Intracoelomic Injection Research Articles

Francisella sciaenopsi sp. nov. isolated from diseased red drum Sciaenops ocellatus in Florida, USA.

Piscine francisellosis is one of the most important bacterial diseases affecting various fish species worldwide. Francisella orientalis, F. noatunensis, and F. salimarina (F. marina) have been reported as etiological agents of disease in fish. A Francisella sp. was isolated from several diseased red drum Sciaenops ocellatus experiencing morbidity in Florida, USA, in 2008. In this study, molecular and phenotypic characterization of the recovered isolate was conducted. Phenotypically, the isolate showed a biochemical reaction profile distinct from that of F. orientalis and F. salimarina. Although the 16S rRNA sequence of this isolate shared 99.61% identity to the type strain of F. philomiragia O#319LT, whole genome analysis (average nucleotide identity <95%; digital DNA-DNA hybridization <70%) and a multilocus sequence analysis of 8 concatenated housekeeping genes in comparison with other Francisella spp. indicated that this isolate was a novel Francisella species, more closely related to F. orientalis. Immersion, intracoelomic injection, and co-habitation challenges using a Nile tilapia Oreochromis niloticus fingerling model of infection were done to investigate virulence in a piscine model. Variably pigmented granulomas and pigmented macrophage aggregates were observed in the kidneys and spleens of the challenged fish, but no mortality was recorded during the 15 d challenge period, suggesting that this novel Francisella sp. might be an opportunistic pathogen of fish. Based on the phenotypic and genotypic differences from other Francisella spp. observed in this study, we propose the name Francisella sciaenopsi sp. nov. for this novel isolate.

Pyrethroid-associated nephrotoxicity in channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus, at a public aquarium.

Over the course of an approximately 11-month period, an outdoor, freshwater, mixed species, recirculating, display system at a public aquarium experienced intermittent mortalities of channel catfish (Ictalurus punctatus) and blue catfish (I. furcatus). Catfish acutely presented for abnormal buoyancy, coelomic distention, and protein-rich coelomic effusion. Gross lesions typically involved massive coelomic distension with protein-rich effusion, generalized edema, and gastric hemorrhage and edema. Microscopically, primary lesions included renal tubular necrosis, gastric edema with mucosal hemorrhages, and generalized edema. Aerobic culture and virus isolation could not recover a consistent infectious agent. Intracoelomic injection of coelomic effusion and aspirated retrobulbar fluid from a catfish into naïve zebrafish (bioassay) produced peracute mortality in 3 of 4 fish and nervous signs in the fourth compared with 2 saline-injected control zebrafish that had - no mortality or clinical signs. Kidney tissue and coelomic effusion were submitted for gas chromatography tandem mass spectrometry by multiple reaction monitoring against laboratory standards, which detected the presence of multiple pyrethroid toxins, including bioallethrin, bifenthrin, trans-permethrin, phenothrin, and deltamethrin. Detection of multiple pyrethroids presumably reflects multiple exposures with several products. As such, the contributions of each pyrethroid toward clinical presentation, lesion development, and disease pathogenesis cannot be determined, but they are suspected to have collectively resulted in disrupted osmoregulation and fluid overload due to renal injury. Pesticide-induced toxicoses involving aquarium fish are rarely reported with this being the first description of pyrethroid-induced lesions and mortality in public aquarium-held fish.

Evaluation of three inactive vaccines against Veronaea botryosa infection in white sturgeon (Acipenser transmontanus)

Veronaea botryosa is the etiological agent of a systemic phaeohyphomycosis known as “fluid belly” in white sturgeon (Acipenser transmontanus). Fluid belly is a critical disease affecting sturgeon aquaculture and the caviar industry for which there are no commercially available vaccines or approved antifungal treatments to manage outbreaks. The primary aim of this study was to investigate the effect of a V. botryosa [conidia], a V. botryosa [mold], and a Saccharomyces cerevisiae [yeast] formalin-killed vaccine on sturgeon immune responses to fungal challenge. Immunization consisted of an initial intracoelomic injection with one of the three treatment preparations, followed by a vaccine booster four weeks later by the same route and dose. Experimental challenge by intramuscular injection with a virulent V. botryosa conidia suspension followed after another four weeks. Non-challenged control fish received injections of PBS. The inactivated vaccines proved safe for white sturgeon fingerlings. Sturgeon immunized with either V. botryosa [mold] or S. cerevisiae [yeast] exhibited a significantly different pro-inflammatory response upon challenge with V. botryosa compared to non-immunized fish. Challenged fish developed clinical signs similar to those reported during natural outbreaks of fluid belly. Positive control treatments (those not immunized but challenged with V. botryosa) experienced the highest mortality; however, survival curves were similar amongst all treatments (p < 0.05). Furthermore, the S. cerevisiae [yeast] vaccine resulted in comparatively lower fungal persistence and fewer lesions following histological analysis. Further efforts evaluating the potential of Saccharomyces spp. as a vaccine candidate against fluid belly are warranted.

SpaB-positive Erysipelothrix rhusiopathiae, a novel teleost pathogen isolated from cultured barramundi.

Members of the genus Erysipelothrix are emergent pathogens of cultured eels, as well as several characid and cyprinid species. Since 2013, E. rhusiopathiae has been reported from diseased barramundi (Lates calcarifer) cultured in North America; we recovered 8 E. rhusiopathiae isolates from diseased fish during different outbreaks from the same farm. The E. rhusiopathiae isolates from barramundi were compared phenotypically and genetically to E. piscisicarius isolates characterized from ornamental fish and E. rhusiopathiae recovered from aquatic and terrestrial animals. All barramundi isolates were PCR-positive for the surface protective antigen type B (spaB) gene, and shared ≥ 99.7% sequence similarity among concatenated multilocus sequence analysis gene sequences, indicating a high degree of genetic homogeneity. These isolates were > 99% similar to other spaB-positive isolates from marine invertebrates and marine mammals, consistent with findings for other spa types. The spaA and spaB isolates shared < 98% similarity, as well as < 90% similarity with spaC-positive E. piscisicarius. Similar clonality among the spaB isolates was observed using repetitive element palindromic PCR. In experimental intracoelomic injection challenges conducted to fulfill Koch postulates, 67% of exposed tiger barbs (Puntigrus tetrazona) died within 14 d of challenge. Our study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and the emergence of members of the genus Erysipelothrix as nascent fish pathogens.

Investigation of euthanasia techniques in four species of cockroaches

While cockroaches are commonly exhibited in zoos and museums, studied in research laboratories, and even kept as pets, scientifically based guidelines for their euthanasia are lacking. This study assessed euthanasia techniques in four species of cockroaches (Dubia (Blaptica dubia), red runner (Shelfordella lateralis), Madagascar hissing (Gromphadorhina portentosa), and giant cave (Blaberus giganteus)). In an initial pilot study, two hundred fifty adult Dubia cockroaches were exposed in groups of ten to a cotton ball soaked with 2 mL of isoflurane in a 1 L air-tight chamber. Thirty minutes beyond loss of any individual movement, groups were exposed to one of the following secondary treatments: freezing at −18 °C or −80 °C from 0.25 to 24 hours; immersion in 10% neutral buffered formalin, 70% isopropyl alcohol, or reverse osmosis water for 0.25 or 0.5 hours; or intracoelomic injection of potassium chloride (456 mEq/kg) or pentobarbital-based euthanasia solution (3.9 g/kg). A control group remained in the air-tight isoflurane chamber for 24 hours. Following all treatments, cockroaches were monitored for an additional 24 hours for spontaneous movement. Irreversible loss of movement was considered synonymous with irreversible loss of consciousness (death). Across all species, isoflurane anesthesia followed by either 70% isopropyl alcohol immersion for 0.25 or 0.5 hours or isoflurane exposure for 24 hours resulted in euthanasia in 100% of cockroaches. This study is the first evaluation of American Veterinary Medical Association-recommended euthanasia protocols in cockroaches.

POTASSIUM CHLORIDE ADMINISTERED VIA FOUR ROUTES FOR EUTHANASIA OF ANESTHETIZED GOLDFISH (CARASSIUS AURATUS).

Immersion overdose in tricaine methanesulfonate (MS-222) is ineffective for euthanasia of adult goldfish (Carassius auratus), and investigation of alternative techniques is warranted. This study evaluated potassium chloride (KCl) administered via four routes for goldfish euthanasia. Thirty clinically healthy adult goldfish (17.2-41.9 g) were anesthetized via immersion in buffered MS-222 (300 mg/L) for 5 min beyond anesthetic induction. Fish were then randomly administered one of five treatments: KCl (333 mg/mL, 4.5 mmol/mL, 4.5 mEq/mL) via intracardiac injection at 10 mEq/kg (IC), intracoelomic injection (ICe) or bilateral topical delivery over the gill filaments (T) at 100 mEq/kg, or 90-min immersion at 4,500 mEq/L (saturated solution) dissolved in induction solution (water [W]), or no treatment (control [C]). Following treatment, all fish were moved to anesthetic-free freshwater. Serial heart rates were collected via Doppler device until sound cessation or recovery. Median (range) time to perform treatments was 5.3 (1.2-8.0) min, 3 (3-3) s, and 10 (10-10) s in IC, ICe, and T, respectively. Doppler cessation occurred in 6/6, 6/6, 6/6, 6/6, and 0/6 fish in median (range) times of 3 (0-210), 18 (10-45), 118 (90-390), and 150 (60-240) min in IC, ICe, T, W, and C, respectively; this was significantly different between groups (P = 0.002). Following or during treatment, 1/6, 2/6, 6/6, and 4/6 fish in IC, ICe, T, and W, respectively, exhibited intermittent transient jerking movements. Median (range) time to recovery in C was 5.5 (3.5-6.5) min. All administration routes were effective, but time to Doppler cessation varied and transient movements were noted. Preliminary follow-up research determined that return to MS-222 induction solution following ICe KCl treatment abolished movements. ICe KCl at 100 mEq/kg was technically simple and resulted in Doppler sound cessation in a clinically relevant time frame in 6/6 anesthetized goldfish. Return to MS-222 immersion following KCl administration is recommended.

Investigation of Potassium Chloride for Euthanasia of Anesthetized African Clawed Frogs (Xenopus laevis).

Euthanasia is frequently performed in amphibians, but techniques are currently limited in number and variable in effectiveness. The current study examined the use of potassium chloride (KCl) for euthanasia of anesthetized African clawed frogs (Xenopus laevis). Twenty adult, female African clawed frogs were anesthetized by immersion in buffered tricaine methanesulfonate (MS-222) for 5 min beyond loss of righting reflex. Frogs were then randomly assigned to receive one of 4 treatments: KCl via intracardiac injection (10 mEq/kg; IC, n = 5), intracoelomic injection (100 mEq/kg; ICe, n = 5), immersion (4,500 mEq/L; IMS, n = 5), or no treatment (C, n = 5). After treatment, serial heart rate was measured via Doppler device until either the loss of Doppler sounds, a 60-min endpoint (IC, ICe, IMS), or recovery (C). Times to loss of righting reflex, loss of Doppler sounds, and/or recovery were recorded. Plasma potassium concentrations were measured immediately after Doppler sound cessation in frogs in IC (n = 1), ICe (n = 2), and IMS (n = 5). Injection failure occurred in 1 IC frog, and 1 Ice frog regained spontaneous movement 4 min after treatment administration. Data from these 2 frogs were not included in statistical analysis. Doppler sound cessation occurred in 4 of 4, 4 of 4, 0 of 5, and 0 of 5 frogs in IC, ICe, IMS, and C, respectively. Median (range) times to Doppler sound cessation in IC and ICe were 6 (0 to 16) s and 18 (10 to 25) min, respectively. Plasma potassium concentration was greater than 9.0 mmol/L in sampled frogs. Intracardiac KCl at 10 mEq/kg and intracoelomic KCl at 100 mEq/kg were effective for euthanasia of anesthetized African clawed frogs. Return to MS-222 solution after KCl administration may be warranted to prevent unintended, premature anesthetic recovery prior to death.

Comparative Evaluation of Booster Vaccine Efficacy by Intracoelomic Injection and Immersion with a Whole-Cell Killed Vaccine against Lactococcus petauri Infection in Rainbow Trout (Oncorhynchus mykiss).

Lactococcus petauri is an important emergent bacterial pathogen of salmonids in the USA. The purpose of this study was to evaluate the protection conferred to rainbow trout (Oncorhynchus mykiss) against L. petauri by formalin-killed vaccines in immersion and injectable forms, as well as the enhanced protection afforded by booster vaccination. In the first challenge, fish were immunized via intracoelomic injection (IC) or immersion (Imm) routes alone. Approximately 418 degree days (Temperature in degree Celsius × days post-immunization) (dd) Imm, or 622 dd IC post-vaccination, fish were challenged via IC with wild-type L. petauri. In the second experiment, initial Imm vaccination was followed by booster vaccination via Imm or IC routes 273 dd post-immunization along with appropriate PBS controls. The various vaccination protocol efficacies were evaluated by challenging fish with L. petauri by cohabitation with diseased fish 399 dd post-booster administration. A relative percent survival (RPS) of 89.5% and 28% was recorded in the IC and Imm single immunization treatments, respectively. In the second study, an RPS of 97.5%, 10.2%, 2.6% and -10.1% plus approximately 0%, 50%, 20%, and 30% bacterial persistence was recorded in the Imm immunized + IC boosted, Imm immunized + mock IC boosted, Imm immunized + Imm boosted, and Imm immunized + mock Imm boosted treatments, respectively. Only the Imm immunized + IC injection boosted treatments provided significant protection when compared to unvaccinated and challenged treatments (p < 0.05). In conclusion, although both Imm and IC vaccines appear safe for trout, the inactivated Imm vaccines seem to provide only mild and temporary protection against lactococcosis; whereas IC immunized trout develop a significantly stronger protective response in both challenges.

Tilapia lake virus (TiLV) causes severe anaemia and systemic disease in tilapia.

Tilapia lake virus disease (TiLVD) is an emerging disease in tilapia that is associated with mass mortality affecting global tilapia aquaculture. In this study, red hybrid tilapias (Oreochromis spp.) were experimentally infected by intracoelomic injection with Tilapia lake virus (TiLV) to gain a better understanding of the clinicopathological changes during infection. Pale bodies and gill were observed in infected fish after 7 days of post-challenge (dpc) associated with severe anaemia. Further haematological analysis in TiLV-infected fish revealed decreased levels of haemoglobin and haematocrit at 3 dpc. Common pathological findings included pale and friable liver, pale intestine with catarrhal content, and dark and shrunken spleen in TiLV-infected fish at 7 dpc and 14 dpc. Histologically, reduced numbers of red blood cells and accumulation of melano-macrophage centre in the spleen were found in infected fish at 3 dpc, and severe lesions were more commonly observed at 7 and 14 dpc. Lymphocyte infiltration, syncytial cell formation and multifocal necrotic hepatitis were the prominent pathological findings in the liver of infected fish. The severity of pathological changes was associated with TiLV-infection with higher viral loads and with the expression pattern of pro-inflammatory cytokines and antiviral genes, including interferon regulatory factor 1 (irf1), interleukin (il-8), radical s-adenosyl methionine domain containing 2 (rsad2) and mx. Our study provides a comprehensive analysis of the haematological profile and pathological changes in tilapia during TiLV infection. Overall, lesions present in various organs, together with alteration of host immune response in TiLV-infected fish, indicate the systemic infection of this virus. The knowledge gained from this study improves our understanding of how TiLV causes pathological and haematological changes in tilapia.

Temperature-associated virulence, species susceptibility and interspecies transmission of a Lactococcus petauri strain from rainbow trout.

Lactococcus petauri is an important emergent aquaculture pathogen in the USA. To better understand environmental conditions conducive to piscine lactococcosis and the susceptibility of fish species, laboratory-controlled challenges were used as models of infection. Rainbow trout Oncorhynchus mykiss maintained at 13 or 18°C were challenged by intracoelomic (ICe) injection with 101, 103 or 105 colony-forming units per fish (CFU fish-1) and monitored for 21 d. At 13°C, trout experienced mortalities of 7, 7 and 0%, and bacterial persistence of 0, 20 and 0% in survivors, respectively. When exposed to the same bacterial doses, trout maintained at 18°C experienced mortalities of 59, 84 and 91%, and bacterial persistence of 60, 66 and 0% in survivors, confirming a significant role of temperature in the pathogenesis of lactococcosis. Additionally, the susceptibility of rainbow trout, Chinook salmon Oncorhynchus tshawytscha, white sturgeon Acipenser transmontanus, Nile tilapia Oreochromis niloticus, and koi Cyprinus carpio to infection by L. petauri was compared using ICe challenges at 18°C. Trout and salmon experienced 96 and 56% cumulative mortality, respectively, and 17% of surviving salmon remained persistently infected. There were no mortalities in the other fish species, and no culturable bacteria recovered at the end of the challenge. However, when surviving fish were used in further cohabitation trials, naïve trout housed with previously exposed tilapia exhibited 6% mortality, demonstrating that non-salmonids can become sub-clinical carriers of this pathogen. The data obtained provide useful information regarding temperature-associated virulence, fish species susceptibility, and potential carrier transmission of L. petauri that can be used in the development of better management practices to protect against piscine lactococcosis.

Efficacy of a multidose acyclovir protocol against cyprinid herpesvirus 3 infection in koi (Cyprinus carpio).

To evaluate the effect of a multidose acyclovir protocol on koi herpesvirus (KHV) viral load and mortality in a cohabitation challenge. 180 koi fish. Forty fish (shedders) were immersed in a 0.5 KHV plaque-forming units/mL static bath for 8 hours. Mock shedders were treated similarly but exposed to cell culture media. KHV shedders were then transferred into 8 tanks (5 shedders per tank) containing 10 naïve fish (cohabitants) each. Fish in the acyclovir group (AT) received a 10 mg/kg acyclovir intracoelomic injection 1, 3, and 6 days after the first confirmed KHV mortality. Positive controls (PC) were treated similarly but received sterile saline injections. Negative controls (NC) were exposed to mock shedders. Morbidity and mortality were evaluated daily for 50 days post-challenge. Quantitative PCR was used to determine viral load in the gill biopsies of shedders and cohabitants collected at days 19 (T1), 22 (T2), 25 (T3), 34 (T4), and 50 (T5) post-challenge. Survival curves analyzed by the Gehan-Breslow-Wilcoxon method revealed a delayed onset of mortalities and a significantly lower KHV load at T2 and T3 detected in AT cohabitant fish (P = .042) compared to PC group. However, there were no significant differences in overall mortality or viral loads at T5. The acyclovir protocol used in this study did not control viral infection or mortality at the end of the 50-day trial. Shorter intervals between injections could improve outcomes, but the additional stress inflicted by handling should be considered. Exploring other therapeutic alternatives and doses is warranted.

Cross-protective efficacy of a live-attenuated Edwardsiella ictaluri vaccine against heterologous Edwardsiella piscicida isolates in channel and channel × blue catfish hybrids.

Edwardsiella piscicida is a growing problem for catfish aquaculture in the southeastern United States, particularly in channel (Ictalurus punctatus) x blue (I. furcatus) catfish hybrids. Research has shown E.piscicida isolates recovered from farmed catfish in Mississippi form at least five discrete phyletic groups, with no apparent differences in virulence in channel and hybrid catfish. Laboratory trials have shown a live-attenuated E.ictaluri vaccine (340X2) cross-protects against at least one E.piscicida isolate (S11-285) in channel and hybrid catfish, although it is unknown if this protection exists for other E.piscicida variants. To this end, channel and hybrid catfish were immunized by immersion with E.ictaluri 340X2. Thirty days later, fish were challenged by intracoelomic injection with representative E.piscicida variants from each phyletic group. Relative percent survival (RPS) for hybrids ranged from 54.7% to 77.8%, while RPS in channels ranged from 80.5% to 100%. A second study investigated whether channel and hybrid catfish exposed to heterologous E.piscicida isolates were similarly protected against wild-type E.ictaluri. Fish were exposed by bath immersion to representative E.piscicida isolates from each phyletic group. Thirty days post-immunization, fish were challenged by immersion with wild-type E.ictaluri isolate S97-773. Regardless of variant, previous exposure to heterologous E.piscicida isolates significantly improved survival following E.ictaluri challenge. These findings suggest the presence of shared and conserved antigens among E.piscicida and E.ictaluri that could be exploited by application of polyvalent or cross-protective vaccines.

Development and efficacy of Streptococcus iniae live-attenuated vaccines in Nile tilapia, Oreochromis niloticus

Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n = 9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and WT strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p < 0.05). Selected strains also showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated cross-reactivity against heterologous bacteria. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival up to 95.05%.

Virulence, immunogenicity and live vaccine potential of aroA and phoP mutants of Edwardsiella piscicida in zebrafish

Vaccination remains the most effective approach for prevention and control of infectious diseases in aquaculture. Edwardsiella piscicida is a causative agent of edwardsiellosis leading to mass mortality in a variety of fish species, leading to huge economic losses in the aquaculture industry. In this study, we have deleted the aroA and phoP genes in E. piscicida and investigated the phenotype, degrees of attenuation, immunogenicity, and ability to confer immune protection in zebrafish host. Our vaccine strain χ16028 with genotype ΔaroA11 ΔphoP12, showed significantly reduced growth, motility, biofilm formation and intracellular replication compared to the wild-type strain J118. In this regard, χ16028 exhibited retarded colonization and attenuation phenotype in zebrafish. Studies showed that χ16028 induced TLR4 and TLR5 mediated NF-kB pathway and upregulated cytokine gene expression i.e., TNF-α, IL-1β, IL-6, IL-8 and type-I IFN in zebrafish. Zebrafish immunized by intracoelomic injection (i.c.) with χ16028 showed systemic and mucosal IgM responses and protection against the wild-type E. piscicida i.c. injection challenge. However, the protection was only 25% in zebrafish following i.c. challenge. We speculate that our vaccine strain might be very attenuated; a booster dose may trigger better immune response and increase the percentage of survival to a more significant level.

Isolation, Identification and Characterization of a Novel Megalocytivirus from Cultured Tilapia (Oreochromis spp.) from Southern California, USA.

Simple SummaryAquaculture is the world’s fastest-growing food production sector, with tilapia (Oreochromis spp.) among the most important cultured species. First reported in red seabream, Pagrus major, in 1990, an increasing number of megalocytiviruses are responsible for losses that threaten the production and economic sustainability of important cultured fish species, including tilapia. In the current study, we describe an epizootic in tilapia fingerlings from Southern California, USA and report the discovery of a novel megalocytivirus associated with the mortalities. In spring 2019, diseased four-month-old tilapia (Oreochromis spp.) from an aquaculture farm in Southern California, USA were received for diagnostic evaluation with signs of lethargy, anorexia, abnormal swimming, and low-level mortalities. At necropsy, non-specific external lesions were noted including fin erosion, cutaneous melanosis, gill pallor, and coelomic distension. Internal changes included ascites, hepatomegaly, renomegaly, splenomegaly, and multifocal yellow-white nodules in the spleen and kidney. Cultures of spleen and kidney produced bacterial colonies identified as Francisella orientalis. Homogenized samples of gill, brain, liver, spleen, and kidney inoculated onto Mozambique tilapia brain cells (OmB) developed cytopathic effects, characterized by rounding of cells and detaching from the monolayer 6–10 days post-inoculation at 25 °C. Transmission electron microscopy revealed 115.4 ± 5.8 nm icosahedral virions with dense central cores in the cytoplasm of OmB cells. A consensus PCR, targeting the DNA polymerase gene of large double-stranded DNA viruses, performed on cell culture supernatant yielded a sequence consistent with an iridovirus. Phylogenetic analyses based on the concatenated full length major capsid protein and DNA polymerase gene sequences supported the tilapia virus as a novel species within the genus Megalocytivirus, most closely related to scale drop disease virus and European chub iridovirus. An intracoelomic injection challenge in Nile tilapia (O. niloticus) fingerlings resulted in 39% mortality after 16 days. Histopathology revealed necrosis of head kidney and splenic hematopoietic tissues.

Isolation and characterization of Lactococcus garvieae from rainbow trout, Onchorhyncus mykiss, from California, USA.

Lactococcus garvieae is an emergent bacterial pathogen of salmonid fish in North America that causes acute infections particularly at water temperatures above 15°C. During 2020, L. garvieae was detected in rainbow trout, Onchorhyncus mykiss, cultured in Southern California and the Eastern Sierras. Infected fish exhibited high mortalities and nonspecific clinical signs of lethargy, erratic swimming, dark skin pigmentation, and exophthalmia. Macroscopic changes included external and internal hemorrhages, mainly in the eyes, liver, coelomic fat, intestine, and brain. Histological examination revealed splenitis, branchitis, panophthalmitis, hepatitis, enteritis, and coelomitis, with variable degrees of tissue damage among evaluated fish. Pure colonies of L. garvieae were isolated from infected trout and specific PCR primers for L. garvieae confirmed the preliminary diagnosis. Multilocus sequence analysis showed that the strains recovered from diseased trout represent a novel genetic group. Isolates were able to form biofilms within 24 h that increased their resistance to disinfection by hydrogen peroxide. Laboratory challenge methods for inducing lactococcosis in steelhead trout, O. mykiss, were evaluated by intracoelomic injection with serial dilutions of L. garvieae. The median lethal dose 21 days post challenge was ∼20 colony-forming units/fish. Experimentally infected trout presented similar clinical signs, gross changes, and microscopic lesions as those with natural disease, fulfilling Koch's postulates and demonstrating the high virulence of the recovered strains.

EVALUATION OF POTASSIUM CHLORIDE ADMINISTERED VIA THREE ROUTES FOR EUTHANASIA OF ANESTHETIZED KOI (CYPRINUS CARPIO ).

Immersion in tricaine methanesulfonate (MS-222) is insufficient for euthanasia in at least one species of fish. The current study investigated the effectiveness of potassium chloride (KCl) to euthanize anesthetized koi (Cyprinus carpio). Twenty-eight healthy koi were anesthetized via immersion in 500 mg/L of buffered MS-222 for 10-12 min, manually removed to room air, and randomly administered 10 mEq/kg KCl (333 mg/ml) via one of three routes-intracardiac injection (IC) (n = 7), intracoelomic injection (ICe) (n = 7), or topical instillment over the gill filaments bilaterally (T) (n = 7)-or received no treatment (control, C) (n = 7). A Doppler ultrasonic flow detector was placed over the heart, and sounds were assessed continuously from immediately prior to treatment until 5 min posttreatment and every 5 min thereafter until Doppler sound cessation, resumption of operculation, or 30 min. Time to Doppler sound cessation or resumption of operculation was recorded. Doppler sound cessation occurred in 7/7 fish in IC (median 0.08, range 0-2.75 min) and 1/7 fish in T (10 min). In T, ICe, and C, 6/7, 7/7, and 7/7 fish, respectively, maintained Doppler sounds to 30 min. All fish in ICe (7/7) and C (7/7) resumed operculation with median (range) times of 22 (7-30) min and 16 (9-29) min from treatment, respectively. Intracardiac KCl at 10 mEq/kg rapidly ceases Doppler sounds and is a successful technique for euthanasia of anesthetized koi. Intracoelomic and topical KCl at 10 mEq/kg were not effective for euthanasia of koi.

First Isolation of a Novel Aquatic Flavivirus from Chinook Salmon (Oncorhynchus tshawytscha) and Its In Vivo Replication in a Piscine Animal Model.

The first isolation of a flavivirus from fish was made from moribund Chinook salmon (Oncorhynchus tshawytscha) from the Eel River, California, USA. Following the observation of cytopathic effect in a striped-snakehead fish cell line, 35-nm virions with flaviviral morphology were visualized using electron microcopy. Next-generation sequencing and rapid amplification of cDNA ends obtained the complete genome. Reverse transcriptase quantitative PCR (RT-qPCR) confirmed the presence of viral RNA in formalin-fixed tissues from the wild salmon. For the first time, in vivo replication of an aquatic flavivirus was demonstrated following intracoelomic injection in a Chinook salmon model of infection. RT-qPCR demonstrated viral replication in salmon brains up to 15 days postinjection. Infectious virus was then reisolated in culture, fulfilling Rivers' postulates. Only limited replication occurred in the kidneys of Chinook salmon or in tissues of rainbow trout (Oncorhynchus mykiss). The proposed salmon flavivirus (SFV) has a 10.3-kb genome that encodes a rare dual open reading frame, a feature uncharacteristic of classical flaviviruses. Phylogenetic analysis places SFV in a basal position among a new subgroup of recently recognized aquatic and bat flaviviruses distinct from the established mosquito-borne, tick-borne, insect-only, and unknown-vector flavivirus groups. While the pathogenic potential of the virus remains to be fully elucidated, its basal phylogeny and the in vivo infection model will allow SFV to serve as a prototype for aquatic flaviviruses. Ongoing field and laboratory studies will facilitate better understanding of the potential impacts of SFV infection on ecologically and economically important salmonid species.IMPORTANCE Chinook salmon are a keystone fish species of great ecological and commercial significance in their native northern Pacific range and in regions to which they have been introduced. Threats to salmon populations include habitat degradation, climate change, and infectious agents, including viruses. While the first isolation of a flavivirus from wild migrating salmon may indicate an emerging disease threat, characterization of the genome provides insights into the ecology and long evolutionary history of this important group of viruses affecting humans and other animals and into an expanding group of recently discovered aquatic flaviviruses.

Cross‐protective potential of a live‐attenuated Edwardsiella ictaluri vaccine against Edwardsiella piscicida in channel (Ictalurus punctatus) and channel × blue (Ictalurus furcatus) hybrid catfish

AbstractEdwardsiella piscicida has emerged as an important pathogen of channel × blue catfish hybrids cultured in the southeastern United States. With increasing popularity of hybrid catfish and growing concern over E. piscicida, the crossprotective potential of a live‐attenuated E.ictaluri vaccine was evaluated. An immunization bath of E. piscicida (3 × 107 CFU/ml) protected channel catfish against immersion challenge of wild‐type E. ictaluri (1 × 107 CFU/ml) across multiple temperatures and feeding regimens (relative percent survival [RPS]: 89–100%). Channel catfish immunized orally with a live‐attenuated E. ictaluri vaccine/feed admix (3 × 107 CFU/g of feed) were protected against intracoelomic injections of both high (HD) (7.4x105 CFU/fish; RPS = 85%) and low doses (LD) (1.5 × 105 CFU/fish; RPS = 88%) of E. piscicida. Because of limited feed consumption during vaccination, oral immunization of hybrids was followed by a 30‐min immersion bath with cultured vaccine (5 × 106 CFU/ml). Combinatory oral/bath vaccination was ineffective against HD E. piscicida but was protective against LD E. piscicida (48% RPS) and E. ictaluri (96% RPS). Finally, naïve catfish that survived LD E. piscicida injections were protected against E. ictaluri immersion challenge (2 × 107 CFU/ml; RPS: Hybrids 55%; Channels 75%). Results suggest a live‐attenuated E. ictaluri vaccine can also protect channel and hybrid catfish against E. piscicida.

Xenobiotics Result in Hormonal and Enzymatic Dysregulations in the Red Mussel Mytilus galloprovincialis (Lamarck, 1819) (Bivalvia, Mytilidae)

&lt;b&gt;Background and Objective:&lt;/b&gt; The contaminants in a marine ecosystem like mercury and synthetic hormones can disrupt the regulation of natural endocrine and reproductive systems of most organisms. This study aims to study the effect of organic and inorganic mercury on the viscera of &lt;i&gt;Mytilus galloprovincialis&lt;/i&gt; after intracoelomic injection of 17α-ethinylestradiol, 17β-estradiol and Dichlorodiphenyltrichloroethane (DDT) and check the histological changes in the gonads. &lt;b&gt;Materials and Methods:&lt;/b&gt; Mussels are collected during June-August, 2018 from Ras el tin beach of the Mediterranean Sea of Alexandria, Egypt. This study aims to: test the effect of 17α-ethinylestradiol, 17β-estradiol and DDT on vitellogenin (VTG) synthesis, enzymes dysfunction through intracoelomic injection of methyl mercury in a 0.75 μg/0.1 mL and mercury chloride to a 75 μg/0.1 mL. Gonads are studied histologically in control and treated mussels. Water-administered E2 and EE2 at 120 μL dose induced VTG expression in males 14 days exposure. &lt;b&gt;Results:&lt;/b&gt; The relative concentration of VTG in the induced groups increases significantly as compared to the control. Alterations in the gonadal tissues and the maturation stages of the mussels are observed. The imposex mussels are characterized by concomitant secondary male sexual characteristics and the female gonad shows testicular structure. Superoxide Dismutase (SOD) activity in mussel digestive glands differed significantly (p = 0.002) after 72 hrs of MeHg exposure. &lt;b&gt;Conclusion:&lt;/b&gt; Significant correlation can be observed between the activities of Glutathione S-Transferases (GST) and Glutathione Reductase (GR) in the digestive glands of mussels treated with MeHg, the enzyme activities of digestive glands treated with HgCl&lt;sub&gt;2&lt;/sub&gt; and between Superoxide Dismutase&lt;i&gt;-&lt;/i&gt;Catalase (SOD-CAT), SOD-GR and GST-GR.