Acipenserid herpesvirus 2 genome and partial validation of a qPCR for its detection in white sturgeon Acipenser transmontanus.

White sturgeon Acipenser transmontanus is the primary species used for caviar and sturgeon meat production in the USA. An important pathogen of white sturgeon is acipenserid herpesvirus 2 (AciHV-2). In this study, 4 archived isolates from temporally discrete natural outbreaks spanning the past 30 yr were sequenced via Illumina and Oxford Nanopore Technologies platforms. Assemblies of approximately 134 kb were obtained for each isolate, and the putative ATPase subunit of the terminase gene was selected as a potential quantitative PCR (qPCR) target based on sequence conservation among AciHV-2 isolates and low sequence homology with other important viral pathogens. The qPCR was repeatable and reproducible, with a linear dynamic range covering 5 orders of magnitude, an efficiency of approximately 96%, an R2 of 0.9872, and an analytical sensitivity of 103 copies per reaction after 35 cycles. There was no cross-reaction with other known viruses or closely related sturgeon species, and no inhibition by sturgeon DNA. Clinical accuracy was assessed from white sturgeon juveniles exposed to AciHV-2 by immersion. Viral culture (gold standard) and qPCR were in complete agreement for both cell culture negative and cell culture positive samples, indicating that this assay has 100% relative accuracy compared to cell culture during an active outbreak. The availability of a whole-genome sequence for AciHV-2 and a highly specific and sensitive qPCR assay for detection of AciHV-2 in white sturgeon lays a foundation for further studies on host-pathogen interactions while providing a specific and rapid test for AciHV-2 in captive and wild populations.

DEXMEDETOMIDINE AND MIDAZOLAM INTRAMUSCULAR SEDATION IN BROWNBANDED BAMBOO SHARKS (CHILOSCYLLIUM PUNCTATUM).

Bamboo sharks are some of the most common elasmobranch species in zoos and aquaria and are frequently sedated for medical exams, treatments, and research. This study investigated the use of an IM sedation protocol of a single dose of dexmedetomidine (0.05 mg/kg) and midazolam (2.0 mg/kg) in brownbanded bamboo sharks (Chiloscyllium punctatum). Sharks were serially monitored every 5 min for heart rate, branchial beats, righting reflex, coelomic response, cloacal response, pelvic fin reflex, response to noxious stimulus, voluntary movement, and ability to swim. This sedation dose was effective at rapidly and significantly decreasing responses to tactile and noxious stimuli with minimal respiratory depression and was quickly reversible with atipamezole (0.5 mg/kg) and flumazenil (0.05 mg/kg). Sedated sharks developed a mild metabolic acidosis evidenced by a significant increase in lactic acid (mean < 0.37 mmol/L presedation, 4.2 mmol/L after reversal) and decrease in blood pH (mean 7.464 presedation, 7.277 after reversal); however, clinical intervention was not required. This protocol should be further investigated in different elasmobranch species but is promising for providing sedation for noninvasive procedures in brownbanded bamboo sharks.

Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

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

OBJECTIVE: To evaluate the effect of a multidose acyclovir protocol on koi herpesvirus (KHV) viral load and mortality in a cohabitation challenge. ANIMALS: 180 koi fish. PROCEDURES: 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. RESULTS: 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. CLINICAL RELEVANCE: 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.

Pharmacokinetic and Efficacy Study of Acyclovir Against Cyprinid Herpesvirus 3 in Cyprinus carpio.

Cyprinid Herpesvirus 3 (CyHV-3), more commonly known as Koi Herpesvirus (KHV), is a re-emergent virus causing acute systemic infection with high mortality rates in koi fish (Cyprinus carpio). Survivors from outbreaks can become latent carriers, with viral reactivation under stressful conditions and permissible temperatures. No vaccines or treatments are currently available in the United States. Acyclovir has been shown effective in vitro against KHV. This study aimed to evaluate the cytotoxicity of acyclovir and cidofovir to koi fin (KF1) cells, the efficacy of a single antiviral intracoelomic dose in a koi fingerling cohabitation challenge, and the pharmacokinetics of the effective antiviral. Initially, a lactate dehydrogenase release-based assay revealed no significant acyclovir or cidofovir cytotoxicity to KF1 cells for 24 h at up to 1,500 µM. In laboratory-controlled challenges, KHV associated mortalities occurred 2 weeks post-infection. At this point, fish were treated with an antiviral (10 mg/kg acyclovir or 5 mg/kg cidofovir) or sterile phosphate-buffered solution. Morbidity and mortality were monitored for 30 days. A significant cumulative mortality reduction (p ≤ 0.05), and a 3-day mortality delay were detected in the acyclovir-treated group. Similar viral loads were detected in gills recovered from mortalities throughout the challenge and surviving fish at the end of the challenge regardless of treatment. For pharmacokinetic analysis, blood was collected at various timepoints after acyclovir administration. Liquid chromatography tandem mass spectrometry plasma analysis indicated a 141 µM peak plasma concentration at 0.75 h, a 14 h half-life, and a 0.05/h elimination rate constant. Histopathology of target tissues detected no evidence of acyclovir toxicity. Results suggest that a single 10 mg/kg dose of acyclovir administered intracoelomically to koi fingerlings is safe and reduces cumulative mortality during a KHV mortality event. However, multiple doses are probably required for effective treatment of pet fish.