Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins.

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm-raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive-sequence-mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep-PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep-PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.

Myxobolus neurophilus Guilford 1963 (Myxosporea: Myxobolidae): a common parasite infecting yellow perch Perca flavescens (Mitchell, 1814) in Saskatchewan, Canada.

The goal of this study was to identify a myxosporidian parasite infecting the central nervous system of yellow perch Perca flavescens (Mitchell, 1814) observed while investigating a fish kill in Saskatchewan, Canada. Fish were collected from seven different lakes, from two distinct watersheds. Sixty-four per cent (54/86) of yellow perch contained myxozoan pseudocysts located throughout the spinal cord and brain. Myxospores measured 16.5 µm (range 16.2-16.8) long and 8.2 µm (range 7.9-8.4) wide and contained two pyriform, mildly dissymmetrical, polar capsules measuring 7.7 µm (range 7.3-8.1) long and 2.7 µm (range 2.4-3.0) wide. The polar capsules each contained a single polar filament, with 7-9 turns per polar filament coil. Sequencing of the 18S SSU rDNA gene demonstrated >99% similarity to Myxobolus neurophilus. In 60% of infected fish, there was a mild to moderate, non-suppurative myelitis or encephalitis, or both, associated with myxospores. Axonal degeneration was present in rare cases. These findings extend the geographical distribution of M. neurophilus and suggest it may be widespread in yellow perch populations in Saskatchewan.

Telomerase expression and telomere length in immortal leukocyte lines from channel catfish.

Normal channel catfish leukocytes readily undergo spontaneous in vitro immortalization yielding functionally active diploid cell lines. Since telomerase activation appears to be a critical step in the establishment of immortal mammalian cells, studies were undertaken to determine if and when telomerase expression occurs during the in vitro immortalization process of channel catfish leukocytes. To this end, freshly isolated peripheral blood leukocytes (PBL) from normal fish were shown to exhibit low to undetectable levels of telomerase activity and within four days after culture initiation showed dramatic increases in telomerase activity which typically remained high for at least four weeks. This activity then declined, concomitant with decreases in cellular proliferation and increases in cell death. Cells which escaped this culture "crisis" re-expressed high levels of telomerase activity indefinitely. Although telomerase activity was expressed early in the immortalization process, clonal cell lines derived from these cultures had relatively short telomeres. These results suggest that telomerase expression in catfish leukocytes is activation-induced, and its expression does not necessarily stabilize telomere length until a critically, albeit ill-defined, short length is reached.

MHC class II A genes in the channel catfish (Ictalurus punctatus).

In order to characterize the Major histocompatibility complex (MHC) class II A genes of the channel catfish (Ictalurus punctatus) a cDNA library was screened and PCR was performed. Four different full-length cDNA sequences for MHC class II A genes were obtained from a clonal B cell line derived from an outbred fish. Two different genomic sequences and corresponding cDNAs were obtained from a presumably homozygous gynogenetic catfish. The A genes have five exons and four phase one introns. The first exon encodes the 5' untranslated region (UTR) and leader peptide; the second and third exons encode the alpha1 and alpha2 domains, respectively. The connecting peptide, transmembrane and cytoplasmic domains, as well as part of the 3' UTR, are encoded by the fourth exon and the rest of the 3' UTR is encoded by the fifth exon. Southern blot analyses using an exon three probe revealed two to four hybridizing fragments with considerable restriction fragment length polymorphisms evident among randomly selected outbred channel catfish. These findings are consistent with the presence of at least two functional polymorphic MHC class II A gene loci. An unusual aspect of the channel catfish MHC class II alpha chain is its lack of N-linked glycosylation sites.

Structure of the catfish IGH locus: analysis of the region including the single functional IGHM gene.

The catfish IGH locus is large ( approximately 1 Mb) and complex, having undergone multiple internal duplications and transpositions. To define the structure of the locus that contains the single expressed IGHM gene, two overlapping bacterial-artificial-chromosome (BAC) clones spanning the most 3' end of the channel catfish immunoglobulin heavy (IGH) chain locus have been completely sequenced. The analyses created a contig of 257,153 bp containing 55 VH, 6 D, 12 JH genes and the IGH constant region genes encoding the functional secreted and membrane forms of IgM and the membrane form of IgD. This analysis revealed three major features. First, no C-region genes were found aside from the previously described IGHM1 and IGHD1, with the latter gene being the most 3' C-region gene of the catfish IGH locus. There was no evidence in the region sequenced for genes that could encode an Ig class similar to the IgZ/IgT described in zebrafish, trout and pufferfish. Second, there are a high number of VH pseudogenes, 28 out of 55 (51%). In contrast, the entire zebrafish IGH locus has 40 functional VH genes and eight pseudogenes (17%). Third, an internal duplication of a 52.4-kb block of VH genes has occurred. These observations suggest that the IGH locus of teleost fish varies significantly from species to species in the diversity of C-region genes as well as the numbers of genes encoding V regions.

Environmental effects on fish immune mechanisms.

Environmental stress factors which influence fish immune (and likely many other physiological) functions can be divided into two broad, but not mutually exclusive, categories, namely those which occur naturally and those which are artificial. Natural environmental stress factors include season, temperature, salinity and photoperiod as well as social stress factors such as crowding and hierarchy. In general, artificial environmental stress factors are man made, and mainly involve pollutants such as acid rain, heavy metals and organic compounds. The available data indicate that regardless of which immune parameters are assessed, both natural and artificial environmental stress factors appear to suppress immune functions. Of the numerous environmental stress factors considered, pollutants, handling/confinement and low temperature are probably the best studied forms in fish. All three forms of stress factors have been shown to suppress components of both the innate (non-specific) and adaptive arms of the immune system. Since immune responses which protect against invading pathogens frequently involve interactions between both the innate and adaptive arms of the immune system, it seems reasonable to conclude that either acute or chronic exposure to stress factors may predispose fish to infectious diseases. Signalling mechanisms responsible for the effects of these various stress factors on immunity in fish are poorly understood, although elevated serum ACTH and cortisol levels appear to be involved in some cases. A better understanding of the mechanism(s) resulting in immunosuppression should facilitate future in vivo manipulations to reduce susceptibility to disease in aquaculture situations.