Immunization with recombinant aerolysin and haemolysin protected channel catfish against virulent Aeromonas hydrophila

Aeromonas hydrophila is emerging as one of the major concerns in catfish aquaculture in the Southeastern United States due to recent outbreaks of motile aeromonad septicaemia (MAS) caused by virulent clonal isolates. There is no effective vaccine currently available for the prevention of MAS. In this study, two virulence‐associated proteins of A. hydrophila, aerolysin and haemolysin, were heterologously expressed in Escherichia coli cells. Recombinant aerolysin (rArl) and haemolysin (rHly) were used to immunize catfish. Both rArl‐ and rHly‐induced humoral immune response as evidenced by immunoblotting and cell agglutination; immunized fish had significantly less mortality as compared to control fish upon challenge with virulent A. hydrophila. When a mixture of rArl and rHly was used to immunize the fish, significantly higher relative per cent survival (RPS) was obtained. Sustained RPS of 71–78% were observed at 2–5 weeks post immunization. The results of this study indicated that immunization against aerolysin and haemolysin had significant impact on the establishment of pathogenesis by A. hydrophila, suggesting that these two proteins could serve as general immunogens for future development of recombinant protein vaccines.     

The role and mechanism of icmF in Aeromonas hydrophila survival in fish macrophages

Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF‐RNAi was constructed by shRNA and RT‐qPCR confirmed the expression of icmF was down‐regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF‐RNAi. The intracellular survival rate of the wild‐type strain was 92.3%, while the survival rate of icmF‐RNAi was only 20.58%. The escape rate of the wild‐type strain was 20%, while that of the icmF‐RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H₂O₂. The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence‐related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages.     

Development of real‐time and quantitative QCM immunosensor for the rapid diagnosis of Aeromonas hydrophila infection

Since bacterial infection cause a significant economic loss in fish farms, it is necessary to develop rapid diagnostic tools. Interests on label‐free biosensors have been raised for the rapid detection of aquatic pathogenic bacteria but have not been extensively studied yet. Here we report a quartz crystal microbalance (QCM) immunosensor system for the rapid and simple detection of Aeromonas hydrophila, a pathogen for fish and human, in comparison with a conventional indirect ELISA method. In QCM immunosensor system, an antibody against A. hydrophila was covalently cross‐linked to the gold surface of sensor chip and bacterial attachment was monitored as real‐time frequency shifts within 5 min. The frequency shifts were very positively related to the amounts of bacterial cells between 6.25 and 100 μg corresponding to 6 × 10⁶ to 10⁸ CFU with a high specificity. The QCM immunosensor was also able to detect bacterial cells in fish tissue extract in a dose‐dependent manner. Indirect ELISA also showed the dose‐dependent reaction and the amplified signal may allow a lower detection limit. However, QCM immunosensor system showed a more linear and reliable standard curve with R² value of almost 1 (0.9999). Moreover, detection of the bacteria was much quicker and simpler.     

Multilocus sequence analysis revealed a high genotypic diversity of Aeromonas hydrophila infecting fish in Uganda

A multilocus sequence analysis (MLSA) was carried out to delineate Aeromonas hydrophila from fish in Uganda. Five housekeeping genes including recA, gyrB, metG, gltA and pps; and the 16S rRNA gene were amplified and sequenced from a total of nine A. hydrophila isolates. The obtained sequences were edited, and consensus sequences generated for each gene locus. The housekeeping gene sequences were concatenated and phylogenetic analysis performed in MEGA version 7.0.2. Pairwise distances ranged from 0.000 to 0.118, highest within the gltA gene locus and lowest within the 16S rRNA gene. The average evolutionary diversity within isolates from the same source ranged between 0.002 and 0.037, and it was 0.033 between the different sources. Similar tree topologies were obtained from the different gene loci with recA, metG and gyrB being more consistent in discriminating isolates according to sources while the 16S rRNA gene had the lowest resolution. The concatenated tree had the highest discriminatory power. This study revealed that A. hydrophila strains infecting fish in Uganda are of diverse genotypes suggesting different sources of infection in a given outbreak. Efforts to minimize spread of the bacteria across sources should be emphasized to control infections of mixed genotypes.     

Indigenous AHL‐degrading bacterium Bacillus firmus sw40 affects virulence of pathogenic Aeromonas hydrophila and disease resistance of gibel carp

Interrupting quorum sensing represents a novel anti‐infective strategy to combat bacterial pathogen, and biodegradation of quorum sensing signal AHLs has been proved to be an efficient way to control pathogenic Gram‐negative bacteria in aquaculture. In this study, the effect of Bacillus firmus sw40 as efficient AHL‐degrading strain on virulence of fish pathogen Aeromonas hydrophila and disease resistance of gibel carp Carassius auratus gibelio was investigated. The results demonstrated that in vitro the B. firmus sw40 extracellular production (ECP) was able to significantly decrease protease production, haemolytic activity and biofilm formation in A. hydrophila. Dietary administration of B. firmus sw40 (10⁹ CFU/g) for 4 weeks significantly reduced the inflammatory cytokines TNF‐1a, TNF‐2a and IFN‐γ genes expression, antioxidant parameter MDA and GSH levels in serum and increased antioxidant enzyme SOD activity. Besides, B. firmus sw40 could significantly increase the survival of gibel carp with pathogenic A. hydrophila infection.     

Extracts of Oliviera decumbens and Satureja khuzestanica as immunostimulants affect some innate immunity indices of Cyprinus carpio against Aeromonas hydrophila infection

In this study, the effect of extracts of two herbs (Oliviera decumbens and Satureja khuzestanica) on immune response of carp was investigated. At the beginning of experiment, fish were divided to two groups including vaccinated (using vaccine developed against A. hydrophila) and non‐vaccinated. Fish in both groups were fed diets containing O. decumbens and S. khuzestanica and combination of two herbal extracts for 5 weeks. Control fish (negative control) and fish vaccinated only (positive control) were fed basal diets without supplements of herbal extracts. Lysozyme activity, antibody titre, complement activity and bactericidal activity in serum were measured. After 5 weeks feeding, fish were infected with A. hydrophila and mortalities were recorded. In both experimental groups, no significant differences were found in terms of alternative haemolytic complement (ACH50) activity and antibody titres of Serum. In non‐vaccinated fish group, lysozyme and bactericidal activity of fish fed S. khuzestanica or combination of O. decumbens and S. khuzestanica was higher compared with control and other experimental treatments. In vaccinated fish group, the lysozyme and bactericidal activity was not significant in all treatments compared with control group. The results of this study showed that feeding non‐vaccinated and vaccinated carp with only S. khuzestanica or in combination with O. decumbens enhance only some immunity indices including lysozyme and bactericidal activity in non‐vaccinated fish and these extracts have no immunological stimulatory role on vaccinated individuals.     

Dietary supplementation with low molecular weight sodium alginate improves growth,haematology, immune reactions and resistance against Aeromonas hydrophila in Clarias gariepinus

A few studies have illustrated the effects of sodium salt derived from alginic acid on different fish species. However, little is known about the effect of sodium alginate on catfish (Clarias gariepinus). Therefore, this study was performed to assess the use of low molecular weight sodium alginate ( LMWSA) in C. gariepinus. A total of 180 apparently healthy C. gariepinus with a mean body weight of 45 g were randomly divided into three equal groups (D1, D2 and D3). D1 the control group received a control diet, while D2 and D3 received 1% and 3% LMWSA, respectively, for 8 weeks. A challenge test against Aeromonas hydrophila was performed on 15 randomly selected catfish for 15 days. At the end of the experiment, catfish in D3 that received a diet of 3% LMWSA showed significant increases in the final body weight, weight gain and thermal‐unit growth coefficient compared with those in D2 and D1. There was a significant decrease in the erythrogram in D1 after the 4‐day pathogen challenge. A leucogram revealed leucocytosis, heterophilia and lymphocytosis in catfish in D2 and D3 compared with those in D1. After the 4‐day challenge, the following changes took place: lysozyme, nitric oxide, phagocytic activity and the respiratory burst were significantly elevated in catfish that received LMWSA and were more pronounced in D3 than in D1. The mortalities of catfish have been stopped after pathogen challenge from 8‐day in D1 and D2 where at 6‐day in D3. Thus, administration of 1% and 3% LMWSA enhances the growth, immune response and resistance of C. gariepinus against A. hydrophila.     

Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida

A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10⁴ ± 1 × 10⁴ CFU g^?1 (without enrichment) and 40 ± 10 CFU g^?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.     

A new potential therapeutic remedy against Aeromonas hydrophila infection in rainbow trout (Oncorhynchus mykiss) using tetra,Cotinus coggygria

Different antibiotic‐based drugs are being used for the treatment of Aeromonas hydrophila infection in rainbow trout, and several studies emphasize the use of medicinal plants as immunostimulants for prophylactic measure against Aeromoniasis disease. However, therapeutic effects of aqueous methanolic extracts of tetra (Cotinus coggygria) against A. hydrophila in rainbow trout were not investigated. Four different concentrations of tetra extract (0 [control], 4, 8 and 12 mg/100 µl) and also two different positive control groups (florfenicol and doxycycline antibiotics) were administered orally using feeding needles to individual rainbow trout, Oncorhynchus mykiss of all experimental groups twice a day after intramuscular inoculation of A. hydrophila. The study period was for 10 days. On 0th, 3rd, 7th and 10th day, blood and tissues were collected from the fish and changes in humoral immune responses, haematology and immune‐related gene expressions were determined. In the study, superoxide radical production was decreased generally in all experimental groups except in 12 mg tetra and florfenicol treatments compared to control (p < .05). Lysozyme activity was generally decreased (p < .05), or no differences were observed in all experimental groups compared to the control. Myeloperoxidase activity was significantly increased in florfenicol‐treated fish group on 7th day (p < .05). Generally, myeloperoxidase activity showed an increase in almost all tetra‐treated groups. Haematological parameters increased but were not significantly high enough in treatments. Almost all immune‐related gene expressions were significantly enhanced on 3rd and 10th day of the study. Survival rate of 53.33% was found in control group. There were no significant differences in survival between control and 4 mg tetra‐treated group (p > .05). All the other groups' survival rate was significantly increased compared to control. The highest survival rate was found in florfenicol group (80%). In 12 mg tetra‐, doxycycline‐ and 8 mg tetra‐treated groups, survival rate was recorded as 74.44%, 70% and 70%, respectively. Our results suggest that tetra methanolic extract is an effective therapeutic remedy against A. hydrophila infection in rainbow trout at the dose of 24 mg/32.34 g body weight/day.     

Immune effects of bathing European eels in live pathogenic bacteria,Aeromonas hydrophila

The specific and non‐specific immune parameters and protection of European eels (Anguilla anguilla) were evaluated after bathing eels with Aeromonas hydrophila. Two hundred eels were distributed into two equal groups and bathed with Phosphate‐buffered saline (Control group) or 1.0 × 10⁷ cfu mL^?1 A. hydrophila (Test group) for 1 h respectively. Then, eels were bled aseptically from the caudal sinus on 1, 4, 7, 14 and 28 days post treatment. The blood cells were used to evaluate the cellular immunity and the serum was used to determine the titres of specific antibody as well as the activities of superoxide dismutase (SOD) and lysozyme. Eels from both groups were challenged by intraperitoneal injection of 1.0 × 10⁷ cfu of A. hydrophila on 28 and 42 days post bathing. The results show that eels bathed in A. hydrophila significantly (P < 0.05) enhanced the proliferation of different types of blood cells and the serum titres of anti‐A. hydrophila antibody. The Relative Percent Survival (RPS) after challenge on 28 and 42 days post bathing in Test group vs. Control group was 40% and 50% respectively. These results suggest that bathing European eels in A. hydrophila would positively affect specific as well as non‐specific immune parameters and protect against A. hydrophila infection in freshwater farming.     

Microcystis aeruginosa (Kütz) incorporated diets increase immunity and survival of Indian major carp Labeo rohita (Ham.) against Aeromonas hydrophila infection

The aim of this study was to evaluate dietary dosages of Microcystis on the immune response and disease resistance against infections due to the opportunistic pathogen Aeromonas hydrophila in Labeo rohita fingerlings. Labeo rohita fingerlings were fed diet containing 0 (Control), 0.5, 1.0 and 5 g Microcystis powder kg^?1 dry diet for 90 days. Three replicate groups of fish averaging 20 ± 2 g were fed for 3 months daily. At 30 days interval, samples were assayed for different biochemical [serum total protein, albumin, globulin, albumin:globulin (A:G) ratio] and immunological (superoxide anion production, lysozyme and serum bactericidal activity) parameters. The results demonstrate that fish fed Microcystis showed increased levels of lysozyme, serum bactericidal activity, serum protein and albumin (P ≤ 0.05) as compared with the control group. After 90 days, fish were challenged with A. hydrophila and mortality (%) was recorded up to day 10 postchallenge. The group fed 1.0 g Microcystis kg^?1 dry diet showed the highest percentage survival (72%). These results indicate that Microcystis aeruginosa stimulates the immunity and makes L. rohita more resistant to infection by A. hydrophila when fed in dried form in feed.     

Probiotic efficiency of Bacillus sp. in Labeo rohita challenged by Aeromonas hydrophila: assessment of stress profile,haemato‐biochemical parameters and immune responses

Fish are always susceptible to a wide variety of deadly pathogens which cause a huge loss in aquaculture industries. In this investigation, we have demonstrated the in vivo probiotic efficiency of Bacillus sp. MVF1 (GenBank Acc. No. KP256503) in Labeo rohita challenged with pathogenic strain of Aeromonas hydrophila (MTCC 1739). To check the probiotic potential of the selected bacterial strain, fish were divided into four groups: control, D1, D2 and D3. A total of 100 days (70 days probiotic feeding + 71th day sampling and 28 days challenged test + 29th day sampling) of feeding trial was conducted. To establish the probiotic potential of Bacillus sp. MVF1, certain haematological parameters (haemoglobin, total erythrocyte and leucocyte count), serum biochemical parameters (total protein, albumin and globulin), immune parameters (serum lysozyme and total IgM levels) and hepatic stress profile (malondialdehyde production, superoxide dismutase and catalase activity) have been measured. Our results demonstrated that red blood cell number, white blood cell number and haemoglobin content were much higher in D2 group fish compared to other groups and control fish. Similarly, total protein contents, albumin concentration, globulin concentration, lysozyme activity and IgM production were also recorded to be highest in D2 group fish. This finding clearly indicated the probiotic potential of Bacillus sp. MVF1 in L. rohita. Furthermore, our results also demonstrated that 1 × 10⁷ CFU g^?1 feed (D2) provides better immunity compared to 1 × 10⁵ (D1) and 1 × 10⁹ (D3). Due to beneficial effects, the bacterium Bacillus sp. MVF1 might be useful in aquaculture industries to reduce the disease susceptibility.     

Protective effects of basil (Ocimum basilicum) ethanolic extract supplementation diets against experimental Aeromonas hydrophila infection in common carp (Cyprinus carpio)

This study evaluated the effects of basil (Ocimum basilicum) leaves extract supplement on growth, blood parameters and resistance to Aeromonas hydrophila in Cyprinus carpio fingerlings. Basil leaves were mixed thoroughly with feed at 0, 100, 200, 400, 800 and 1600 mg kg^?1 of diet and were fed in triplicate group for 2 months. At the end of this period, growth performance and survival were determined. Then fish were challenged intraperitoneally with A. hydrophila and mortalities were recorded up to 10 days post challenge. Results indicated that WBC, RBC, Ht, Hb, total protein, albumin and globulin were higher (P < 0.05) in fish fed diets containing O. basilicum compared with the control. Highest specific growth rate values were observed in 400 mg kg^?1 group. Feed conversion ratio was significantly lower (P < 0.05) in fingerlings fed 400 and 800 mg O. basilicum kg^?1 dry diet. After 10 days post challenge, total protein, WBC, RBC and Hb were significantly higher in 400 mg kg^?1 compared with other groups. These results reveal that a dietary O. basilicum leaves extract of 400 mg kg^?1 fed for 60 days leads to increased growth performance and survival rate as well as improved feeding efficiency in common carp fingerlings rendering them more resistant against infection by A. hydrophila.     

Comparative genomic analysis of five high drug‐resistance Aeromonas hydrophila strains induced by doxycycline in laboratory and nine reference strains in Genbank

Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal haemorrhagic septicaemia in fish and shellfish. Doxycycline, one of the second generation tetracyclines, has been used in fish farming to fight against infectious diseases caused by A. hydrophila due to its broad‐spectrum antimicrobial activity and lower cost. However, progressive increase in resistance of Aeromonas strains to doxycycline aroused serious concern. In this report, drug‐resistant A. hydrophilaAH10 strains were induced and selected by using a consecutive batch culture system in Mueller‐Hinton Broth (MHB) supplemented with varying concentrations of doxycycline. Five isolates (AH101‐105) were obtained from the bacterial culture induced by 25 μg/ml doxycycline for drug‐resistance analysis. Minimal inhibitory concentrations (MIC) values of all five isolates were 50 times higher than that of the parental strain AH10. All of them also displayed high‐level resistance to sulphonamides and amides. We sequenced five isolates and performed comparative genomic analysis of these draft genomes with nine A. hydrophila complete genomes from GenBank. Results showed that the pan‐genome of 14 strains contains 4,730 genes, 3,056 genes of which present in all strains. The drug‐resistance genes also showed significant difference in these genomes, which indicated dangers of indiscriminate use of antibiotics in aquaculture and the necessity of understanding the variation of antibiotic resistance of A. hydrophila. Pan‐genome analysis further revealed that no specific SNP (single nucleotide polymorphism) or InDel (insertion and deletion variation) was identified in any functional gene locus among the genomes of AH10 mutated strains, in contrast, significant CNVs (copy number variations) and SV (structure variations) for gene groups were identified in all the mutant genomes.     

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

Natural co‐infection of cultured Nile tilapia Oreochromis niloticus with Aeromonas hydrophila and Gyrodactylus cichlidarum experiencing high mortality during summer

Aeromonas hydrophila and Gyrodactylus cichlidarum are common pathogens that induce significant economic losses in farm‐reared Nile tilapia. Nowadays, the sudden appearance of fish mortalities was exaggerated due to mixed and multiple infections. During summer 2016, mass mortality among earthen pond‐farmed Nile tilapia was reported. Clinico‐pathological, bacteriological and parasitological examinations have been demonstrated. As well, the water quality parameters were assessed. The clinical and histopathological findings of the moribund and recently dead fish were characterized by generalized septicaemic signs. The water quality parameters were significantly elevated over the permissible levels, whereas there was an elevation in nitrite (0.04 mg/L), un‐ionized ammonia (0.8 mg/L), hydrogen sulphide levels (153.1 mg/L) and organic matter content (3.79 mg/L). A. hydrophila was identified based on phenotypic characterization, API 20E features and the homology of 16S rRNA gene sequence analysis. In addition, PCR data confirmed the presence of aerolysin (aerA) and haemolysin (hly) genes in the identified A. hydrophila isolates. Gene sequencing and phylogenetic analysis based on 16S rRNA sequence confirmed that A. hydrophila H/A (accession No. MN726928) of the present study displayed 98%–99% identity with the 16S rRNA gene of A. hydrophila. Furthermore, the monogenetic trematode, G. cichlidarum was identified in the wet mounts from the skin and gills of the examined fish with a high infestation rate. In this context, it was reported that the synergistic co‐infection of A. hydrophila and G. cichlidarum with deteriorated water quality parameters could induce exaggerated fish mortalities during hot weather.     

Investigation on protective effect of recombinant protein (OmpTS) of Aeromonas hydrophila in Common carp (Cyprinus carpio)

The outer membrane protein of Aeromonas hydrophila is a potential candidate for vaccine development. In this study, after cloning and expression of ompTS, 270 common carp, weighing 44 ± 5.7 g divided into five groups, were injected intraperitoneally twice with 3‐week intervals. Groups included the following: PBS, PBS plus Freund's adjuvant, recombinant protein, recombinant protein plus Freund's adjuvant and 20 fish as negative control. Two weeks after the second injection, 30 fish of each group were challenged with a dose of 2 × LD₅₀ of Aeromonas hydrophila and RPS was measured. The antibody level was measured using ELISA test. The protection of recombinant protein in the immunized fish with and without adjuvant, respectively, was about 82.61% and 78.26% (the protection of recombinant protein electroeluted from an SDS–PAGE with and without adjuvant, respectively, was about 78.62% and 69.57%). The average of antibody level in recombinant protein with and without adjuvant was significantly higher than the PBS group (p < .05). The ability of recombinant ompTS to increase the antibody level and to protect the fish from challenge by A. hydrophila demonstrated that recombinant ompTS protein injection can be used to immunize common carp against A. hydrophila infection.     

The in vitro efficacy of oxytetracycline against re‐isolated pathogenic Aeromonas hydrophila carrying the cytolytic enterotoxin gene through hybrid catfish,Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) in Thailand

Aeromonas hydrophila is a pathogen infecting farmed hybrid catfish, Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) which incurs substantial economic losses in Thailand. The study aimed at a genetic tracking of A. hydrophila infection and the in vitro assessment of the efficacy of antibiotics against its virulent strains. Five clinical strains from catfishes and Nile tilapia were employed. They were 3‐passage re‐isolated through healthy hybrid catfish and the cytolytic enterotoxin gene (AHCYTOEN) of individuals was traced. Each of the re‐isolates at a dose of ~6.67 × 10⁵ CFU/g was intraperitoneally injected into ~15 g‐healthy hybrid catfish and their pathogenicity were observed for 7 days. It was found that AHCYTOEN was carried over whereas typical signs of motile aeromonas septicaemia were found in the specimens. The bacterial strains of Nile tilapia origin did not induce mortality but those of catfish origins (80%–100% rate of mortality). The strains were susceptible to the tetracycline antibiotics, and oxytetracycline produced MIC₅₀ and MBC as low as 0.007–0.031 μg/ml and 1–8 μg/ml respectively. As oxytetracycline specifically inhibited pathogenic A. hydrophila in vitro, it is recommended that an appropriate dosage regimen of the drug should be established.