Characteristics of Vibrio parahaemolyticus isolates obtained from crayfish (Procambarus clarkii) in freshwater
Introduction
Vibrio parahaemolyticus is a leading cause of foodborne gastroenteritis worldwide, and is often associated with the consumption of raw or undercooked seafood (Okuda et al., 1997). Infections caused by V. parahaemolyticus produce symptoms such as diarrhea, nausea, vomiting, fever, and chills (Makino et al., 2003). Vibrio parahaemolyticus was first identified in 1950 during the study of a foodborne disease in Japan (Fujino et al., 1953), since then the reported incidence of V. parahaemolyticus infections has increased sharply (Okuda et al., 1997, González-Escalona et al., 2005, Li et al., 2014, Haendiges et al., 2014). The CDC (Centers for Disease Control and Prevention) reported that 57% of Vibrio spp. infections from 1997 to 2006 were caused by V. parahaemolyticus, and an estimated 4500 cases of V. parahaemolyticus infection occur each year in the United States (Iwamoto et al., 2010). However, the number of reported cases may be much lower than the real incidence as a result of under-reporting. Europe used to be considered as a low-risk area for V. parahaemolyticus infection before 2001, however, data suggest that the incidence of V. parahaemolyticus infections in Spain is higher than previously thought (Martinez-Urtaza et al., 2005).
The freshwater crayfish, Procambarus clarkii, is a freshwater crustacean resembling a small lobster, which has been cultivated extensively for food and has become one of the most important commercially cultivated crustaceans in the Chinese aquaculture industry (Wang et al., 2005, Xiao et al., 2014). Hubei province, in the middle of China, is one of the main crayfish exporters. The total export of crayfish from Hubei was worth 85,960,000 US dollars in 2013, while the domestic consumption was in excess of 100,000 tons, and is still increasing (http://www.hubei.gov.cn/zwgk/bmdt/201311/t20131128_480471.shtml). Wang and Lv reported two cases of foodborne disease caused by eating crayfish, and isolated V. parahaemolyticus from related live crayfish and the vomitus of the patients (Wang and Lv, 2011), confirming that V. parahaemolyticus from freshwater crayfish could cause foodborne disease. Thereafter, an outbreak of 375 cases of V. parahaemolyticus infection caused by undercooked frozen crayfish was reported in Jiangxi province in 2011, China, and V. parahaemolyticus carrying the virulence genes tdh and trh was isolated from boiled crayfish, rectal swabs, and stools (Sun et al., 2012). Thus survey of V. parahaemolyticus from crayfish was required since the high consumption of crayfish in many countries and the potential risk of infection mentioned above (Holdich, 1993, Taugbøl and Skurdal, 1999, USITC, 2003, Mu et al., 2007, Richert and Sneddon, 2007, NBSC, 2013, FAO (Food and Agriculture Organization), 2014, Patoka et al., 2014).
To date, pathogenic V. parahaemolyticus has been considered a natural inhabitant of marine environments and has frequently been involved in outbreaks associated with eating seafood (Centers for Disease Control and Prevention, 1993, Centers for Disease Control and Prevention, 1996, Daniels et al., 2000). However, few published papers have reported the existence of V. parahaemolyticus in freshwater (Mishra et al., 2010, Nelapati and Krishnaiah, 2010). Furthermore, there have been limited studies on the reasons for the existence of and the characterization of V. parahaemolyticus in freshwater products. Duckweed and small marine fish used as baits may contaminate freshwater crayfish, but there have been no detailed reports (Chen et al., 2009). Alternatively, Bean proposed that crayfish served as a vehicle for V. parahaemolyticus, though there has been lack of information on V. parahaemolyticus isolates (Bean et al., 1988). It is therefore necessary to determine the phenotypic and phylogenetic characteristics of V. parahaemolyticus isolates to help reevaluate the risk posed by V. parahaemolyticus from consuming freshwater crayfish and other aquatic products.
The complete genome sequence of V. parahaemolyticus RIMD 2210633 provides an insight into this organism (Makino et al., 2003). In addition to the classical virulence genes tdh and trh, other virulence-related genes also play important roles in its pathogenesis. T3SS proteins and urease, as well as genes involved in biofilm formation, are known to be involved in V. parahaemolyticus pathogenicity and survival (Park et al., 2000, Park et al., 2004, Nakaguchi et al., 2003), while urease-positive clinical isolates of V. parahaemolyticus and a possible role of this enzyme in bacterial virulence have also been reported (Eko, 1992). T3SS1 was mainly related to cytotoxicity, while T3SS2 contributed to enterotoxicity, and the effectors of these two sets of T3SS were shown to be critical in pathogenesis (Hiyoshi et al., 2010, Park et al., 2004). Further studies demonstrated that T6SS1 was predominant in clinical isolates, and Chao et al., 2009, Chao et al., 2010 reported that 77.5% of pandemic or pathogenic strains harbored complete or partial T6SS1 genes, while 54% of the nonpathogenic strains only possessed partial T6SS genes, suggesting that T6SS1 might play a role in enhancing pathogenicity. In addition, a putative virulence-associated DNA methyltransferase (MTase) gene, carried on a 22-kb pathogenicity island-like element sited on chromosome I, was also included in the screened genes.
Multilocus sequence typing (MLST) is an important genetic fingerprinting technique for determining the global epidemiology of bacterial pathogens (Jolley, 2001). González-Escalona (González-Escalona et al., 2008) reported the first MLST scheme for V. parahaemolyticus using sequences of internal fragments of seven housekeeping genes from two chromosomes. They revealed by sequence typing that V. parahaemolyticus pandemic strains with an international distribution were sequence type (ST) 3, and that clonal complex three (CC3), consisting of ST3 and its single-locus variants, was in a dominant position. Further studies confirmed that ST3 and CC3 had worldwide distributions (Theethakaew et al., 2013, Yu et al., 2011). MLST thus provides a valid approach for elucidating the evolution of V. parahaemolyticus from different sources.
In this study, we isolated V. parahaemolyticus from freshwater crayfish and determined the diversity of the isolates by MLST analysis. Data on pandemic strains, mainly derived from the MLST database, were collected to analyze the relationships between all isolates and pandemic strains. We also investigated the potential pathogenic characteristics of V. parahaemolyticus isolates from freshwater crayfish by detecting virulence-related genes and evaluating the virulence of V. parahaemolyticus in zebrafish and mouse infection models. This study will provide necessary information for the survey of V. parahaemolyticus and will help us get better insight into V. parahaemolyticus from freshwater crayfish and other aquatic products.
Section snippets
Collection of samples and identification of isolates
Crayfish samples were collected from seven crayfish-culturing areas in Hubei province, China, in 2013 and 2014. After collection, all samples were immediately transported to our laboratory and washed with tap water until it ran clear. The crayfish were sacrificed using MS222 (Syndel) and dissected under sterile conditions to separate the gills, hindgut, stomach, and cephalothorax, respectively. According to National Standards of the People's Republic of China (GB 4789.7-2013), Vibrio
Identification of isolates
The prevalence of V. parahaemolyticus was detected in crayfish at seven areas in Hubei province throughout the study. A total of 96 V. parahaemolyticus isolates were identified. The distribution of V. parahaemolyticus in various crayfish organs was detected. V. parahaemolyticus was frequently found in the gills and hindgut, but was rarely detected in the stomach. However, the tail portion without hindgut was free of V. parahaemolyticus. To examine the persistence of V. parahaemolyticus in
Discussion
Although V. parahaemolyticus clearly poses a threat in terms of food safety and public health, its virulence repertoire remains incompletely understood. In this study, we screened a series of virulence and virulence-related genes and revealed a very low incidence of the classical virulence genes tdh and/or trh, consistent with food-source and environmental isolates (Martinez-Urtaza et al., 2005, DePaola et al., 2003). However, there were high incidences of some other virulence-related genes in
Acknowledgments
This research was supported by the Special Fund for Agro-scientific Research in the Public Interest (2014BBB016), Natural Science Foundation of Hubei Province (2015CKC901), the National Natural Science Foundation of China (31402341, 31471660, 31502080), the Fundamental Research Funds for the Central Universities (52902-0900201537), the China Postdoctoral Science Foundation (2015T80819) and the Open Project Program of Jiangsu Key Laboratory of Zoonosis (R1605).
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These authors contributed equally to this work.