyticus K6 antigen (Denka Seiken, Tokyo, Japan). According to the manufacturer's protocol, K6 V. parahaemolyticus was concentrated in 0.1 mL PBS with 2% NaCl. A loopful (10 μL) of concentrate by IMS method was streaked onto CV agar, and incubated at 37°C for 18 h.

Characteristics of isolates

The suspected V. parahaemolyticus colonies on CV agar were tested; specifically, isolates from tdh- and trh-negative enrichment cultures were tested for the V. parahaemolyticus–specific sequences of the toxR gene (Kim et al., 1999). Cultures (0.1 mL) of isolates in tryptic soy broth (Oxoid, Hampshire, UK) containing 2% NaCl were incubated at 37°C for 18 h, and then centrifuged at 5000×g for 10 min. After the supernatant was removed, the pellet was resuspended in 0.1 mL sterile distilled water and heated at 100°C for 5 min. After centrifugation at 10,000×g for 10 min, the supernatant was used as template DNA. toxR-positive isolates were inoculated in triple sugar iron (Oxoid) and nutrient broth (Difco, BD, Franklin Lakes, NJ) containing 0%, 3%, 7%, and 10% NaCl for biochemical characteristics tests. After incubation at 37°C for 20–24 h, strains showing alkaline slant and acid butt reactions in the triple sugar iron medium, and no growth in nutrient broth with 0% and 10% NaCl were identified as V. parahaemolyticus (Kaysner and DePaola, 2004). Isolates from the tdh-positive and trh-positive enrichment cultures were tested for tdh and trh (both trh1 and trh2) by PCR assays (Tada et al., 1992), respectively. DNA was extracted from the cultures by the method described above. Tdh- and/or trh-positive isolates were tested for biochemical characteristics as described above. tdh- and/or trh-positive V. parahaemolyticus serotypes were analyzed using antiserum against K and O antigens of the organism (K Serum Set and Group O Serum Set, Denka Seiken). Additionally, the isolates were identified as pandemic strains by a group-specific PCR assay (Matsumoto et al., 2000).

PFGE analysis

Thirty-five strains of tdh- and/or trh-positive V. parahaemolyticus were analyzed using pulsed-field gel electrophoresis (PFGE) with NotI and SfiI restriction enzymes (Parsons et al., 2007). PFGE was performed with a gradient of 6 V/cm at 14°C using a CHEF-DR II apparatus (Bio-Rad Laboratories, CA). Plugs digested by NotI and SfiI were run on a 2-block program (Block 1: 4–8 s for 11 h, Block 2: 8–50 s for 9 h) and a single-block program (10–35 s for 18 h), respectively. A dendrogram was constructed from the PFGE profiles as for the Jaccard similarity coefficient, and an unweighted-pair group method with arithmetic mean clustering analysis was performed using Fingerprinting II (Bio-Rad). One hundred percent concordance was considered to indicate identical patterns. SfiI profiles were analyzed for strains producing the same pattern as NotI restriction profiles.

Information on V. parahaemolyticus outbreaks and sporadic infections

A questionnaire regarding V. parahaemolyticus outbreaks and sporadic infections spanning the period 1998 through 2007 was sent from the National Institute of Health Sciences to various prefectures and cities in Japan. The questionnaire requested such information as the date and place of occurrence, number of cases, type of foods implicated, and details of the V. parahaemolyticus isolates (serotype and whether they harbored tdh and/or trh genes). Responses from 12 prefectures/cities were received by the National Institute of Health Sciences. Information on tdh- and/or trh-positive V. parahaemolyticus and the serotypes of the isolates from patients was analyzed.

Statistical analysis

The incidence of outbreaks and sporadic infections caused by tdh-negative and trh-positive strains was compared to prevalence of tdh-negative and trh-positive seafood with the chi-square test.

Results

Of the 74 samples obtained in 2007–2008, 4 (5.4%), 9 (12.2%), and 3 (4.1%) were tdh positive and trh negative, and tdh negative and trh positive, and both tdh and trh positive, respectively (Table 1). Of the 177 samples obtained in 2010, 9 (5.1%), 33 (18.6%), and 10 (5.6%) were tdh positive and trh negative, and tdh negative and trh positive, and both tdh and trh positive, respectively (Table 2). The number of tdh-negative and trh-positive samples was almost two to four times higher than those of tdh-positive and trh-negative, and both tdh- and trh-positive samples. Short-neck clams showed higher frequencies of tdh-negative and trh-positive, and both tdh- and trh-positive V. parahaemolyticus than hen clam in 2007–2008 (Table 1), and any of the four bivalve species studied in 2010. Seven, 20, and 1 strain of tdh-positive and trh-negative, tdh-negative and trh-positive, and both tdh- and trh-positive V. parahaemolyticus were isolated from tdh- and/or trh-positive samples, respectively (Table 2). Pathogenic V. parahaemolyticus was isolated in short-neck clams more frequently than the other bivalve species studied. Bloody clams and hen clams also showed high isolation rates of tdh-negative and trh-positive V. parahaemolyticus. In hen clams, one of two tdh-positive and trh-negative strains were isolated from a tdh- and trh-positive sample.

Most of the tdh-positive strains were identified as the serotype O3:K6 pandemic strain (Table 3), and an isolate of nonpandemic strain O11:K5 was also detected. The tdh-negative and trh-positive strains were all trh2 positive and belonged to 14 different serotypes (including untypable): O1:K32, O4:K12, O4:K49, O5:K30, O4:KUT, O5:K17, O11:K22, O1:KUT, O3:KUT, O10:KUT, O11:KUT, OUT:K49, OUT:K50, and OUT:KUT. In addition, one strain harboring both tdh and trh1 was OUT:KUT.

Table 3.

Characteristics of Vibrio parahamolyticus in Commercial Bivalve Samples in Japan

	Origin			Strain
						PFGE pattern
tdh- and/or trh-positive strain (number of strains)	Seafood	Sample no.	Region	Serotype (no. of isolates)	GS-PCR	NotI	SfiI
tdh	Hen clam	NH-19	North	O3:K6(4)	+	4	9
(24)		NH-17	Central	O3:K6(7)	+	4	9
				O11:K51(1)	−	3	20
	Short-neck clam	SS-11	Central	O3:K6(4)	+	5	6
		SS-12	Central	O3:K6(3)	+	5	6
		SS-14	Central	OUT:K6(1)	+	5	8
		MS-17	Western	O3:K6(3)	+	4	10
		KS-12	South	O3:K6(1)	+	4	7
trh2	Bloody clam	NB-4	North	O4:KUT(1)	−	16	5
(61)				O10:KUT(13)	−	11	22
		NB-7	North	O4:KUT(2)	−	18	27
				OUT:K50(1)	−	18	27
		NB-3	Western	O1:K32(1)	−	8	21
				O11:KUT(1)	−	7	23
		SB-2	Western	O4:K49(1)	−	20	4
	Hen clam	NH-1	North	O4:K12(2)	−	14	13
		NH-3	Central	O1:KUT(1)	−	17	25
				O4:KUT(1)	−	17	25
		NH-17	Central	O5:K30(1)	−	1	11
				OUT:KUT(1)	−	15	16
		NH-18	Central	OUT:KUT(3)	−	15	16
		FH-5	Central	OUT:KUT(7)	−	14	15
	Short-neck clam	AS-2	Central	OUT:KUT(1)	−	15	16
		SS-2	Central	OUT:K49(1)	−	20	3
				O4:K49(2)	−	20	3
				OUT:KUT(2)	−	NT	NT
		SS-11	Central	OUT:KUT(1)	−	18	25
		MS-4	Central	OUT:KUT(2)	−	17	26
		MS-11	Central	OUT:KUT(1)	−	12	2
		MS-16	Western	OUT:KUT(1)	−	14	14
		KS-2	South	O5:K17(2)	−	19	18
				O3:KUT(5)	−	9	17
		KS-8	South	OUT:KUT(1)	−	7	23
		KS-10	South	O11:KUT(2)	−	2	19
		KS-12	South	OUT:KUT(2)	−	13	24
	Rock oyster	AR-5	North	O11:K22(2)	−	6	1
tdh and trh1 (1)	Short-neck clam	SS-12	Central	OUT:KUT(1)	−	10	12

GS-PCR, group-specific polymerase chain reaction assay (Matsumoto et al., 2000); PFGE, pulsed-field gel electrophoresis, NT, not tested.

In the analysis of the characteristics of V. parahaemolyticus strains, tdh-positive serotype O3:K6 isolates from two hen clam samples obtained from the north and central regions of Japan showed the same PFGE pattern using NotI and SfiI restriction enzymes (NotI:4 and SfiI:9) (Table 3). The isolates from two short-neck clam samples also showed the same PFGE pattern (NotI:5 and SfiI:6). There were 22 distinct PFGE patterns in tdh-negative and trh2-positive isolates. Six patterns were detected in O1:KUT, O4:K49, O4:KUT, O11:KUT, OUT:K49, OUT:K50 or OUT:KUT strains from two or three different samples. A tdh-positive and trh1-positive isolate was consistent with NotI:10 and SfiI:12.

Data on the serotypes of isolates and the presence and absence of tdh and trh genes were analyzed from 330 outbreaks and sporadic infections. tdh positive and trh negative strains caused the majority of V. parahaemolyticus infections (89.4%) (Table 4). A small proportion of infections were trh positive, particularly, 1.2% were tdh negative and trh positive and 3.0% were tdh positive and trh positive (Table 4). A few (0.6%) V. parahaemolyticus infections were tdh negative and trh negative. Thus, the incidence of infections caused by tdh-negative and trh-positive strains (1.2%) was significantly lower (p<0.01) than the prevalence of tdh negative- and trh-positive strains in seafood (12.2% in 2007–2008 and 18.6% in 2010, Tables 1 and 2). The most common serotype among the 468 tdh-positive and trh-negative isolates was O3:K6 (62.6%, 293 isolates) followed by O1:K25 (30 isolates), O4:K8 (23 isolates), and O4:K68 (23 isolates). O6:K18 (7 isolates), O1:KUT (7 isolates), and O1:K1 (4 isolates) were the major serotypes for the tdh- and trh-positive isolates (26 isolates). The tdh-negative and trh-positive isolates (12 isolates) were O4:K53 (3 isolates), O1:K56 (2 isolates), OUT:KUT (2 isolates), O1:KUT, O3:K43, O6:K18, O6:K28, and O10:K66. The PFGE analysis of clinical strains from the prefecture would be helpful to clarify the relation with the seafood strains.

Table 4.

Harboring of TDH and TRH Genes and Serotypes of Vibrio parahamolyticus Caused Outbreaks in 1998–2007

Type of tdh- and trh-harboring	Number of outbreaks and sporadic infections (%)	Serotypes (number of outbreaks and sporadic infections) ^a						Total number of serotypes	Total number of outbreaks and sporadic infections ^a
tdh+/trh−	295 (89.4)		O3:K6(243)	O4:K68(19)	O1:K25(18)	O1:K56(11)	O4:K8(10)	23	345
			O4:K11(6)	O4:K9(5)	O1:KUT(4)	O3:K29(4)	O4:K55(4)
			O4:K13(3)	O8:K41(3)	O2:K3(2)	O3:K57(2)	OUT:KUT(2)
			O1:K1(1)	O2:K28(1)	O3:K5(1)	O3:KUT(1)	O4:K6(1)
			O4:K10(1)	O5:KUT(1)	O9:K44(1)
tdh−/trh+	4 (1.2)		O1:KUT(1)	O4:K53(1)	O6:K28(1)	OUT:KUT(1)		4	4
tdh+/trh+	10 (3.0)		O6:K18(4)	O1:K1(2)	O1:KUT(2)	O3:K6(1)	O5:K17(1)	5	6
tdh−/trh−	2 (0.6)		O2:K28(1)	O10:K24(1)				2	2
tdh+/trh− & tdh−/trh+	3 (0.9)	tdh+/trh−	O3:K6(3)	O4:K68(1)				2	2
		tdh−/trh+	O1:K56(1)	O4:K53(1)	O6:K18(1)	OUT:KUT(1)		4	4
tdh+/trh− & tdh+/trh+	8 (2.4)	tdh+/trh−	O3:K6(6)	O2:K3(2)	O1:K25(1)	O1:K41(1)	O3:K29(1)	12	18
			O4:K8(1)	O4:K9(1)	O4:K11(1)	O4:K55(1)	O4:K68(1)
			O5:K15(1)	O8:K21(1)
		tdh+/trh+	O6:K18(3)	O1:KUT(2)				2	5
tdh+/trh− & tdh−/trh−	8 (2.4)	tdh+/trh−	O3:K6(7)	O4:K8(3)	O4:K11(2)	O1:K25(1)	O1:KUT(1)	8	17
			O2:K3(1)	O3:K29(1)	O4:K9(1)
		tdh−/trh−	O3:K7(2)	O4:K4(2)	O1:K33(1)	O3:K54(1)	O3:KUT(1)	14	16
			O4:K8(1)	O4:K10(1)	O4:K11(1)	O4:K12(1)	O4:KUT(1)
			O5:KUT(1)	O8:KUT(1)	O10:KUT(1)	O11:KUT(1)
tdh−/trh+& tdh+/trh+	0
tdh−/trh+& tdh−/trh−	0
tdh+/trh+& tdh−/trh−	0
Total	330^b

A part of outbreaks and sporadic infections included two or more serotypes.

Forty-six sporadic outbreaks were included.

Discussion

Few reports analyze seafood contaminated with trh-positive V. parahaemolyticus and the occurrence of infections with the bacterium in the same region. Velazquez-Roman et al. (2012) reported that tdh-positive and trh-negative, tdh-negative and trh-positive, and both tdh- and trh-positive strains constitutes 34%, 1%, and 17% of environmental samples, and 86%, 1%, and 6.5% of clinical strains in northwest Mexico, respectively. This suggests that the frequency of trh-positive V. parahaemolyticus in the environment and the possibility of infections with trh-positive strains are low. Vuddhakul et al. (2006) investigated virulent and pandemic strains of V. parahaemolyticus from 230 samples of molluscan shellfish in Hat Yai City of southern Thailand between 2000 and 2002 and detected tdh and trh in 4.8% and 0.9% of strains, respectively. The characteristics of 865 clinical V. parahaemolyticus isolates from the main hospital in Hat Yai City between 2000 and 2005 were also analyzed by Vuddhakul's team (Wootipoom et al., 2007). The tdh-positive and trh-negative, tdh-negative and trh-positive, and both tdh- and trh-positive strains were found to comprise 83.1%, 1.8%, and 6.2% of all strains, respectively. These data support low frequencies of seafood contamination and infections with tdh-negative and trh-positive strains.

In France, 46%, 36%, and 9% of the 11 clinical strains studied were tdh positive and trh negative, tdh negative and trh positive, and both tdh and trh positive, respectively (Robert-Pillot et al., 2004). The report also showed that no tdh-positive and trh-negative V. parahaemolyticus was isolated from the total 135 strains obtained from the domestic environment and 130 strains obtained from seafood, whereas tdh-negative and trh-positive and both tdh- and trh-positive strains were isolated in 5.3% and 0.4% of samples, respectively. The results indicate that trh-positive V. parahaemolyticus causes infection just as the tdh-positive bacterium does, but the frequency of tdh-negative and trh-positive strains in the environment as well as in seafood is higher than that of tdh-positive and trh-negative strains. Thus, the virulence of tdh-positive strains appears higher than that of the trh-positive strains in studies using a small number of clinical strains.

In this study, outbreaks and sporadic infections with tdh-negative and trh-positive V. parahaemolyticus (1.2%) were much less frequent than those with the tdh-positive and trh-negative bacterium (89.4%) in epidemiological data from 1998 to 2007. However, the frequency of trh-positive samples (12.2% in 2007–2008 and 18.6% in 2010) in bivalve samples was 2–4 times higher than that of tdh-positive samples (5.4% in 2007–2008 and 5.1% in 2010). We focused on four species of bivalves as the target seafood population in this study. Because tdh is present in these bivalves (Hara-Kudo et al., 2003, 2012), they are considered as important seafood in the control of foodborne diseases caused by V. parahaemolyticus. The short-neck clam is one of the most popular seafoods in Japan, and bloody clam, hen clam, and oysters are often consumed raw as sashimi (raw fish fillet) and sushi (vinegary rice ball with raw fish fillet) in Japan. It is suggested that people consume seafood contaminated with greater amounts of trh-positive V. parahaemolyticus than tdh-positive strains; however, infections with trh-positive V. parahaemolyticus are less common than those with tdh-positive strains. There are many reports of tdh-positive V. parahaemolyticus infection. However, reports of foodborne infections caused by tdh-negative and trh-positive V. parahaemolyticus are limited (Ikeda et al., 1997; Thongjun et al., 2013).

Conclusions

The results of this study suggest that the TRH has a lesser role than TDH in V. parahaemolyticus pathogenicity; however, one cannot deny its pathogenicity.

Footnotes

Acknowledgments

We are thankful to Fumio Gondaira for providing anti-K6 immunomagnetic beads. This research was supported by a grant from the Ministry of Health, Labour, and Welfare, Japan. We appreciate the following governments of prefectures Okinawa, Kagoshima, Kumamoto, Oita, Fukuoka, Hiroshima, Tokyo Metropolitan, Saitama, and Hokkaido, as well as the cities of Nagasaki, Fukuoka, and Kitakyusyu for their cooperation in responding to the questionnaire on V. parahaemolyticus outbreaks.

Disclosure Statement

No competing financial interests exist.

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