Betanodavirus meningoencephalitis in an Atlantic blue marlin.

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans.

Flavobacterium covae is the predominant species of columnaris-causing bacteria impacting the Channel Catfish industry in the southeastern United States.

OBJECTIVE: Columnaris disease is a leading cause of disease-related losses in the catfish industry of the southeastern United States. The term "columnaris-causing bacteria" (CCB) has been coined in reference to the four described species that cause columnaris disease: Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry; however, there is a lack of knowledge of which CCB species are most prevalent in farm-raised catfish. The current research objectives were to (1) sample columnaris disease cases from the U.S. catfish industry and identify the species of CCB involved and (2) determine the virulence of the four CCB species in Channel Catfish Ictalurus punctatus in controlled laboratory challenges. METHODS: Bacterial isolates or swabs of external lesions from catfish were collected from 259 columnaris disease cases in Mississippi and Alabama during 2015-2019. The DNA extracted from the samples was analyzed using a CCB-specific multiplex polymerase chain reaction to identify the CCB present in each diagnostic case. Channel Catfish were challenged by immersion with isolates belonging to each CCB species to determine virulence at ~28°C and 20°C. RESULT: Flavobacterium covae was identified as the predominant CCB species impacting the U.S. catfish industry, as it was present in 94.2% (n = 244) of diagnostic case submissions. Challenge experiments demonstrated that F. covae and F. oreochromis were highly virulent to Channel Catfish, with most isolates resulting in near 100% mortality. In contrast, F. columnare and F. davisii were less virulent, with most isolates resulting in less than 40% mortality. CONCLUSION: Collectively, these results demonstrate that F. covae is the predominant CCB in the U.S. catfish industry, and research aimed at developing new control and prevention strategies should target this bacterial species. The methods described herein can be used to continue monitoring the prevalence of CCB in the catfish industry and can be easily applied to other industries to identify which Flavobacterium species have the greatest impact.

Complete Genome Sequences of Francisella marina Strains E95-16 and E103-15, Isolated from Maricultured Spotted Rose Snapper (Lutjanus guttatus) on the Pacific Coast of Central America.

In 2015 and 2016, a previously unrecognized Francisella sp. was isolated from disease outbreaks in maricultured spotted rose snapper (Lutjanus guttatus) on the Pacific coast of Central America. Polyphasic analysis demonstrated these bacteria differed from any known Francisella spp. Here, the complete genomes from the recently described Francisella marina strains are released.

Virulence and immunogenicity of blue catfish alloherpesvirus in channel, blue and blue × channel hybrid catfish.

Blue catfish alloherpesvirus (BCAHV) is a novel virus isolated from the blue catfish (Ictalurus furcatus). To date, the ultrastructure, virulence and immunogenicity of BCAHV have not been reported. Given the importance of blue catfish in producing channel ♀ (I. punctatus) × â™‚ blue (I. furcatus) catfish hybrids and the increasing demand for hybrid catfish in the US catfish industry, the susceptibility of blue, channel and hybrid catfish to BCAHV was assessed. Further, the cross-protective potential of BCAHV against Ictalurid herpesvirus 1 (IcHV1) was investigated in channel and hybrid catfish that survive BCAHV exposure. Neutralization assays revealed BCAHV is refractive (neutralization index [NI] = 0) to anti-IcHV1 monoclonal antibody Mab 95, compared to IcHV1 (NI = 1.8). Exposure of blue catfish fingerling to 1.3 × 105 TCID50 /L BCAHV produced cumulative mortality of 51.67 ± 0.70% and pathologic changes similar to those of channel catfish virus disease. No mortality was observed in channel or hybrid catfish. Twenty-eight days post-challenge, surviving channel and hybrid catfish were exposed to 9.4 × 104 TCID50 /L IcHV1 (LC50 dose), resulting in 100% relative per cent survival compared to naïve cohorts. These data provide baseline information for BCAHV and lay the groundwork for future studies. Data also identify BCAHV as a potential vaccine candidate against IcHV1. Based on host range and immunogenicity evaluations, in addition to genome sequence data from previous studies, BCAHV should be given consideration as a new species of Ictalurivirus.

Potential of Double-crested Cormorants ( Phalacrocorax auritus), American White Pelicans ( Pelecanus erythrorhynchos), and Wood Storks ( Mycteria americana) to Transmit a Hypervirulent Strain of Aeromonas hydrophila between Channel Catfish Culture Ponds.

Aeromonas hydrophila is a Gram-negative bacterium ubiquitous to freshwater and brackish aquatic environments that can cause disease in fish, humans, reptiles, and birds. Recent severe outbreaks of disease in commercial channel catfish ( Ictalurus punctatus) aquaculture ponds have been associated with a hypervirulent Aeromonas hydrophila strain (VAH) that is genetically distinct from less virulent strains. The epidemiology of this disease has not been determined. Given that research has shown that Great Egrets ( Ardea alba) can shed viable hypervirulent A. hydrophila after consuming diseased fish, we hypothesized that Double-crested Cormorants ( Phalacrocorax auritus), American White Pelicans ( Pelecanus erythrorhynchos), and Wood Storks ( Mycteria americana) could also serve as a reservoir for VAH and spread the pathogen during predation of fish in uninfected catfish ponds. All three species, when fed VAH-infected catfish, shed viable VAH in their feces, demonstrating their potential to spread VAH.

Differential gene expression following TLR stimulation in rag1-/- mutant zebrafish tissues and morphological descriptions of lymphocyte-like cell populations.

In the absence of lymphocytes, rag1-/- mutant zebrafish develop protective immunity to bacteria. In mammals, induction of protection by innate immunity can be mediated by macrophages or natural killer (NK) cells. To elucidate potential responsive cell populations, we morphologically characterized lymphocyte-like cells (LLCs) from liver, spleen and kidney hematopoietic tissues. In fish, these cells include NK cells and Non-specific cytotoxic cells (NCCs). We also evaluated the transcriptional expression response of select genes that are important indicators of NK and macrophage activation after exposure to specific TLR ligands. The LLC cell populations could be discriminated by size and further discriminated by the presence of cytoplasmic granules. Expression levels of mx, tnfα, ifnγ, t-bet and nitr9 demonstrated dynamic changes in response to intra-coelomically administered ß glucan (a TLR2/6 ligand), Poly I:C (a TLR3 ligand) and resiquimod (R848) (a TLR7/8 ligand). Following TLR 2/6 stimulation, there was a greater than 100 fold increase in ifnγ in liver, kidney and spleen and moderate increases in tnfα in liver and kidney. TLR3 stimulation caused broad up regulation of mx, down-regulation of tnfα in kidney and spleen tissues and up regulation of nitr9 in the kidney. Following TLR 7/8 stimulation, there was a greater than 100 fold increase in ifnγ in liver and kidney and t-bet in liver. Our gene expression findings suggest that LLCs and macrophages are stimulated following ß glucan exposure. Poly I:C causes type I interferon response and mild induction of LLC in the kidney and R-848 exposure causes the strongest LLC stimulation. Overall, the strongest NK like gene expression occurred in the liver. These differential effects of TLR ligands in rag1-/- mutant zebrafish shows strong NK cell-like gene expression responses, especially in the liver, and provides tools to evaluate the basis for protective immunity mediated by the innate immune cells of fish.

Detection of Antigenic Variants of Subtype H3 Swine Influenza A Viruses from Clinical Samples.

A large population of genetically and antigenically diverse influenza A viruses (IAVs) are circulating among the swine population, playing an important role in influenza ecology. Swine IAVs not only cause outbreaks among swine but also can be transmitted to humans, causing sporadic infections and even pandemic outbreaks. Antigenic characterizations of swine IAVs are key to understanding the natural history of these viruses in swine and to selecting strains for effective vaccines. However, influenza outbreaks generally spread rapidly among swine, and the conventional methods for antigenic characterization require virus propagation, a time-consuming process that can significantly reduce the effectiveness of vaccination programs. We developed and validated a rapid, sensitive, and robust method, the polyclonal serum-based proximity ligation assay (polyPLA), to identify antigenic variants of subtype H3N2 swine IAVs. This method utilizes oligonucleotide-conjugated polyclonal antibodies and quantifies antibody-antigen binding affinities by quantitative reverse transcription-PCR (RT-PCR). Results showed the assay can rapidly detect H3N2 IAVs directly from nasal wash or nasal swab samples collected from laboratory-challenged animals or during influenza surveillance at county fairs. In addition, polyPLA can accurately separate the viruses at two contemporary swine IAV antigenic clusters (H3N2 swine IAV-α and H3N2 swine IAV-ß) with a sensitivity of 84.9% and a specificity of 100.0%. The polyPLA can be routinely used in surveillance programs to detect antigenic variants of influenza viruses and to select vaccine strains for use in controlling and preventing disease in swine.

Zebrafish Sensitivity to Botulinum Neurotoxins.

Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins.

POTENTIAL FOR GREAT EGRETS (ARDEA ALBA) TO TRANSMIT A VIRULENT STRAIN OF AEROMONAS HYDROPHILA AMONG CHANNEL CATFISH (ICTALURUS PUNCTATUS) CULTURE PONDS.

Aeromonas hydrophila is a gram-negative, rod-shaped, facultative, anaerobic bacterium that is ubiquitous in freshwater and slightly brackish aquatic environments and infects fish, humans, reptiles, and birds. Recent severe outbreaks of disease in commercial channel catfish (Ictalurus punctatus) aquaculture ponds have been associated with a highly virulent A. hydrophila strain (VAH), which is genetically distinct from less-virulent strains. The epidemiology of this disease has not been determined. Given that A. hydrophila infects birds, we hypothesized that fish-eating birds may serve as a reservoir for VAH and spread the pathogen by flying to uninfected ponds. Great Egrets (Ardea alba) were used in this transmission model because these wading birds frequently prey on farmed catfish. Great Egrets that were fed VAH-infected catfish shed VAH in feces demonstrating their potential to spread VAH.

Antibody response of channel catfish after channel catfish virus infection and following dexamethasone treatment.

Channel catfish virus (CCV, Ictalurid herpesvirus 1) and CCV disease have been extensively studied. Yet, little is known about CCV-host interaction after resolution of the primary infection. In order to determine potential recrudescence of CCV from latency, we established latency by exposing channel catfish juveniles with CCV or a thymidine kinase-negative recombinant (CCVlacZ) at a dose that caused less than 20% mortality. Then, we evaluated antibody response by serially sampling the same fish at 0 (pre-infection), 30, 60 and 90 d post challenge (DPC). We then attempted to induce viral recrudescence by intramuscular administration of dexamethasone and sampled the fish at 2, 4, 7, or 10 d post treatment. Recrudescence was evaluated by leukocyte co-cultivation and cell culture of tissue homogenates but no virus was detected. Western blot data demonstrated the highest number of seropositive fish by 30 DPC and a secondary antibody induction after dexamethasone treatment. The antigen specificity of the secondary response corresponded to viral proteins with molecular masses similar to those recognized by the same fish by 30 DPC. The recognized proteins were predominantly large, ranging from approximately 90 to >200 kDa. Expression analysis of selected virus genes at 90 DPC and following dexamethasone treatment demonstrated occasional immediate-early virus gene expression in peripheral blood leukocytes. Early and late gene expression was rarely detected. The combined data suggest restricted re-activation of CCV in our experimental system. Primary and secondary responses and virus gene expression were demonstrated in CCVlacZ-exposed fish but were less frequent than in CCV-exposed fish.

The pathology associated with visceral toxicosis of catfish.

Visceral toxicosis of catfish (VTC) syndrome was recognized in the late 1990 s and recently has been associated with exposure to Clostridium botulinum type E neurotoxin. Tentative diagnosis is based on clinical presentation and gross findings, and is confirmed by bioassay. In April 2009, channel catfish (Ictalurus punctatus) from 2 different farms presented with abnormal swimming behavior and mortalities. Nine fish were submitted to the Aquatic Research and Diagnostic Laboratory (Stoneville, Mississippi) for evaluation. Bacterial cultures from these fish were negative. Necropsy findings included intestinal intussusceptions, ascites, pale proximal intestines with engorged serosal blood vessels, splenic congestion, and a reticular pattern to the liver. Significant histopathologic findings were limited to cerebral, splenic, and hepatic congestion, splenic lymphoid depletion and perivascular edema, vascular dilation and edema of the gastrointestinal tract, and perivascular edema in the anterior and posterior kidneys. Intoxication from C. botulinum type E neurotoxin was suspected based on the clinical signs and lack of gross and microbiological evidence of an infectious disease process. The toxicosis was confirmed with a positive bioassay using serum collected from the submitted fish.

Expression analysis of selected immune-relevant genes in channel catfish during Edwardsiella ictaluri infection.

Channel catfish Ictalurus punctatus were intraperitoneally challenged with the bacterium Edwardsiella ictaluri (the causative agent of enteric septicemia of catfish), and the expression of genes presumed to function in the inducible innate defense was evaluated. End-binding protein 1 (EB1), beta1-integrin, natural-resistance-associated macrophage protein (Nramp), heat shock protein 70 (Hsp70), serum amyloid P (SAP), and transferrin gene expression profiles were determined using quantitative reverse-transcriptase-polymerase chain reaction on liver, anterior kidney, spleen, and gut. Fish were subsampled at 0, 24, 48, 72, and 96 h after bacterial or phosphate-buffered-saline injection. Posterior kidney sampling demonstrated increasing bacterial counts at 24-48 h postinjection (hpi), followed by a plateau to 96 hpi. The transferrin and SAP transcripts were liver specific. The other genes were expressed in all four tissues. In bacterially infected fish, expression of EB1 (anterior kidney, spleen, and liver), Hsp70 (anterior kidney and spleen), and Nramp (spleen and gut) significantly increased by 48 hpi. Transferrin was strongly up-regulated and SAP was downregulated by 72 hpi, indicating positive and negative acute-phase reactants, respectively. The data indicate a substantial response of innate immunity effector cells by 48 hpi, followed by suppression of bacterial growth and induction of the acute-phase response. This suggests that the 48-72-hpi time frame is critical in our model for evaluating the effectiveness of innate defenses.

A Gateway recombination herpesvirus cloning system with negative selection that produces vectorless progeny.

Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines.

An overlapping bacterial artificial chromosome system that generates vectorless progeny for channel catfish herpesvirus.

Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild-type (wt) virus are extremely valuable tools for elucidating the roles of specific genes in virus pathophysiology as well as for making vaccines. Ictalurid herpesvirus 1 (channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here, we describe the cloning of the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BACs). These clones allowed us to regenerate vectorless wt CCVs with a phenotype that is indistinguishable from that of the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full-length CCV BAC by replacing the CCV ORF5 with the BAC cassette and cotransfecting CCO cells. The viral progeny that we used to transform Escherichia coli and the resulting BAC had only one of the 18-kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of the generation of an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants.

A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene.

BACKGROUND: Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. RESULTS: We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus) from cell culture and lymphocystis disease virus (iridovirus) and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1). The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. CONCLUSION: The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified viruses according to relatedness to sequences of reference viruses and the submission of the sequence data to GenBank will build the database to make the BLAST analysis a valuable resource readily accessible by most diagnostic laboratories. We demonstrated the utility of this assay on viruses that infect fish and birds. These hosts are phylogenetically distant from mammals yet, sequence data suggests that the assay would work equally as well on mammalian counterparts of these groups of viruses. Furthermore, we demonstrated that obtaining genetic information on routine diagnostic samples has great potential for revealing new virus strains and suggesting the presence of new species.

Identification and initial characterization of a putative Mycoplasma gallinarum leucine aminopeptidase gene.

Aminopeptidases (APN) may play a role in host colonization of M. gallinarum. Characterization of endogenous APN activity suggests that the leucine APN (LAP) of M. gallinarum is a metallo-aminopeptidase activated by Mn2+ and is present in the cytosol and possibly associated with the inner leaflet of the membrane. A 1.36-kb open reading frame (ORF) identified from overlapping genomic phage clones showed 68% nucleotide identity and 51% amino acid identity with the M. salivarium LAP gene. This ORF is expressed as a 1.5-kb monocistronic transcript and is present as a single copy in M. gallinarum. This gene sequence was modified to account for codon usage, and expression in E. coli produced a 51-kDa protein, which compares well with the product predicted from the ORF. This ORF is a strong candidate for contributing the LAP activity of M. gallinarum protein extracts.

Susceptibility of channel catfish fry to Channel Catfish Virus (CCV) challenge increases with age.

Susceptibility of channel catfish to Channel Catfish Virus Disease (CCVD) has been generally considered to be inversely related to age. However, in experimental immersion challenges, we found that channel catfish fry, 3 to 8 d post hatch (dph), are most resistant to CCV and susceptibility increases with age. Initial studies involved 2 spawns that had high CCV carrier percentage. To determine if the resistance seen in the fry was related to the CCV carrier status of the parents, we selected 4 spawns from CCV negative parents and 2 spawns from CCV positive parents and immersion challenged them at 8, 23, 36 and 60 dph with 0, 2.5 x 10(4) or 2.5 x 10(6) plaque forming units (PFU) of CCV l(-1). Survivors of the low-dose exposed groups were rechallenged at 120 dph with 2.5 x 10(6) PFU CCV l(-1). Each brood demonstrated increasing susceptibility to CCVD with age and only the fish that were initially exposed at 60 dph developed protective immunity. Time course assays evaluating tissue levels of virus in channel catfish exposed to CCV at 7, 21 and 42 dph suggested that the resistance was an early event in the infection process. The resistance in fry was most pronounced in fish from CCV positive spawns and was correlated to neutralizing antibody titers in the maternal parent in the 8 dph challenge. However, other factors may be involved because all groups displayed the initial resistance and subsequent susceptibility to CCVD. The age effect may be an important influence on the progression of CCVD outbreaks and indicates the need to consider age for experimental challenges. Additionally, we documented the level of vertical transmission of CCV. Fry from the 4 positive spawns had a CCV prevalence of 40 to 75 %.

Isolation and characterization of channel catfish natural resistance associated macrophage protein gene.

Natural resistance associated macrophage protein 1 (Nramp1) affects the ability of macrophages to kill pathogens. We cloned Nramp cDNA of channel catfish to identify potential molecular markers for disease resistance. Three different Nramp transcripts were identified: NrampCa-2912 nucleotides (nt), NrampCb-3245 nt, and NrampCc-3721 nt. At the 5' end, the transcripts have a common 2263 nt sequence containing the open reading frame. The differences are in the 3' untranslated region resulting from alternative splicing and polyadenylation. NrampCc is the predominant form expressed. The deduced 550 amino acid sequence of the channel catfish Nramp (NrampC) has high homology to Nramp from other vertebrates and a predicted conserved structure. The NrampC contains the 12 transmembrane domains, and the consensus transport motif. Post-transcriptional processing is also conserved. Phylogenetic analysis grouped NrampC with other fish Nramps and closer to Nramp2 than to Nramp1 of mammals. However, the catfish transcript does not contain an iron-responsive regulatory-protein binding site, a characteristic of Nramp2, and, like Nramp1, NrampC expression is induced in macrophage-rich tissues after exposure to lipopolysaccharide and in a macrophage cell line when stimulated. Thus NrampC is structurally closer to mammalian Nramp2 but may function similar to Nramp1.