Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non‐host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non‐ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.     

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

Salinity a significant environmental factor for Vibrio harveyi virulence in Fenneropenaeus indicus

The effect of ambient salinity on the haemolymph variables of Fenneropenaeus indicus and its susceptibility to Vibrio harveyi infection under salinity stress has been studied. Adult shrimps were acclimated to 5‰ (hypo osmotic), 25‰ (iso osmotic) and 35‰ (hyper osmotic) salinity levels and the animals were injected with a mid logarithmic culture of V. harveyi at sub lethal level and haemolymph parameters were analysed. Haemolymph proteins, intracellular superoxide anion production, phenoloxidase (PO), alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were found to be at elevated level both at 5‰ and 35‰ post challenge. The haematological responses showed a progressive increase (P < 0.05) up to post challenge day 5 (PCD 5) followed by a considerable decline at all salinities with the lowest being at 35‰. The alterations in the variables were higher in shrimps held at 5‰. However, the V. harveyi infection was severe in animals held at 35‰. The reduction in the parameters could be correlated with the decrease in survival rate of shrimps at 35‰ with a concurrent increase in V. harveyi at this salinity. Multiple regression analysis revealed that ACP (P < 0.001), haemocyte protein HCP (P < 0.001) and PO (P < 0.05) could explain 91% variability in the shrimp survival. These parameters may be used as effective shrimp health indicators. It is evident from the study that ambient salinity alters the haemolymph variables, modulates the virulence in V. harveyi and makes the shrimps more vulnerable to infection at higher salinity. The virulence of V. harveyi is increased at 35‰ salinity as being evidenced from the high mortality at this salinity. The study emphasizes the importance of salinity as an important environmental factor both in terms of host susceptibility and virulence of the pathogen.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Comparative genomic analysis of different virulence strains reveals reasons for the increased virulence of Aeromonas veronii

Aeromonas veronii is an important zoonotic and aquatic agent. More and more cases have shown that it has caused huge economic losses in the aquaculture industry in addition to threatening human health. But the reasons for the increasing virulence of A. veronii are still unclear. In order to further understand the reasons for the increased virulence of A. veronii, we conducted a comparative analysis of the genomes of A. veronii with different virulence. The analysis revealed that there are multiple virulence factors, such as those related to fimbriae, flagella, toxins, iron ion uptake systems and type II, type III and type VI secretion systems in the virulent strain TH0426 genome. And comparative analysis showed that there were two complete type III secretion systems (API1 and API2), of which the API2 and iron ion transport system were unique to the TH0426 strain. In addition, TH0426 strain also has unique functional gene clusters, which may play important roles in terms of resisting infection, adapting to different environments and genetic evolution. These particular virulence factors and gene clusters may be the important reasons for the increased virulence. These insights will provide a reference for the study of the pathogenesis of A. veronii.     

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes – a novel early defence strategy in larval fish

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A‐gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod‐like formations. The engulfed structures were positive for protein A‐colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread‐like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.     

Virulence‐associated factors in Vibrio cholerae non‐O1/non‐O139 and V. mimicus strains isolated in ornamental fish species

During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non‐O1/non‐O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South‐East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix‐assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization‐associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR⁺, rtxA⁺ and hlyAET⁺ genotype. Interestingly, the V. mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.     

Development of a two‐step,non‐probed multiplex real‐time PCR for surveilling Vibrio anguillarum in seawater

Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. Vibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah1, empA and rpoN of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL^?1 of V. anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.     

Mucus piRNAs profiles of Vibrio harveyi-infected Cynoglossus semilaevis: A hint for fish disease monitoring

The half-smooth tongue sole, Cynoglossus semilaevis, is an important cultured flatfish species. Vibrio harveyi is a common pathogen to this fish, which may result in great economic loss to C. semilaevis culture industry. piRNAs, a non-coding RNAs with 26–32 nt, have been regarded as promising biomarkers for cancer diagnosis and fish diseases. Here, we extracted the RNA from mucus of C. semilaevis and constructed the differential expression profiles of piRNAs between the sick fish (MS) and healthy fish (MC). We identified 45,696 differentially expressed piRNAs including 22,735 up-regulated piRNAs and 22,961 down-regulated piRNAs in MS group compared with MC group. The GO enrichment and KEGG pathway enrichment analyses of the differential piRNAs were carried out. The result showed immunity-related target genes mainly involved in immune system process, response to stimulus, cell killing, immune system, infectious diseases and cell growth and death. The 10 most differentially expressed piRNAs were chosen to perform the qRT-PCR, while only seven piRNAs were consistent with the sequence result. Compared with MC group, the expression levels of piR-mmu-72173>piR-rno-62831>piR-xtr-704880, piR-dme-15546979, piR-mmu-49941660, piR-mmu-29283297 and piR-mmu-1758399 were significantly lower, and piR-gga-10574 and piR-gga-134812 were significantly higher in MS group. These piRNAs may be potential biomarkers during the V. harveyi infection of C. semilaevis. This study could provide a new method to identify the infection status of C. semilaevis and understand better about the innate and adaptive immune system in C. semilaevis during bacterial infection.     

Identification and characterization of pathogens associated with fin erubescence and ulceration of cultured Scophthalmus maximus

Scophthalmas maximus is a precious and main aquacultural species in northern China with an annual output value of more than 1.5 billion yuan. However, with the expansion of breeding range, increased stocking density and variety degradation, a variety of bacterial, parasitic and viral diseases have often occurred and worsened. Among them, bacterial disease is the most serious in farmed S. maximus. Since 2010, an epidemic bacterial disease with primary symptoms of lighter colour of lateral and caudal fins, spotty bleeding, fin base bleeding, abdominal swelling and redness and lesions of internal organs has put broken in S. maximus cultured in Weihai, Longkou and Haiyang, Shandong Province, China. The disease occurred mostly at the seedling and early development stages and propagated very fast, resulting in high mortality. These S. maximus died with obvious surface ulceration in only 3–4 days, causing great economic losses to the aquaculture industry. Beginning in 2010, we conducted epidemiological investigation in farmed S. maximus and isolated two different pathogens from the body of the diseased S. maximus from different farms. In this paper, we observed their morphology, conducted phylogenetic analysis based on their physiological and conventional biochemical characteristics and 16S rRNA gene sequences, preliminarily analysed their pathogenesis and investigated the histopathological changes of the diseased S. maximus. We believed that Vibrio anguillarum and V. parahaemolyticus were the pathogenic bacteria for the epidemic disease in S. maximus.