Larry A. Hanson

Small subunit ribosomal RNA sequence of Henneguya exilis (class Myxosporea) identifies the actinosporean stage from an oligochaete host.

ABSTRACT Several transmission studies, as well as recent molecular data, have indicated that the two classes Myxosporea and Actinosporea represent different life cycle stages of Myxozoa. To evaluate the life cycles of myxozoa in catfish aquaculture systems, the small subunit (18S) ribosomal RNA gene sequences of Henneguya exilis, a myxosporean from channel catfish Ictalurus punctatus, and an actinosporean (previously designated as Aurantiactinomyxon janiszewskai) from the aquatic oligochaete Dero digitata were determined. The sequences were identical, indicating that H. exilis and the actinosporean are alternate life stages of a single species. This is the first report identifying the actinosporean stage of the genus Henneguya.

Small Subunit Ribosomal RNA Sequences Link the Cause of Proliferative Gill Disease in Channel Catfish to Henneguya n. sp. (Myxozoa: Myxosporea)

Abstract In a previous study the small subunit ribosomal RNA (SSU rRNA) gene of Aurantiactinomyxon ictaluri, the actinosporean stage (actinospore) of the myxozoan associated with proliferative gill disease (PGD), was sequenced. In this study bath exposures of channel catfish Ictalurus punctatus to A. ictaluri isolated from the aquatic oligochaete Dero digitata were used to study the subsequent development of this parasite and to confirm the SSU rRNA gene sequences of each life stage. On day 7 postexposure, typical PGD organisms were observed in the gills, and at 3 months postinfection, cysts containing the newly identified myxozoan Henneguya sp. spores were observed in the gills. The sequence of the SSU rRNA genes of the actinospore and myxospore life stages demonstrated their identity. This is the first molecular evidence that A. ictaluri, isolated from D. digitata, can infect catfish and produce the typical PGD organisms in the gills. These data provide further proof that the actinospore A. ictaluri is ...

Goldfish Hematopoietic Necrosis Herpesvirus (Cyprinid Herpesvirus 2) in the USA: Molecular Confirmation of Isolates from Diseased Fish

Abstract Moribund goldfish Carassius auratus from private collections and breeding facilities were tested for hematopoietic necrosis herpesvirus 2 (also known as cyprinid herpesvirus 2 (CyHV-2)) by degenerate polymerase chain reaction (PCR) of the viral polymerase gene followed by sequencing. The degenerate PCR method produced a fragment with a DNA sequence that was more than 99% identical to the sequence of CyHV-2. Histology and electron microscopy showed that the moribund fish had severe necrosis in the hematopoietic tissues of the kidney and spleen and that herpesvirus particles were present. In the three cases studied, the presence of the virus was associated with high mortality (up to 80%) that could not be attributed to any other cause. The CyHV-2 is very difficult to isolate in cell culture, but our PCR and case studies demonstrate that this virus has a wide geographic distribution in the United States and that goldfish hematopoietic necrosis herpesvirus disease is likely to be an important, but ra...

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

BackgroundMany 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.ResultsWe 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.ConclusionThe 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.

Nitrite-Induced Predisposition of Channel Catfish to Bacterial Diseases

Abstract Channel catfish, Ictalurus punctatus, continuously exposed to 6 mg/L (ppm) nitrite and intraperitoneally injected with Aeromonas hydrophila had lower bacterial median lethal doses (LD50) than control fish. The clearance rate of injected A. hydrophila was reduced in fish chronically exposed to 5 mg/L (ppm) nitrite. Flexibacter columnaris infections occurred spontaneously in channel catfish exposed to 5 mg/L (ppm) nitrite for 7 days. None of the control fish became infected with F. columnaris.

Characterization of rag1 mutant zebrafish leukocytes

BackgroundZebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag 1 mutants).ResultsDifferential counts of peripheral blood leukocytes (PBL) showed that rag 1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag 1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes.Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag 1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag 1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the rag 1 mutants and 26% in the wild-types.Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated rag 1 mutant kidney hematopoietic tissue expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer (NK) cell lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) transcript expression, while wild-type kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig transcript expression.ConclusionOur study demonstrates that in comparison to wild-type zebrafish, rag 1 mutants have a significantly reduced lymphocyte-like cell population that likely includes Non-specific cytotoxic cells (NCC) and NK cells (and lacks functional T and B lymphocytes), a similar macrophage/monocyte population, and a significantly increased neutrophil population. These zebrafish have comparable leukocyte populations to SCID and rag 1 and/or 2 mutant mice, that possess macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes. Rag 1 mutant zebrafish will provide the platform for remarkable investigations in fish and innate immunology, as rag 1 and 2 mutant mice did for mammalian immunology.

Persistence of Largemouth Bass Virus Infection in a Northern Mississippi Reservoir after a Die-Off

Abstract Approximately 3,000 adult largemouth bass Micropterus salmoides were found dead in September 1998 in Sardis Reservoir, Mississippi. Investigation of largemouth bass electrofished 1 month after the reported losses revealed a high level of infection with largemouth bass virus (LMBV) in the population. Subsequent sampling on five separate occasions during the following year revealed that approximately one-half of the fish were positive for LMBV 7 months after the die-off. On the last sampling, 13 months after the die-off, approximately one-third of bass cultured positive for LMBV. The most common finding associated with LMBV infection was a yellow waxy substance in the swim bladder that consisted of erythrocytes and eosinophils in a fibrin clot indicating previous hemorrhage. In nearly every case, fish that had the swim bladder lesion cultured positive for LMBV. Predisposition to infection did not correlate with the gender or size of the fish. Sympatric white bass Morone chrysops, white crappies Pom...

Small Subunit rRNA Gene Comparisons of Four Actinosporean Species to Establish a Polymerase Chain Reaction Test for the Causative Agent of Proliferative Gill Disease in Channel Catfish

Abstract Proliferative gill disease (PGD) causes high morbidity and mortality in cultured channel catfish Ictalurus punctatus. The presence of the myxozoan Aurantiactinomyxon ictaluri (class Actinosporea) is strongly associated with PGD. This parasite, shed as an actinospore from the aquatic oligochaete Dero digitata, infects channel catfish by an undetermined route. Several other actinosporeans have been identified that are shed from D. digitata isolated from catfish ponds, including those designated A. mississippiensis, Helioactinomyxon sp., and the actinospore stage of Henneguya exilis. By the use of multiple sequence alignment of polymerase chain reaction (PCR)-amplified small subunit ribosomal RNA (SSU rRNA) genes of A. ictaluri, A. mississippiensis, and H. exilis, we identified two variable regions. The largest variable region was PCR amplified, sequenced from the Helioactinomyxon sp., and used in addition to the other three sequences in multiple-sequence alignment comparison to develop PCR primers ...

Mutation tryptophan to leucine at position 222 of haemagglutinin could facilitate H3N2 influenza A virus infection in dogs

An avian-like H3N2 influenza A virus (IAV) has recently caused sporadic canine influenza outbreaks in China and Korea, but the molecular mechanisms involved in the interspecies transmission of H3N2 IAV from avian to canine species are not well understood. Sequence analysis showed that residue 222 in haemagglutinin (HA) is predominantly tryptophan (W) in the closely related avian H3N2 IAV, but was leucine (L) in canine H3N2 IAV. In this study, reassortant viruses rH3N2-222L (canine-like) and rH3N2-222W (avian-like) with HA mutation L222W were generated using reverse genetics to evaluate the significance of the L222W mutation on receptor binding and host tropism of H3N2 IAV. Compared with rH3N2-222W, rH3N2-222L grew more rapidly in MDCK cells and had significantly higher infectivity in primary canine tracheal epithelial cells. Tissue-binding assays demonstrated that rH3N2-222L had a preference for canine tracheal tissues rather avian tracheal tissues, whereas rH3N2-222W favoured slightly avian rather canine tracheal tissues. Glycan microarray analysis suggested both rH3N2-222L and rH3N2-222W bound preferentially to α2,3-linked sialic acids. However, the rH3N2-222W had more than twofold less binding affinity than rH3N2-222L to a set of glycans with Neu5Aca2-3Galb1-4(Fuca-)-like or Neu5Aca2-3Galb1-3(Fuca-)-like structures. These data suggest the W to L mutation at position 222 of the HA could facilitate infection of H3N2 IAV in dogs, possibly by increasing the binding affinities of the HA to specific receptors with Neu5Aca2-3Galb1-4(Fuca-) or Neu5Aca2-3Galb1-3(Fuca-)-like structures that are present in dogs.

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.