Development of neutralizing antibody responses in muskellunge, Esox masquinongy (Mitchill), experimentally exposed to viral haemorrhagic septicaemia virus (genotype IVb)

A complement‐dependent 50% plaque neutralization test was used to assess the neutralizing antibody response in sera of muskellunge, Esox masquinongy, experimentally infected with viral haemorrhagic septicaemia virus (VHSV, genotype IVb) by immersion. Groups of muskellunge were challenged with varying concentrations of VHSV: Group 1 with 10² plaque‐forming units (pfu) mL^?1, Group 2 with 4 × 10³ pfu mL^?1, Group 3 with 10⁵ pfu mL^?1 and Group 4 with 0 pfu mL^?1. The fish were held at a temperature of 11 ± 1 °C and were sampled over a 20‐week period. Neutralizing antibodies were not detected in sera of any of the negative control fish throughout the study. Low neutralizing titres were detected in Groups 1–3 by 6 days post‐infection (p.i.). Neutralizing titres of <80 were not detected again until 3, 4 and 7 weeks p.i. for Groups 2, 3 and 1, respectively, with peak titres for those groups occurring 16, 11 and 17 weeks p.i., respectively. VHSV was detected in serum for up to 11 weeks p.i. Results of this study show that survivors can be detected by a serological technique, despite being virus negative. This may benefit the investigation of VHSV IVb distribution in the Great Lakes and the study of host immune responses to this emerging sublineage.     

Experimental Infection of Rainbow Trout,Oncorhynchus mykiss,and Hybrid Striped Bass,Morone chrysops ♂ × Morone saxatilis ♀, with Viral Hemorrhagic Septicemia Virus Genotype IVb

The emergence of a new sublineage of viral hemorrhagic septicemia virus (VHSV) within the Laurentian Great Lakes has caused concern for aquaculture in the United States. Because of the occurrence of VHSV in a new geographic location, new host species have been identified and the complete host range has not been determined. This study confirmed the high resistance of rainbow trout, Oncorhynchus mykiss, to VHSV type IVb infection. In addition, the experimental susceptibility of hybrid striped bass, Morone chrysops ♂ × Morone saxatilis ♀, to VHSV type IVb infection was examined but determined to be highly dependent on age of fish and exposure temperature. No mortality was observed in adult fish infected via intraperitoneal (IP) injection at 15 C, whereas yearling fish infected via IP injection under the same conditions experienced 20.8% mortality. Among yearling fish infected via IP injection, mortality increased to 100% when exposure to VHSV occurred at 10 C. An LD₅₀ for yearling hybrid striped bass exposed to VHSV at 10 C by IP injection was determined to be 1.4 × 10⁴ pfu (SE = 2.1). Thus, at 10 C, yearling hybrid striped bass experience a high mortality when exposed to VHSV IVb by IP injection.     

Differential viral haemorrhagic septicaemia virus genotype IVb infection in fin fibroblast and epithelial cell lines from walleye,Sander vitreus (Mitchill), at cold temperatures

A cell line, WE‐cfin11e, with an epithelial‐like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast‐like cell line, WE‐cfin11f, and compared with WE‐cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens‐1 (ZO‐1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE‐cfin11e stained for ZO‐1 and only WE‐cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE‐cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE‐cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE‐cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism.     

Development of a walleye cell line and use to study the effects of temperature on infection by viral haemorrhagic septicaemia virus group IVb

A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a walleye caudal fin and used to study the intersection of thermobiology of walleye, Sander vitreus (Mitchill), with the thermal requirements for replication of viral haemorrhagic septicaemia virus (VHSV) IVb. WE-cfin11f proliferated from 10 to 32 °C and endured as a monolayer for at least a week at 1–34 °C. WE-cfin11f adopted an epithelial shape and did not proliferate at 4 °C. Adding VHSV IVb to cultures at 4 and 14 °C but not 26 °C led to cytopathic effects (CPE) and virus production. At 4 °C, virus production developed more slowly, but Western blotting showed more N protein accumulation. Infecting monolayer cultures at 4 °C for 7 days and then shifting them to 26 °C resulted in the monolayers being broken in small areas by CPE, but with time at 26 °C, the monolayers were restored. These results suggest that at 26 °C, the VHSV IVb life cycle stages responsible for CPE can be completed, but the production of virus and the initiation of infections cannot be accomplished.     

Evaluation of sodium percarbonate as a bath treatment for amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a major health challenge for Atlantic salmon aquaculture globally. While freshwater bathing for 2 hr is effective in reducing infection severity, there is need for more rapid and lower cost alternatives. To this end, a combination of sodium percarbonate (SPC) in freshwater was examined for its treatment efficacy. Initial in vitro studies showed a reduction in amoeba viability when exposed for 30 min to freshwater containing >500 mg/L SPC. Subsequently, AGD‐affected salmon were bathed for 30 min in 16°C freshwater containing 100, 500 or 1,000 mg/L SPC, or for 2 hr in 16°C freshwater to mimic industry practice. Treatment at the highest SPC concentration caused extensive gill damage and substantial mortality. Neither occurred to a significant extent at lower SPC concentrations. Gill pathology of surviving fish 10 days post‐treatment (dpt) was comparable to or more severe than pre‐treatment, and significantly (p < .001) more severe than in 2 hr freshwater bathed fish. N. perurans DNA was confirmed by qPCR in all treatment groups at 10 dpt. The data indicate that a 30‐min exposure to SPC in freshwater is not a suitable alternative to existing freshwater treatment of AGD.     

Low pathogenicity of flounder iridovirus (FLIV) and the absence of cross‐protection between FLIV and rock bream iridovirus

The genus Megalocytivirus is known to infect a wide range of cultured marine fish. In this study, we examined the pathogenicity of FLIV (Megalocytivirus from olive flounder, genotype III) and RBIV (Megalocytivirus from rock bream, genotype I) to their homologous and heterologous host species. Olive flounder (7.5 ± 1.3 cm) injected with FLIV [major capsid protein (MCP) gene copies, 6.8 × 10³–6.5 × 10⁶/fish] at 24 °C did not die until 90 days post‐infection (dpi). The average virus replication in the spleen peaked (1.27 × 10⁶/fish) at 20 dpi. Rock bream (6.5 ± 1.5 cm) injected with FLIV (8.8 × 10⁵ and 6.5 × 10⁶/fish of MCP copies) showed no mortality until 50 dpi. The rock bream that survived after FLIV infection were rechallenged with RBIV at 50 dpi had 100% mortality, showing that there is no cross‐protection between FLIV and RBIV. Temperature shifting (26 °C and 20 °C at 12 h intervals) did not cause FLIV‐specific mortality into olive flounder, but higher virus copies were observed in the fish exposed to higher stocking density. This study demonstrates that FLIV and RBIV have different antigenic and pathogenic characteristics and that FLIV has low pathogenicity to olive flounder.     

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p < .05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.     

Identification and pathogenicity study of emerging fish pathogens Acinetobacter junii and Acinetobacter pittii recovered from a disease outbreak in Labeo catla (Hamilton, 1822) and Hypophthalmichthys molitrix (Valenciennes, 1844) of freshwater wetland in West Bengal,India

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD₅₀ of 1.24 × 10⁵ and 1.88 × 10⁷ cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.     

Susceptibility of isogeneic ginbuna Carassius auratus langsdorfii Temminck et Schlegel to cyprinid herpesvirus‐2 (CyHV‐2) as a model species

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus‐2 (CyHV‐2), has affected the commercial production of the goldfish Carassius auratus and gibelio carp Carassius auratus gibelio. High water temperature treatments are reported to reduce the mortality rate of infected goldfish and elicit immunity in the survivors. To define the mechanism by which this intervention induces resistance, clonal ginbuna Carassius auratus langsdorfii, which is closely related to both species and has been used in fish immunology, may represent a promising model species. In this study, we investigated the susceptibility of clonal ginbuna strains to CyHV‐2 and the effect of high water temperature treatment on infected ginbuna and goldfish. Experimental intraperitoneal infection with CyHV‐2 at 25 °C caused 100% mortality in ginbuna strains, which was accompanied by histopathological changes typical of HVHN. Both infected ginbuna S3n strain and goldfish, exposed to high temperature for 6 days [shifting from 25 °C (permissive) to 34 °C (non‐permissive)], showed reduced mortalities after the 1st inoculation, and subsequent 2nd virus challenge to 0%, indicating induction of immunity. It was concluded that ginbuna showed a similar susceptibility and disease development in CyHV‐2 infection compared to goldfish, suggesting that ginbuna can be a useful fish model for the study of CyHV‐2 infection and immunity.     

Ca. Branchiomonas cysticola,Ca. Piscichlamydia salmonis and Salmon Gill Pox Virus transmit horizontally in Atlantic salmon held in fresh water

Elucidation of the role of infectious agents putatively involved in gill disease is commonly hampered by the lack of culture systems for these organisms. In this study, a farmed population of Atlantic salmon pre‐smolts, displaying proliferative gill disease with associated Candidatus Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Atlantic salmon gill pox virus (SGPV) infections, was identified. A subpopulation of the diseased fish was used as a source of waterborne infection towards a population of naïve Atlantic salmon pre‐smolts. Ca. B. cysticola infection became established in exposed naïve fish at high prevalence within the first month of exposure and the bacterial load increased over the study period. Ca. P. salmonis and SGPV infections were identified only at low prevalence in exposed fish during the trial. Although clinically healthy, at termination of the trial the exposed, naïve fish displayed histologically visible pathological changes typified by epithelial hyperplasia and subepithelial inflammation with associated bacterial inclusions, confirmed by fluorescent in situ hybridization to contain Ca. B. cysticola. The results strongly suggest that Ca. B. cysticola infections transmit directly from fish to fish and that the bacterium is directly associated with the pathological changes observed in the exposed, previously naïve fish.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.     

Effects of stocking density and feeding duration in cage‐cum‐pond‐integrated system on growth performance,water quality and economic benefits of Labeo victorianus (Boulenger 1901) culture

We evaluated the effect of varying cage stocking density (60, 90 and 120 fish m^?3) and feeding duration (10, 30 and 60 min) in a cage‐cum‐pond‐integrated system on growth performance, water quality and economic benefits in Labeo victorianus culture. Interactions between stocking density and feeding duration significantly (P < 0.05) affected the fish growth performance and yields in the cages‐cum‐pond system. Stocking density of 60 fish m^?3 resulted in the highest growth in cages and in ponds regardless of the feeding duration, but produced lower yields than at stocking density 90 fish m^?3_. The lowest Apparent Food Conversion Ratio (AFCR) in cages occurred at stocking density of 60 fish m^?3 and feeding duration of 30 min. Growth performance in the open ponds declined with increased feeding duration of the caged fish. Survival in cages and in the open ponds decreased with increased cage density, but was not affected by feeding duration. Low dissolved oxygen were recorded, at stocking density of 120 fish m^?3, the lowest DO occurred when feeding of caged fish lasted 60 min. Growth performance, water quality and economic benefits in Labeo victorianus culture positively respond to interaction between stocking density and feeding durations.     

Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia,Oreochromis niloticus (L.)

Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non‐A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport‐induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 × 10⁶ CFU fish^?1) and A. veronii (8.9 × 10⁶ CFU fish^?1) killed 100% of experimental fish within 24 h, while a 10‐fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100‐fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation.     

Effect of repeated exposure to AQUI‐S® on the viability and growth of Neoparamoeba perurans

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28‐day period to AQUI‐S^® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post‐smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non‐attached amoeba in the culture was assessed with a vital stain. AQUI‐S^® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.