Linda M. Pote

Small subunit ribosomal RNA sequence of Henneguya exilis (class Myxosporea) identifies the actinosporean stage from an oligochaete host.

ABSTRACT Several transmission studies, as well as recent molecular data, have indicated that the two classes Myxosporea and Actinosporea represent different life cycle stages of Myxozoa. To evaluate the life cycles of myxozoa in catfish aquaculture systems, the small subunit (18S) ribosomal RNA gene sequences of Henneguya exilis, a myxosporean from channel catfish Ictalurus punctatus, and an actinosporean (previously designated as Aurantiactinomyxon janiszewskai) from the aquatic oligochaete Dero digitata were determined. The sequences were identical, indicating that H. exilis and the actinosporean are alternate life stages of a single species. This is the first report identifying the actinosporean stage of the genus Henneguya.

Small Subunit Ribosomal RNA Sequences Link the Cause of Proliferative Gill Disease in Channel Catfish to Henneguya n. sp. (Myxozoa: Myxosporea)

Abstract In a previous study the small subunit ribosomal RNA (SSU rRNA) gene of Aurantiactinomyxon ictaluri, the actinosporean stage (actinospore) of the myxozoan associated with proliferative gill disease (PGD), was sequenced. In this study bath exposures of channel catfish Ictalurus punctatus to A. ictaluri isolated from the aquatic oligochaete Dero digitata were used to study the subsequent development of this parasite and to confirm the SSU rRNA gene sequences of each life stage. On day 7 postexposure, typical PGD organisms were observed in the gills, and at 3 months postinfection, cysts containing the newly identified myxozoan Henneguya sp. spores were observed in the gills. The sequence of the SSU rRNA genes of the actinospore and myxospore life stages demonstrated their identity. This is the first molecular evidence that A. ictaluri, isolated from D. digitata, can infect catfish and produce the typical PGD organisms in the gills. These data provide further proof that the actinospore A. ictaluri is ...

Small Subunit rRNA Gene Comparisons of Four Actinosporean Species to Establish a Polymerase Chain Reaction Test for the Causative Agent of Proliferative Gill Disease in Channel Catfish

Abstract Proliferative gill disease (PGD) causes high morbidity and mortality in cultured channel catfish Ictalurus punctatus. The presence of the myxozoan Aurantiactinomyxon ictaluri (class Actinosporea) is strongly associated with PGD. This parasite, shed as an actinospore from the aquatic oligochaete Dero digitata, infects channel catfish by an undetermined route. Several other actinosporeans have been identified that are shed from D. digitata isolated from catfish ponds, including those designated A. mississippiensis, Helioactinomyxon sp., and the actinospore stage of Henneguya exilis. By the use of multiple sequence alignment of polymerase chain reaction (PCR)-amplified small subunit ribosomal RNA (SSU rRNA) genes of A. ictaluri, A. mississippiensis, and H. exilis, we identified two variable regions. The largest variable region was PCR amplified, sequenced from the Helioactinomyxon sp., and used in addition to the other three sequences in multiple-sequence alignment comparison to develop PCR primers ...

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.

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.

Etiological and Epizootological Factors Associated with Outbreaks of Proliferative Gill Disease in Channel Catfish

Abstract Water, sediment, and channel catfish Ictalurus punctatus were sampled from seven farms experiencing proliferative gill disease (PGD) outbreaks in spring 1992. At each farm, samples were collected from the pond that experienced the outbreak (PGD-positive pond) and from another pond where no PGD was observed (control pond). Seven species of Actinosporea were detected in the oligochaete populations of the ponds examined The only actinosporean species significantly (P ≤ 0.05) correlated with clinical PGD outbreaks was Aurantiactinomyxon sp. (type 1). Population densities of aquatic oligochaetes in the pond sediments, including Dero digitata, the host of Aurantiactinomyxon sp., were surveyed during the PGD outbreaks and over the following year. Population densities of all oligochaetes, densities of D. digitata, and proportions of the total oligochaete population represented by D. digitata were all significantly higher (P ≤ 0.05) in ponds experiencing clinical outbreaks of PGD than in control ponds. Af...

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.

MORPHOLOGIC AND MOLECULAR IDENTIFICATIONS OF DIGENETIC TREMATODES IN DOUBLE-CRESTED CORMORANTS (PHALACROCORAX AURITUS) FROM THE MISSISSIPPI DELTA, USA

Abstract Increasing numbers of Double-crested Cormorants (Phalacrocorax auritus) in the Mississippi River Delta, USA, have been observed over the past few decades. This piscivorous bird is a definitive host for numerous digenetic trematodes, some of which may cause pathology in a fish host. We conducted a 2-yr survey of intestinal trematodes in 35 Double-crested Cormorants collected in the Mississippi Delta. We counted gastrointestinal trematodes, identified them to species using morphometric and molecular techniques, and sequenced the 18S and cytochrome oxidase I (COI) genes. We collected 4,909 trematodes, representing five digenetic species: Drepanocephalus spathans, Hysteromorpha triloba, Pseudopsilostoma varium, Austrodiplostomum ostrowskiae, and Ascocotyle longa. The most prevalent trematode of the Double-crested Cormorants was D. spathans (91%), followed by H. triloba (78%), P. varium (74%), A. ostrowskiae (57%), and A. longa (29%). Among these, the life cycles are only known for H. triloba and A. longa. Novel DNA sequences of the COI gene were obtained for D. spathans, A. ostrowskiae, P. varium, and A. longa adults. Using these DNA sequences, the identification and confirmation of the larval stages of these parasites in the fish and snail hosts will be possible.

Communications: Motile Stage of Aurantiactinomyxon sp. (Actinosporea: Triactinomyxidae) Isolated from Dero digitata Found in Channel Catfish Ponds during Outbreaks of Proliferative Gill Disease

Abstract Dero digitata infected with Aurantiactinomyxon sp. were obtained from a pond with channel catfish Ictalurus punctatus during an outbreak of proliferative gill disease. These worms were maintained until actinosporeans were released. Aurantiactinomyxon sp. was exposed to channel catfish gills then observed microscopically. After exposure to gills, the sporoplasm of the actinosporean began to change and a motile stage was observed. During the motile stage, pseudopodia-like structures retracted and protruded. In the final stage, all movement ceased, and the sporoplasm rounded up and detached from the valves. This motile stage is a possible link to the penetration of Aurantiactinomyxon sp. into channel catfish gills.

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.