Veronii Infection Research Articles

Identification of Aeromonas veronii isolated from largemouth bass Micropterus salmoides and histopathological analysis

Aeromonas veronii is abundant in aquatic environment and could cause diseases in various kinds of aquatic animals. However, few cases of A. veronii infection were reported in largemouth bass (Micropterus salmoides), which has been widely farmed and become an important economic fish species in China in recent years. In this study, a strain of Gram-negative bacteria was isolated from the diseased largemouth bass, and was tentatively named strain HN1903. According to the results of biochemical characteristics, 16S rRNA sequencing analyses, and artificial infection experiment, the isolate was identified as A. veronii. The virulence of strain HN1903 was tested by challenge experiments, and the value of LD50 with intraperitoneal injection was 3.72 × 104 CFU/g fish weight. Eight virulent genes, including aerA, gcaT, act, fla, lip, ser, hlyA, and ahyB were screened by PCR assay. Antibiotic resistance results showed that the isolate was susceptible to gentamicin and cefotaxime, and moderately sensitive to florfenicol and streptomycin based on the 12 antimicrobial agents tested. Histopathology examination indicated the necrosis in liver, kidney, and gill, inflammatory cellular infiltration in kidney, and hemosiderin granules increase in spleen. In this study, A. veronii was isolated, identified, and its pathogenicity was clarified. The findings in this study could provide the strategy of fish disease control, preventing the outbreak of A. veronii infection.

Transcriptome analysis of the spleen provides insight into the immunoregulation of Cyprinus carpio koi under Aeromonas veronii infection

Koi carp (Cyprinus carpio koi) is an important cultured freshwater ornamental fish. It has a rich color and high economic value. However, owing to epidemic infectious diseases in recent years, its cultivation has been restricted. Information about the interaction between koi carp and Aeromonas veronii is limited. To better understand the immune response of koi carp to A. veronii infection, we performed transcriptome sequencing of the spleen of koi carp after artificial A. veronii infection. Consequently, a total of 72,694 unigenes with a N50 length of 2011 bp were obtained. Differential expression analysis showed that there were 373 significantly differentially expressed genes (DEGs) 24 h after infection, of which 240 genes were upregulated and 133 genes were downregulated. The expression levels of NLRP3, CD11b, CD11c, ASC, and IL-1β, which are closely related to inflammasomes, were significantly upregulated. Further annotation and enrichment analysis indicated that DEGs were principally enriched in the phagosome, cell adhesion molecules, IL-17 signaling pathway, and antigen processing and presentation. The above mentioned molecules and signaling pathways may play an important role in the elimination of foreign, invading pathogens. To verify the accuracy of RNA-seq data, 10 genes related to immune function were selected for RT-qPCR experiments, and the results showed that their expression is highly consistent with the RNA-seq data. Additionally, the expression patterns of five differentially expressed inflammasome-related genes were also verified by RT-qPCR. A splenic transcriptome of koi carp between the control group and the A. veronii-immunized group was obtained, which provided new clues to the molecular mechanism of host–A. veronii interactions and provided a basis for further study on the gene expression and functional genomics of koi carp.

Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii.

Aeromonas veronii is one of the main pathogens causing various diseases in humans and animals. It is currently difficult to eradicate drug-resistant A. veronii due to the biofilm formation by conventional antibiotic treatments. In this study, a marine peptide-N6NH2 and its analogs were generated by introducing Orn or replacing with D-amino acids, Val and Pro; their enzymic stability and antibacterial/antibiofilm ability against multi-drug resistant (MDR) A. veronii ACCC61732 were detected in vitro and in vivo, respectively. The results showed that DN6NH2 more rapidly killed A. veronii ACCC61732 and had higher stability in trypsin, simulated gastric/intestinal fluid, proteinase K, and mouse serum than the parent peptide-N6NH2. DN6NH2 and other analogs significantly improved the ability of N6NH2 to penetrate the outer membrane of A. veronii ACCC61732. DN6NH2, N6PNH2 and V112N6NH2 protected mice from catheter-associated biofilm infection with MDR A. veronii ACCC61732, superior to N6NH2 and CIP. DN6NH2 had more potent efficacy at a dose of 5 μmol/kg (100% survival) in a mouse peritonitis model than other analogs (50–66.67%) and CIP (83.33%), and it inhibited the bacterial translocation, downregulated pro-inflammatory cytokines, upregulated the anti-inflammatory cytokine, and ameliorated multiple-organ injuries (including the liver, spleen, lung, and kidney). These data suggest that the analogs of N6NH2 may be a candidate for novel antimicrobial and antibiofilm agents against MDR A. veronii infections.

Transcriptome analysis of the Macrobrachium nipponense hepatopancreas provides insights into immunoregulation under Aeromonas veronii infection

The oriental river prawn Macrobrachium nipponense is a commercially important freshwater shrimp that is widely farmed in China. Aeromonas veronii is a conditional pathogen of farmed shrimp, which has caused huge economic losses to the industry. Therefore, there is urgency to study the host-pathogen interactions between M. nipponense and A. veronii to screen individuals with antimicrobial resistance. In this study, we examined the hepatopancreas of moribund M. nipponense infected with A. veronii and healthy individuals at both the histopathological and transcriptomic levels. We showed that A. veronii infection resulted in tubular necrosis of the M. nipponense hepatopancreas. Such changes likely affect assimilation, storage, and excretion by the hepatopancreas, which could ultimately affect the survival and growth of infected individuals. Among the 61,345 unigenes obtained through RNA sequencing and de novo transcriptome assembly, 232 were differentially expressed between the two groups. KEGG and GO analyses revealed that these differentially expressed genes were implicated in pathways, including PPAR, PI3K/AKT, and AMPK signaling. The results of this study will contribute to an analysis of the immune response of M. nipponense to A. veronii infection at the transcriptomic level. Furthermore, the RNA-seq data generated here provide an important genomic resource for research on M. nipponense in the absence of a reference genome.

Aeromonas veronii associated with red gill disease and its induced immune response in Macrobrachium nipponense

Aeromonas veronii is a freshwater bacterium associated with many diseases in aquatic animals. In this study, the pathogenic of A. veronii was isolated from diseased Macrobrachium nipponense showing signs of weakness, poor appetite and red gills in some farms in Jiangsu province, China. The pathogenicity of A. veronii to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. veronii NJ1 was pathogenic to M. nipponense, and the LD50 was 4.5 × 106 CFU/ml at 7 d post infection. Detection virulence factors of the A. veronii NJ1 showed that A. veronii NJ1 produced caseinase, lipase, amylase, lecithinase, gelatinase and haemolysin. Detection of virulence-related genes showed that A. veronii NJ1 was positive for aerolysin (aer), outer membrane protein (ompA), adhesin (aha), flagellin (flaA, flgH, flgM, flgN), lipoidase (lip), cytotonic enterotoxins (act, alt, ast), serine protease (ser), DNases (exu), haemolysin (hlyA), type IV pilus (tapA) and fimbrillin (fim). Antibiotic sensitivity testing indicated A. veronii NJ1 was susceptible to quinolones, but resistant to penicillins. Additionally, the immune-related gene expression in M. nipponense was evaluated, and the results showed that anti-lipopolysaccharide factor (ALF1, ALF2, ALF3), Cusrin1, Cusrin2, Cusrin3, Haemocyanin, i-lysozyme and Prophenoloxidase were significantly upregulated from 6 to 24 hr after A. veronii infection. The results of our study suggested that A. veronii was an aetiological element in the mass mortalities of M. nipponense and this study provided preliminary insights into the diversity in the immune response of M. nipponense to the A. veronii infection.

Nano spirulina dietary supplementation augments growth, antioxidative and immunological reactions, digestion, and protection of Nile tilapia, Oreochromis niloticus, against Aeromonas veronii and some physical stressors.

The current study evaluated the effects of nano delivery of Spirulina platensis on growth performance, digestive enzymes, and biochemical, immunological, and antioxidative status, as well as resistance to Aeromonas veronii and some physical stressor challenges in Nile tilapia, Oreochromis niloticus. Three experimental fish groups (n = 270) with mean weights of 26 ± 0.30 g and mean lengths of 10 ± 0.5 cm were used; the first additive-free basal diet served as the control group, whereas the following two groups were supplemented with spirulina nanoparticles (SPNP) at 0 (control), 0.25, and 0.5%/kg diet for 4 weeks. Following the feeding trial, fish were challenged with hypoxia, cold stresses, and pathogenic bacteria (A. veronii) infection (9 × 108 CFU/ml). SPNP supplementation, especially 0.5%, (p < 0.05) significantly increased growth performance (specific growth rate % day-1, feed conversion ratio, and length gain rate %), immunological (plasma lysozyme and liver nitrous oxide) antioxidants (superoxide dismutase, catalase, and glutathione peroxidase in liver), biochemical (aspartate aminotransferase, alanine transaminase, glucose, and cortisol concentrations in plasma) assays, and digestive enzymes (lipase and amylase in plasma). The expression of liver's heat shock protein 70 (HSP70) and interleukin 1, beta (IL-1β) genes showed a significant upregulation outline of 0.5% SPNP > 0.25% SPNP > 0% SPNP compared with the control. Protection in the incorporated fish groups exposed to A. veronii was 100% compared with the control group, which showed 50% cumulative mortalities. In conclusion, dietary SPNP supplementation improved growth performance, antioxidant activity, immune response, digestive enzymes, related gene expression, and resistance of Nile Tilapia to hypoxia, cold, and A. veronii infection. Thus, SPNP could be used as a natural therapy for controlling those stressors.

Comparative analysis of microbial community structure between healthy and Aeromonas veronii-infected Yangtze finless porpoise

BackgroundThe gut microbiota is a complex ecosystem, which is essential for the metabolism, health and immunity of host. Many diseases have been shown to be closely related to the alteration of intestinal flora. Aeromonas veronii as a conditioned pathogen can cause disease in Yangtze finless porpoise through intestinal infections. However, it is not clear whether the disease caused by Aeromonas veronii is related to changes of intestinal flora. In the current study, the diversity and composition of gut microbiota in the healthy and Aeromonas veronii-infected Yangtze finless porpoise were evaluated by high-throughput sequencing to further investigate the potential association between intestinal flora alteration and pathogen invasion.ResultsA total of 127,3276 high-quality sequences were achieved and 2465 operational taxonomic units (OTUs) were in common among all samples. The results of alpha diversity showed that there was no obvious difference in richness and diversity between healthy and Aeromonas veronii-infected Yangtze finless porpoise. Firmicutes, Bacteroidetes and Proteobacteria were the most dominant phyla in all samples. In addition, the healthy Yangtze finless porpoise exhibited higher abundance of Firmicutes and Fusobacteria than Aeromonas veronii-infected Yangtze finless porpoise, while, the level of Proteobacteria was decreased. At the genus level, Paeniclostridium and Paraclostridium were the predominant bacteria genera in the CK (healthy Yangtze finless porpoise) group. In the DIS (Aeromonas veronii-infected Yangtze finless porpoise) group, Lactobacillus and unidentified_Enterobacteriaceae were the dominant bacteria genera and the proportion of Paeniclostridium, Paraclostridium, Terrisporobacter, Cetobacterium, Candidatus Arthromitus, Terrabacter and Dechloromonas were reduced.ConclusionsIn conclusion, our results showed that Aeromonas veronii infection can alter the gut microbiota of the Yangtze finless porpoise by affecting the number of harmful bacteria and beneficial bacteria.

An effective live attenuated vaccine against Aeromonas veronii infection in the loach (Misgurnus anguillicaudatus)

Aeromonas veronii is a major pathogenic bacterium in humans and animals. When it causes outbreaks, there are enormous economic losses to the aquaculture industry. An effective live attenuated vaccine strain, ΔhisJ, was obtained in our previous studies by gene knockout in Aeromonas veronii TH0426 using the suicide vector pRE112. Here, we evaluated whether the live attenuated vaccine ΔhisJ was suitable for prevention of Aeromonas veronii infection by injection and immersion in loaches. Compared with that of the TH0426 wild-type strain, the virulence of the live vaccine was significantly weakened. Vaccine safety assessment results also indicated that 1 × 107 CFU/mL live vaccine was safe and did not induce clinical symptoms or obvious pathological changes. Additionally, after challenging loaches with Aeromonas veronii TH0426, the relative percent survival of the IN3 injection group was 65.66%, and that of the IM group was 50.78%. Our data show that the live attenuated vaccine ΔhisJ can improve the immune protection rate of loaches. Furthermore, increased enzyme activity parameters (SOD, LZM, ACP, and AKP) in the skin mucus, increased enzyme activity parameters (SOD, LZM, ACP, AKP, and GPx) in the serum, increased specific IgM antibodies and cytokine IL-1β contents in the serum, and increased cytokine (IL-15, pIgR, IL-1β, and TNF-α) expression in the liver and spleen were observed. These data are the first to indicate that the live attenuated vaccine ΔhisJ is suitable for the development of a safe and effective vaccine against Aeromonas veronii infection in loach aquaculture.

Effects of dietary Lactobacillus plantarum C20015 on growth, immunity, and disease resistance in koi carp

Koi carp (Cyprinus carpio) are well-known and highly valuable fish in the international ornamental fish community that have been plagued by Aeromonas veronii infections in recent years. Some A. veronii infections are serious enough to cause death. Lactobacillus plantarum, a probiotic, is one of the most environmentally friendly and safe alternatives to antibiotics for use as a feed additive for aquatic animals such as fish. The present study investigated the effects of L. plantarum C20015 on growth performance, immune responses, and eradication of A. veronii infection in koi carp. During a 6-week feeding experiment in an indoor aquarium and a 14-day A. veronii artificial infection experiment, a control diet group was fed with untreated feed twice a day, while experimental groups were given different concentrations of L. plantarum in their feed. The results indicated that the specific growth rate and percent weight gain of fish were significantly higher in the experimental groups than in the control diet group (P < 0.05). The activity of superoxide dismutase (SOD), peroxidase (POD), and lysozyme (LZM) and the levels of IgM antibody were significantly higher in the L. plantarum–supplemented groups than in the control diet group, and the enzymatic activity in the 108 CFU/g C20015 group was higher than that in the 107 CFU/g C20015 and 109 CFU/g C20015 groups (P < 0.01). In contrast, alkaline phosphatase (AKP) activity in the experimental groups was the highest at 42 days. All fish in the control diet group died 7 days after A. veronii infection. After 14 days, the relative percent survival (%, RPS) of the fish in the L. plantarum–supplemented groups was significantly higher than that of the fish in the control group, and the 108 CFU/g C20015 group had the highest RPS. Therefore, we first reported that fish fed with 108 CFU/g of L. plantarum C20015 could promote food intake and growth for koi carp.

Aeromonas veronii Infection in Commercial Freshwater Fish: A Potential Threat to Public Health.

Simple SummaryAeromonas veronii is an important aquatic zoonotic agent. In this study, A. veronii was isolated from healthy fish, and the relationship between the pathogenicity and virulence genes of A. veronii was investigated by molecular identification. The aim of this study was to ensure the safety of freshwater products by evaluating the infection status in edible freshwater fish. Aeromonas veronii is an important pathogen causing freshwater fish sepsis and ulcer syndrome. An increasing number of cases have demonstrated its significance as an aquatic zoonotic agent. The purpose of this study was to ensure the safety of freshwater products by evaluating the infection status of edible freshwater fish. In this experiment, we isolated A. veronii from several species of apparently healthy freshwater fish, including Carassius auratus, Cyprinus carpio, Ctenopharyngodon idella, and Silurus asotus. A. veronii was identified through bacterial staining, culture characteristics, and 16S rDNA gene sequence. In addition, polymerase chain reaction (PCR) was used to investigate the distribution of seven major virulence genes, including aerolysin (aer: 88.51%), cytotoxic enterotoxin (act: 71.26%), serine proteinase (ser: 54.02%), adhesin (Aha: 40.23%), phospholipase (lip: 45.98%), nuclease (exu: 51.72%), and quorum sensing-controlled virulence factor (LuxS: 59.77%). In total, 496 strains of Aeromonas were isolated, including 87 strains of A. veronii. The isolates of A. veronii were Gram-negative, rod-shaped bacteria, and the colonies are yellow on Rimler-Shotts (RS) medium and showed greater than 99% homology with A. veronii ATCC35624 according to analyses of the 16S rDNA sequence. Nearly 50% of the A. veronii isolates carried at least four or more virulence genes, 25% of the isolates carried at least five types of virulence genes, and 59.77% isolates carried the LuxS gene, and the isolates carrying more virulence genes were found to be more virulent. These results are of great significance for further improving the food safety assessment of freshwater aquatic products.

First report of tetracycline-resistant Aeromonas veronii infection in Amur catfish (Silurus asotus) cultured in Korea

First report of tetracycline-resistant Aeromonas veronii infection in Amur catfish (Silurus asotus) cultured in Korea

Oral Administration of Lactobacillus Casei Expressing Flagellin A Protein Confers Effective Protection against Aeromonas Veronii in Common Carp, Cyprinus Carpio.

Aeromonas veronii is a pathogen capable of infecting humans, livestock and aquatic animals, resulting in serious economic losses. In this study, two recombinant Lactobacillus casei expressing flagellin A (FlaA) of A. veronii, Lc-pPG-1-FlaA (surface-displayed) and Lc-pPG-2-FlaA (secretory) were constructed. The immune responses in fish administered with recombinant L. casei were evaluated. The two recombinant L. casei were orally administered to common carp, which stimulated high serum IgM and induced higher ACP, AKP, SOD and LYZ activity. Using qRT-PCR, the expression of IL-10, IL-8, IL-1β, TNF-α and IFN-γ in the tissue of fish immunized with recombinant L. casei was significantly (p < 0.05) upregulated, which indicated that recombinant L. casei could activate the innate immune system to trigger the cell immune response and inflammatory response. Furthermore, recombinant L. casei was able to survive the intestinal environment and colonize in intestine mucosal. The study showed that after being challenged by A. veronii, fish administered with Lc-pPG-1-FlaA (70%) and Lc-pPG-2-FlaA (50%) had higher survival rates compared to Lc-pPG and PBS, indicating that recombinant L. casei might prevent A. veronii infection by activating the immune system to trigger immune responses. We demonstrated that flagellin as an antigen of vaccine, is acceptable for preventing A. veronii infection in fish. The recombinant L. casei expressing FlaA may be a novel mucosal vaccine for treating and controlling A. veronii.

Effect of Bacillus velezensis on Aeromonas veronii-Induced Intestinal Mucosal Barrier Function Damage and Inflammation in Crucian Carp (Carassius auratus).

Aeromonas veronii is an emerging aquatic pathogen causing hemorrhagic septicemia in humans and animals. Probiotic is an effective strategy for controlling enteric infections through reducing intestinal colonization by pathogens. Here we report that the consumption of Bacillus velezensis regulated the intestinal innate immune response and decreased the degree of intestinal inflammation damage caused by the A. veronii in Crucian carp. In this study, we isolated four strains of B. velezensis, named C-11, S-22, L-17 and S-14 from apparently healthy Crucian carp, which exerted a broad-spectrum antimicrobial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens. B. velezensis isolates showed typical Bacillus characteristics by endospore staining, physiological and biochemical test, enzyme activity analysis (amylase, protease, and lipase), and molecular identification. Here, Bacillus-containing dietary was orally administrated to Crucian carp for 8 weeks before A. veronii challenge. Immunological parameters and the expression of immune-related genes were measured at 2, 4, 6, 8, and 10 weeks post-administration. The results showed that B. velezensis was found to promote the increase in the phagocytic activities of peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs), as well as the increase in interleukin 1β (IL-1β), IL-10 and tumor necrosis factor α (TNF-α) concentration of serum. Lysozyme levels (113.76 U/mL), ACP activity (25.32 U/mL), AKP activity (130.08 U/mL), and SOD activity (240.63 U/mL) were maximum (P < 0.05) in the B. velezensis C-11 treated group at 8 week. Our results showed that Crucian carp fed with the diet containing B. velezensis C-11 and S-22 developed a strong immune response with significantly higher (P < 0.05) levels of IgM in samples of serum, mucus of skin and intestine compared to B. velezensis L-17 and S-14 groups. Moreover, B. velezensis spores appeared to show no toxicity and damage in fish, which could inhabit the gut of Crucian carp. B. velezensis restrained up-regulation of pro-inflammation cytokines (IL-1β, IFN-γ, and TNF-α) mRNA levels in the intestine and head kidney at final stage of administration, and the expression of IL-10 was increased throughout the 10-week trial. A. veronii infection increased the population of inflammatory cells in the intestinal villi in the controls. In contrast, numerous goblet cells and few inflammatory cells infiltrated the mucosa in the B. velezensis groups after challenge with A. veronii. Compared with A. veronii group, B. velezensis could safeguard the integrity of intestinal villi. The highest post-challenge survival rate (75.0%) was recorded in B. velezensis C-11 group. The present data suggest that probiotic B. velezensis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection.IMPORTANCEIn this work, four Bacillus velezensis strains isolated from apparently healthy Crucian carp, which exhibited a broad-spectrum antibacterial activity especially the fish pathogens. Administration of B. velezensis induced the enhancement of the intestinal innate immune response through reducing intestinal colonization by pathogens. The isolation and characterization would help better understand probiotic can be recognized as an alternative of antimicrobial drugs protecting human and animal health.

Aeromonas veronii caused disease and mortality in channel catfish in Vietnam

Channel catfish (Ictalurus punctatus) are one of the most commercially important fish species cultured in Vietnam, and has been a major driver of Vietnam's rise to become a leading country for fisheries exports in the world. Since 2016, there have been several outbreaks of a contagious disease which in channel catfish has resulted in severe levels of economic loss. In this study, bacterial isolates from diseased fish were sampled and analyzed from 22 farms in four Northern Vietnam provinces experiencing outbreaks. Isolates were identified as Aeromonas veronii based on their bacteriological characteristics, 16S rRNA sequence analysis, and presence of Aeromonas veronii specific rpoB gene by PCR examinations. Subsequently, A. veronii isolates were tested for their virulence to fish via an experimental infection challenge, and their susceptibility to 21 antibiotics was also assessed. Results showed that A. veronii is pathogenic and has probably contributed to the mass mortality of this channel catfish species in Vietnam. Notably, A. veronii also exhibited resistance to four common antibiotics including amoxicillin, bicozamycin, lincomycin, and vancomycin. To our knowledge, this is the first report of outbreaks associated with A. veronii infection in channel catfish in a Southeast Asian country. This report could be used to alert aquaculture farmers and managers in this region to develop an appropriate biosecurity strategy for protecting catfish and other vulnerable species to A. veronii infection, and find alternative solutions to help mitigate occurences of the disease.

Role of Myeloperoxidase of northern snakehead (Channa argus) in Aeromonas veronii infection

Myeloperoxidase (MPO) is a ferrous lysosomal protein with many immune functions that belongs to the heme peroxidase enzyme. In this study, the functions of MPO in the northern snakehead (Channa argus) were investigated by cloning an MPO cDNA sequence with a full length of 3181 bp. Homology analysis showed that northern snakehead MPO gene had the highest (81%) homology with mandarin fish (Siniperca chuatsi). In healthy northern snakehead, the MPO gene was expressed in the head-kidney, kidney, heart, gill, spleen, liver, and muscles but not midgut. After the northern snakehead was infected with Aeromonas veronii, the MPO gene expression varied in different tissues with low level in spleen, liver, gill and muscle, fluctuated in kidney and head-kidney and showed high level in heart. The result indicated that MPO might play an important role in the antimicrobial immune response of the northern snakehead.

Aeromonas veronii caused disease and pathological changes in Asian swamp eel Monopterus albus

More and more diseases were occurred in the aquaculture of Asian swamp eel. Bacterial isolates were collected from the pathogenic organs of diseased Asian swamp eels. A dominant strain, Aeromonas veronii JL-01, was isolated and identified from the liver and intestine tissue of diseased Asian swamp eels. The symptoms of fish infected artificially with A. veronii were the same as those observed under natural infection. The mortality rate was 40%–80% and infected Asian swamp eels died 2–3 days after infection. Drug sensitivity tests showed that the isolated bacterium was highly sensitive to seven kinds of antibiotics, including cephalosporins, aminoglycoside, quinolones, tetracyclines (except Tetracycline), pheniclos, nitrofurans and sulfonamides. The bacterium was resistant to penicillins (except Piperacillin), macrolides, polypeptides and lincomycin. The histological changes in Asian Swamp Eel induced by A. veronii infection mainly occurred in the liver, intestines and kidney. A. veronii showed strong pathogenicity towards the Asian swamp eel, causing tissue damage and death. Gentamycin, doxycycline and sulfamethoxazole could be used for preventing and treat this disease.

Transcriptome analysis of the spleen provides insight into the immunoregulation of Mastacembelus armatus under Aeromonas veronii infection

Mastacembelus armatus, also known as the zigzag eel, is an economically important species of freshwater fish that is very popular with consumers as a high-grade table fish in China. Recently, the wild population of this fish has declined gradually due to overfishing and various types of ecological imbalance. Meanwhile, the aquaculture of this spiny eel has flourished in southern China. To understand the immune response of zigzag eel to Aeromonas veronii, we carried out transcriptome sequencing of zigzag eel spleens after artificial bacterial infection. After assembly, 110,328 unigenes were obtained with 44.42% GC content. A total of 27,098 unigenes were successfully annotated by four public protein databases, namely, Nr, UniProt, KEGG and KOG. Differential expression analysis revealed the existence of 1278 significantly differentially expressed unigenes at 24 h post infection, with 767 unigenes upregulated and 511 unigenes downregulated. After GO and KEGG enrichment analyses, many immune-related GO categories and pathways were significantly enriched. The typical significantly enriched pathways included toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway. In addition, 40,027 microsatellites (SSRs) and 52,716 candidate single nucleotide polymorphisms (SNPs) were identified from the infection and control transcriptome libraries. Overall, this transcriptomic analysis provided valuable information for studying the immune response of zigzag eels against bacterial infection.

Oral immunization with recombinant Lactobacillus casei expressing flaB confers protection against Aeromonas veronii challenge in common carp, Cyprinus carpio

Aeromonas veronii is an important type of gram-negative pathogen of human-livestock-aquatic animal and causes great economic losses in the aquaculture industry. Vaccination is an effective method of defence against A. veronii. There are many factors that restrict the use of vaccination, and the development of new oral vaccines is urgently needed. The selection of suitable antigens is of great significance for the development of aquaculture vaccines. Bacterial flagellin can specifically bind to TLR5 and induce the release of cytokines from the organism, which could be used in the development of vaccines. In this study, we constructed two recombinant Lactobacillus casei (L. casei) (surface-displayed or secretory) expressing the flaB of A. veronii and evaluated the effect of immune responses in common carp. The flaB gene (900 bp) of A. veronii was subcloned into the L. casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secretory). Western blot and immunofluorescence assays confirmed the expression of the recombinant flaB protein. Common carp immunized with Lc-pPG-1-flaB and Lc-pPG-2-flaB via oral administration route exhibited induction of antibody expression and innate immune responses. The results indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB can induce high levels of IgM, ACP, AKP, LZM and SOD activity in organisms, and Lc-pPG-1-flaB can induce even higher levels. The recombinant L. casei may effectively induce humoral immunity and increase the serum immunological index. Furthermore, leukocytes phagocytosis percentage and index of the recombinant L. casei were enhanced. The results of qRT-PCR showed that recombinant L. casei can significantly increase the expression of IL-10, IL-β, IFN-γ and TNF-α in the tissues of immunized common carp, compared with control groups. Viable recombinant L. casei strains, which were delivered directly survived throughout the intestinal tract. Common carp that received Lc-pPG-1-flaB (66.7%) and Lc-pPG-2-flaB (53.3%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our work indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB had beneficial effects on immune response and enhanced the disease resistance of common carp against A. veronii infection. The combination of flaB delivery and the Lactic acid bacteria (LAB) approach may be a promising method for the development of oral vaccines for treating A. veronii. In future research, we will focus on the colonization ability of LAB in the intestines and on the impact of these bacteria on intestinal flora.

Aeromonas veronii and aerolysin are important for the pathogenesis of motile aeromonad septicemia in cyprinid fish.

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.

EPSP of L. casei BL23 Protected against the Infection Caused by Aeromonas veronii via Enhancement of Immune Response in Zebrafish.

Aquaculture is the fastest-growing food production sector in the world, and it supplies nearly 50% of the global food fish supply. However, disease outbreaks have become a major problem in the fish farming industry. The beneficial contribution of probiotic bacteria to aquatic animals’ health has been widely described, and they have been widely used in aquaculture for disease control and growth promotion. However, the action of probiotic bacterial components and mechanisms underlying protection against pathogens afforded by probiotic bacteria remain poorly understood. In the present study, we pre-colonized zebrafish larvae (before hatching) with 17 potential probiotic bacterial strains and screened for those possessing anti-infective effects against Aeromonas veronii. We found that Lactobacillus casei BL23 significantly increased the survival of zebrafish larvae upon A. veronii infection. Using a germ-free (GF) zebrafish model and gut microbiota transplant experiment, we showed that L. casei BL23 per se has anti-infective effects in zebrafish larvae, which does not involve microbiota. Furthermore, we identified an exopolysaccharide-protein complex (EPSP) extracted from L. casei BL23 cells, which consisted of a 40–45 KD size protein and an exopolysaccharide composed of α-Rha, α-Glc, β-GlcNAc, and β-GalNAc. EPSP significantly increased the survival rate of GF zebrafish at a dose of 10–20 μg/ml after A. veronii infection (P < 0.01). In addition, the EPSP induced a higher expression of TLR1 and TLR2, and modulated the expression profile of pro-inflammatory and anti-inflammatory cytokines in zebrafish liver (ZFL) cells. Our data indicated that the anti-infective effect of EPSP from L. casei BL23 was mediated by enhancement of immune responses in zebrafish, which might involve the TLR1/TLR2 signal pathway.