Elsevier

Aquaculture

Volume 503, 30 March 2019, Pages 322-329
Aquaculture

Protective effectiveness of feeding phage cocktails in controlling Vibrio parahaemolyticus infection of sea cucumber Apostichopus japonicus

https://doi.org/10.1016/j.aquaculture.2019.01.006Get rights and content

Highlights

  • Two bacteriophages lytic to Vibrio parahaemolyticus were isolated.

  • Skim milk was considered to be a better lyophilized powder protectant during phage feeding experiment.

  • This study monitored the efficiency of feeding phages against Vibrio parahaemolyticus infection.

Abstract

Vibrio parahaemolyticus is a common zoonotic bacterium that causes infections and disease in both sea cucumbers (Apostichopus japonicus) and humans, potentially greatly impacting both aquaculture and human health. In this study, two phages with the ability to control V. parahaemolyticus were isolated and designated as vB_VpaP_VP-ABTNL-1 (PVP1) and vB_VpaS_VP-ABTNL-2 (PVP2). Morphological analyses showed that PVP1 belonged to C3 phage group Podoviridae while PVP2 belonged to the family Siphoviridae. In vitro phage-treated cultures showed significant (P < 0.05) inhibition of growth for V. parahaemolyticus VP-ABTNL when compared to untreated groups. Subsequently, we determined that skim milk might be the best protective agent, as the dissolution time and moisture content were both significantly (P < 0.05) higher than other agents tested. We also explored the utility of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumbers against V. parahaemolyticus infection. The antibiotic group had a significant(P < 0.05)difference with MOI = 1, but not with the two phage treatments (MOI = 10, MOI = 100), indicating an increase of phage protection with MOI = 10 and MOI = 100 against V. parahaemolyticus VP-ABTNL. There were no significant differences in weight gain, wall-to-wall ratio, feeding rate or food conversion efficiency among sea cucumbers fed the two phage treatments compared with those fed the non-supplemented diet (P > 0.05). Hence, feeding phage cocktails might be another optimal therapeutic agents to treat V. parahaemolyticus infections in sea cucumber aquaculture.

Introduction

In recent years, sea cucumbers (Apostichopus japonicus) have been recognized as not only having important economic benefits, but that of overall nutritional value (Gu et al., 2010). Artificial rearing of sea cucumbers has become one of the pillar industries of China's aquaculture industry since 2004 (Gu et al., 2010; Liu et al., 2010). In 2016, the total sea cucumber production exceeded nine billion dollars. However, the increased market demand has necessitated scaling up of breeding facilities, yet the lag of breeding technology has led to various diseases and high mortality of sea cucumbers, causing huge losses to aquaculture facilities.

Generally, Vibrio parahaemolyticus is recognized as the leading causal agent of human acute gastroenteritis caused by consumption of raw or undercooked seafood. In China, especially coastal areas, food poisoning cases caused by V. parahaemolyticus have become the highest of all microbial food poisonings (Lan et al., 2009; Shen et al., 2011), and since 2012, V. parahaemolyticus is the leading pathogen causing diarrhea in Liaoning Province, China. At the same time, as a zoonotic pathogen widely disseminated in estuarine, marine and coastal environments, V. parahaemolyticus was also determined to be the cause of vibriosis in A. japonicus aquaculture, especially in juveniles with reduced adhesion, body contraction and collapses (Wang et al., 2012).

In order to control infections in juvenile sea cucumbers and guarantee the seafood safety, large amounts of sanitizers and antibiotics are frequently used throughout the rearing lines, increasing pollution and antibiotic residues in aquatic water, thus allowing the antibiotic resistant bacteria to develop (Oliveira et al., 2012; Wong et al., 2015). Bacterial diseases affecting sea cucumbers are usually caused by vibriosis, such as V. splendidus, V. alginolyticus, V. cyclitrophicus, and V. parahaemolyticus (Kalatzis et al., 2018). Our previous studies have focused on phage therapies to control V. alginolyticus, V. cyclitrophicus and V. splendidus infections in A. japonicas (Zhang et al., 2015; Li et al., 2016a; Li et al., 2016b). Aimed to ensure the comprehensiveness of phages in preventing and controlling bacterial infections in sea cucumbers, there is an urgent need to develop alternative approaches to defend against V. parahaemolyticus infections in sea cucumber aquaculture.

Bacteriophages are obligatory parasites infecting bacteria (Duckworth, 1976). As natural antimicrobials, bacteriophages provide favorable features in that they have no effect on commensal microflora and no relation to antibiotic resistance (Barrow and Soothill, 1997; Smith and Huggins, 1982). Phage therapy is both an old and an attractive new strategy to treat and control bacterial infections and are potentially viable to any impacted field, including aquaculture (Endersen et al., 2014). Prior works have documented the effectiveness of bacteriophages in controlling several types of Vibrio infections across fish, shrimp, and shellfish (Crothers-Stomps et al., 2010; Letchumanan et al., 2016; Doss et al., 2017; Plaza et al., 2018). This study aims to evaluate the lytic abilities of novel isolated phages in vitro and the potential protective effectiveness of phage cocktail feedstuff on treating V. parahaemolyticus infections in sea cucumbers.

Section snippets

Bacterial strain and animals

The bacterial strain used in the current study was V. parahaemolyticus VP-ABTNL, first isolated from diseased sea cucumbers previously procured from a hatchery in Dalian Bay (Dalian, China). The bacterium was isolated via thiosulfate citrate bile salt-sucrose agar medium (TCBS agar, Difco, USA). After subsequent culture in 2216E liquid medium (5 g peptone and 2 g yeast extract per liter), at 26 °C for 12 h, the VP-ABTNL strain was then stored as a bacterial stock suspension at −80 °C. Genetic

Pathogenicity test of the isolate Vibrio parahaemolyticus

The host bacterial strain was identified by sequencing 16S rDNA and the obtained sequence of 1462 bp was submitted to NCBI under accession number of MG589511, showing 99% identity to V. parahaemolyticus strain CM12 (EU660326) and 99% identity to V. parahaemolyticus strain V7 (EU636231).

The LD50s of VP-ABTNL were 1.57 × 105 CFU/mL and 7.60 × 104 CFU/g in coelomic injections (Fig. 1A) and immersion challenges (Fig. 1B), respectively, indicating the high virulence of VP-ABTNL to sea cucumbers.

Discussion

Aquaculture, including the sea cucumber industry, has traditionally faced many problems of water pollution, destruction of water micro-ecological balances, and food safety caused by antibiotic residues. This study is the first time to observe the effects of using phage cocktails as a promising alternative method to control V. parahaemolyticus infections in sea cucumbers. Since 2006, semi-intensive culturing methods have made tremendous contributions to the development of the sea cucumber

Conclusions

This study was the first to evaluate the protective effectiveness of feeding phages for sea cucumbers against Vibrio parahaemolyticus. The overall results of this experiment indicate that phage feeding treatment increased the survival of sea cucumbers infected with V. parahaemolyticus VP-ABTNL and there is no effect on the normal growth of sea cucumbers. Skim milk might become an encouraging agent for phage lyophilization before mixing with sea cucumber feedstuff. As of now, our team has

Acknowledgments

This work was supported by the National Public Science and Technology Research Funds Projects of Ocean (Grant No. 201405003), and we appreciate the partial bacteria support from Dalian Ocean University (Dalian, China).

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