Draft Genome Sequence of Non-Vibrio parahaemolyticusAcute Hepatopancreatic Necrosis Disease Strain KC13.17.5, Isolated from Diseased Shrimp in Vietnam

Hidehiro Kondo,a Phan Thi Van,b Lua T. Dang,b and Ikuo Hironoa

Abstract

GENOME ANNOUNCEMENT

Cultured shrimp have a high economic value. Shrimp production had been steadily increasing until 2011, when the outbreak of a new disease, called early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND), caused severe damage to the industry (1, 2). AHPND causes mortality of as high as 100%, and the causative agent of AHPND is a strain of Vibrio parahaemolyticus. The sequenced genomes of strains from Thailand and Mexico have a plasmid that might contain putative virulence genes (3, 4). Virulent strains can be identified by PCR using primers based on the virulence genes (5).

Because the virulence genes were on a plasmid, virulence might be transferred not only among V. parahaemolyticus strains but also to different bacterial species. In Vietnam, an AHPND strain, KC13.17.5, was isolated and identified to be Vibriosp., but the 16S rRNA gene sequence showed that it was not V. parahaemolyticus. To further characterize this strain, its genome was sequenced.

Bacterial DNA was prepared according to Sambrook and Russell (6). A mate-pair library was generated from DNA using an Illumina Nextera XT DNA sample preparation kit. The library was sequenced using Illumina MiSeq and MiSeq reagent kit version 2 (300 cycles). Sequence data were assembled with CLC Genomics Workbench version 6.5.1, and then the scaffolds were analyzed on the RAST server (7). BLASTn searches of the genome were used to find sequences homologous to known plasmid-like and putative virulence genes (8).

The genome of the strain was assembled into 42 scaffolds. Annotation by the RAST server identified 5,288 genes. By further characterization, the closest neighbor of this strain was predicted to be Vibrio harveyi ATCC BAA-1116. A homology search using the predicted genes also showed the higher similarity to the genes of V. harveyi and Vibrio campbellii than those of V. parahaemolyticus.

We previously found that contig 4 of TUMSAT_D06_S3 (63 kbp) was highly conserved among 3 AHPND strains but was not conserved in 3 non-AHPND strains (3). This sequence is likely a plasmid. In the present study, some of the scaffolds, such as scaffold 21 (29 kbp) and scaffold 27 (16 kbp), were almost identical to contig 4 of TUMSAT_D06_S3. The putative virulence genes were identified and used to diagnose AHPND by PCR (5). Because scaffold 25 (3.8 kbp) was identical to the virulence genes, we speculate that this strain was made virulent by acquiring the plasmid.

Here, we sequenced an AHPND-causing species of Vibrio that was not V. parahaemolyticus. This strain also possesses the putative toxin genes and related plasmid-like sequences. The strain was closest to V. harveyi, meaning that the toxin genes could be transmitted to different Vibrio species. Further research on how the genes are transmitted across species is needed to prevent the spread of AHPND.

ACKNOWLEDGMENTS

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