Phylogenomic Characterization of Ranavirus Isolated from Wild Smallmouth Bass (Micropterus dolomieu).

In September 2021, 14 smallmouth bass (SMB; Micropterus dolomieu) with skin lesions were collected from Green Bay waters of Lake Michigan and submitted for diagnostic evaluation. All the skin samples tested positive for largemouth bass virus (LMBV) by conventional PCR. The complete genome of the LMBV (99,328 bp) isolated from a homogenized skin sample was determined using an Illumina MiSeq sequencer. A maximum likelihood (ML) phylogenetic analysis based on the 21 core iridovirus genes supported the LMBV isolated from SMB (LMBV-WVL21117) as a member of the species Santee-Cooper ranavirus. Pairwise nucleotide comparison of the major capsid protein (MCP) gene showed that LMBV-WVL21117 is identical to other LMBV reported from the United States and nearly identical to doctor fish virus and guppy virus 6 (99.2%) from Southeast Asia, as well as LMBV isolates from China and Thailand (99.1%). In addition, ML phylogenetic analysis based on the MCP gene suggests three genotypes of LMBV separated by region: genotype one from the United States, genotype two from Southeast Asia, and genotype three from China and Thailand. Additional research is needed to understand the prevalence and genetic diversity of LMBV strains circulating in wild and managed fish populations from different regions.

Mycobacterium kansasii Infection in a Farmed White-Tailed Deer (Odocoileus virginianus) in Florida, USA.

A 7-year-old farmed white-tailed deer doe was transported to a Levy County, Florida property and began to decline in health, exhibiting weight loss and pelvic limb weakness. The doe prematurely delivered live twin fawns, both of which later died. The doe was treated with corticosteroids, antibiotics, gastric cytoprotectants, and B vitamins but showed no improvement. The doe was euthanized, and a post mortem examination was performed under the University of Florida's Cervidae Health Research Initiative. We collected lung tissue after the animal was euthanized and performed histological evaluation, using H&E and Ziehl-Neelsen (ZN) staining, and molecular evaluation, using conventional PCR, followed by Sanger sequencing. The microscopic observations of the H&E-stained lung showed multifocal granuloma, while the ZN-stained tissue revealed low numbers of beaded, magenta-staining rod bacteria inside the granuloma formation. Molecular analysis identified the presence of Mycobacterium kansasii. This isolation of a non-tuberculous Mycobacterium in a white-tailed deer emphasizes the importance of specific pathogen identification in cases of tuberculosis-like disease in farmed and free-ranging cervids. We report the first case of M. kansasii infection in a farmed white-tailed deer (Odocoileus virginianus) in Florida. Although M. kansasii cases are sporadic in white-tailed deer, it is important to maintain farm biosecurity and prevent farmed cervids from contacting wildlife to prevent disease transmission.

Genomic characterization of two ranavirus isolates identified from a gopher frog (Lithobates capito) and a striped newt (Notophthalmus perstriatus) during a mass mortality event in Florida.

Two ranavirus isolates were recovered from anuran and salamander samples collected during an amphibian mass mortality event in North-Central Florida in 2021. Phylogenetic analyses of the full genomes confirmed that the two isolates were nearly identical and strains of the species Frog virus 3.

Convolutional Neural Networks in the Inspection of Serrasalmids (Characiformes) Fingerlings.

Aquaculture produces more than 122 million tons of fish globally. Among the several economically important species are the Serrasalmidae, which are valued for their nutritional and sensory characteristics. To meet the growing demand, there is a need for automation and accuracy of processes, at a lower cost. Convolutional neural networks (CNNs) are a viable alternative for automation, reducing human intervention, work time, errors, and production costs. Therefore, the objective of this work is to evaluate the efficacy of convolutional neural networks (CNNs) in counting round fish fingerlings (Serrasalmidae) at different densities using 390 color photographs in an illuminated environment. The photographs were submitted to two convolutional neural networks for object detection: one model was adapted from a pre-trained CNN and the other was an online platform based on AutoML. The metrics used for performance evaluation were precision (P), recall (R), accuracy (A), and F1-Score. In conclusion, convolutional neural networks (CNNs) are effective tools for detecting and counting fish. The pre-trained CNN demonstrated outstanding performance in identifying fish fingerlings, achieving accuracy, precision, and recall rates of 99% or higher, regardless of fish density. On the other hand, the AutoML exhibited reduced accuracy and recall rates as the number of fish increased.

A novel bluetongue virus serotype 2 strain isolated from a farmed Florida white-tailed deer (Odocoileus virginianus) arose from reassortment of gene segments derived from co-circulating serotypes in the Southeastern USA.

Bluetongue disease is a reportable animal disease that affects wild and farmed ruminants, including white-tailed deer (WTD). This report documents the clinical findings, ancillary diagnostics, and genomic characterization of a novel reassortant bluetongue virus serotype 2 (BTV-2) strain isolated from a dead Florida farmed WTD in 2022. Our analyses support that this BTV-2 strain likely stemmed from the acquisition of genome segments from co-circulating BTV strains in Florida and Louisiana. In addition, our analyses also indicate that genetically uncharacterized BTV strains may be circulating in the Southeastern USA; however, the identity and reassortant status of these BTV strains cannot be determined based on the VP2 and VP5 genome sequences. Hence, continued surveillance based on complete genome characterization is needed to understand the genetic diversity of BTV strains in this region and the potential threat they may pose to the health of deer and other ruminants.

Ranavirus (Frog Virus 3) Infection in Free-Living Three-Toed Box Turtles (Terrapene mexicana triunguis) in Missouri, USA.

Frog virus 3 (FV3) and related ranaviruses are emerging infectious disease threats to ectothermic vertebrate species globally. Although the impact of these viruses on amphibian health is relatively well studied, less is understood about their effects on reptile health. We report two cases of FV3 infection, 11 mo apart, in three-toed box turtles (Terrapene mexicana triunguis) from a wildlife rehabilitation center. Case 1 had upper respiratory signs upon intake but had no clinical signs at the time of euthanasia 1 mo later. Case 2 presented for vehicular trauma, had ulcerative pharyngitis and glossitis, and died overnight. In case 1, we detected FV3 nucleic acid with qPCR in oral swabs, kidney, liver, spleen, and tongue. In case 2, we detected FV3 in an oral swab, an oral plaque, heart, kidney, lung, liver, spleen, and tongue. We also detected FV3 nucleic acid with in situ hybridization for case 2. For both cases, FV3 was isolated in cell culture and identified with DNA sequencing. Histopathologic examination of postmortem tissue from case 1 was unremarkable, whereas acute hemorrhagic pneumonia and splenic necrosis were noted in case 2. The difference in clinical signs between the two cases may have been due to differences in the temporal course of FV3 disease at the time of necropsy. Failure to detect this infection previously in Missouri reptiles may be due to lack of surveillance, although cases may also represent a novel spillover to box turtles in Missouri. Our findings reiterate previous suggestions that the range of FV3 infection may be greater than previously documented and that infection may occur in host species yet to be tested.

Characterization of two novel reassortant bluetongue virus serotype 1 strains isolated from farmed white-tailed deer (Odocoileus virginianus) in Florida, USA.

Hemorrhagic diseases caused by epizootic hemorrhagic disease virus or by bluetongue virus (BTV) are the most important orbivirus diseases affecting ruminants, including white-tailed deer (WTD). Bluetongue virus is of particular concern for farmed WTD in Florida, given its lethality and its wide distribution throughout the state. This study reports the clinical findings, ancillary diagnostics, and genomic characterization of two BTV serotype 1 strains isolated from two farmed WTD, from two different farms in Florida in 2019 and 2022. Phylogenetic and genetic analyses indicated that these two novel BTV-1 strains were reassortants. In addition, our analyses reveal that most genome segments of these strains were acquired from BTVs previously detected in ruminants in Florida, substantiating their endemism in the Southeastern U.S. Our findings underscore the need for additional research to determine the genetic diversity of BTV strains in Florida, their prevalence, and the potential risk of new BTV strains to WTD and other ruminants.

Report of amoebic disease in a colony of Western honey bees (Apis mellifera).

The amoeba Malpighamoeba mellificae is the etiologic agent of amoebic (amoeba) disease of Western honey bees (Apis mellifera). M. mellificae damages the Malpighian tubules, which is believed to weaken and kill the host bee. Here, the authors describe the detection of this organism in a honey bee colony in the Yukon Territory, Canada. The Malpighian tubules of 14% (7/50) of the adult worker bees were discolored dark brown. Fifteen bees screened using conventional polymerase chain reaction for the 18S gene of M. mellificae were positive for the pathogen. Histologically, the lumens of Malpighian tubules were packed with amoebae, causing dilation of the tubules and attenuation and loss of the tubular epithelium. This phylogenetic analysis places M. mellificae in a new clade, a sister group to the Entamoebidae. This work provides a foundation for further investigation into the distribution, prevalence, and pathology associated with M. mellificae infection.

Myxobolus lentisuturalis infection in a farmed population of goldfish Carassius auratus from the USA.

Myxobolus lentisuturalis is a myxozoan parasite of piscine muscle that has been described in goldfish Carassius auratus and Prussian carp Carassius gibelio. This report documents a naturally occurring infection of M. lentisuturalis in a population of farmed goldfish in the USA. Postmortem examination was performed on 4 affected goldfish. Gross findings included large cystic cavities along the dorsal midline filled with caseous exudate. Histopathology revealed myxozoan plasmodia and spores in the epaxial muscles with varying degrees of granulomatous and necrotizing myositis accompanied by lymphohistiocytic meningoencephalitis. Spore morphology and dimensions were consistent with M. lentisuturalis, as observed by light microscopy. PCR and sequence analysis of the small subunit ribosomal DNA of infected muscle samples from 2 goldfish confirmed the parasite to have 99-100% nucleotide identity to M. lentisuturalis sequences recovered from similar cases of this parasite infecting goldfish in China and Italy and Prussian carp in China. This is the first reported case of M. lentisuturalis in the USA and furthers the understanding of the pathogenicity of this under-described parasite.

Characterization of an alloherpesvirus from wild lake sturgeon Acipenser fulvescens in Wisconsin (USA).

In the spring of 2017, 2 adult lake sturgeon (LS) Acipenser fulvescens captured from the Wolf River, Wisconsin (USA), presented with multiple cutaneous plaques that, upon microscopic examination, indicated proliferative epidermitis. Ultrastructural examination of affected keratinocytes revealed particles in the nucleus having a morphology typical of herpesviruses. A degenerate PCR assay targeting the DNA polymerase catalytic subunit (pol) gene of large double-stranded DNA viruses generated amplicons of the anticipated size from skin samples, and sequences of amplicons confirmed the presence of a novel alloherpesvirus (lake sturgeon herpesvirus, LSHV) related to acipenserid herpesvirus 1 (AciHV1). The complete genome (202660 bp) of this virus was sequenced using a MiSeq System, and phylogenetic analyses substantiated the close relationship to AciHV1. A PCR assay targeting the LSHV DNA packaging terminase subunit 1 (ter1) gene demonstrated the presence of the virus in 39/42 skin lesion samples collected from wild LS captured in 2017-2019 and 2021 in 4/4 rivers in Wisconsin. Future efforts to isolate LSHV in cell culture would facilitate challenge studies to determine the disease potential of the virus.

Characterization of a Novel Reassortant Epizootic Hemorrhagic Disease Virus Serotype 6 Strain Isolated from Diseased White-Tailed Deer (Odocoileus virginianus) on a Florida Farm.

We report an outbreak of a novel reassortant epizootic hemorrhagic disease virus serotype 6 (EHDV-6) in white-tailed deer (WTD) on a Florida farm in 2019. At necropsy, most animals exhibited hemorrhagic lesions in the lung and heart, and congestion in the lung, liver, and spleen. Histopathology revealed multi-organ hemorrhage and congestion, and renal tubular necrosis. Tissues were screened by RT-qPCR and all animals tested positive for EHDV. Tissues were processed for virus isolation and next-generation sequencing was performed on cDNA libraries generated from the RNA extracts of cultures displaying cytopathic effects. Six isolates yielded nearly identical complete genome sequences of a novel U.S. EHDV-6 strain. Genetic and phylogenetic analyses revealed the novel strain to be most closely related to a reassortant EHDV-6 strain isolated from cattle in Trinidad and both strains received segment 4 from an Australian EHDV-2 strain. The novel U.S. EHDV-6 strain is unique in that it acquired segment 8 from an Australian EHDV-8 strain. An RNAscope® in situ hybridization assay was developed against the novel U.S. EHDV-6 strain and labeling was detected within lesions of the heart, kidney, liver, and lung. These data support the novel U.S. reassortant EHDV-6 strain as the cause of disease in the farmed WTD.

A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida, USA.

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

Effect of water temperature on frog virus 3 disease in hatchery-reared pallid sturgeon Scaphirhynchus albus.

Ranaviruses are large double-stranded DNA viruses within the genus Ranavirus (family Iridoviridae) that are being detected with increasing frequency among aquacultured and wild fishes. In the USA, multiple sturgeon hatcheries have experienced ranavirus epizootics resulting in significant morbidity and mortality in young-of-year (YOY). Significant economic losses have resulted from repeated outbreaks of frog virus 3 (FV3), the type species for the genus Ranavirus, in YOY pallid sturgeon Scaphirhynchus albus reared at a hatchery within the Missouri River Basin. Water temperature and stocking density are known to influence the severity of ranavirus disease in ectothermic vertebrates. To determine the effect of water temperature on ranavirus disease in hatchery-raised S. albus, we conducted FV3 challenges at 2 temperatures (17 and 23°C) and compared cumulative survival over a 28 d study period. A mean (±SE) survival rate of 57.5 ± 13.2% was observed in replicate tanks of sturgeon maintained at 23°C, whereas no mortality was observed among sturgeon maintained at 17°C. In a second challenge study, we compared the effect of water temperature on disease progression by regularly sampling fish over the study period and evaluating lesions by histopathology and in situ hybridization, and by assessing viral titer and load in external and internal tissues using virus isolation and qPCR, respectively. Results suggest that temperature manipulation may be an effective mitigation strategy that sturgeon hatcheries can employ to minimize ranavirus-associated disease.

Genome Sequence of Rotavirus A from a Florida Racing Pigeon (Columba livia domestica).

The complete coding sequence of a rotavirus A strain was determined from a dead racing pigeon in Florida. It was found to be most closely related to a rotavirus A strain isolated from a dead racing pigeon in California.

The complete mitochondrial genome of the Jaguar Loach (Yasuhikotakia splendida).

Herein, we present the complete mitochondrial genome of the Jaguar Loach, Yasuhikotakia splendida. The sequence was determined from an aquarium specimen using a next-generation sequencing approach. The annotated Y. splendida mitogenome was 16,695 bp in length and contained 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 non-coding control region. The Y. splendida mitogenome displayed an A + T bias with an overall base composition of 32.0% A, 24.7% T, 27.6% C, and 15.7% G. Maximum Likelihood and Bayesian phylogenetic analyses, based on the aligned mitogenome sequences of 22 botiid loach species from each of the 8 genera and 3 outgroups, generated nearly identical trees that supported the Jaguar Loach as the sister species to the Skunk Loach, Y. morleti.

Genome Sequence of a Yunnan Orbivirus Isolated from a Dead Florida White-Tailed Deer (Odocoileus virginianus).

We report the complete coding sequences of a Yunnan orbivirus isolated from a dead white-tailed deer (Odocoileus virginianus) in Florida in 2019. The prevalence of Yunnan orbivirus and its role in disease among farmed white-tailed deer remain to be determined.

Investigation of a Cyprinid Herpesvirus 1 Disease Episode in a Group of Pond-Reared Koi.

Fifteen adult koi (a variant of Common Carp Cyprinus carpio) simultaneously developed white cutaneous proliferations affecting up to 30% of their bodies. The onset of these lesions (in September 2014) was associated with their return to a remodeled backyard water garden after temporarily being maintained in a plastic swimming pool. A single water temperature taken during the outbreak read 21°C on November 17, 2014. The water garden had no extrinsic heat source, with average ambient temperatures ranging from 9.4 to 26.4°C during the outbreak (September 2014-January 2015). Representative skin biopsies were obtained from two fish; the histologic features included severe epidermal hyperplasia, dysplasia, keratinocyte apoptosis, decreased and haphazardly distributed goblet cells with the absence of club cells, keratinocyte hydropic degeneration, and moderate infiltration by lymphocytes and eosinophilic granular cells. Ultrastructural findings included intranuclear nonenveloped hexagonal nucleocapsids and abundant cytoplasmic-enveloped virions morphologically consistent with the Alloherpesviridae family. Polymerase chain reaction was performed on formalin-fixed, paraffin-embedded shavings from the two biopsied koi targeting the thymidine kinase gene of cyprinid herpesvirus 1 (CyHV-1). Together with the aforementioned findings, these results are consistent with an outbreak of CyHV-1 in a population of adult koi.

Development of a PCR assay to detect cyprinid herpesvirus 1 in koi and common carp.

Cyprinid herpesvirus 1 (CyHV1) infects all scaled and color varieties of common carp Cyprinus carpio, including koi. While it is most often associated with unsightly growths known as 'carp pox,' the underlying lesion (epidermal hyperplasia) can arise from a variety of disease processes. CyHV1-induced epidermal hyperplasia may occur transiently in response to water temperature, and thus histopathology cannot be used in isolation to assess CyHV1 infection status. To address this problem, here we describe a PCR assay targeted to the putative thymidine kinase gene of CyHV1. The PCR assay generates a 141 bp amplicon and reliably detects down to 10 copies of control plasmid DNA sequence (analytic sensitivity). The PCR does not cross-detect genomic DNA from cyprinid herpesvirus 2 and 3 (analytic specificity). The CyHV1 PCR effectively detected viral DNA in koi and common carp sampled from various locations in the UK, USA, Brazil, and Japan. Viral DNA was detected in both normal appearing and grossly affected epidermal tissues from koi experiencing natural epizootics. The new CyHV1 PCR provides an additional approach to histopathology for the rapid detection of CyHV1. Analysis of the thymidine kinase gene sequences determined for 7 PCR-positive carp originating from disparate geographical regions identified 3 sequence types, with 1 type occurring in both koi and common carp.

Hematology, biochemical profile and thyroid hormones of four species of freshwater stingrays of the genus Potamotrygon / Hematologia, perfil bioquímico e hormônios da tireoide em quatro espécies de arraias de água doce do gênero Potamotrygon

Potamotrygonidae is a family of freshwater stingray endemic in South America. We determined the hematological and serum biochemistry parameters of Potamotrygon motoro and Potamotrygon falkneri from Parana Basin and Potamotrygon orbignyi and Potamotrygon scobina from the Amazon Basin. Blood was collected from 55 specimens of Potamotrygon sp., and these parameters were evaluated: red blood cell count, hematocrit percent, hemoglobin concentration, leucocytes count, trombocytes count, total protein concentration, albumin, uric acid, urea, creatinine, triglycerides, cholesterol, high-density lipoprotein HDL, calcium, phosphorus, calcium/phosphorus relationship, creatine kinase CK, gamma-glutamyl transpeptidase GGT, Aspartate transaminase AST, alanine transaminase ALT, globulin, albumin/globulin relationship, alkaline phosphatase AP, sodium, chloride, potassium, triiodothyronine T3, and thyroxine T4 hormones. The hematological indices were similar for all species, except for levels of heterophils in P. orbignyi and P. scobina that were higher. Calcium, phosphorus, calcium/phosphorus relationship, sodium, potassium, chloride, AST, ALT, GGT, CK, albumin/globulin relationship, uric acid, creatinine, urea, triglycerides, HDL, T3, T4 levels had no significant difference between the species. These results suggest that there is low influence of habitat and feeding habits. AP, total protein, albumin, globulin, glucose, and total cholesterol had significant differences between the studied species. These results support the theory that stingrays migrated from the Atlantic Ocean, adapting to different conditions over time and placed themselves geographically distant from each other.

Potamotrygonidae e um grupo de raias endêmicas da América do Sul distribuídas nas principais bacias hidrográficas brasileiras, mas informações sobre suas variáveis hematológicas e bioquímicas são escassas. Este estudo teve como objetivo determinar estas variáveis em Potamotrygon motoro e P. falkneri na Bacia do Rio Paraná, Estado do Paraná e de P. orbignyi e P. scobina do Rio Piririm, na Bacia Amazônica, Estado do Amapá. Foi capturado um total de 53 espécimes de Potamotrygon spp. para colheita de sangue e avaliações do hemograma e variáveis bioquímicas séricas. Os valores do hemograma foram próximos para as quatro espécies e os resultados com diferenças significativas entre Potamotrygon falkneri, P. motoro, P. orbigni e P. scobina ocorreram quanto ao número de eritrócitos e heterófilos. Apesar da diversidade geográfica os resultados apresentaram pouca interferência dos diferentes habitats nas variáveis avaliadas. Os níveis de proteínas, globulinas, relação albumina:globulina, acido úrico, creatinina, ureia, HDL-C, relação cálcio:fósforo, sódio, cloreto, AST, ALT, creatinoquinase (CK), fosfatase alcalina, hormônios triiodotironina (T3) e tiroxina (T4) não diferiram entre as espécies de Potamotrygon. Porem, os níveis de albumina, glicose, colesterol total, triglicerídeos e gama glutamil transferase (GGT) apresentaram diferenças. Este fato sugere pouca interferência do habitat nas variáveis avaliadas apesar da diversidade geográfica de origem das raias de vida livre.