Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality

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Abstract

A mass mortality of clam, Meretrix meretrix, occurred in Jiangsu Province of China in the late September of 2007. Of the isolates obtained from the diseased clams, MM21 had the strongest virulence to the clam in the virulence test, with a LD50 value of ∼6 × 106 CFU ml−1. MM21 was identified as Vibrio parahaemolyticus by the VITEK 2 Compact system and 16S rDNA sequencing. Detection of virulence-associated genes by PCR indicated that MM21 was positive for toxR and tlh, and negative for tdh. Compared with control group, histiocytes from MM21-infected clams displayed a variety of cytopathological changes by transmission electron microscopy examination, which included increased lipid droplets in hepatocytes, deposition of granules in the mantle, excessive secretion in the gill. The results of our study suggested that MM21 may have been an etiological element in the mass mortalities of hard clam (M. meretrix) in Jiangsu Province of China in 2007.

Introduction

The clam Meretrix meretrix is an important commercial species in the coastal areas of South and Southeast Asia, including China, Korea, Japan, and India (Liu et al., 2006). In recent years, disease outbreaks have occurred and caused heavy economic losses. Mortality outbreaks of M. meretrix along the coast of Jiangsu Province in China often happen during August and September. The temperature of shallow water in Jiangsu Province of China when the mortality happened ranged from 24 °C to 27 °C, which are the optimum temperatures for many Vibrio species (Farmer, 2006).

Vibrio is a major threat to aquaculture of marine animals (Cheng et al., 2004). Many bacterial diseases in adult bivalves are provoked by Gram-negative bacteria, mostly the genus Vibrio (Paillard et al., 2004). Elston et al. (1982) described Vibrio infections associated with growth depression and mortality in cultured oysters and clams, including oyster Crassostrea virginica, Ostrea edulis, and clam Mercenaria mercenaria. In the past decades, many shellfish, such as scallop Argopecten purpuratus (Riquelme et al., 1995), Pecten maximus (Lambert et al., 1998), clam Ruditapes philippinarum (Allam et al., 2000), R. decussates (Gómez-León et al., 2005) and oyster C. gigas (Lacoste et al., 2001, Waechter et al., 2002, Le Roux et al., 2002) have been reported to experience disease associated with Vibrio species.

A heavy mortality of clam, M. meretrix, occurred in Jiangsu Province of China in the late September of 2007. This disease with a sharp increase of mortality was similar to the disease induced by Vibrio in summer (Lacoste et al., 2001, Travers et al., 2008). The aim of this study was to isolate the pathogen from the diseased clams, characterize its pathogenic effect, including investigating the histopathological changes of the host infected by the pathogen, and understanding its pathogenesis and pathogenicity further.

Section snippets

Bacterial isolation and preliminary virulence screening

The clams were collected from Rudong of Jiangsu Province, China, in the late September, 2007 when heavy mortality occurred. Symptoms of the diseased clams were tissue color variation (darker and lackluster), tissue edema and increased mucus.

Fifteen diseased clams in total were used to isolate bacteria. They were divided into three groups, each of which contained five clams. Hepatopancreas of every five clams were removed under aseptic conditions and homogenized in PBS. The homogenates were

Bacterial isolation and preliminary virulence screening

Twenty-one isolates were isolated on TSAYE agar plates. All these isolates were used in the preliminary virulence screening experiments to determine their virulence to M. meretrix.

At 6 days post-immersion challenge, MM21 induced about 50% mortality which was the highest, followed by MM17 with 40% mortalities. MM11 and MM5 induced about 30% mortalities. The mortalities induced by other isolates were lower than 30%, mostly lower than 10% (such as MM2). At 96 h post-injection challenge, MM21 was

Discussion

A heavy mortality of clam, M. meretrix, occurred in Jiangsu Province of China in the late September of 2007. Twenty-one isolates were obtained from diseased clams. In order to find out the potential pathogen related to this mortality, these isolates were applied to virulence test. Laboratory experiments results indicated that MM21 was the most virulent one of the total isolates. The LD50 value of MM21 at 24 h post-infection was ∼6 × 106 CFU ml−1. 16S rDNA sequence analysis and biochemical analysis

Acknowledgments

We would like to thank Prof. Li Sun for her help in language correction and kind technical assistance. This work was financially supported by the Chinese National High-Tech R&D Program (2006AA10A410), the National Science and Technology Pillar Program (2006BAD09A02).

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