Cross-protective efficacy of a live-attenuated Edwardsiella ictaluri vaccine against heterologous Edwardsiella piscicida isolates in channel and channel × blue catfish hybrids.

Edwardsiella piscicida is a growing problem for catfish aquaculture in the southeastern United States, particularly in channel (Ictalurus punctatus) x blue (I. furcatus) catfish hybrids. Research has shown E. piscicida isolates recovered from farmed catfish in Mississippi form at least five discrete phyletic groups, with no apparent differences in virulence in channel and hybrid catfish. Laboratory trials have shown a live-attenuated E. ictaluri vaccine (340X2) cross-protects against at least one E. piscicida isolate (S11-285) in channel and hybrid catfish, although it is unknown if this protection exists for other E. piscicida variants. To this end, channel and hybrid catfish were immunized by immersion with E. ictaluri 340X2. Thirty days later, fish were challenged by intracoelomic injection with representative E. piscicida variants from each phyletic group. Relative percent survival (RPS) for hybrids ranged from 54.7% to 77.8%, while RPS in channels ranged from 80.5% to 100%. A second study investigated whether channel and hybrid catfish exposed to heterologous E. piscicida isolates were similarly protected against wild-type E. ictaluri. Fish were exposed by bath immersion to representative E. piscicida isolates from each phyletic group. Thirty days post-immunization, fish were challenged by immersion with wild-type E. ictaluri isolate S97-773. Regardless of variant, previous exposure to heterologous E. piscicida isolates significantly improved survival following E. ictaluri challenge. These findings suggest the presence of shared and conserved antigens among E. piscicida and E. ictaluri that could be exploited by application of polyvalent or cross-protective vaccines.

Genetic variability of Edwardsiella piscicida isolates from Mississippi catfish aquaculture with an assessment of virulence in channel and channel × blue hybrid catfish.

The bacterium Edwardsiella piscicida causes significant losses in global aquaculture, particularly channel (Ictalurus punctatus) × blue (I. furcatus) hybrid catfish cultured in the south-eastern United States. Emergence of E. piscicida in hybrid catfish is worrisome given current industry trends towards increased hybrid production. The project objectives were to assess intraspecific genetic variability of E. piscicida isolates recovered from diseased channel and hybrid catfish in Mississippi; and determine virulence associations among genetic variants. Repetitive extragenic palindromic sequence-based PCR (rep-PCR) using ERIC I and II primers was used to screen 158 E. piscicida diagnostic case isolates. A subsample of 39 E. piscicida isolates, representing predominant rep-PCR profiles, was further characterized using BOX and (GTG)5 rep-PCR primers, virulence gene assessment and multilocus sequence analysis (MLSA) targeting housekeeping genes gyrb, pgi and phoU. The MLSA provided greater resolution than rep-PCR, revealing 5 discrete phylogroups that correlated similarly with virulence gene profiles. Virulence assessments using E. piscicida representatives from each MLSA group resulted in 14-day cumulative mortality ranging from 22% to 54% and 63 to 72% in channel and hybrid fingerlings, respectively. Across all phylogroups, mortality was higher in hybrid catfish (p < .05), supporting previous work indicating E. piscicida is an emerging threat to hybrid catfish aquaculture in the south-eastern United States.

Arrested Development of Henneguya ictaluri (Cnidaria: Myxobolidae) in ♀ Channel Catfish × â™‚ Blue Catfish Hybrids.

Henneguya ictaluri is the etiologic agent of proliferative gill disease (PGD) in farm-raised Channel Catfish Ictalurus punctatus and hybrid catfish in the southeastern United States, and significant annual losses are attributed to this disease. Research suggests that H. ictaluri infection dynamics in Blue Catfish I. furcatus and hybrid catfish (Channel Catfish × Blue Catfish) differ from those in Channel Catfish. Two separate infectivity trials were conducted to investigate H. ictaluri development in Channel Catfish, Blue Catfish, and their hybrids. On two separate occasions with two different year-classes, fish were exposed to pond water containing H. ictaluri actinospores and sampled weekly for 12 weeks (trial 1) or 14 weeks (trial 2). In trial 1, the presence of H. ictaluri was evaluated histologically and by quantitative PCR of fish tissues, including gills, blood, anterior kidney, brain, heart, liver, posterior kidney, spleen, and stomach. Henneguya ictaluri DNA was detected in significantly higher concentrations throughout multiple organ systems in the Channel Catfish compared to the hybrid catfish and Blue Catfish, with the gills having higher quantities. Myxospores were observed in Channel Catfish gill tissue at 8 weeks postexposure. No myxospores were observed in Blue Catfish or hybrid catfish. The second trial focused on gills only and yielded similar results, with Channel Catfish having significantly greater H. ictaluri DNA quantities than hybrids or Blue Catfish across all time points. Myxospores were observed in Channel Catfish beginning at 6 weeks postexposure and were found in 36% (58/162) of Channel Catfish sampled for molecular and histological analysis during weeks 6-14. Myxospores in hybrid catfish were sparse, with single pseudocysts observed in two hybrid catfish (1.2%) at 14 weeks postexposure. These results imply arrested development of H. ictaluri in hybrid catfish. As such, culture of hybrid catfish may be an effective management strategy to minimize the burden of PGD.

Comparative Phenotypic and Genotypic Analysis of Edwardsiella Isolates from Different Hosts and Geographic Origins, with Emphasis on Isolates Formerly Classified as E. tarda, and Evaluation of Diagnostic Methods.

Edwardsiella spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as Edwardsiella tarda 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 Edwardsiella 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 Edwardsiella spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of Edwardsiella spp. and advantages of employing alternative single-copy genes gyrB and sodB for molecular identification and classification of Edwardsiella were demonstrated. Last, sodB sequencing confirmed that isolates previously defined as typical motile fish-pathogenic E. tarda are synonymous with Edwardsiella piscicida, while atypical nonmotile fish-pathogenic E. tarda isolates are equivalent to Edwardsiella anguillarum Fish-nonpathogenic E. tarda isolates are consistent with E. tarda 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 Edwardsiella isolates in clinical and research settings.

Validation of Fermentation and Processing Procedures for the Commercial-Scale Production of a Live, Attenuated Edwardsiella ictaluri Vaccine for Use in Channel Catfish Aquaculture.

Mortality associated with Edwardsiella ictaluri infection is a serious impediment to the commercial production of fingerling Channel Catfish Ictalurus punctatus. A patented, live, attenuated, orally delivered vaccine has been developed that offers exceptional protection against E. ictaluri infection in both laboratory and small-scale pond trials. Further vaccine development is contingent on the successful completion of large-scale field trials that accurately reflect industry conditions. This current work focuses on the validation of fermentation protocols and the optimization of downstream processing procedures to produce sufficient quantities of vaccine to conduct commercial-scale field trials. Eight vaccine serials were produced from a master seed stock (S97-773-340X2) in a 50-L floor model fermenter over two consecutive years. Following fermentation, cells were harvested, concentrated 10-fold, and cryogenically stored (-74°C). To assess processing protocols and determine shelf life of cryogenically stored vaccine, serials were tested for cell viability and vaccine potency at various intervals over 24 months. There were no significant differences in cell viability between the fresh vaccine and the stored frozen product. All serials provided a high level of protection (77-100% relative percent survival) against E. ictaluri infection in juvenile Channel Catfish and exhibited excellent poststorage viability. This data demonstrates that the live, attenuated, orally delivered vaccine can be stored at -74°C for at least 2 years with no reduction in cell viability or vaccine potency. Received May 17, 2016; accepted January 19, 2017.

Myxobolus ictiobus n. sp. and Myxobolus minutus n. sp. (Cnidaria: Myxobolidae) from the gills of the smallmouth buffalo Ictiobus bubalus Rafinesque (Cypriniformes: Catostomidae).

The smallmouth buffalo Ictiobus bubalus Rafinesque (Catostomidae) is native to North American waterways and occasionally grown in pond aquaculture. Species of Myxobolus Bütschli, 1882 have been reported from the gills, integument, and intestinal tract of buffalo fish, although there is ambiguity in some host records. In the summer of 2013, thirteen adult smallmouth buffalo were seined from a 0.1-acre (0.04-hectare) experimental research pond at the Thad Cochran National Warmwater Aquaculture Center in Stoneville, Mississippi, USA, and examined for the presence of parasitic infection. Two previously unknown species of Myxobolus were observed parasitising the gills. Plasmodia of the two species differed from each other in both size and shape. Morphologically the two species were distinct from one another and from other Myxobolus spp. previously reported from buffalo fish. Myxospores of Myxobolus ictiobus n. sp. were spherical and measured 12.7-14.5 (13.9 ± 0.4) µm in length and 10.7-13.6 (12.5 ± 0.7) µm in width with a thickness of 10.3-14.8 (12.6 ± 2.3) µm. Polar capsules measured 5.6-7.4 (6.6 ± 0.4) µm in length and 3.7-4.9 (4.5 ± 0.8) µm in width and each contained a coiled polar filament with 5-6 turns. Myxospores of Myxobolus minutus n. sp. were circular in shape and measured 7.4-9.6 (8.6 ± 0.7) µm in length and 7.5-9.9 (8.8 ± 0.7) µm in width with a thickness of 6.5-7.3 (6.7 ± 0.3) µm. Polar capsules measured 3.6-4.9 (4.3 ± 0.3) µm in length and 2.8-3.8 (3.3 ± 0.3) µm and each contained a coiled polar filament with 5-6 turns. Supplemental 18S rRNA gene sequencing identified unique sequences for each isolate. Phylogenetic analysis of 18S rRNA sequences demonstrated a strong clustering of both isolates with other species of Myxobolus from cypriniform fish.

Oral Vaccination of Channel Catfish against Enteric Septicemia of Catfish Using a Live Attenuated Edwardsiella ictaluri Isolate.

Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are typically administered by immersion when fry are transferred from the hatchery to rearing ponds. While this approach is a practical method of mass delivery, this strategy administers vaccines to very young fish, which lack a fully developed immune system. To circumvent this limitation, an oral vaccination strategy was evaluated as a means of immunizing catfish at the fingerling stage of production, when fish possess a more complete immune arsenal. A virulent E. ictaluri isolate (S97-773) was attenuated by successive passage on media containing increasing concentrations of rifamycin. In laboratory trials, cultured vaccine was diluted and mixed with feed (100 mL diluted vaccine/454 g feed). This mixture was then fed to Channel Catfish Ictalurus punctatus fingerlings. Two separate dilutions of cultured vaccine (1:10 and 1:100) were used to create the vaccine-feed mixture, equating to estimated doses of 5 × 107 and 5 × 106 CFU/g of feed, respectively. After 30 d, catfish were exposed by immersion (1 × 106 CFU/mL) to the virulent parental strain of E. ictaluri. The target dose (1:100 dilution, ∼5 × 106 CFU/g of feed) offered exceptional protection (relative percent survival = 82.6-100%). In addition, negligible deaths occurred in fish vaccinated at 10 times the target dose (1:10 dilution, ∼5 × 107 CFU/g of feed). In pond trials, antibody production increased 18-fold in orally vaccinated fish. When compared with nonvaccinated controls, vaccination significantly improved survival, feed fed, feed conversion, biomass produced, and total harvest. This research demonstrates Channel Catfish can be successfully immunized in a commercial setting against E. ictaluri with a single dose of an orally delivered, live attenuated, E. ictaluri vaccine.

Small subunit ribosomal RNA sequence links the myxospore stage of Henneguya mississippiensis n. sp. from channel catfish Ictalurus punctatus to an actinospore released by the benthic oligochaete Dero digitata.

There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 µm (range = 40.7-61.6 µm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 µm (14.4-19.3 µm) in length and 5.0 ± 0.3 µm (4.5-5.5 µm) in width. The two polar capsules were 6.2 ± 0.4 µm (5.8-7.0 µm) long and 5.0 ± 0.3 µm (4.5-5.5 µm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 µm (22.9-40.6 µm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4% similarity (100% coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.

Real-time polymerase chain reaction assays for the detection and quantification of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella piscicida-like species in catfish tissues and pond water.

Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida-like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published primers, for E. tarda, E. piscicida, and E. piscicida-like sp. to provide rapid quantitative confirmatory tests for these phenotypically ambiguous bacteria. The qPCR assays were shown to be repeatable and reproducible, with high degrees of sensitivity and specificity. Each assay showed a linear dynamic range covering 8 orders of magnitude and a sensitivity limit of 5 copies of target DNA in a 15-µL reaction. In addition, each assay was found specific to their respective targets with no observed amplification from nontarget organisms, including the closely related E. ictaluri and E. hoshinae. Under the conditions used in this study, the 3 assays had a quantifiable limit ranging from 10(3) (E. piscicida) to 10(2) (E. piscicida-like and E. tarda) colony forming units in kidney tissue biopsies (approximately 25 mg), pond water samples (35 mL), and broth culture (20 µL). In experimental challenges, the assays were able to detect their respective targets in both clinically and subclinically infected channel catfish (Ictalurus punctatus) fingerlings. In addition to quantifying target bacteria from various substrates, the assays provide rapid identification, differentiation, and confirmation of the phenotypically indistinguishable E. tarda, E. piscicida, and E. piscicida-like sp., a valuable tool for diagnostic assessments.

Chronic pathology and longevity of Drepanocephalus spathans infections in juvenile Channel Catfish.

Drepanocephalus spathans (Digenea: Echinostomatidae) is a common parasite of the double-crested cormorant Phalacrocorax auritus. The cercariae of D. spathans have been shown infective to juvenile Channel Catfish Ictalurus punctatus. The developing metacercariae concentrate in the cranial regions, often occluding blood vessels at the base of the branchial arch, occasionally resulting in death. The purpose of this study was to determine how long metacercariae of D. spathans persist in experimentally challenged Channel Catfish. Two separate infectivity trials were conducted. In both trials, metacercariae persisted at least 49 d postinfection, although prevalence and intensity of infection decreased over time. In the first trial, juvenile catfish (1-3 g) were exposed over three consecutive days to 100, 100, and 80 cercariae/fish/d, respectively. Fish were sampled 7 d after the final exposure, and metacercariae were observed in 83.3% (five of six) of challenged fish. At 21 d postexposure, metacercariae were present in only 50% of exposed fish (three of six). No metacercaria were observed in fish sampled at 35 d, however, metacercariae were present in one of six (16.7%) fish sampled 49 and 70 d postexposure, respectively. A second challenge consisted of a 24-h pooled exposure of 500 cercariae per fish. Again, metacercariae were present in most (six of seven; 85.7%) fish at 7 d postexposure. At 21 d postexposure, metacercariae were only evident in one of seven (14.3%) sampled fish. No metacercariae were present in any fish at 35 d postchallenge, yet one of seven (14.3%) fish was positive at 49 d postchallenge. The second study was terminated at 63 d postchallenge, as all fish sampled (n = 14) were negative for metacercariae. These data suggest that cercariae of D. spathans are infective to juvenile Channel Catfish, although the infection appears short lived as metacercariae rarely persisted longer than 2 months.

Molecular and morphological characterization of myxozoan actinospore types from a commercial catfish pond in the Mississippi delta.

The actinospore diversity of infected Dero digitata was surveyed (May 2011) from a channel catfish (Ictalurus punctatus) production pond in the Mississippi Delta region for the elucidation of unknown myxozoan life cycles. At present, only 2 myxozoan life cycles have been molecularly confirmed in channel catfish, linking the actinospore stage from an aquatic oligochaete (D. digitata ) and the myxospore stage from the catfish. In this study D. digitata (n = 2,592) were isolated from oligochaetes collected from the bottom sediment of a channel catfish production pond. After 1 wk of daily observation, a total of 6 genetically different actinospore types were observed. The collective groups were classified as 2 aurantiactinomyxons, 2 helioactinomyxons, 1 raabeia, and 1 triactinomyxon. Overall prevalence of myxozoan infections in the isolated oligochaetes was 4.4%. Actinospores were photographed and measured for morphological characterization. Four previously undescribed actinospore types were identified and characterized molecularly and morphologically. Phylogenetic analysis revealed the raabeia and one of the helioactinomyxon (type 1) actinospores were closely related to the group of myxozoans known to parasitize ictalurids in North America. To date, no myxospores have been linked to the newly sequenced actinospores reported in this survey. The morphological and molecular data generated from this study will assist in the identification of myxospore counterparts for these actinospore stages and aid in the elucidation of unknown myxozoan life cycles in closed production systems.

Comparative susceptibility of Channel Catfish, Blue Catfish, and their hybrid cross to experimental challenge with Bolbophorus damnificus (Digenea: Bolbophoridae) cercariae.

The digenetic trematode Bolbophorus damnificus has been implicated in significant losses in catfish aquaculture since the late 1990s. The complex life cycle sequentially involves the American white pelican Pelecanus erythrorhynchos, the marsh rams horn snail Planorbella trivolvis, and Channel Catfish Ictalurus punctatus. Research supports anecdotal reports from the industry, suggesting that the hybrid of Channel Catfish×Blue Catfish I. furcatus is less susceptible to disease agents that have been historically prohibitive to Channel Catfish production, namely the gram-negative bacteria Edwardsiella ictaluri and Flavobacterium columnare, as well as the myxozoan parasite Henneguya ictaluri. This current research compared the susceptibility of Channel Catfish, Blue Catfish, and their hybrid cross to an experimental challenge by B. damnificus. Fish were exposed to 0, 100, 200, and 400 B. damnificus cercariae per fish, and the numbers of metacercariae per fish were determined 14 d postchallenge. Metacercariae were recovered from all challenged fish. There were no significant differences among fish groups challenged with the same dose, suggesting Channel and Blue Catfish and their hybrid are comparably susceptible to B. damnificus infection. As such, it is recommended that producers raising hybrid catfish remain diligent in controlling populations of the snail intermediate host to prevent production losses attributed to B. damnificus, especially when loafing pelicans have been observed at the aquaculture operation.

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).

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.

A real-time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi.

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.

A duplex real-time polymerase chain reaction assay for differentiation between Bolbophorus damnificus and Bolbophorus type II species cercariae.

A duplex quantitative real-time polymerase chain reaction (qPCR) assay was developed to differentiate between Bolbophorus damnificus and Bolbophorus type II species cercariae. Both trematode species are prevalent throughout the commercial catfish industry, as both infect the ram's horn snail, Planorbella trivolvis, which is commonly found in catfish ponds. Identification of cercaria to species is important in catfish disease challenge experiments, as only B. damnificus has been shown to have negative impacts on channel catfish. Oligonucleotide primers and fluorescence resonance energy transfer hydrolysis probes were designed to amplify the 18S small subunit ribosomal DNA gene of each species. The quantification cycle indicative of the number of cercariae in the sample prep was determined, and standard curves correlating to cercaria numbers were established. For both species, the assay was found to be highly repeatable and reproducible, with a linear dynamic range covering 7 orders of magnitude. The sensitivity limit of the assay was approximately 1/256th of a cercaria, regardless of species, and there was no remarkable interference between the 2 assays when run simultaneously within the same reaction. In a field study, identification of cercaria by the duplex real-time qPCR assay was in complete agreement with previously established end-point PCR protocols, demonstrating the assay to be a more rapid, quantifiable means of parasite identification.

Variation in susceptibility to Henneguya ictaluri infection by two species of catfish and their hybrid cross.

Proliferative gill disease (PGD) in channel catfish Ictalurus punctatus is caused by the myxozoan parasite Henneguya ictaluri. There is no effective treatment for PGD, and mortalities can exceed 50% in severe outbreaks. One approach to controlling losses would be to utilize a less susceptible ictalurid species in pond culture; alternatively, one could identify the traits that convey resistance and exploit them in a selective breeding program. Challenge studies have found less severe inflammatory responses in the gill tissue of blue catfish I. furcatus and fewer mortalities than in channel catfish. However, it remains unclear whether infection and subsequent plasmodial development progress the same way in the two species. To investigate this, we compared the dynamics of H. ictaluri infection in blue catfish, channel catfish, and channel catfish x blue catfish hybrids in continuous long-term (5-7-d) and short-term (24-h) pond challenges. After long-term challenge, 66.2% of the channel catfish and 63.6% of the hybrid catfish developed characteristic PGD lesions, compared with 3.7% of the blue catfish. Quantitative polymerase chain reaction analysis detected H. ictaluri in larger percentages of channel and hybrid catfish than blue catfish (98.7% and 95.7% versus 45.9%), with significantly greater parasite DNA equivalents in channel and hybrid catfish than blue catfish. Similar findings were obtained in the short-term exposures. Histologically, channel and hybrid catfish developed severe PGD accompanied by large numbers of developing plasmodia. While mild PGD was observed in some blue catfish, the progression of lesions lagged behind that in channel and hybrid catfish. Most importantly, developing plasmodia were not observed in blue catfish, and parasite DNA was not detected 14 d after removal from the source of infection. Our findings indicate that the resistance of blue catfish to H. ictaluri infection can be overcome by large numbers of infective actinospores but that infection appears to be eliminated before plasmodial development occurs.

Application of a real-time PCR assay for the detection of Henneguya ictaluri in commercial channel catfish ponds.

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.

A real-time polymerase chain reaction assay for the detection of the myxozoan parasite Henneguya ictaluri in channel catfish.

Proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya ictaluri, is the most prevalent parasitic infection affecting commercial channel catfish (Ictalurus punctatus) aquaculture. There are currently no effective chemotherapeutic or biological control measures for PGD, which often peaks during the spring and fall when water temperatures are between 16-25 degrees C. The current diagnostic techniques of gross examination of gill clip wet mounts and histopathology are subject to false-negatives during the early stages of infection, and the quantifiable nature of end-point polymerase chain reaction (PCR) is subjective. Consequently, a rapid and more sensitive quantitative real-time PCR assay was developed for the detection of H. ictaluri during the early stages of infection in channel catfish. A 23 base-pair TaqMan probe was designed based on previously published H. ictaluri PCR protocols. The sensitivity of the assay was the equivalent of a single H. ictaluri actinospore, and in a pond challenge study, quantitative real-time PCR proved to be more sensitive than gross examination, microscopic examination of gill clip wet mounts, and histopathologic examination of gill tissue sections. Future applications of this assay will focus on developing methodologies to be used in conjunction with current pond-monitoring protocols to evaluate potential treatments and better manage this significant seasonal disease.