Susceptibility of Six Bacterial Pathogens of Channel Catfish to Six Antibiotics

Abstract

Two hundred eight bacterial isolates from diseased channel catfish Ictalurus punctatus were screened for susceptibility to apramycin, enrofloxacin, cephalothin, oxytetracycline, sulfadimethoxine-ormetoprim, and tilmicosin by the Kirby-Bauer disk diffusion method. Bacteria evaluated were Edwardsiella ictaluri, E. tarda, Aeromonas sp., A. hydrophila, A. sobria, and Pseudomonas sp. All bacteria were most susceptible to enrofloxacin (99.0%) and apramycin (97.6%), but only 86.5% were susceptible to sulfadimethoxine-ormetoprim, 84.1%o to oxytetracycline, and 75.5% to cephalothin. Edwardsiella ictaluri, E. tarda, A. hydrophila, and A. sobria were 100% susceptible to enrofloxacin. Aeromonas sp., E. ictaluri and E. tarda were 100% susceptible to apramycin. Resistance was detected in all bacterial species to cephalothin, oxytetracycline, sulfadimethoxine-ormetoprim, and tilmicosin. Testing E. ictaluri against sulfadimethoxine-ormetoprim gave larger zones of inhibition on Mueller-Hinton medium than on brain-heart infusion agar.     

Communications: Efficacy of Norfloxacin for the Control of Edwardsiella tarda Infection in the Flounder Paralichthys olivaceus

Abstract

Efficacy of norfloxacin was evaluated in laboratory and field studies for control of Edwardsiella tarda infection in the flounder Paralichthys olivaceus. In disc diffusion tests, five E. tarda strains showed high sensitivity to norfloxacin. Minimum inhibitory concentrations of norfloxacin against the five isolates were approximately 0.016–0.031 μg/mL. In laboratory studies in which flounder were challenged with E. tarda, a significant (P < 0.05) reduction in mortality was observed among fish receiving norfloxacin in feed at 100 mg/kg body weight (or more) daily for 3 d compared with mortality among nonmedicated controls. Similar results were achieved when 100 mg norfloxacin/kg body weight was used in field trials.     

Growth Inhibition of Selected Aquatic Bacteria by Tannic Acid and Related Compounds

Abstract

Tannic acid, propyl gallate, methyl gallate, and gallic acid were tested for their inhibitory effects on selected aquatic microorganisms by the well assay technique. Tannic acid, propyl gallate, and methyl gallate in deionized water inhibited the growth of Aeromonas hydrophila, A. sobria, Edwardsiella iclaluri, E. tarda, Pseudomonas fluorescens, and Escherichia coli, but gallic acid did not. When 500-μg/mL concentrations of these four compounds were tested in sterilized fish pond water at pH 7.0 and with a low bacterial inoculum of 10³–10⁴ colony-forming units (CFU) per milliliter, they inhibited the growth of P. fluorescens and (except for tannic acid) E. coli. Pseudomonas fluorescens inoculated at 10³–10⁴ CFU/mL in pond water was inhibited by methyl gallate, propyl gallate, and tannic acid concentrations as low as 50 μg/mL, but a gallic acid contration of 100 μg/mL was required for inhibition. Escherichia coli was inhibited by methyl gallate and propyl gallate at 250 μg/mL and by gallic acid at 500 μg/mL, but it was not inhibited by tannic acid at concentrations up to 500 μg/mL in water of pH 7.0. Tannic acid (500 μg/mL) did inhibit E. coli growth at pH 4.5.     

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.     

Biofilm Formation on Aquaculture Substrates by Selected Bacterial Fish Pathogens

Abstract

The objective of this study was to determine whether common bacterial catfish pathogens could attach and colonize surfaces commonly found in aquaculture facilities. In addition, we evaluated the role of calcium in biofilm formation. Attachment to polystyrene plates was used to quantify biofilm formation by five bacterial pathogens (i.e., Flavobacterium columnare, Aeromonas hydrophila, Edwardsiella ictaluri, E. tarda, and E. piscicida). Flavobacterium columnare and A. hydrophila formed thick biofilms that were enhanced by calcium supplementation. Biofilm formation was significantly lower in all Edwardsiella species tested and calcium had little to no effect on Edwardsiella biofilm formation. Attachment to natural and artificial surfaces was quantified by a standard plate count method. Scanning electron microscopy (SEM) was used to confirm biofilm formation on the substrates. Flavobacterium columnare formed biofilm on the liner, flexible PVC, and nets. Bamboo prevented F. columnare attachment and inhibited cell growth. Aeromonas hydrophila and E. ictaluri formed biofilm on all materials tested, although significant differences were found among substrates. While E. ictaluri failed to form biofilm on microtiter polystyrene plates, it was able to colonize and multiply on all aquaculture materials tested. Our results demonstrated that common bacterial pathogens had the potential of colonizing surfaces and may use biofilm as reservoirs in fish farms.

Received July 19, 2016; accepted January 19, 2017 Published online April 13, 2017     

Comparison of Two Biochemical Test Systems with Conventional Methods for the Identification of Bacteria Pathogenic to Warmwater Fish

Abstract

One hundred seven Aeromonas spp., 26 Edwardsiella ictaluri, 6 E. tarda, 12 Plesiomonas shigelloides, and 6 Pseudomonas spp. (131 piscine isolates and 26 reference isolates) were studied with 36 biochemical tests from the Minitek system, 20 tests from the API 20E system, and corresponding standard tube tests. Isolates were incubated at 25°C. Arginine dihydrolase, ornithine decarboxylase, mannose, and citrate showed less than 95% agreement between the Minitek system and the tube tests. Arginine dihydrolase, lysine decarboxylase, nitrite reductase, Voges-Proskauer, and citrate showed less than 95% agreement between the API 20E system and the tube tests. The 26 reference isolates were examined with the three systems and were incubated at both 25 and 37°C. There were no major differences between tests run at 25 and 37°C except with nine Aeromonas spp. that did not grow well at 37°C. Both the Minitek and API 20E systems will reproduce standard biochemical tube test results with at least 95% accuracy when used to test warmwater fish pathogens incubated at 25°C. However, the numerical identification databases for both the Minitek and API 20E systems were not usable for identifying fish pathogens.     

Antibiotic Resistance of Aeromonas spp. Isolated from Tropical Fish Imported from Singapore

Abstract

To determine the scope of antibacterial resistance among Aeromonas spp., bacterial cultures were taken from a variety of tropical fish species imported from Singapore. The samples were plated on Rimler-Shotts medium for bacterial isolation and identified with both the API20E and Nonfermenter Test Strip systems. Aeromonas hydrophila, A. sobria, and A. caviae were identified in monomicrobic and concomitant infections. Following identification of bacterial isolates, 11 antimicrobials routinely used in the tropical fish industry and 1 new fluoroquinolone were tested for their effectiveness against Aeromonas spp. with the Kirby-Bauer disk-diffusion technique. Aeromonas sobria proved to be the most resistant, often showing susceptibility to only 3 of 12 test drugs. Aeromonas hydrophila was consistently the least resistant. Based on these results, antimicrobial resistance appears to be a rapidly emerging problem in the pet fish industry.     

Infections of Edwardsiella tarda among Brook Trout in Quebec

Abstract

Acute bacterial septicemia is commonly diagnosed in brook trout Salvelinus fontinalis of Quebec, Canada. The agents most commonly isolated include Aeromonas salmonicida (furunculosis), Aeromonas hydrophila (motile aeromonad septicemia), and Pseudomonas species. Septicemia in brook trout caused by the gram-negative bacterium Edwardsiella tarda was diagnosed for the first time in the province of Quebec from two different fish farms producing stock for fee fishing establishments. Affected fish displayed nonspecific lesions associated with bacterial septicemia including hemorrhages on the gills and viscera and exophthalmia. Stress-associated immunosuppression due to an increase in summer water temperatures and lack of precipitation were considered as primary causes of these disease outbreaks.     

Effect of Common Aquaculture Chemicals against Edwardsiella ictaluri and E. tarda

Abstract

Edwardsiellosis is an important bacterial infection of freshwater and marine fishes. Edwardsiella ictaluri causes enteric septicemia of catfish, and E. tarda causes emphysematous putrefactive disease of catfish and fish gangrene in various species; these diseases have considerable economic effects on the aquaculture industry. In addition, E. tarda is an important zoonotic pathogen. Thus, the reduction or elimination of these pathogens from an aquarium or aquaculture facility is imperative. This study examined a variety of commercially available chemicals for their ability to reduce or eliminate E. ictaluri and E. tarda from the aquatic environment. The various concentrations of chemicals were tested in vitro in microcentrifuge tubes with a known concentration of bacteria at room temperature. In this study, ethyl alcohol (30, 50, or 70%), benzyl-4-chlorophenol/phenylphenol (1%), sodium hypochlorite (50, 100, 200, or 50,000 mg/L), n-alkyl dimethyl benzyl ammonium chloride (1:256), povidone iodine (50 or 100 mg/L), glutaraldehyde (2%), and potassium peroxymonosulfate/sodium chloride (1%) were effective disinfectants, as each reduced or eliminated the number of detectable organisms within 1 min of contact time. However, neither Chloramine-T (15 mg/L) nor formalin (250 mg/L) substantially reduced bacterial counts even after 60 min of contact time.     

Experiments on Virulence Dose and Portals of Entry for Aeromonas hydrophila in Walking Catfish

Abstract

Aeromonas hydrophila, isolated from chevron snakehead Ophicephalus (=Channa) striatus affected with epizootic ulcerative syndrome (EUS), was injected intramuscularly into healthy walking catfish Clarias batrachus at varying 10-fold serial dilutions from 10⁸ to 0 colony-forming units (cfu) per fish. Only 10⁶ or more cfu/mL induced dermomuscular lesions. Initial healing of lesions was observed by day 7 but complete healing was not apparent until day 16. Experiments were also conducted on possible portals of entry of A. hydrophila into walking catfish: Intramuscular (IM) injection, gastric gavage, fish food, and immersion of injured fish in rearing water inoculated with the test bacteria. Injuries were caused by skin or muscle cut, dermal scraping or incision, fish bite, and cohabitation of fish with golden snails Ampullarius sp. Only IM injection treatment induced dermomuscular pathology in the test catfish. This suggests that a localization of A. hydrophila to a level of 10⁶ cfu/mL in the musculature must be established for dermal lesions to develop.     

Decreased Resistance to Edwardsiella ictaluri Infection in Channel Catfish Intraperitoneally Administered an Oil Adjuvant

Abstract

The effects of intraperitoneal injection of squalene, an oil adjuvant, on nonspecific mortality of channel catfish Ictalurus punctatus and on their resistance to experimental Edwardsiella ictaluri infection were studied. Yearling channel catfish were assigned to control (N = 22) or squalene (N = 25) treatment groups, and mortality was monitored for 14 d following treatment. On day 14 both groups were infected with E. ictaluri, the causative agent of enteric septicemia of catfish, and mortality was monitored for an additional 11 d. Before infection, mortality did not differ between groups. After E. ictaluri infection, fish that received squalene were at a substantially higher risk of dying than control fish (relative risk after squalene treatment = 6.86). These results suggest that intraperitoneal administration of squalene, although not directly toxic, decreased resistance to E. ictaluri infection.     

Quantitative and Qualitative Bacterial Flora of Giant Freshwater Prawn Macrobrachium rosenbergii Cultured in Earthen Ponds in Saudi Arabia

Abstract

Quantitative and qualitative analyses of bacterial flora associated with the digestive tract of the giant freshwater prawn Macrobrachium rosenbergii cultured in earthen ponds of Saudi Arabia were carried out. Bacterial counts and flora of prawn-culture pond water, sediment, and prawn carapaces, along with important physicochemical parameters, were investigated, and the isolates were identified to the genus or species level. Total viable counts (TVC; mean ± SD) varied between (1.0 ± 2.3) × 10⁴ and (1.7 ± 0.8) × 10⁵ colony-forming units (cfu) per milliliter in pond water, (5.8 ± 1.7) × 10⁷ and (1.1 ± 2.9) × 10⁹ cfu/g in sediment, (1.5 ± 0.9) × 10⁵ and (1.6 ± 2.4) × 10⁶ cfu/cm² in the carapace, and (9.1 ± 1.5) × 10⁶ and (8.7 ± 1.8) × 10⁷ cfu/g in the digestive tract of freshwater prawns. The bacterial flora was predominantly gram-negative, accounting for 80% of total isolated strains. Altogether, 21 bacterial species of 16 genera were identified. Aeromonas hydrophila, Shewanella putrefaciens, other Aeromonas spp., Vibrio spp., and Enterococcus spp. were the most abundant bacterial species (prevalence ≥10%) in pond water; A. hydrophila, S. putrefaciens, Vibrio spp., and Pseudomonas spp. were the most abundant in sediment; S. putrefaciens, A. hydrophila, Vibrio spp., Enterococcus spp., and Aeromonas spp. were the most abundant on the prawn carapace; and A. hydrophila, S. putrefaciens, Vibrio spp., Enterococcus spp., and Pasteurella spp. were the most abundant in the digestive tracts. In every population studied, Aeromonas spp., S. putrefaciens, Enterococcus spp., Vibrio spp., Pasteurella spp., Chryseomonas spp., and Pseudomonas spp. were present.     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

Survival of Largemouth Bass Iridovirus in Frozen Fish

Abstract

Juvenile largemouth bass Micropterus salmoides were infected with largemouth bass iridovirus by injection and frozen at ?10°C, and the visceral organs and tissues (liver, kidney, spleen, gonads, swim bladder, and muscle) of individual fish were periodically assayed for virus for 155 d postfreezing. The virus was isolated from 35 of 39 (89.7%) of the frozen fish. However, virus loss occurred more rapidly in fish that were dead when frozen than in fish that were alive when frozen. Overall, largemouth bass iridovirus was stable in frozen tissue when the fish were fresh upon freezing.     

Serological Analysis of Thermolabile Antigens of Vibrio anguillarum

Abstract

Serological analysis of thermolabile antigens of representative strains belonging to the J-O-1, J-O-2, and J-O-3 serotypes of Vibrio anguillarum, which are the major serotypes of thermostable antigens (O-antigens) in Japan, showed that the strains belonging to each O-serotype possessed a common thermolabile antigen designated as k-1. In addition to the k-1 antigen, strains of the J-O-1 serotype of biovar II V. anguillarum possessed another antigen designated as k-2. These findings were confirmed by examining additional strains belonging to each O-serotype. The k-1 antigen was found in all the strains of J-O-4, J-O-5, J-O-6, J-O-7, and J-O-8 serotypes, which are minor serotypes in Japan. The same antigen was also found in some strains of V. metschnikovii, Aeromonas hydrophila, and A. salmonicida.     

Pathogenesis of and strategies for preventing Edwardsiella tarda infection in fish

Edwardsiella tarda is one of the serious fish pathogens, infecting both cultured and wild fish species. Research on edwardsiellosis has revealed that E. tarda has a broad host range and geographic distribution, and contains important virulence factors that enhance bacterial survival and pathogenesis in hosts. Although recent progress in edwardsiellosis research has enabled the development of numerous, highly effective vaccine candidates, these efforts have not been translated into a commercialized vaccine. The present review aims to provide an overview of the identification, pathology, diagnosis and virulence factors of E. tarda in fish, and describe recent strategies for developing vaccines against edwardsiellosis. The hope is that this presentation will be useful not only from the standpoint of understanding the pathogenesis of E. tarda, but also from the perspective of facilitating the development of effective vaccines.     

Effective,appropriate and simple culture,egg hatching and cryopreserving of the nematode Cheilospirura hamulosa

1. Successful invasion by nematode parasites is associated with several factors including egg hatching at the right time in their hosts. To determine a simple and appropriate medium for culture and egg hatching of the highly pathogenic species of the Acuariidae family, Cheilospirura hamulosa were cultured in three different media. In addition the viability of C. hamulosa eggs was determined after storage in frozen infected gizzards.

2. Eggs removed from the uteri of the female worms in infected gizzards were pooled and washed in distilled water and screened under a stereo dissecting microscope. Eggs were counted and cultured in three different media, nutrient agar, normal saline 0.9% and Bearman, at room temperature. Additionally, 10 infected gizzards were kept at ?20°C for 2 and 8 months.

3. After 4–5 d there had been no growth in the nutrient agar medium, whereas 11% of the cultured eggs in the Bearman medium contained larvae 2–3 d after culturing. In 0.9% normal saline medium the two polar knobs appeared on the two poles of the eggs at 2 d post cultivation, and 74% of the eggs contained a larva on the third day. Mature larvae gradually exited from the eggs.

4. Eggs collected from female worms in gizzards frozen at ?20°C were cultured in the same three culture media at room temperature. Larvae were visible in the eggs after 2–3 d in the Bearman and 0.9% normal saline media and hatched thereafter.

5. The 0.9% normal saline medium is recommended for egg hatching and cultivation of C. hamulosa due for simplicity, efficacy and cost effectiveness. Moreover, freezing of the infected gizzards at ?20°C is proposed for long-term storage of the eggs.     

Behavior of Rocinela angustata (Isopoda,Aegidae), an Ectoparasite of Alaskan Marine Fishes

Abstract

Observations were made on the feeding and host selection behavior of the aegid isopod Rocinela angustata. Host susceptibility was assessed by placing isopods into laboratory tanks containing 14 common species of Alaska marine fishes. Five species of Sebastes and Blepsias bilobus were attacked within a few minutes of the introduction. Five species—Enophrys bison, Eumicrotremus orbis, Hexagrammos lagocephalus, Pleuronectes asper, and Pholis laeta—were attacked 10–24 h after introduction of the isopods. Three species—Podothecus acipenserinus, Liparis gibbus, and Lycodes brevipes—were not successfully attacked, even when held in isolation with an isopod for 14 d. Starved isopods isolated with B. bilobus attached to fish within 1 h and remained attached 4–14 d. The isopods reattached 1–4 d after detachment. Isopod digestive tracts cleared within 24 h after detachment.     

Evaluation of Zebrafish Danio rerio as a Model for Enteric Septicemia of Catfish (ESC)

Abstract

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 × 10³, 6 × 10⁴, or 6 × 10⁵ colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 × 10⁴ CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 × 10² CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 × 10⁵, 1.16 × 10⁶, and 1.16 × 10⁷ CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.