Lester H. Khoo

The epidemiology of bacterial diseases in food-size channel catfish.

Enteric septicemia of catfish (ESC) and columnaris are the most economically important bacterial diseases affecting the channel catfish Ictalurus punctatus industry in the United States. Although these two diseases have been extensively researched, little is known about their prevalence and epidemiology in production systems. In 1997, a two-part survey of catfish producers in Alabama, Arkansas, Louisiana, and Mississippi was conducted to estimate the proportion of ponds and catfish operations that have these diseases and to develop information on the risk factors associated with reporting an occurrence. The response rates to the two phases of the survey were 65.6% and 75.3%, respectively. Overall, 78.1% of all operations and 42.1% of all ponds experienced problems with ESC/columnaris. Higher percentages of large operations and ponds on large operations experienced these problems. The most frequently reported average loss per outbreak of the two diseases was 200-2,000 lb (1 lb = 0.454 kg) per outbreak. Univariate analysis and multivariable regression modeling of the survey data identified three possible risk factors associated with ESC/columnaris, namely, operation size, stocking density, and feeding rate. Conversely, operations that produced their own fingerlings and those that drained ponds at intervals of 3 years or less were less likely to report losses. The associations identified in this study do not establish firm causal relationships, but they do generate hypotheses about managerial and environmental interactions that represent substantial risks to production.

Efficient assembly and annotation of the transcriptome of catfish by RNA-Seq analysis of a doubled haploid homozygote.

BackgroundUpon the completion of whole genome sequencing, thorough genome annotation that associates genome sequences with biological meanings is essential. Genome annotation depends on the availability of transcript information as well as orthology information. In teleost fish, genome annotation is seriously hindered by genome duplication. Because of gene duplications, one cannot establish orthologies simply by homology comparisons. Rather intense phylogenetic analysis or structural analysis of orthologies is required for the identification of genes. To conduct phylogenetic analysis and orthology analysis, full-length transcripts are essential. Generation of large numbers of full-length transcripts using traditional transcript sequencing is very difficult and extremely costly.ResultsIn this work, we took advantage of a doubled haploid catfish, which has two sets of identical chromosomes and in theory there should be no allelic variations. As such, transcript sequences generated from next-generation sequencing can be favorably assembled into full-length transcripts. Deep sequencing of the doubled haploid channel catfish transcriptome was performed using Illumina HiSeq 2000 platform, yielding over 300 million high-quality trimmed reads totaling 27 Gbp. Assembly of these reads generated 370,798 non-redundant transcript-derived contigs. Functional annotation of the assembly allowed identification of 25,144 unique protein-encoding genes. A total of 2,659 unique genes were identified as putative duplicated genes in the catfish genome because the assembly of the corresponding transcripts harbored PSVs or MSVs (in the form of pseudo-SNPs in the assembly). Of the 25,144 contigs with unique protein hits, around 20,000 contigs matched 50% length of reference proteins, and over 14,000 transcripts were identified as full-length with complete open reading frames. The characterization of consensus sequences surrounding start codon and the stop codon confirmed the correct assembly of the full-length transcripts.ConclusionsThe large set of transcripts assembled in this study is the most comprehensive set of genome resources ever developed from catfish, which will provide the much needed resources for functional genome research in catfish, serving as a reference transcriptome for genome annotation, analysis of gene duplication, gene family structures, and digital gene expression analysis. The putative set of duplicated genes provide a starting point for genome scale analysis of gene duplication in the catfish genome, and should be a valuable resource for comparative genome analysis, genome evolution, and genome function studies.

Implication of Lateral Genetic Transfer in the Emergence of Aeromonas hydrophila Isolates of Epidemic Outbreaks in Channel Catfish

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Preliminary Assessment of the Tolerance and Efficacy of Florfenicol against Edwardsiella ictaluri Administered in Feed to Channel Catfish

Abstract A tolerance study was conducted to determine the palatability of florfenicol to channel catfish Ictalurus punctatus. Four tanks of fish (20 fish/tank) were assigned to each of five treatments distinguished by the amount of florfenicol given in feed per kilogram of body weight, namely, 0, 10, 20, 40, or 100 mg. Fish were fed at a rate of 2.5% of body weight per day for 10 consecutive days. On day 11, all surviving fish were euthanatized, counted, and weighed as a group. Florfenicol- medicated feed was palatable to fish at doses of 10, 20, 40, and 100 mg for 10 consecutive days. All 400 fish were necropsied and examined by histopathology, and no treatment-related changes were observed. In a separate exploratory efficacy study, four tanks (20 fish/tank) were assigned to each of the following treatments: (1) not challenged with Edwardsiella ictaluri and fed unmedicated feed, (2) challenged with E. ictaluri and fed unmedicated feed, (3) challenged with E. ictaluri and fed florfenicol at 10 mg per kilo...

Bolbophorus confusus Infections in Channel Catfish in Northwestern Mississippi and Effects of Water Temperature on Emergence of Cercariae from Infected Snails

Abstract The digenetic trematode tentatively identified as Bolbophorus confusus has recently been implicated as a cause of severe mortalities and reduced production in commercially raised catfish in the lower Mississippi Valley. Our study documents the prevalence of B. confusus infections in channel catfish Ictalurus punctatus from fish farms located in northwestern Mississippi. As of August 2000, 821 ponds had been sampled (approximately 20–40 fish per pond) from 32 farms in eight Mississippi counties. Of these ponds, 262 yielded at least one fish with metacercariae characteristic of B. confusus. The overall impact on farms and the industry cannot be fully evaluated from these data, but evidence suggests that infection with this trematode is becoming widespread, with the more severely affected farms being in close proximity to pelican roosting or resting sites. To evaluate some of the long-term implications of this trematode for farm management, two laboratory studies were conducted to determine the rela...

Complete Genome Sequence of a Channel Catfish Epidemic Isolate, Aeromonas hydrophila Strain ML09-119.

ABSTRACT Aeromonas hydrophila is a Gram-negative, rod-shaped, mesophilic bacterium that infects both aquatic poikilothermic animals and mammals, including humans. Here, we present the complete genome sequence of Aeromonas hydrophila strain ML09-119, which represents a clonal group of A. hydrophila isolates causing outbreaks of bacterial septicemia in channel catfish since 2009.

Morphologic, Pathologic, and Genetic Investigations of Bolbophorus Species Affecting Cultured Channel Catfish in the Mississippi Delta

Trematodes belonging to the genus Bolbophorus have recently been reported as the cause of substantial morbidity and mortality in cultured channel catfish Ictalurus punctatus in Mississippi and Louisiana. Previous investigators identified only a single species, B. confusus. In this investigation, genetic techniques were used to identify all stages of the parasite in all of its hosts. The 18s rRNA genes from specimens collected in Mississippi were sequenced and compared; this analysis revealed that there are two distinct species, B. damnificus (previously identified as B. confusus) and another, undescribed species. (Phylogenetic analysis indicated that a third species, B. levantinus, is also closely related to the Mississippi species.) Species-specific polymerase chain reaction assays capable of identifying and differentiating between these two parasites were developed. Both species were found to infect the first intermediate host (the rams horn snail Planorbella trivolvis) in commercial channel catfish ponds, but only B. damnificus was recovered from the fish themselves. The new, unidentified Bolbophorus species was determined to be highly pathogenic to a number of fish species. The contribution of B. damnificus to disease in cultured channel catfish remains undetermined. Future investigations of these parasites must now take into account the presence of two distinct species.Trematodes belonging to the genus Bolbophorus have recently been reported as the cause of substantial morbidity and mortality in cultured channel catfish Ictalurus punctatus in Mississippi and Louisiana. Previous investigators identified only a single species, B. confusus. In this investigation, genetic techniques were used to identify all stages of the parasite in all of its hosts. The 18s rRNA genes from specimens collected in Mississippi were sequenced and compared; this analysis revealed that there are two distinct species, B. damnificus (previously identified as B. confusus) and another, undescribed species. (Phylogenetic analysis indicated that a third species, B. levantinus, is also closely related to the Mississippi species.) Species-specific polymerase chain reaction assays capable of identifying and differentiating between these two parasites were developed. Both species were found to infect the first intermediate host (the rams horn snail Planorbella trivolvis) in commercial channel catfish ponds, but only B. damnificus was recovered from the fish themselves. The new, unidentified Bolbophorus species was determined to be highly pathogenic to a number of fish species. The contribution of B. damnificus to disease in cultured channel catfish remains undetermined. Future investigations of these parasites must now take into account the presence of two distinct species.

Edwardsiella piscicida identified in the southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR

A new Edwardsiella taxon was recently described from fishes of Europe and Asia. Phenotypically similar to E. tarda, extensive genetic and phenotypic characterization determined this new strain does not belong to any established Edwardsiella taxa, leading to the adoption of a new taxon, E. piscicida. Concurrent research in the USA also identified 2 genetically distinct taxa within the group of organisms traditionally classified as E. tarda. Comparisons of gyrB sequences between US isolates and E. piscicida from Europe and Asia identified several US isolates with >99.6% similarity to the gyrB sequence of the E. piscicida type strain (ET883) but <87% similarity to the E. tarda type strain (ATCC #15947). A discriminatory PCR was developed for the identification of E. tarda and 2 genetic variants of E. piscicida (E. piscicida and E. piscicida-like species). Using these PCR assays, a survey was conducted of 44 archived bacterial specimens from disease case submissions to the Aquatic Research and Diagnostic Laboratory (Stoneville, MS, USA) between 2007 and 2012. All 44 isolates, originally identified phenotypically and biochemically as E. tarda, were identified as E. piscicida by PCR. Repetitive sequence-mediated PCR (rep-PCR) analysis of these archived specimens suggests they are largely homogenous, similar to what has been observed for E. ictaluri. The gyrB sequence data, coupled with the E. piscicida specific-PCR and rep-PCR data, confirms that E. piscicida has been isolated from fish disease cases in the southeastern USA. Moreover, our survey data suggests E. piscicida may be more prevalent in catfish aquaculture than E. tarda.

Genetic Sequence Data Identifies the Cercaria of Drepanocephalus spathans (Digenea: Echinostomatidae), A Parasite of the Double-Crested Cormorant (Phalacrocorax auritus), with Notes on its Pathology in Juvenile Channel Catfish (Ictalurus punctatus)

Abstract: An unidentified xiphidio-type cercaria, previously thought inconsequential to catfish health, was found to be released from marsh rams-horn snails (Planorbella trivolvis) inhabiting ponds on a commercial catfish operation in the Mississippi Delta. A preliminary challenge of cohabiting channel catfish (Ictalurus punctatus) with snails actively shedding the unidentified cercariae resulted in death of some fish. A second cohabitation trial yielded similar results, as did a third challenge of 250 cercariae/fish. Histopathology revealed developing metacercariae concentrated in the cranial region, especially within the branchial chamber, with several metacercariae at the base of the branchial arches within, or adjacent to, blood vessels, possibly the proximate cause of death. Genetic sequence analysis of the 18S small subunit ribosomal DNA (ssDNA), 28S large subunit rDNA (lsDNA), and cytochrome oxidase (Cox1) genes all matched the cercariae to Drepanocephalus spathans (Digenea: Echinostomatidae), a parasite of the double-crested cormorant (Phalacrocorax auritus), a piscivorous bird endemic on most catfish farms. This is the first commentary regarding pathology of D. spathans in juvenile channel catfish as well as the first report of the marsh rams-horn snail as an intermediate host in the D. spathans life cycle. The data presented here suggest this parasite could have limiting effects on catfish production, further supporting the need for adequate snail control programs to reduce trematode prevalence on commercial catfish operations.