Identification of envelope protein orf10 of channel catfish herpesvirus

Channel catfish virus (CCV) is a viral pathogen of fry and fingerling channel catfish and can cause significant commercial loss. Previous studies have shown that the CCV virion contains at least 25 predicted structural proteins, including viral protein 10, which is encoded by the orf10 gene of the CCV. In this paper, the orf10 gene was expressed in Escherichia coli and used to produce a specific antibody. Western blot analysis confirmed that open reading frame 10 is an envelope protein. A viral neutralization assay demonstrated that open reading frame 10 antiserum was able to inhibit CCV infection of channel catfish ovary cells, suggesting that viral protein 10 is likely to play an important role in the CCV infection of channel catfish ovary cells.     

Systemic herpes-like virus in catfish Ictalurus melas (Italy) differs from Ictalurid herpesvirus 1 (North America)

A herpesvirus was isolated during 2 occurrences of mass mortality among adult catfish Ictalurus melas raised in different farms in northern Italy. The agent replicated in the channel catfish ovary (CCO) cell line from channel catfish I. punctatus, inducing a cytopathic effect similar to that caused by Ictalurid herpesvirus 1 (also referred to as channel catfish herpesvirus, CCV). The new herpesvirus, designated I. melas herpesvirus (IcmHV) did not react with polyclonal rabbit or monoclonal antibodies directed to CCV in either neutralization or indirect immunofluorescence assays. The virions of IcmHV possessed a hexagonal nucleocapsid of 107 nm in diameter surrounded by an envelope with a diameter of 227 nm (n = 20) typical for members of the family Herpesviridae. Virions of IcmHV purified from infected CCO cells contained 17 polypeptides ranging in size from 17.5 to 175 kDa and most differed in molecular weight from those found for CCV. The IcmHV was also distinct from CCV when compared by restriction fragment length polymorphisms (RFLP) of genomic DNA following digestions with the endonucleases Kpn I and Sac I. Lastly, the virulence of IcmHV for channel catfish fry and juveniles, respectively, was demonstrated by experimental infections induced by bath exposure or intraperitoneal injection that resulted in 78 to 96% cumulative mortality in groups of exposed fish. Preventing the introduction of this agent into geographic regions where significant channel catfish production occurs should be a high priority.     

Poly I:C inhibits the expression of channel catfish virus immediate-early gene ORF 1 at early times after infection

Channel catfish virus (CCV) is a herpesvirus that infects channel catfish fry and fingerlings. Previous research has demonstrated that Type I interferons inhibit the expression of immediate-early (IE) genes of some mammalian herpesviruses. However, CCV is distantly related to the mammalian herpesviruses and Type I interferons from higher vertebrates exhibit only 20% similarity to fish interferons. In this work we demonstrate that treatment of channel catfish ovary (CCO) cells, a fibroblast-like cell line, with poly I:C, a known inducer of Type I interferons, results in inhibition of expression of the CCV IE gene ORF 1. Thus, although the genes involved have diverged, the mechanism appears to be conserved. If this paradigm holds true for other CCV IE-Type I interferon interactions, it could have important implications for the impact of CCV on the host immune system.     

Fate of intraperitoneally injected fluorescent microspheres in developing Ictalurus punctatus

Fluorescent microspheres (FMS) were injected intraperitoneally into channel catfish fry at 2 days post hatch (dph), 1, 2, 3, 4 and 8 weeks post hatch (wph). The FMS were observed in the vasculature almost immediately after injection in all age groups except 2 dph. Fluorescent microspheres were observed within mononuclear phagocytes in the vasculature after 0.16 dph in all age groups. Fluorescent microspheres were first phagocytized in the coelomic cavity immediately after injection, while the majority of coelomic FMS were phagocytized between 0.16 and 1 dph for all ages. Enzyme cytochemical staining indicated that both polymorphonuclear (neutrophilic granulocytes) and mononuclear phagocytes had phagocytized FMS in the coelomic cavity and organs, with a predominance of FMS found in mononuclear phagocytic cells in all age groups across all sample periods. The predominant organs associated with the observed cellular responses were the posterior kidney, spleen, and anterior kidney. Splenic organization and melanomacrophage development and activity were more pronounced as the fish aged from 2 wph on. Particulate clearance rates were faster in the 2 dph and 1 wph fish than the older ages of fish. These results suggest that to facilitate particulate retention, channel catfish should be vaccinated at 4 wph or older.     

Resistance of the European catfish (Silurus glanis) to channel catfish virus

European catfish (Silurus glanis) fingerlings (2 to 4 g each) were tested for susceptibility to channel catfish virus (CCV). They had supported CCV replication at 2 days after intraperitoneal injection with 0.1 ml of saline containing 10⁵ TCID₅₀. Homogenized visceral organs (liver, kidney and spleen) contained 10⁴ TCID₅₀/0.1 ml at 2 days post inoculation (PI) but at 4 days the titer decreased to 10¹ TCID₅₀. Bathing European catfish in CCV yielded only one positive sample with à titer of 10^0.83 TCID₅₀ per 0.1 ml of tissue. No clinical signs of CCV developed and no virus related deaths occurred.     

Humoral immune responses of channel catfish (Ictalurus punctatus) fry and fingerlings exposed toEdwardsiella ictaluri

The ability of channel catfish to develop antibody responses to Edwardsiella ictaluri was evaluated. Fish were produced and reared under specific pathogen free (SPF) conditions. At the ages of 1, 2, 3 and 4 weeks and 2, 3, 4, 5, and 6 months post-hatch, a group of these naive fish were given a primary immersion exposure to E. ictaluri (mean doses of 6·4×10⁴ cfu ml⁻¹of tank water). Each group received a secondary immersion exposure 4 weeks after the primary exposure and were sampled 2 weeks after each exposure. Control groups were exposed to sterile culture medium. Specific antibody titres were first detected in fish exposed at 4 weeks post-hatch at an average weight of 85 mg. A secondary response was first demonstrated by fry that received a primary exposure at 4 weeks post-hatch and a secondary exposure at 8 weeks post-hatch. However, a true boosting effect was first demonstrated in fish that received their primary exposure at 2 months of age. Fish given a primary exposure before 4 weeks of age and a secondary exposure failed to produce a significant antibody response, even though they were the same age at secondary exposure as fish that produced strong antibody responses upon primary exposure. This phenomenon suggests that immunological tolerance was induced and indicates that channel catfish may not be capable of generating a humoral immune response before four weeks of age.     

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.     

Catfish hybrid Ictalurus punctatus × I. furcatus exhibits higher resistance to columnaris disease than the parental species

We present experimental data on susceptibility to columnaris disease, caused by the bacterium Flavobacterium columnare, in hybrid catfish (female channel catfish Ictalurus punctatus × male blue catfish I. furcatus) (C×B). Under our experimental conditions, C×B hybrids were significantly more resistant to columnaris disease caused by the highly virulent strain of F. columnare BGFS-27 (genomovar II) than channel catfish and blue catfish. Channel and blue catfish cumulative mortalities after immersion challenge were 74 and 87%, respectively, whereas mortality in the C×B hybrid was 31%. Susceptibility to the strain ARS-1 (genomovar I) was lower among all catfishes, although channel catfish was the least resistant species at 32% cumulative mortality. By contrast, C×B hybrid and blue catfishes were strongly resistant to the ARS-1 strain, with <10% mortality. Our data suggest enhanced disease resistance of the C×B hybrid to columnaris disease.     

Fate of fluorescent microspheres in developing Ictalurus punctatus following prolonged immersion

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes (p < 0.05). Significantly higher FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs (p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.     

Predator Avoidance of Transgenic Channel Catfish Containing Salmonid Growth Hormone Genes

Transgenic channel catfish (Ictalurus punctatus) containing salmonid growth hormone genes can grow 33% faster than normal channel catfish under aquaculture conditions. However, before transgenic catfish are released and utilized by the private sector, their genetic impact on the natural environment must be examined. Predator avoidance is one of the major fitness traits determining potential environmental risk. To determine the predator avoidance ability and growth performance of transgenic catfish in a natural habitat, various densities of transgenic and nontransgenic channel catfish were communally stocked in 0.04-ha earthen ponds without supplemental feeding. Largemouth bass (Micropterus salmoides) and green sunfish (Lepomis cyanellus) were stocked as predators. Nontransgenic fry had better predator avoidance than transgenic channel catfish when data were pooled (p < .01). When data were not pooled, nontransgenic catfish had better predator avoidance in six trials and transgenic individuals had better predator avoidance in four trials. There was no difference in predator avoidance in three trials. Overall predator avoidance was also better for nontransgenic individuals (p < .01) when the fish were evaluated as 3.5-g fingerlings, more clearly than as fry, as transgenic individuals were more vulnerable in 3 of 4 trials at this life stage. There was no significant difference in growth performance between transgenic and nontransgenic channel catfish in ponds without supplemental feeding. These findings indicate that transgenic channel catfish could be used for commercial aquaculture without affecting the natural environment. Although transgenic channel catfish may be released to nature by accident, any ecological effect would be unlikely because the increased susceptibility of transgenic channel catfish to predators would most likely decrease or eliminate the transgenic genotype. Received March 8, 1999; accepted June 3, 1999.     

斑点叉尾鮰病毒实时荧光LAMP检测方法的建立

斑点叉尾鮰病毒(Channel catfish virus, CCV)属于大DNA病毒, 是一种能引起斑点叉尾鮰(Letalurus punetaus)病毒性疾病的重要病原。研究以编码产物为磷酸激酶的CCV ORF77基因为靶标, 设计了能识别靶基因上的8个独立区域的6条特异引物, 利用基于环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了用于CCV的实时荧光LAMP快速检测方法。结果显示, 建立的实时荧光LAMP检测方法在63℃恒温反应45min条件下, 反应大约12min后出现明显的峰值, 最低检测限度为100个拷贝的病毒质粒DNA, 检出时间为35min; 并且具有良好的特异性, 与白斑综合症病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、锦鲤疱疹病毒(KHV)、真鲷虹彩病毒(RSIV)、传染性脾肾坏死病毒(ISKNV)和新加坡石斑鱼虹彩病毒(SGIV)等常见水生动物病原DNA均没有交叉反应; 同一样品于试验内及试验间重复性试验发现, 20个平行样品的扩增曲线基本重合, 表现出良好的重复性。通过对实际样品的检测结果显示, 实时荧光LAMP检测方法能够特异性的检出CCV。因此, 研究建立的CCV实时荧光LAMP检测方法操作简单、检测快速、特异性好、灵敏度高、结果可靠, 能为斑点叉尾鮰养殖现场和实验室的斑点叉尾鮰疱疹病毒病病原的快速诊断和实时监测方面提供技术支撑。     

A standardized microsatellite marker panel for parentage and kinship analyses in channel catfish,Ictalurus punctatus

This research was designed to produce a standardized set of microsatellite loci for parentage and kinship analyses in channel catfish, the leading species of US aquaculture. Three panels of five to six markers each were developed that contained a total of two dinucleotide‐, eight trinucleotide‐ and seven tetranucleotide‐microsatellite loci respectively. The loci had a range of nine to 31 alleles per locus in an outbred population. Based on the allele frequencies measured in commercial randomly bred broodstock, the combined probability of non‐exclusion of an unrelated candidate parent pair was 5.36e‐18. The combined probability of non‐exclusion of unrelated identical genotypes was 2.58e‐08. The microsatellite panels were validated by parentage and kinship evaluation in three populations. A total of 697 spawns were collected from matings of outbred broodstock over three spawning seasons, and parents were determined unambiguously for all but three spawns. Genotype analysis also enabled the identification of half‐sibling and full‐sibling families produced by pond spawning. In a second experiment, parentage was unambiguously determined in nine spawns from a population consisting of broodstock derived from only four families. A third experiment demonstrated that all but one of 374 individuals from 10 full‐sibling families could be assigned to a family after coculture in an earthen pond for 1 year. The standardized microsatellite panels enable the development of pedigreed catfish populations and large‐scale performance evaluations in common environments to support the genetic improvement of cultured catfish through selective breeding.     

Efficacy of a modified live Flavobacterium columnare vaccine in fish

Flavobacterium columnare is an aquatic bacterium that is responsible for columnaris disease. This aquatic pathogen has a worldwide distribution and is highly infectious to both warm and cold water fish. A modified live F. columnare vaccine was developed by repeated passage of a virulent strain on increasing concentrations of rifampicin that resulted in attenuation. Here we report vaccination/challenge trials to evaluate efficacy and safety. In separate laboratory trials, immersion vaccination of channel catfish (Ictalurus punctatus) fry between 10 to 48 days post hatch (DPH) with experimental vaccine or licensed product resulted in relative percent survival (RPS) between 57–94% following challenge. Similarly, a vaccination/challenge trial using largemouth bass (Micropterus salmoides) fry at 10 DPH was performed using various doses of licensed product under laboratory conditions. Results demonstrated safety of the vaccine and significant protection following challenge with RPS values between 74–94%, depending on vaccine dose. Together, these trials demonstrate the vaccine administered to early life-stage channel catfish and largemouth bass is safe and reduces mortality following challenge with F. columnare.     

Differences in mortality, growth, lysozyme, and toll-like receptor gene expression among genetic groups of catfish exposed to virulent Edwardsiella ictaluri

Survivorship to ESC (enteric septicemia of catfish) varies among and within strains of commercially raised catfish, however the immunological basis for differences in susceptibility is not well-understood. We assessed the effect of pathogen challenge with Edwardsiella ictaluri on five genetic groups of catfish by measuring both phenotypic response (mortality, pathogen levels, specific growth rate), and three measures of immune response, including lysozyme activity and mRNA expression of two toll-like receptors (TLR3 and TLR5). Both mortality and pathogen loads, in addition to non-specific immune response, consistently ranged from the least susceptible Blue catfish (24%, 3.4 x 10(2)+/-9.3 x 10(1)cell-equivalents/mg, 13.2+/-3.2U/mL tissue, respectively) to the most susceptible 103 channel catfish (65%, 1.1x10(4)+/-6.4 x 10(3)cell-equivalents/mg tissue, 67.3+/-28.7U/mL, respectively). Similarly, specific growth rate was reduced in exposed fish, compared to non-exposed controls, only in the most susceptible genetic groups (P=0.0051). Trends in mRNA expression levels were apparent in each tissue type for both genes. In kidney, differences were evident in expression of both TLR3 and TLR5 mRNA between strains early and late in challenge (P<0.01). TLR5 mRNA showed significant downregulation in all strains on days 1 and 4 (P=0.0001). In spleen, all strains had elevated levels of TLR3 (P=0.0050) and TLR5 mRNA (P<0.0001) only 1day post-exposure. In stomach, only one strain (103 x RR) showed upregulation (P=0.0063) throughout challenge. The relationship of phenotypic (mortality and growth) and immune responses measured here, suggests that variation in susceptibility to ESC is a function of differences in innate immune response. Understanding these differences will be crucial for enhancing the immune system through selective breeding and in developing disease management protocols for channel catfish.     

Histophathology and electron microscopy of channel catfish virus in infected channel catfish,Ictalurus punctatus (Rafinesque)*

A histologic and electron microscopic study was made on selected organs from channel catfish Ictalurus punctatus (Rafinesque) fingerlings that were experimentally infected with channel catfish virus (CCV). Histopathology was characterized by necrosis and haemorrhage in kidney and liver, and haemorrhage in the spleen and gastrointestinal tract. Virus replication occurred in nuclei of cells in the kidney, liver and spleen. Intranuclear inclusion bodies consisting of geometric crystalline arrays and lamellar structures were associated with virus replication.     

Effect of dietary cortisol administration on growth and reproductive success of channel catfish

The effect of cortisol administration on reproductive performance was investigated in channel catfish Ictalurus punctatus broodfish. Cortisol was added to a commercial catfish feed by dissolving in ethanol and spraying the feed to yield a dietary concentration of 150 mg kg⁻¹ feed. The cortisol diet and the control (no cortisol) diet were offered at a rate of 1% of biomass to three replicate ponds each containing 28 female and 14 male broodfish, respectively, three times per week for 11 weeks. Spawning began 10 days after the start of the experiment, and continued for 10 weeks. In fish fed cortisol, body mass and the hepato‐somatic index were reduced ( P  ≤ 0·02) and concentrations of plasma cortisol and glucose were significantly higher ( P  ≤ 0·0003) compared to those of controls. The relative frequency of spawning was similar between the two treatments; however, cortisol‐fed channel catfish had an average of 47·1% more spawns than the control‐fed fish. On average, there were 25·5 spawns per pond in the treated groups compared to 12·3 spawns per control pond ( P  = 0·10). No effect was observed on egg production, with individual egg mass, fecundity, and hatching success being similar ( P  ≥ 0·27) for both treatments. Despite the observed negative effects of cortisol on somatic and hepatic growth, the increased reproductive output coupled with no observable effects on the eggs or hatching success demonstrates that cortisol does not suppress channel catfish reproduction.     

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.     

Fish cell lines: Establishment and characterization of three new cell lines from grass carp (Ctenopharyngodon idella)

Summary Three new cell lines were established from tissues of the grass carp,Ctenopharyngodon idella. Derived from the fin, snout, and swim bladder of two apparently healthy diploid fry, these cell lines have been designated GCF, GCS-2, and GCSB, respectively. The cells grew at temperatures between 24° and 36° C with optimal growth at 32° C and have been subcultured more than 50 times since their initiation in August 1986. Two of the lines remained diploid or pseudodiploid after 38 passages. The cells were tested for microbial contamination, and plating efficiencies were determined. The three cell lines were sensitive toRhabdovirus carpio (RVC), infectious hematopoietic necrosis virus (IHNV), golden shiner virus (GSV), chum salmon virus (CSV), and infectious pancreatic necrosis virus serotype VR299 IPNV). They were refractory to channel catfish virus (CCV), channel catfish reovirus (CRV), chinook salmon paramyxovirus (CSP), and an Ab serotype of IPNV. This work is a result of research sponsored by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U.S. Department of Commerce, under grant NA85AA-D-5G-095, and was undertaken while the first author was on leave from the Department of Fisheries, Huazhong Agricultural University, Wuhan, China. Salary support was provided by the People's Republic of China. Oregon Agricultural Experiment Station Technical Paper 8952.     

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.