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.

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

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

Animal drug shortages limit veterinary therapeutic options and introduce artifacts in antimicrobial sales reporting.

Supply chain issues disrupt veterinary care and cause downstream consequences that alter the practice of veterinary medicine. Antimicrobials are just 1 class of pharmaceuticals that have been impacted by supply chain issues over the last couple of years. Since February 2021, 2 sponsors/manufacturers of penicillin products have reported shortages in the active pharmaceutical ingredient. With the release of the 2021 Summary Report on Antimicrobials Sold or Distributed for Use in Food-Producing Animals by the FDA, a key finding was a 19% decrease in penicillin sales and distribution from 2020 to 2021. Herein, we provide our clinicians' professional perspective regarding how drug shortages, specifically that of penicillin, might contribute to misconstrued patterns in antimicrobial use and what can be done by veterinarians and the FDA to minimize the impact of an antimicrobial drug shortage on animal health and well-being.

Quality control ranges for testing broth microdilution susceptibility of Flavobacterium columnare and F. psychrophilum to nine antimicrobials.

A multi-laboratory broth microdilution method trial was performed to standardize the specialized test conditions required for the fish pathogens Flavobacterium columnare and F. psychrophilum. Nine laboratories tested the quality control (QC) strains Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 against 10 antimicrobials (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole) in diluted (4 g l-1) cation-adjusted Mueller-Hinton broth incubated at 28 and 18°C for 44-48 and 92-96 h, respectively. QC ranges were set for 9 of the 10 antimicrobials. Most of the minimal inhibitory concentration (MIC) distributions (16 of 18, 9 drugs at both temperatures) for A. salmonicida ATCC 33658 were centered on a single median MIC ± 1 two-fold drug dilution resulting in a QC range that spanned 3 dilutions. More of the E. coli ATCC 25922 MIC distributions (7 of 16) were centered between 2 MIC dilutions requiring a QC range that spanned 4 dilutions. A QC range could not be determined for E. coli ATCC 25922 against 2 antimicrobials at the low temperature. These data and their associated QC ranges have been approved by the Clinical and Laboratory Standards Institute (CLSI), and will be included in the next edition of the CLSI M49-A Guideline. This method represents the first standardized reference method for testing fish pathogenic Flavobacterium spp.

Epidemiological Cutoff Values for Standard Broth Microdilution Susceptibility Testing of Flavobacterium columnare Isolated from Fishes.

Flavobacterium columnare, the causative agent of columnaris disease in a large variety of freshwater fish, is a major problem in commercial aquaculture. A limited number of antimicrobial therapies are available to control this disease; therefore, these agents must be used judiciously. To facilitate effective monitoring for changes in susceptibility, the Clinical Laboratory Standards Institute (CLSI) has a standard broth microdilution test method specific for F. columnare. However, there are no CLSI-approved criteria (termed epidemiological cutoff values [ECVs]) to interpret results. Nevertheless, researchers have developed provisional ECVs based on testing by one laboratory. To satisfy CLSI data requirements, three laboratories used the standard method to generate additional antimicrobial susceptibility data against ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, oxolinic acid, oxytetracycline, sulfadimethoxine/ormetoprim, and sulfamethoxazole-trimethoprim using 109 F. columnare isolates. The new data combined with previously published data from 120 F. columnare isolates were analyzed and ECVs proposed to CLSI. Of the 10 antimicrobials, ECVs were approved for ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, oxolinic acid, and oxytetracycline, which were published in the 2020 edition of the CLSI document VET04 performance standards. These ECVs will help microbiologists categorize decreased antimicrobial susceptibility among F. columnare and will help in surveillance efforts to ensure judicious antimicrobial use.

Epidemiological Cutoff Values for Standard Broth Microdilution and Disk Diffusion Susceptibility Testing of Aeromonas hydrophila Isolated from Fish.

Aeromonas hydrophila and other closely related Aeromonas species cause motile aeromonad septicemia, a common fish disease. The disease affects many aquaculture sectors potentially requiring antimicrobial treatments. Therefore, researchers and laboratory diagnosticians need criteria called epidemiological cutoff values (ECVs) to determine whether a bacterial isolate has developed decreased susceptibility to an antimicrobial. To generate ECVs for this bacterium, we assembled a diverse collection of 245 isolates previously identified as A. hydrophila from fish. Using rpoD sequencing, we confirmed that 97 of the 245 isolates were A. hydrophila. We allocated the isolates among three laboratories and tested their susceptibility against eight antimicrobials using standard Clinical and Laboratory Standards Institute (CLSI) disk diffusion and broth microdilution methods. The resulting frequency distributions were statistically analyzed to determine wild-type cutoff estimates, which, along with scatterplots, were used to estimate potential ECVs. In collaboration with the CLSI, aquaculture working group, we proposed ECVs for six of the eight antimicrobials tested. Subsequently, the CLSI Subcommittee on Veterinary Antimicrobial Susceptibility Testing reviewed our data and approved the ECVs to be added to the 2020 edition of the VET04 performance standards for antimicrobial susceptibility testing of aquatic bacteria.

The AVMA's definitions of antimicrobial uses for prevention, control, and treatment of disease.

Recent state and federal legislative actions and current recommendations from the World Health Organization seem to suggest that, when it comes to antimicrobial stewardship, use of antimicrobials for prevention, control, or treatment of disease can be ranked in order of appropriateness, which in turn has led, in some instances, to attempts to limit or specifically oppose the routine use of medically important antimicrobials for prevention of disease. In contrast, the AVMA Committee on Antimicrobials believes that attempts to evaluate the degree of antimicrobial stewardship on the basis of therapeutic intent are misguided and that use of antimicrobials for prevention, control, or treatment of disease may comply with the principles of antimicrobial stewardship. It is important that veterinarians and animal caretakers are clear about the reason they may be administering antimicrobials to animals in their care. Concise definitions of prevention, control, and treatment of individuals and populations are necessary to avoid confusion and to help veterinarians clearly communicate their intentions when prescribing or recommending antimicrobial use.

Detection of botulinum type E toxin in channel catfish with visceral toxicosis syndrome using catfish bioassay and endopep mass spectrometry.

Visceral toxicosis of catfish (VTC) is a syndrome characterized by sudden mortality in apparently healthy market- and brooder-sized catfish (Ictalurus punctatus). This paper reports the design of a catfish neutralization assay to detect botulinum in catfish with VTC and verification by Endopep mass spectrometry (Endopep-MS). Sera from 6 affected catfish were incubated with botulinum antitoxin serotypes A, B, C, D, E, or F. For each serum sample, 3 experimental fingerlings were injected intracoelomically with each serotype-serum mixture and placed separately in an aquarium. Three fish were injected with VTC-affected serum only, and 3 fish were injected with unaffected serum only and also placed in separate aquaria. Signs of morbidity and mortality were seen in fish injected with sera combined with serotype A, B, C, or D, as well as in positive controls. No morbidity or mortality was seen in fish injected with sera combined with antitoxin serotypes E or F or negative control serum. Sera from affected and unaffected catfish were sent to Centers for Disease Control and Prevention for detection and differentiation of botulinum neurotoxin. Aliquots of 0.5 ml of sera were incubated with magnetic beads coated with antibodies to botulinum, and the beads were subjected to the Endopep-MS reaction. Sera from affected catfish tested positive for botulinum E. Sera from 34 unaffected catfish tested negative for botulinum. Although there was not enough botulinum present in affected samples to obtain exact quantification, the estimated quantity of botulinum E in these sera samples was between 0.01 and 0.5 mouse LD50/ml.

Zebrafish Sensitivity to Botulinum Neurotoxins.

Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins.

Efficacy of Florfenicol for Control of Mortality Associated with Edwardsiella ictaluri in Three Species of Catfish.

The efficacy of florfenicol for control of mortality associated with Edwardsiella icatluri was studied in fingerlings of Channel Catfish Ictalurus puntatus (Delta strain), Blue Catfish I. furcatus (D&B strain), and a hybrid catfish (Delta strain Channel Catfish × D&B strain Blue Catfish). On day 0, fish were immersion challenged in 65-L aquaria. For each of the three species of catfish, 10 aquaria were randomly assigned to two treatment groups, either treated with florfenicol at 0 mg/kg of body weight (unmedicated feed) or at 10 mg/kg (medicated feed). Fish were treated for 10 consecutive days, monitored for mortality during this treatment period, and observed for 14 d afterwards. Post observation, all survivors were humanely euthanized in tricaine methanesulfonate, cultured for E. ictaluri, and examined for gross pathology. The mean cumulative percent mortality from enteric septicemia of catfish (ESC) challenge among the three genotypes of catfish did not differ between Blue Catfish, hybrid, and Channel Catfish in treated or control groups. However, the florfenicol-treated fish had a significantly lower mean cumulative mortality (6%) than the controls (78%). All genotypes of catfish tested were responsive to treatment with florfenicol-medicated feed for control of mortality associated with ESC. There were no significant differences in mortality associated with hybrid catfish, blue catfish, and Channel Catfish (Delta strain).

Gastrointestinal torsion in a channel catfish (Ictalurus punctatus).

A case of gastrointestinal torsion with dilatation in a farm-raised channel catfish (Ictalurus punctatus) was examined at the Thad Cochran National Warmwater Aquaculture Center (Stoneville, Mississippi, USA). The affected fish was a gravid female broodfish, which displayed pale gills and a markedly distended abdomen. Internal examination revealed that the gastrointestinal tract and ovaries were rotated around each other four times in a counterclockwise direction as viewed in right lateral recumbency. The catfish had a markedly distended gastrointestinal tract, pale liver, hypoplastic spleen, hypoplastic swim bladder, and high volume of ascitic fluid. Blood analysis indicated multiple abnormalities, including severe anemia and metabolic acidosis. The etiology of the torsion was uncertain; however, the presence of a hypoplastic swim bladder most likely allowed for increased movement of the gastrointestinal tract and ovaries. When examining cases of abdominal distention in fish, gastrointestinal torsion can be considered among the differential diagnoses.

The effects of a sublethal dose of botulinum serotype e on the swimming performance of channel catfish fingerlings.

Abstract Visceral toxicosis of catfish (VTC) is a disease of cultured Channel Catfish Ictalurus punctatus in the Mississippi Delta region and surrounding states. The etiology of VTC is associated with botulinum serotype E (BoNT/E), which causes blockage of acetylcholine release at the neuromuscular junction, leading to weakness and paralysis of skeletal muscles (including those involved in swimming). This study attempted to determine if sublethal exposure to purified BoNT/E caused reductions in swimming performance and metabolism of Channel Catfish. Catfish swimming performance was assessed on stocker-sized Channel Catfish (mean weight ± SD, 62.35 ± 2.5 g) with 10 sham-injected fish and 10 fish injected with a sublethal dose of BoNT/E. A modified Blazka-type swim chamber was used to assess swimming performance. We injected Channel Catfish with either 0.015% trypsin or 400 pg purified BoNT/E digested with 0.015% trypsin intracoelomically, then acclimated an individual catfish in the swim chamber for 17 h prior to the swimming trial. Water temperature was maintained at ∼28°C, and dissolved oxygen (DO) was between 4 and 7 mg/L. A critical swimming speed (Ucrit) protocol was followed, and DO and temperature were monitored every 2 min throughout the swim trial. Cost of transport was calculated from the oxygen consumption at each test speed (10-70 cm/s). There was a statistical difference between the Ucrits (P = 0.0034), but no differences were found between the cost of transports (P = 0.67) of the sham-injected and BoNT/E groups. There was a difference in the cost of transport as it relates to the speeds tested (P < 0.0001), cost of transports being highest at low speeds and decreasing as speed increased. These results indicate that botulinum E interferes with the swimming speed of the catfish, which could contribute to the mortality from the disease of VTC and potentially make the fish more susceptible to predation. Received September 20, 2013; accepted February 14, 2014.

Zebrafish (Danio rerio) bioassay for visceral toxicosis of catfish and botulinum neurotoxin serotype E.

Visceral toxicosis of catfish (VTC), a sporadic disease of cultured channel catfish (Ictalurus punctatus) often with high mortality, is caused by botulinum neurotoxin serotype E (BoNT/E). Presumptive diagnosis of VTC is based on characteristic clinical signs and lesions, and the production of these signs and mortality after sera from affected fish is administered to sentinel catfish. The diagnosis is confirmed if the toxicity is neutralized with BoNT/E antitoxin. Because small catfish are often unavailable, the utility of adult zebrafish (Danio rerio) was evaluated in BoNT/E and VTC bioassays. Channel catfish and zebrafish susceptibilities were compared using trypsin-activated BoNT/E in a 96-hr trial by intracoelomically administering 0, 1.87, 3.7, 7.5, 15, or 30 pg of toxin per gram of body weight (g-bw) of fish. All of the zebrafish died at the 7.5 pg/g-bw and higher, while the catfish died at the 15 pg/g-bw dose and higher. To test the bioassay, sera from VTC-affected fish or control sera were intracoelomically injected at a dose of 10 µl per zebrafish and 20 µl/g-bw for channel catfish. At 96 hr post-injection, 78% of the zebrafish and 50% of the catfish receiving VTC sera died, while no control fish died. When the VTC sera were preincubated with BoNT/E antitoxin, they became nontoxic to zebrafish. Histology of zebrafish injected with either VTC serum or BoNT/E demonstrated renal necrosis. Normal catfish serum was toxic to larval zebrafish in immersion exposures, abrogating their utility in VTC bioassays. The results demonstrate bioassays using adult zebrafish for detecting BoNT/E and VTC are sensitive and practical.

Phenotypic and genotypic heterogeneity among Streptococcus iniae isolates recovered from cultured and wild fish in North America, Central America and the Caribbean islands.

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Lactococcosis in Silver Carp.

An adult Silver Carp Hypophthalmichthys molitrix with a focally extensive skin lesion near the caudal peduncle and mild iridial hemorrhage was submitted to the Aquatic Research and Diagnostic Laboratory (ARDL) in Stoneville, Mississippi, as part of a fish kill investigation. Touch impressions of this musculoskeletal lesion revealed small cocci (∼1 µm) in pairs or chains within an inflammatory milieu. A pure Gram-positive cocci isolate was obtained from the brain, while cultures of the kidney and muscle yielded multiple bacterial colony types, including the Gram-positive cocci seen in the brain. This brain isolate was characterized biochemically and identified as Lactococcus spp. Analysis of the near complete 16S small subunit ribosomal DNA (SSU rDNA) and DNA gyrase subunit B (gyrB) gene sequences revealed the bacterium to be L. lactis subsp. lactis (SSU rDNA: 100% identity, 1,372/1,372 bp; gyrB: 99.7% identity, 1,779/1,785 bp). Comparatively, at the gyrB locus the case isolate shared less than 90% similarity to L. lactis subsp. cremoris (1,599/1,781 bp) and less than 80% homology to L. garvieae (1409/1775 bp). Histopathological examination confirmed a severe meningoencephalitis, a moderate mononuclear myositis, and a mild interstitial nephritis. We believe this represents the first report of a natural infection by L. lactis subsp. lactis in Silver Carp.

Determination of the median lethal dose of botulinum serotype E in channel catfish fingerlings.

The median lethal dose of botulinum serotype E in 5.3-g channel catfish Ictalurus punctatus fingerlings was determined. Five tanks (five fish/tank) were assigned to each of the following treatment groups: 70, 50, 35, 25, or 15 pg of purified botulinum serotype E. Fish were injected intracoelomically and observed for 96 h. Administration of the toxin resulted in initial hyperactivity followed by erratic swimming, paresis, and death. The cumulative mortality by treatment group was 100% at 70 pg, 96% at 50 pg, 100% at 35 pg, 88% at 25 pg, and 56% at 15 pg. The median lethal dose was calculated as 13.7 pg/fish (equivalent to a 0.81 median lethal dose for mice Mus musculus) using a logistic regression model. All fish were necropsied; lesions included exophthalmia, ascites, splenic congestion, intussusception of the intestines, congested spleen, and blanching of the intestinal tract. The resultant clinical signs and lesions were similar to those noted in the syndrome of visceral toxicosis of catfish. This study indicates that channel catfish are more sensitive to the effects of botulinum serotype E than laboratory mice, and the signs and lesions of visceral toxicosis of catfish were replicated by injecting catfish with the toxin.

The pathology associated with visceral toxicosis of catfish.

Visceral toxicosis of catfish (VTC) syndrome was recognized in the late 1990 s and recently has been associated with exposure to Clostridium botulinum type E neurotoxin. Tentative diagnosis is based on clinical presentation and gross findings, and is confirmed by bioassay. In April 2009, channel catfish (Ictalurus punctatus) from 2 different farms presented with abnormal swimming behavior and mortalities. Nine fish were submitted to the Aquatic Research and Diagnostic Laboratory (Stoneville, Mississippi) for evaluation. Bacterial cultures from these fish were negative. Necropsy findings included intestinal intussusceptions, ascites, pale proximal intestines with engorged serosal blood vessels, splenic congestion, and a reticular pattern to the liver. Significant histopathologic findings were limited to cerebral, splenic, and hepatic congestion, splenic lymphoid depletion and perivascular edema, vascular dilation and edema of the gastrointestinal tract, and perivascular edema in the anterior and posterior kidneys. Intoxication from C. botulinum type E neurotoxin was suspected based on the clinical signs and lack of gross and microbiological evidence of an infectious disease process. The toxicosis was confirmed with a positive bioassay using serum collected from the submitted fish.

Efficacy of florfenicol for control of mortality caused by Flavobacterium columnare infection in channel catfish.

The studied in channel catfish Ictalurus punctatus fingerlings held in 80-L aquaria. Nonabraded fish were challenged by immersion on day 0. Thirty 80-L tanks were randomly assigned in equal numbers to two treatment groups, one in which fish were fed a commercial diet without florfenicol (unmedicated feed) and one in which they were fed a diet containing 10 mg of florfenicol/kg of body weight (medicated feed) for ten consecutive days. Mortality was monitored during the treatment period and a 14-d posttreatment observation period. At the end of the posttreatment period, all fish were euthanized, examined for gross lesions, and cultured for F. columnare. Significantly fewer fish fed the medicated diet died (8.0%) than fish fed the unmedicated diet (54.2%). Flavobacterium columnare was cultured from 15.0% of the medicated fish, compared with 68.9% of the unmedicated fish. The gross lesions in the fish were consistent with columnaris disease, and F. columnare was cultured from 99.5% of the dead fish. No differences were observed in weight gain and appetence between the medicated and unmedicated groups. For the F. columnare strain used in this study, the minimal inhibitory concentration of florfenicol ranged from 0.5 to 1.0 mg/mL in the 30 bacterial cultures obtained from infected fish, and the mean disk diffusion zone of inhibition was 40 mm. There were no adverse effects among the medicated fish. Administration of florfenicol at a dosage of 10 mg/kg body weight for 10 d was efficacious and safe for the control of mortality from F. columnare infection in channel catfish.