The use of polymerase chain reaction assay to diagnose proliferative gill disease in channel catfish (Ictalurus punctatus).

To assess the potential of a polymerase chain reaction (PCR) assay as a diagnostic tool in the detection of proliferative gill disease (PGD) in channel catfish (Ictalurus punctatus), PCR assays were compared with the traditional diagnostic methods of gill wet mounts and histology. A PCR assay using primers for Aurantiactinomyxon ictaluri, the actinospore associated with PGD, was performed with tissues from fish from commercial ponds. Using histology as the "gold standard," the sensitivity, specificity, and accuracy of the PCR assay were all >90%. In comparison, the wet mount examinations had a lower sensitivity and specificity. Using the chi-square test and a test for strength of association, there was a significant, strong association between results obtained by PCR and those obtained by the other 2 methods. These results demonstrate that the PCR assay is a good diagnostic tool for the detection of PGD.     

Induction and Evaluation of Proliferative Gill Disease in Channel Catfish Fingerlings

Abstract

Proliferative gill disease (PGD) was first reported in channel catfish Ictalurus punctatus at commercial farms in 1981 and is caused by the myxozoan parasite Henneguya ictaluri. The disease affects the gills and is characterized by severe branchial inflammation, epithelial hyperplasia, lamellar fusion, and lysis of chondrocytes. Presumptive diagnosis is based on the presence of lytic areas in the cartilage of the primary lamellae on microscopic examination and is confirmed histologically by the presence of the organism. In these trials, PGD was induced by exposing channel catfish fingerlings to fresh or aged infectious water collected from a pond containing fish diagnosed with severe PGD. The severity of disease was graded by histological scoring and microscopic examination of wet mounts to determine the percentage of gill filaments containing chondrolytic lesions. Exposure of fish to infectious pond water was shown to produce pathological lesions consistent with PGD, and the percentage of gill filaments containing chondrolytic lesions was positively correlated with histological scoring of gill pathology. The number of trophozoite stages in the gills was shown to increase with the severity of the disease. In most cases, however, parasitic cells were not observed in tissue samples with chondrolytic lesions during the early stages of infection. These observations indicate that pathology and lysis of chondrocytes can occur prior to detection of the organism by histopathology. Exposing fish to infectious pond water that was aged for 1 d produced negligible gill pathology and implies that the infectivity of the H. ictaluri actinospore stage is short lived. Removing fish from the source of infection promoted repair of damaged gill tissue; within 14 d of fish transfer to clean water, gill pathology associated with the acute infection was negligible.     

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 specific for A. ictaluri. This PCR specific for A. ictaluri produced 104-base-pair products from a plasmid clone containing the SSU rRNA gene of A. ictaluri, spore DNA of A. ictaluri, and DNA prepared from channel catfish gill and D. digitata infected with A. ictaluri. The PCR assay was able to detect as few as 100 copies of the cloned gene. There was no detectable product from the genomic DNA of H. exilis, A. mississippiensis, or Helioactinomyxon sp., specific pathogen-free channel catfish gill, and noninfected D. digitata. The PCR assay will be useful as a diagnostic tool for PGD in channel catfish and will aid in the elucidation of the life cycle of A. ictaluri.     

Enhanced susceptibility of channel catfish to the bacterium Edwardsiella ictaluri after parasitism by Ichthyophthirius multifiliis

Bacterium Edwardsiella ictaluri and parasite Ichthyophthirius multifiliis (Ich) are two common pathogens of cultured fish. The objective of this study was to evaluate the susceptibility of channel catfish Ictalurus punctatus to E. ictaluri and determine bacterial loads in different fish organs after parasitism by Ich. Fish received the following treatments: (1) infected by I. multifiliis at 5000 theronts/fish and exposed to E. ictaluri; (2) infected by I. multifiliis alone; (3) exposed to E. ictaluri alone; and (4) non-infected control. E. ictaluri in fish organs were quantified by quantitative real-time polymerase chain reaction and reported as genome equivalents per mg of tissue (GEs/mg). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (91.7%) when exposed to E. ictaluri than non-parasitized fish (10%). The bacterial loads in fish infected by 5000 theronts/fish ranged from 6497 to 163,898 GEs/mg which was between 40 and 2000 fold higher than non-parasitized fish (49-141 GEs/mg). Ich infection enhanced the susceptibility of channel catfish to bacterial invasion and increased fish mortality.     

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.     

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.     

Proliferative Gill Disease of Fish from the Tennessee-Tombigbee Waterway,Mississippi

Abstract

Proliferative gill disease (PGD), a condition not previously reported in wild fish, was found in two channel catfish Ictalurus punctatus sampled from the Tennessee-Tombigbee Waterway in Mississippi during June and July 1989. The parasite thought to cause PGD was observed in only one of the fish, but the distinctive lesions associated with this disease were prominent in both of the channel catfish. Organisms resembling the PGD parasite were also found in the gills of 4 of 18 largemouth bass Micropterus salmoides and 6 of 20 bluegills Lepomis macrochirus, but there was little or no host response to these parasites.     

Edwardsiella ictaluri as the causative agent of mortality in cultured Nile tilapia

Edwardsiella ictaluri was consistently isolated from the spleens, livers, and head kidneys of diseased Nile tilapia Oreochromis niloticus from a farm experiencing mortality events in several culture ponds. We describe the first published outbreak of E. ictaluri-induced edwardsiellosis in Nile tilapia. Pure cultures of the isolated bacteria were characterized both biochemically and molecularly. Biochemical analysis was performed using the API-20E and RapID One systems, and antimicrobial susceptibility was determined by the broth microdilution method. Molecular analysis involved sequencing of the 16S rRNA gene, species-specific real-time polymerase chain reaction (PCR), and PCR-mediated genomic fingerprinting (rep-PCR). Pairwise sequence analysis of the 16S rRNA gene identified the case isolates to be a 100% match to E. ictaluri cultured from channel catfish in the southeastern United States. However, rep-PCR analysis identified the case isolates to be genetically different from representative strains isolated from disease outbreaks in cultured channel catfish in Mississippi. Infectivity challenges (intraperitoneal injection and immersion) demonstrated that a representative E. ictaluri strain isolated from tilapia was pathogenic to naive tilapia, reproducing clinical signs and mortality, thereby establishing Koch's postulates.     

A Real-Time Polymerase Chain Reaction Assay of the Bacterium Edwardsiella ictaluri in Channel Catfish

Abstract

A rapid (4.5-h) and sensitive assay based on polymerase chain reaction (PCR) was developed to facilitate the early detection of Edwardsiella ictaluri in channel catfish Ictalurus punctatus. A 129-base-pair fragment of a sequence specific to E. ictaluri was amplified with both standard and real-time (quantitative) PCR. The sensitivity of detection was determined to be as low as the equivalent of 2.5 cells in DNA samples from both E. ictaluri cells and mixtures of blood from noninfected catfish and E. ictaluri cells. Infection levels (as determined by real-time PCR) in blood from experimentally challenged fish were compared with brain–heart-infusion-cultured bacterial colony counts to assess the accuracy of the PCR assay. The PCR-based detection level (the equivalent of 10⁵–10⁸ cells/mL) was comparable to that of traditional culturing techniques (10⁶–10⁷ cells/mL). In future applications, this assay will be applied in a comprehensive breeding program to select channel catfish that are resistant to enteric septicemia of catfish.     

Effect of Indomethacin on the Development of Proliferative Gill Disease

Abstract

After parenteral treatment with the cyclooxygenase inhibitor indomethacin, channel catfish Ictalurus punctatus were exposed to mature spores of an Aurantiactinomyxon sp. demonstrated to be the etiological agent of proliferative gill disease (PGD). Fish that received indomethacin at a dose of 2.0 or 5.0 mg/kg body weight within 0.5 h before exposure to the myxozoan and again at 24 h postexposure had significantly (P < 0.05) less severe gill lesions 7 d after exposure than fish that received the drug vehicle alone. Fish that received 0.5 mg indomethacin/kg had moderately severe lesions. All fish were confirmed to be infected with the organism associated with PGD by microscopic examination of gills 4 or 7 d postexposure. These results suggest that products of the cyclooxygenase pathway (e.g., prostaglandins) participate in the pathophysiologic host response to PGD.     

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCR TaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838-843]. The pCS20 quantitative real-time PCR TaqMan probe was compared to the currently used pCS20 PCR and PCR/(32)P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCR TaqMan probe was the most sensitive assay detecting seven copies of DNA/mul of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/(32)P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCR TaqMan probe assay was the most sensitive and can be performed within 2h it is an effective assay for epidemiological surveillance and monitoring of infected animals.     

Communications: Experimental Production of Proliferative Gill Disease in Channel Catfish Exposed to a Myxozoan-Infected Oligochaete,Dero digitata

Abstract

Specific-pathogen-free fry of channel catfish Ictalurus punctatus raised in well water were exposed to Dero digitata (an oligochaete) collected from ponds where catfish have had proliferative gill disease (PGD) and where D. digitata is known to be infected with the triactinomyxid myxozoan Aurantiactinomyxon sp. These fry developed gill lesions and parasites characteristic of PGD. Fry exposed to suspensions of mature Aurantiactinomyxon spores obtained from squashes of infected D. digitata also developed PGD. Fry exposed to oligochaetes other than Dero spp., non-oligochaete benthic macroinvertebrates, or suspensions of squashes of D. digitata without identifiable myxozoans did not develop PGD.     

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of variable periods of food deprivation on the development of enteric septicemia in channel catfish

Enteric septicemia of catfish (ESC), caused by the bacterium Edwardsiella ictaluri, is the most significant bacterial disease affecting channel catfish Ictalurus punctatus. Withholding feed during outbreaks of ESC is a widely accepted industry practice used to control losses from the disease. Scientific evidence concerning the validity of the practice is contradictory. Two studies were conducted to further evaluate the survival of channel catfish fingerlings following variable periods of feed deprivation before and after exposure to E. ictaluri in controlled aquarium experiments. In the first study, feed was withheld for varying time periods before bacterial challenge. After bacterial challenge, feed was either withheld or fish were fed daily. The second study utilized fish fed daily or fish deprived of feed 7 d before bacterial challenge. Daily feeding was resumed 4, 48, and 96 h after fish were exposed to E. ictaluri. In both experiments, the prechallenge feed treatments did not affect mortality. In contrast, withholding feed after bacterial challenge reduced mortalities by 52% in experiment 1 and by 45% in experiment 2. The highest mortality was observed when fish were fed immediately after immersion exposure and the lowest when fish were completely denied feed or fed daily starting 96 h after challenge. This reduction in mortality occurred when the concentration of E. ictaluri in aquarium water was negligible. These data suggest that when E. ictaluri is present in the water, feeding fish increases mortality by enhancing oral exposure to the pathogen.     

Sphaerospora infection of American catfishes (Ictalurus punctatus and I. nebulosus) in Europe.

The occurrence in Europe of a Sphaerospora species described in North America is reported. Based upon its morphological characteristics, the parasite could be identified with the species S. hankai described from brown bullhead in Canada. This parasite was found to infect channel catfish (Ictalurus punctatus) cultured in farm ponds in Italy and brown bullhead (Ictalurus nebulosus) living in the supply channels of fish ponds in Hungary. The spores and sporogonic developmental stages were situated in the lumen of the renal tubules. In the authors' opinion, S. ictaluri described from channel catfish can be considered synonymous with S. hankai.     

EHP、SHIV双重TaqMan实时荧光定量PCR检测方法的构建及应用

本研究根据GenBank中已有的虾肝肠胞虫(EHP)和虾血细胞虹彩病毒(SHIV)基因的保守序列,设计特异的引物和探针。建立了快速诊断EHP和SHIV的双重TaqMan实时荧光定量PCR检测方法,并对其特异性、敏感性和稳定性进行检测。结果表明：该方法检测限可达10 copies/μL,其敏感性是SYBR Green real-time PCR的10倍,普通PCR法的100倍;对白斑综合征病毒、传染性皮下及造血组织坏死病毒、高致病性副溶血弧菌,以及桃拉综合征病毒的检测结果均为阴性,表明无交叉反应,具有良好特异性;重复性试验结果表明,该方法Ct值的变异系数小于4%,具有良好的稳定性。对37份已知检测结果的样品进行检测,结果符合率为100%。本研究建立的EHP和SHIV检测方法具有快速、特异性强、灵敏度高等优点,适用于对虾隐性感染的早期监测。