Isolation, genetic characterization, and virulence profiling of different Aeromonas species recovered from moribund hybrid catfish (Clarias spp.).

Background and Aim: The high diversity of Aeromonas spp. results in various pathogenicity levels. This group of bacteria causes a serious disease named motile Aeromonas septicemia (MAS) in catfish (Clarias spp.). This study aimed to characterize the species and virulence gene diversity of Aeromonas spp. isolated from diseased catfish. Materials and Methods: Nine Aeromonas spp. were isolated from infected catfish cultivated in Java, Indonesia, and they were identified at the phenotypic and molecular levels (16S rDNA). The virulence genes assessed included aer/haem, alt, ast, flaA, lafA, and fstA. Results: Phylogenetic analysis identified nine isolates of Aeromonas spp.: Aeromonas hydrophila (11.11%), Aeromonas caviae (11.11%), Aeromonas veronii bv. veronii (44.44%), and Aeromonas dhakensis (33.33%). Virulence genes, such as aer/haem, alt, ast, flaA, lafA, and fstA, were detected in all isolates at frequencies of approximately 100%, 66.67%, 88.89%, 100%, 55.56%, and 66.67%, respectively. This study is the first report on A. dhakensis recovered from an Indonesian catfish culture. Furthermore, our study revealed the presence of A. veronii bv veronii, a biovar that has not been reported before in Indonesia. Conclusion: This finding confirms that MAS was caused by multiple species of Aeromonas, notably A. dhakensis and A. veronii bv veronii, within Indonesian fish culture. The presence of these Aeromonas species with multiple virulence genes poses a significant threat to the freshwater aquaculture industry.

A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.

Prevalence, Antibiotics Resistance and Plasmid Profiling of Vibrio spp. Isolated from Cultured Shrimp in Peninsular Malaysia.

Vibrio is the most common bacterium associated with diseases in crustaceans. Outbreaks of vibriosis pose a serious threat to shrimp production. Therefore, antibiotics are commonly used as preventative and therapeutic measures. Unfortunately, improper use of antibiotics leads to antibiotic resistance. Nevertheless, information on the occurrence of Vibrio spp. and antibiotic use in shrimp, particularly in Malaysia, is minimal. This study aimed to provide information on the occurrence of Vibrio spp., its status of antibiotic resistance and the plasmid profiles of Vibrio spp. isolated from cultured shrimp in Peninsular Malaysia. Shrimp were sampled from seven farms that were located in different geographical regions of Peninsular Malaysia. According to the observations, 85% of the shrimp were healthy, whereas 15% were unhealthy. Subsequently, 225 presumptive Vibrio isolates were subjected to biochemical tests and molecular detection using the pyrH gene. The isolates were also tested for antibiotic susceptibility against 16 antibiotics and were subjected to plasmid profiling. Eventually, 13 different Vibrio spp. were successfully isolated and characterized using the pyrH gene. They were the following: V. parahaemolyticus (55%), V. communis (9%), V. campbellii (8%), V. owensii (7%), V. rotiferianus (5%), Vibrio spp. (4%), V. alginolyticus (3%), V. brasiliensis (2%), V. natriegens (2%), V. xuii (1%), V. harveyi (1%), V. hepatarius (0.4%) and P. damselae (3%). Antibiotic susceptibility profiles revealed that all isolates were resistant to penicillin G (100%), but susceptible to norfloxacin (96%). Furthermore, 16% of the isolates revealed MAR of less than 0.2, while 84% were greater than 0.2. A total of 125 isolates harbored plasmids with molecular weights between 1.0 and above 10 kb, detected among the resistant isolates. The resistant isolates were mediated by both chromosomal and plasmid factors. These findings support the use of surveillance data on the emerging patterns of antimicrobial-resistance and plasmid profiles of Vibrio spp. in shrimp farms. The findings from this study can be used to develop a better disease management strategy for shrimp farming.

Effect of an Oral Bivalent Vaccine on Immune Response and Immune Gene Profiling in Vaccinated Red Tilapia (Oreochromis spp.) during Infections with Streptococcus iniae and Aeromonas hydrophila.

Streptococcosis and aeromonasis inflicted by Streptococcus iniae and Aeromonas hydrophila, respectively, have affected tilapia industries worldwide. In this study, we investigated antibody responses and explored the mechanisms of protection rendered by an oral bivalent vaccine in red tilapia following challenges with S. iniae and A. hydrophila. The results of specific IgM antibody response revealed that the IgM titers against S. iniae and A. hydrophila in the bivalent incorporated (BI) vaccine group were significantly higher (p < 0.05) than those in the bivalent spray (BS) vaccine fish and unvaccinated control fish throughout the experiment. Real-time qPCR results also showed that the gene expression of CD4, MHC-I, MHC-II, IgT, C-type lysozyme, IL-1ß, TNF-α, and TGF-ß remained significantly higher (p < 0.05) than that of the controls between 24 and 72 h post-infection (hpi) in both mucosal (hindgut) and systemic (spleen and head−kidney) organs of BI vaccinated fish. Furthermore, the highest relative expression of the TGF-ß, C-type lysozyme, and IgT genes in the BI vaccinated group was observed in the challenged fish's spleen (8.8-fold), head kidney (4.4-fold), and hindgut (19.7-fold) tissues, respectively. The present study suggests that the bivalent incorporated (BI) vaccine could effectively improve the immune function and activate both humoral and cell-mediated immunities in vaccinated red tilapia following the bacterial challenges.

Draft genome sequence of myo-inositol utilizing Aeromonas dhakensis 1P11S3 isolated from striped catfish (Pangasianodon hypopthalmus) in a local fish farm in Malaysia.

A hypervirulent pathotype of A. hydrophila (vAh) is responsible for Motile Aeromonas Septicemia (MAS) and causes mass mortalities among farmed carp and catfish species in the USA and China. One unique phenotype for vAh among other A. hydrophila strains is the ability to utilize myo-inositol as a sole carbon source. While screening for Aeromonas isolates from diseased fish that can grow using myo-inositol as a sole carbon source, A. dhakensis 1P11S3 was isolated from the spleen of striped catfish (Pangasianodon hypopthalmus) displaying clinical MAS symptoms from a freshwater farm in Malaysia. Aeromonas dhakensis is also an important pathogen in aquaculture, and in this study, we report the draft genome sequence for A. dhakensis 1P11S3, that utilize myo-inositol as a sole carbon source.

Comparative Pathogenicity of Aeromonas spp. in Cultured Red Hybrid Tilapia (Oreochromis niloticus × O. mossambicus).

The genus Aeromonas has been recognised as an important pathogenic species in aquaculture that causes motile Aeromonas septicaemia (MAS) or less severe, chronic infections. This study compares the pathogenicity of the different Aeromonas spp. that were previously isolated from freshwater fish with signs of MAS. A total of 124 isolates of Aeromonas spp. were initially screened for the ability to grow on M9 agar with myo-inositol as a sole carbon source, which is a discriminatory phenotype for the hypervirulent A. hydrophila (vAh) pathotype. Subsequently, LD50 of six selected Aeromonas spp. were determined by intraperitoneal injection of bacterial suspension containing 103, 105, and 107 CFU/mL of the respective Aeromonas sp. to red hybrid tilapias. The kidneys, livers and spleens of infected moribund fish were examined for histopathological changes. The screening revealed that only A. dhakensis 1P11S3 was able to grow using myo-inositol as a sole carbon source, and no vAh strains were identified. The LD50-240h of A. dhakensis 1P11S3 was 107 CFU/mL, while the non-myo-inositol utilizing A. dhakensis 4PS2 and A. hydrophila 8TK3 was lower at 105 CFU/mL. Similarly, tilapia challenged with the myo-inositol A. dhakensis 1P11S3 showed significantly (p < 0.05) less severe signs, gross and histopathological lesions, and a lower mortality rate than the non-myo-inositol A. dhakensis 4PS2 and A. hydrophila 8TK3. These findings suggested that myo-inositol utilizing A. dhakensis 1P11S3 was not a hypervirulent Aeromonas sp. under current experimental disease challenge conditions, and that diverse Aeromonas spp. are of concern in aquaculture farmed freshwater fish. Therefore, future study is warranted on genomic level to further elucidate the influence of myo-inositol utilizing ability on the pathogenesis of Aeromonas spp., since this ability correlates with hypervirulence in A. hydrophila strains.

Antibiotic susceptibility and pathogenicity of Aeromonas hydrophila isolated from red hybrid tilapia (Oreochromis niloticus×Oreochromis mossambicus) in Malaysia.

BACKGROUND AND AIM: Aeromonas hydrophila is a major cause of bacterial infections affecting a wide range of warm water fishes worldwide. In Malaysia, A. hydrophila isolations from diseased fishes were previously reported; however, with limited information. The present study investigates the antibiotic susceptibility and pathogenicity of A. hydrophila isolated from farmed red hybrid tilapia (Oreochromis spp.) in Malaysia. MATERIALS AND METHODS: A. hydrophila was biochemically identified and subjected to antibiotic susceptibility tests. The isolate was then intraperitoneally injected into red hybrid tilapia, and the mortality, clinicopathological changes, and LD50 were determined up to 240 h post-infection (hpi). RESULTS: The isolate demonstrated multiple antibiotic resistances (MAR) toward amikacin, ampicillin, cefotaxime, amoxicillin, trimethoprim-sulfamethoxazole, erythromycin, and streptomycin, with a MAR index of 0.5. The experimental infection of A. hydrophila at 105 CFU/mL in the red hybrid tilapia resulted in 100% mortality at 240 hpi. The LD50 was determined at 1.1×104 CFU/mL. Infected fish demonstrated occasional erratic swimming patterns, localized hemorrhages and depigmentation on the body and operculum areas, fin erosion, enlargement of the gall bladder, and hemorrhage in internal organs. Microscopic observation of infected fish revealed brain congestion, tubular necrosis, and glomerular shrinkage in the kidneys, necrosis of hepatocytes, and congestion of blood vessels in the liver. CONCLUSION: The high virulence of A. hydrophila to the red hybrid tilapia emphasizes the importance of active, on-going monitoring of its prevalence in Malaysian tilapia farming.