Channel Catfish Virus: a New Herpesvirus of Ictalurid Fish

Channel catfish virus was studied in ictalurid fish cell culture, the only system of fish, amphibian, avian, and mammalian cells found to be susceptible. Channel catfish virus infection resulted in intranuclear inclusions and extensive syncytium formation. Replication occurred from 10 to 33 C, but not higher. Best growth was from 25 to 33 C, and the amount of virus released nearly equalled the amount which remained cell-associated. The virus was labile to lipid solvents, and indirect determinations with labeled precursors and a metabolic inhibitor showed evidence of deoxyribonucleic acid. Electron microscopy showed progeny virus, about 100 nm in diameter, in various stages of development in cell nuclei by 4 hr. Present also were nuclear masses of exceptionally electron-dense lamellar material, with a unit dimension of 10 to 15 nm. Virus was enveloped at the nuclear membrane and in cytoplasmic vacuoles, resulting in virions having a diameter of 175 to 200 nm. Negative staining demonstrated icosehedral symmetry and 162 capsomeres. Our data indicate that channel catfish virus is a herpesvirus.     

Optimization of Conditions for Production of Channel Catfish Ovary Cells and Channel Catfish Virus DNA

Medium supplements were examined for their effect on the growth of channel catfish ovary cells. It was found that the usual serum supplement of 10% fetal calf serum could be successfully replaced with a combination of 5% fetal calf serum and a mixture of insulin, transferrin, and selenous acid. It was also found that these cells could be grown in a more efficient manner on microcarrier beads. This type of culture produced 14 times the number of cells per milliliter of total medium used compared with the usual tissue culture flasks used for cell growth. The microcarrier system also provided for greater production efficiency of DNA from channel catfish virus, a virus that infects this cell line.     

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 %.     

斑点叉尾鮰NK-lysin基因的cDNA克隆及融合表达质粒构建

以斑点叉尾鮰(Ictalurus punctatus)NK-lysin-type1的成熟肽为研究目标,首先通过RT-PCR和巢式PCR从斑点叉尾鮰的鳃中克隆到编码NK-lysin成熟肽的基因,该基因编码由92个氨基酸残基组成的成熟肽;6个高度保守的半胱氨酸残基位于成熟肽内,它们被推测与NK-lysin的抗菌活性有关。为了进一步构建原核表达系统用于成熟肽的表达,pET-32a(+)被选择作为融合表达质粒,该质粒含有1个trxA融合头基因,与目的片段mNK-lysin连接后形成融合基因trxA-mNK-lysin,进而有助于在工程菌E.coliBL21(DE3)中的可溶性融合表达。通过PCR、EcoR I和HindⅢ双酶切处理以及DNA测序鉴定,证明pET-32a-mNK-lysin重组融合表达质粒已经被成功构建;将其转化至工程菌E.coliBL21(DE3)后的测序结果表明该重组质粒未发生任何DNA变异。     

Molecular and Immunologic Characterization of Gynogenetic Channel Catfish (Ictalurus punctatus)

Second-generation gynogenetic channel catfish were characterized by molecular and immunologic assays to determine if they were isogenic at major histocompatibility complex loci. Southern blot analyses, using channel catfish MHC class II B and class I A gene probes, revealed identical banding patterns among second-generation gynogenetic fish. In contrast, banding patterns from outbred fish differed not only from gynogenetic animals, but also among themselves. Nucleotide sequence analysis of the MHC class II β₁ domain, which encompasses the peptide binding region, from four randomly selected gynogenetic fish showed a single DNA sequence. In contrast, analysis of the same region from three outbred fish showed sequences that differed not only among themselves, but also from those of gynogenetic animals. In cytotoxic assays, peripheral blood leukocytes from outbred fish lysed both gynogenetic and allogeneic targets, whereas those from gynogenetic fish lysed only allogeneic targets. Taken together, these results suggest that this group of second-generation gynogenetic channel catfish is isogenic at MHC loci and may provide an excellent system with which to study cell-mediated immunity in teleosts. Received September 11, 1998; accepted January 14, 1999     

Purification and Quantitation of Channel Catfish (Ictalurus punctatus) Immunoglobulin M

A method is described for purification of channel catfish IgM from immune catfish serum. This should also be a feasible method for purification of IgM from other teleost fish species. The IgM concentration of adult channel catfish was determined to be 11 mg IgM per ml of serum.     

Production of Viable Homozygous, Doubled Haploid Channel Catfish (Ictalurus punctatus)

Production of doubled haploids via mitotic gynogenesis is a useful tool for the creation of completely inbred fish. In order to produce viable doubled haploid channel catfish, we utilized hydrostatic pressure or thermal treatments on eggs fertilized with sperm that had been exposed to ultraviolet light. At 1.5 h post-fertilization, the embryos were exposed to either 590 kg/cm² hydrostatic pressure for 3 min, 37°C for 5 min, or 41°C for 3 min. In the pressure-treated group, only 21 offspring hatched from five spawns with family sizes of one, two, two, four, and 12 offspring each. Eight embryos from the 37°C treatment and 32 embryos from the 41°C treatment survived to hatch. Genotype analysis using microsatellite loci demonstrated all 21 offspring resulting from pressure treatment were homozygous at the 64 loci tested, and none contained alleles unique to the donor male. Eleven of 32 offspring from the 41°C treatment were homozygous at the 18 loci tested, while 21 offspring were heterozygous at six to 12 of these loci. Again, no offspring contained alleles unique to the donor male. However, all eight offspring from the 37°C treatment were heterozygous at multiple loci, and one contained unambiguous paternal alleles. These experiments demonstrated our ability to produce viable homozygous, doubled haploid channel catfish. Doubled haploid catfish can be used to create completely inbred populations for genetic analyses, and homozygous genomic templates will be useful in gene identification and genome characterization.     

Bimodal (Taste/Tactile) Fibers Innervate the Maxillary Barbel in the Channel Catfish

Analysis of single fibers isolated from a branch of the facial/trigeminalcomplex innervating the maxillary barbel of the channel catfish,Ictalurus punctatus, indicated the existence of bimodal (taste/tactile)fibers. Of the 60 single fibers recorded, 14 (23%) respondedto both taste (amino acid) and tactile stimulation, 43 (72%)were responsive to only tactile stimulation and three (5%) respondedonly to taste stimulation. Quinine hydrochloride at a concentrationof 1.0 mM suppressed the mechanosensory activity of the bimodalfibers, but had no effect on the tactile-only fibers. Chem.Senses 22: 477–482, 1997. ¹Current address: Department of Otolaryngology, Kagoshima UniversityMedical School, 8-35-1 Sakuragaoka, Kagoshima 890, Japan ²Current address: Department of Oral Physiology, Ohu UniversitySchool of Detistry, 31-1 Misumido, Tomita, Koriyama, Fukushima963, Japan     

Edwardsiella tarda, a New Pathogen of Channel Catfish (Ictalurus punctatus)

Edwardsiella tarda, an enteric, gram-negative bacterium, causes gas-filled, malodorous lesions in muscle tissue of channel catfish. Incidence and epizootiology of the disease are presented.     

Regulation of the Formyl Peptide Receptor 1 (FPR1) Gene in Primary Human Macrophages

The formyl peptide receptor 1 (FPR1) is mainly expressed by mammalian phagocytic leukocytes and plays a role in chemotaxis, killing of microorganisms through phagocytosis, and the generation of reactive oxygen species. A large number of ligands have been identified triggering FPR1 including formylated and non-formylated peptides of microbial and endogenous origin. While the expression of FPR1 in neutrophils has been investigated intensively, knowledge on the regulation of FPR1 expression in polarized macrophages is lacking. In this study we show that primary human neutrophils, monocytes and resting macrophages do express the receptor on their cell surface. Polarization of macrophages with IFNγ, LPS and with the TLR8 ligand 3M-002 further increases FPR1 mRNA levels but does not consistently increase protein expression or chemotaxis towards the FPR1 ligand fMLF. In contrast, polarization of primary human macrophages with IL-4 and IL-13 leading to the alternative activated macrophages, reduces FPR1 cell surface expression and abolishes chemotaxis towards fMLF. These results show that M2 macrophages will not react to triggering of FPR1, limiting the role for FPR1 to chemotaxis and superoxide production of resting and pro-inflammatory M1 macrophages.