Isolation, characterization, and molecular cloning of cryptic plasmids isolated from Edwardsiella ictaluri

Fifty-five isolates of Edwardsiella ictaluri were examined for the presence of plasmid DNA by a rapid alkaline extraction procedure. All 49 isolates from channel catfish and a single isolate from Bengal danio carried 2 plasmids with molecular masses of approximately 3.2 and 3.7 megadaltons (Mdal). Five E ictaluri isolates from other fish contained 1 to 3 plasmids, which had molecular masses ranging from 2.5 to 45 Mdal. The 2 plasmids (3.2 and 3.7 Mdal) from the type strain of E ictaluri (ATCC 33202) were ligated into pUC19 cloning vectors, and restriction endonuclease maps of each insert were prepared.     

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.     

Effect of Incubation Temperature and Salinity on Expression of the Outer Membrane Protein Profile of Edwardsiella tarda

Abstract

Outer membrane proteins (OMP) of 10 isolates of Edwardsiella tarda were compared by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The OMP profile of the type strain E. tarda ATCC 15947 cultured at 25°C had five major protein bands of 40, 36.5, 34, 28.5, and 25 kDa and a large number of minor proteins ranging in size from approximately 10 to 120 kDa. Differences between the OMP profiles of the isolates of E. tarda included the inconsistent presence of the 34- or 36.5-kDa proteins in five isolates of E. tarda and two major bands of 47 and 44 kDa that were present in only two isolates of E. tarda. There were no differences in the outer membrane protein profiles of 9 out of 10 isolates of E. tarda incubated at a temperature of 25°C compared with those at 35°C. To evaluate the effect of salinity, 10 isolates of E. tarda were cultured in brain heart infusion broth containing 0.5, 1.5, and 3.0% sodium chloride. Reactions of isolates of E. tarda to the different salinity levels were placed into three groups. The first group expressed more or fewer protein bands at 1.5% sodium chloride. The second group lost major bands at 3% salinity, whereas the third group had no change in the OMP profile with salinity. The OMP profile differences and the different reactions to salinity levels suggest that the isolates are heterogeneous.     

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.     

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.     

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.     

Spleen index and mannose-binding lectin levels in four channel catfish families exhibiting different susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.     

Characterization of outer membrane protein-enriched extracts from Pasteurella multocida isolated from turkeys

Outer membrane protein (OMP)-enriched extracts of avian strains of Pasteurella multocida were examined by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Culture medium did not have a significant effect on the OMP profiles of strains of P multocida examined; however, in vivo propagation had an appreciable effect on the OMP profile composition of the reference strain P-1059. Such bacteria, expressed several additional OMP in the 27-kD, 48-kD, 56-kD, 60-kD, 80-kD, and 94-kD molecular mass regions. These OMP were not detected in the electrophorogram of strain P-1059 grown in vitro. The OMP profiles of reference strains of the 16 serotypes of P multocida did not identify any serotype-specific protein markers. Field strains of serotype A:3 had variation in OMP profiles and did not express OMP that all were identical to that expressed by the reference strain P-1059. The live attenuated CU and M9 bacterial vaccine strains expressed strain-specific OMP markers of 48-kD and 45-kD molecular masses, respectively. These strain-specific OMP markers may be used to differentiate these strains from virulent field strains that are of the same serotype and isolated from turkeys that have succumbed to pasteurellosis as a result of vaccine-related reactions or breakdown in immunity.     

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.     

Characterization and comparison of antimicrobial susceptibilities and outer membrane protein and plasmid DNA profiles of Pasteurella haemolytica and certain other members of the genus Pasteurella.

The outer membrane protein (OMP), plasmid, and antimicrobial resistance profiles of Pasteurella haemolytica serotypes 1 through 12, a bovine isolate of P multocida, a chicken isolate of P multocida, and an unidentified Pasteurella species of bovine origin were examined. Isolates of P haemolytica serotypes belonging to the same biotype possessed similar OMP profiles. Biotype A isolates contained 2 prominent OMP of 43 kilodaltons (kD) and 29 kD, whereas biotype-T serotypes contained 3 major OMP of 43, 36, and 25 kD. The major OMP profiles of the 2 P multocida isolates and the unidentified Pasteurella species were different from each other and from P haemolytica isolates. Plasmid DNA screening indicated both plasmid-containing and plasmid-free P haemolytica and P multocida isolates. Multiple drug resistance was found in pasteurellae isolates with and without plasmids. However, a relationship between drug resistance and plasmid isolation was found in 3 of 4 haemolytica serotype 1 field isolates, all of which contained a 2.51-megadalton plasmid and had multiple drug resistance for benzylpenicillin, ampicillin, streptomycin, and tetracycline.     

新肝宝对鲶爱德华氏菌感染黄颡鱼的保护作用研究

为了考察新肝宝对鲶爱德华氏菌感染黄颡鱼的保护作用,采用不同粉碎粒度的新肝宝拌饲投喂7 d,随后用鲶爱德华氏菌腹腔注射感染,观察死亡率、酶学指标及组织病理变化。结果显示,新肝宝组死亡率(48.57%和44.28%)低于阴性对照组(54.29%),溶菌酶及超氧化物歧化酶活性显著高于对照组(P<0.05),肝脏、肾脏、脾脏损伤情况均小于阴性对照组,其中600目作用效果优于200目。结果表明,新肝宝能够提高黄颡鱼非特异性免疫力,增强黄颡鱼对鲶爱德华氏菌的抵抗能力,在一定程度上减轻鲶爱德华氏菌感染导致的肝、脾、肾损伤,其中600目作用效果优于200目。     

Chemotactic and Chemokinetic Responses of Channel Catfish Macrophages to Exoantigen from Edwardsiella ictaluri

Abstract

The ability of Edwardsiella ictaluri to attract macrophages of channel catfish Ictalurus punctatus was investigated. Exoantigen from E. ictaluri was tested for macrophage chemotactic activities both in vitro and in vivo. The exoantigen was chemotactic and chemokinetic for macrophages in vitro. Peritoneal injection of 750 μg of exoantigen protein into normal (E. ictaluri-free) channel catfish induced a marked increase in macrophage accumulations at 24 and 48 h. Neutrophil accumulation did not occur at the injection sites. Edwardsiella ictaluri exoantigen attracts macrophages, and this attraction may play an important role in macrophage responses during E. ictaluri infections.     

Outer membrane protein profiles of Yersinia ruckeri

The outer membrane protein (OMP) profiles of 135 isolates of Yersinia ruckeri, obtained from nine European countries (100 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), and including four reference strains, were examined by SDS-PAGE. Outer membranes were isolated by selective solubilisation of the cytoplasmic membrane with 0.5% (w/v) sodium N-lauroyl sarcosinate (Sarkosyl). Outer membrane proteins were stable after in vitro passage and there was no variation in OMP profiles due to colony selection. With the exception of a 39.5 kDa peptidoglycan-associated protein there was also no variation at different stages of the growth cycle. The 39.5 kDa protein was not produced during logarithmic growth phase but increased in abundance as the stationary phase progressed. Interstrain variation occurred in the possession of a 36.5 or 38 kDa heat-modifiable protein and in the possession of peptidoglycan-associated proteins in the molecular weight range 36.5 to 40.5 kDa. Based on variation of these proteins five OMP-types, designated OMP-types 1-5, were identified among the 135 isolates examined. Outer membrane protein analysis was demonstrated to be useful in epidemiological studies of Y. ruckeri.     

Evidence that Enteric Septicemia of Catfish (ESC) was Present in Arkansas by the Late 1960s: New Insights into the Epidemiology of ESC

Abstract

Enteric septicemia of catfish (ESC), a disease of channel catfish Ictalurus punctatus, was first reported in 1979 based on isolates obtained from 1976 through 1978. Channel catfish that had been preserved in 1970, labeled “nutritional cranial spot,” and stored at the Stuttgart National Aquaculture Research Center were tested with Gram stains, histology, and immunohistochemistry to demonstrate that the specimens were actually infected by Edwardsiella ictaluri (the causative agent of ESC). A reexamination of catfish disease case records has indicated that ESC might have been present in Arkansas in 1969. Investigation of these old records and specimens has led to insights on the discovery and epidemiology of the disease.     

Distribution of Edwardsiella ictaluri in California

Abstract

Wild and domestic populations of channel catfish Ictalurus punctatus were examined to determine the distribution of the disease called enteric septicemia of catfish (ESC) in California. The causative agent of ESC, Edwardsiella ictaluri, was isolated from five separate sites in California. Two of these isolations were from rectal swabs of asymptomatic fish, confirming that a carrier state may exist. Normal-appearing fish with serum antibody titer to E. ictaluri were commonly found in domestic channel catfish populations, suggesting that many fish become infected but recover. Wild channel catfish with antibody to E. ictaluri were also found in major reservoirs and water distribution canals. Edwardsiella ictaluri appears to be widely distributed within California.     

Modified live Edwardsiella ictaluri vaccine, AQUAVAC-ESC, lacks multidrug resistance plasmids

Plasmid-mediated antibiotic resistance was first discovered in Edwardsiella ictaluri in the early 1990s, and in 2007 an E. ictaluri isolate harboring an IncA/C plasmid was recovered from a moribund channel catfish Ictalurus punctatus infected with the bacterium. Due to the identification of multidrug resistance plasmids in aquaculture and their potential clinical importance, we sought to determine whether the modified live E. ictaluri vaccine strain in AQUAVAC-ESC harbors such plasmids, so that the use of this vaccine will not directly contribute to the pool of bacteria carrying plasmid-borne resistance. Antimicrobial sensitivity testing of the E. ictaluri parent isolate and vaccine strain demonstrated that both were sensitive to 15 of the 16 antimicrobials tested. Total DNA from each isolate was analyzed by polymerase chain reaction (PCR) using a set of 13 primer pairs specific for conserved regions of the IncA/C plasmid backbone, and no specific products were obtained. PCR-based replicon typing of the parent isolate and vaccine strain demonstrated the absence of the 18 commonly occurring plasmid incompatibility groups. These results demonstrate that the vaccine strain does not carry resistance to commonly used antimicrobials and provide strong support for the absence of IncA/C and other commonly occurring plasmid incompatibility groups. Therefore, its use should not directly contribute to the pool of bacteria carrying plasmid-borne resistance. This work highlights the importance of thoroughly investigating potential vaccine strains for the presence of plasmids or other transmissible elements that may encode resistance to antibiotics.     

Comparative Analysis of the Outer Membrane Protein Profiles of Isolates of the Pasteurella multocida (B:2) Associated with Haemorrhagic Septicaemia

Outer membrane proteins (OMP) of P. multocida (serotype B:2) field isolates (n = 6) and a vaccine strain (P-52) were extracted by a sarkosyl method and characterized using SDS-PAGE and immunoblotting. About 20 polypeptide bands were observed in the profile of the vaccine strain with MW ranging from 16 to 90 kDa and, based on band thickness and intensity of staining, three polypeptides of MW 31, 33 and 37 kDa were considered to be the major OMPs. The profiles of the field isolates showed minor differences when compared with that of the vaccine strain. The OMP of 33 kDa was only expressed by the vaccine strain. Four field isolates expressed an OMP of 39 kDa, which did not appear in the profiles of the remaining two field isolates and the P-52 strain. Similarly, an OMP of 25 kDa was exclusively seen in the profile of a single isolate. By immunoblotting studies, using anti-P. multocida (P-52) whole-cell hyperimmune serum raised in rabbits as well as buffalo immune sera, it became evident that the polypeptide of 37 kDa was the most antigenic OMP in the profiles of all the isolates, including the P-52 strain. Other polypeptides were either weakly antigenic or visible in the profile of only a few of the isolates. The study thus identified the major OMP of P. multocida (B:2) and suggested that this highly antigenic 37 kDa OMP has potential for further protective and immunodiagnostic studies.