spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as actually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each species using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of spp. and advantages of employing alternative single-copy genes and for molecular identification and classification of were demonstrated. Last, sequencing confirmed that isolates previously defined as typical motile fish-pathogenic are synonymous with, while atypical nonmotile fish-pathogenic isolates are equivalent to Fish-nonpathogenic isolates are consistent with as it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of isolates in clinical and research settings.

One major concern about using adenoviral vectors for repetitive gene delivery to lung epithelial cells is the induction of an immune response to the vector, thus, impeding effective gene transduction. To assess the immune response to the adenoviral vector, repetitive intratracheal (i.t.) gene dosing was performed in CD-1 mice using the replication-deficient adenovirus 5 (Ade5) vector carrying the lacZ gene, and compared to the antibody responses induced by conventional intranasal (i.n.) and intraperitoneal (i.p.) routes of immunization. Kinetics of serum IgG, IgA, and IgM antibody responses to the adenoviral vector and to beta-galactosidase (beta-Gal) were evaluated. Two or three adenoviral vector doses given by i.t., i.n., or i.p. routes resulted in serum IgG titers in excess of 1:200,000, whereas serum IgM and IgA were moderately induced. Analysis of the predominant murine IgG subclass was determined to be IgG2b and IgG2a. To determine the localization of this antibody response, the ELISPOT assay was employed. Lymphocytes were isolated from the lung, the lower respiratory lymph nodes (LRLN), the nasal passages (NP), and the spleen. For i.t- and i.n.-administered mice, the highest IgA spot-forming cell (SFC) response to Ade5 and beta-Gal was located in the NP and in the lung. Both the lung and the LRLN showed elevated numbers of IgG SFCs (4- to 12-fold greater than splenic IgG SFC response) for Ade5 and beta-Gal. This evidence suggests that the lung and associated lymphoid tissues were the source for serum antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

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A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri-specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.

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.

Members of the genus Edwardsiella are important pathogens of cultured and wild fish globally. Recent investigations into the phenotypic and genotypic variation of Edwardsiella tarda have led to the segregation of E. tarda into three distinct taxa: E. tarda, Edwardsiella piscicida, and Edwardsiella anguillarum. In catfish aquaculture in the southeastern USA, E. piscicida has been more commonly associated with disease than E. tarda or E. anguillarum, and recent research has demonstrated E. piscicida to be more pathogenic in channel catfish than E. tarda or E. anguillarum. Anecdotal reports from industry suggest an increased prevalence of E. piscicida associated with the culture of channel (♀) × blue (♂) hybrid catfish. This work investigated the comparative susceptibility of channel catfish, blue catfish, and their hybrid cross to molecularly confirmed isolates of E. tarda, E. piscicida, and E. anguillarum. There was significantly higher mortality in hybrid catfish compared to channel catfish following intracoelomic injection of E. piscicida. To our knowledge, E. piscicida is the first bacterial pathogen to demonstrate increased pathogenicity in hybrid catfish compared to channel catfish. KEYWORDS blue catfish, channel catfish, Edwardsiella, hybrid catfish

Proliferative gill disease (PGD) in channel catfish Ictalurus punctatus is caused by the myxozoan parasite Henneguya ictaluri. Prolonged exposure of channel catfish to the actinospore stage of the parasite results in extensive gill damage, leading to reduced production and significant mortality in commercial operations. A H. ictaluri-specific real-time (Q)PCR assay was used to determine parasite levels in commercial channel catfish ponds and evaluate the risk of losing fish newly stocked into the system. Previous research has shown the H. ictaluri actinospore to be infective for approximately 24 h; therefore, determining the parasite load (ratio of parasite DNA to host DNA) in sentinel fish exposed for 2 separate 24 h periods with a minimum of 1 wk between sampling indirectly represents the rate at which infective actinospores are being released by the oligochaete host and if that rate is changing over time. Alternatively, QPCR analysis of pond water samples eliminates the need for sentinel fish. Water samples collected on 2 separate days, with a minimum of 1 wk between sampling, not only determines the approximate concentrations of actinospores in the pond but if these concentrations are remaining stable. Increases in parasite load (r = 0.69, p = 0.054) correlated with percent mortality in sentinel fish, as did increases in mean actinospore concentrations (r = 0.63, p = 0.003). Both applications are more rapid than current protocols for evaluating the PGD status of a catfish pond and identified actinospore levels that correlate to both high and low risk of fish loss.

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