Virulence assay of rhizoid and non‐rhizoid morphotypes of Flavobacterium columnare in red tilapia,Oreochromis sp., fry

Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris‐like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non‐rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non‐rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post‐challenge as determined by whole fish bacterial loads. At 24 and 48 h post‐challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non‐rhizoid morphotype. The results suggested that an inability of the non‐rhizoid morphotype to persist in tilapia fry may explain lack of virulence.     

Evaluation of a 4‐h static copper sulphate treatment against experimental infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus)

The efficacy of copper sulphate (CuSO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus) was evaluated. Fish were challenged, by waterborne exposure to F. columnare in an ultra‐low flow‐through water delivery system, and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 at 5.5 h post challenge for 4 h. Bacterial challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged with F. columnare and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 post challenge had mortalities of 6.4% and 18.3%, respectively, compared with the positive control, which had 90.2% mortality. The mortality of challenged fish treated with CuSO₄ at 4.2 and 2.1 mg L^?1 was significantly different from the positive control. There was no significant difference between the mortalities of the 4.2 and 2.1 mg L^?1 treatments. The results suggest that CuSO₄ has clear therapeutic value against columnaris infections.     

Sickeningly Sweet: L‐rhamnose stimulates Flavobacterium columnare biofilm formation and virulence

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L‐rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L‐rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L‐rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L‐rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.     

Lack of association between Flavobacterium columnare genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.) × Oreochromis aureus (Steindachner)

Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex‐reversed hybrid tilapia, Oreochromis niloticus (L.) × O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established genomovars of F. columnare: I, II, II‐B, III and I/II. The mean per cent mortality of fish challenged with genomovar I, II and III isolates ranged from 0 to 100, 3.3–78 and 3.3–75%, respectively. The mean per cent mortality of fish challenged with genomovar II‐B ranged from 35 to 96.7%, and the only genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.     

Pre‐challenge and post‐challenge haemato‐immunological changes in Oreochromis niloticus (Linnaeus, 1758) fed argan oil against Lactococcus garvieae

The present study investigated the effects of argan oil, obtained from Argania spinosa, on pre‐ and post‐challenge immuno‐haematological and biochemical responses of Nile tilapia, Oreochromis niloticus. For this purpose, the fish were fed diets containing 0, 0.5%, 1% or 2% argan oil for 45 days. Following 45 days of feeding, fish were challenged with Lactococcus garvieae and mortality was recorded for 15 days. During the pre‐challenge period, significantly higher respiratory burst activity, total white blood cell (WBC), serum lysozyme activity and myeloperoxidase activity were determined in the argan oil‐fed groups. The serum glucose and cholesterol levels decreased whilst total protein and albumin did not change in the groups fed with argan oil‐supplemented diets. After challenge with Lactococcus garvieae, the percentage survival (%) was found to be the highest in the 1% and 2% argan oil‐supplemented feeding groups. Also, there was a significant increase in weight gain, specific growth rate and feed conversion ratio in those fish fed argan oil. The results of this study indicated that after the supplementation of fish diets with argan oil, especially at 1% and 2% concentrations, the immunological, haematological and biochemical values remained similar in both the pre‐ and post‐challenge periods and the immune response against L. garvieae in Nile tilapia was modulated.     

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.     

Disease resistance and immune‐relevant gene expression in golden mandarin fish,Siniperca scherzeri Steindachner,infected with infectious spleen and kidney necrosis virus‐like agent

Infectious spleen and kidney necrosis virus (ISKNV), family Iridoviridae, genus Megalocytivirus, may cause high mortality rates such as those seen in mandarin fish, Siniperca chuatsi. ISKNV has attracted much attention due to the possible environmental threat and economic losses it poses on both cultured and wild populations. We have investigated the pathogenicity of ISKNV‐like agent Megalocytivirus, isolated from infected pearl gourami, in golden mandarin fish, Siniperca scherzeri – a member of the Percichthyidae family – and in another Percichthyidae species, S. chuatsi. Fish were challenged with four different doses of ISKNV‐like agent Megalocytivirus (1, 10, 100 or 1000 μg per fish) over a 30‐day period, and cumulative fish mortalities were calculated for each group. No significant mortality was observed for fish challenged with the lowest dose (1 μg per fish) relative to a control group. However, all other challenged groups showed 100% mortality over a 30‐day period in proportion to the challenge dose. Quantitative real‐time PCR was performed to measure mRNA expression levels for six immune‐related genes in golden mandarin fish following ISKNV‐like agent challenge. mRNA expression levels for IRF1, Mx, viperin and interleukin 8 significantly increased, while mRNA levels for IRF2 and IRF7 remained constant or declined during the challenge period.     

Characterization of spaC‐type Erysipelothrix sp. isolates causing systemic disease in ornamental fish

Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.     

Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim‐sulfadimethoxine and trimethoprim‐sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards – amongst others – ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.     

An outbreak of acute disease and mortality in Atlantic salmon (Salmo salar) post‐smolts in Norway caused by Tenacibaculum dicentrarchi

An outbreak of disease characterized by skin ulcers, fin rot and mortality was observed a few days after the transfer of Atlantic salmon (Salmo salar) from a freshwater smolt production facility to a land‐based seawater post‐smolt site. Dead and moribund fish had severe skin and muscle ulcers, often 2–6 cm wide, particularly caudal to the pectoral fins. Microscopic examination of smears from ulcers and head kidney identified long, slender Gram‐negative rods. Histopathological analysis revealed abundance of long, slender Tenacibaculum‐like bacteria in ulcers and affected fins. Genetic characterization using multilocus sequence analysis (MLSA) of seven housekeeping genes, including atpA, dnaK, glyA, gyrB, infB, rlmN and tgt, revealed that the isolates obtained during the outbreak were all clustered with the Tenacibaculum dicentrarchi‐type strain (USC39/09^T) from Spain. Two bath challenge experiments with Atlantic salmon and an isolate of T. dicentrarchi from the outbreak were performed. No disease or mortality was observed in the first trial. In the second trial with a higher challenge dose of T. dicentrarchi and longer challenge time, we got 100% mortality within 48 hr. This is the first reported outbreak of disease caused by T. dicentrarchi in Norwegian farmed Atlantic salmon.     

Development of attenuated erythromycin‐resistant Streptococcus agalactiae vaccine for tilapia (Oreochromis niloticus) culture

Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re‐attenuated S. agalactiae (named TFJ‐ery) was developed from a natural low‐virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ‐ery were determined. The results indicated that TFJ‐ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ‐ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ‐ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ‐ery at a dose of 5.0 × 10⁷ CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post‐vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post‐vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ‐ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.     

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.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone)

Five different Vibrio parahaemolyticus strains (SH8, SH108, SH58, AH5 and GD10) isolated from the hepatopancreas of moribund shrimp in farms of mainland China were identified and capable of inducing massive mortality of Penaeus (Litopenaeus) vannamei. The immersion challenge results with five isolates indicated variance of virulence, while only GD10 caused massive sloughing of tubule epithelial cells which was recognized as the most significant symptom of AHPND. Differences in immune responses were detected of P. vannamei during 48 h post‐infection (p.i.) by injection or immersion challenge with V. parahaemolyticus (SH8, SH108 and GD10) isolates. When injected SH8 and SH108 isolates, the expression of lysozyme (LSZ) showing statistically significant upregulation at 16 and 48 h p.i. and that of Toll‐like receptors (TLR) showed statistically significant upregulation at 48 h p.i. When immersion challenge with the GD10 isolate, TLR were upregulated after 8 h p.i. challenge with 10⁴ cfu mL^?1; however, LSZ was downregulated when challenged with 10³ cfu mL^?1. The results suggested that LSZ and TLR serve as crucial molecular markers of innate immunity in shrimp against V. parahaemolyticus infection. LSZ is a vital marker for acute bacterial infection, while TLR serves as a crucial marker for chronic infection.     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Vaccination strategies with oral booster for surubim hybrid (Pseudoplatystoma corruscans × P. reticulatum) against haemorrhagic septicaemia

This study evaluated the efficiency of differently prepared vaccines against Aeromonas hydrophila in the hybrid surubim (Pseudoplatystoma corruscans × P. reticulatum). Survival and haemato‐immunological parameters were compared between the treatments: non‐vaccinated fish (C); bacterin‐vaccinated fish (B); bacterin plus oral booster vaccinated fish (B+O); bacterin and toxoid‐vaccinated fish (B+T) and bacterin, toxoid and oral booster‐vaccinated fish (B+T+O). Fourteen‐days vaccinated fish from B+O and B+T+O were fed with an oral booster for 4 days. After 1 week, the fish were intraperitoneally challenged with 2 × 10⁸ CFU mL^?1 of A. hydrophila. Fish from the treatment B+T+O showed the lowest cumulative mortality (11.36%) 96 h after challenge, compared with other treatments (22.72–44.04%), and a relative survival of 74%. Serum immunoglobulin in B+T+O fish was higher than in other treatments. All vaccinated fish showed an increased agglutination titre when compared with non‐vaccinated fish, both before and after challenge. Fish fed with oral booster showed an increase in phagocytic percentage before and after challenge. It can be inferred that the oral booster vaccination was efficient in reducing mortality in hybrid surubim by enhancing the response against haemorrhagic septicaemia due to A. hydrophila infection.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.