Isolation and Molecular Identification of Vibrio natriegens from Diseased Portunus trituberculatus in China

Abstract

In July 2010, heavy mortalities were seen in cultured swimming crabs, Portunus trituberculatus, from Jiangsu Province in China. Two isolates, XA1 and XA2, were isolated from cardiac muscle and somatic muscle of the moribund crabs on ZoBell's marine 2216E agar and were confirmed as the causative agent for the mass mortalities seen on that farm. The morphological and physiological characteristics as well as the biochemical tests of the two isolates were identical and were similar to Vibrio natriegens. Both were gram-negative, oxidase positive, produced acid when fermenting glucose, and failed to grow in the media containing 1% NaCl. Furthermore, the sequencing of the 16S rRNA, gapA, ftsZ, mreB, and topA genes revealed that the two isolates were identical and had a 0.99 homology coefficient with V. natriegens. The Neighbor-Joining tree based on the concatenated sequences of four housekeeping genes (gapA, ftsZ, mreB, and topA) strongly supported that XA1 was most closely related to V. natriegens within the Vibrio harveyi-related clade. In addition, pathogenicity testing using immersion challenge confirmed that strain XA1 was virulent to juvenile crabs, and the LD50 value was 1.09 × 10 6 . This is the first report on V. natriegens as a virulent pathogen for Portunus trituberculatus.

16S rRNA sequences of Exiguobacterium spp. bacteria dominant in a biofloc pond cultured with whiteleg shrimp, Penaeus vannamei

“…is found distributed in salt march mud, estuarine habitats and also widespread in the ocean, andd has a faster growth rate compared with any known non‐pathogenic bacteria (Payne, 1958; Schleicher et al, 2018). However, there is a report on infection with this bacterium of a marine crustacean by Bi et al (2016), who found that V. natriegens strains can produce mortality of ≤85% in the cultured swimming crab, Portunus trituberculatus . Villaseñor et al (2015) identified V. natriegens in biofloc sample cultures, with the capacity to use different carbon and nitrogen sources with high efficiency, making it able to defeat pathogenic bacteria in the carbon and nutrient biofloc environment.…”

Rapid and Sensitive Detection of Vibrio natriegens in Portunus trituberculatus with the Loop-Mediated Isothermal Amplification Test

“…However, it has been verified as a local, highly prevalent, pathogen and has been isolated from aquatic animals and the environment. The pathogenicity of V. natriegens to aquatic animals seems to have been underestimated (Bi et al, 2016;Deng et al, 2004;Li et al, 2009).…”

“…A forward inner primer (FIP), a reverse inner primer (BIP), and two outer primers (F3 and B3) were chosen and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Information relating to the primers is shown in Table 1 and Figure 1. In addition, 16S rDNA primer (27f/1942r) (Bi et al, 2016) and dulplex PCR primer (data not shown) were used to ascertain the specificity and sensitivity of LAMP assays. Optimization for LAMP.…”

“…It comprises a large proportion of crab aquaculture in China, with the total yield exceeding 100,000 tons in 2011 (Lv et al, 2014). However, with the rapid development of intensive crab culture, the sustainability of the crab aquaculture industry has been threatened by outbreaks of diseases, particularly those caused by bacteria in the genus Vibrio (Liu et al, 2007;Yan et al, 2010;Wan et al, 2011;Zhang et al, 2014;Bi et al, 2016).…”

“…natriegens is a Gram-negative, assumed to be non-pathogenic halophile marine bacterium (Payne et al, 1961). However, recent studies have showed that V. natriegens is pathogenic to aquatic animals and mortality rate was greater than 90% (Bi et al, 2016;Deng et al, 2004;Li et al, 2009). Disease has been difficult to control because V. natriegens has the fastest growth rate of any known organism, with a reported doubling time of <10 min (Weinstock et al, 2016).…”