In Vitro Antimicrobial Activity of Sarafloxacin against Clinical Isolates of Bacteria Pathogenic to Fish

Abstract

An in vitro susceptibility assay of sarafloxacin (A-56620), a new 4-quinolone, was performed against five important bacterial species that are pathogenic to fish. A collection of 44 clinical isolates and five corresponding type strains were included in the study. The objectives were to determine the minimal inhibitory concentrations (MICs) of sarafloxacin by a drug microdilution method and to compare the MIC values at two different temperatures, 4 and 15°C. Sarafloxacin was active against all species tested and showed the following mean MIC values at 15 and 4°C, respectively, against the bacterial pathogens investigated: Aeromonas salmonicida subspecies salmonicida, 0.029 and 0.045 μg/mL; atypical A. salmonicida, 0.053 and 0.041 μg/mL; Vibrio anguillarum, 0.085 and 0.054 μg/mL; V. salmonicida, 0.125 and 0.123 μg/mL; and Yersinia ruckeri, 0.023 and 0.031 μg/mL. The MICs ranged from 0.0025 μg/mL (or less) for two strains of A. salmonicida salmonicida to 0.32 μg/mL for one strain of atypical A. salmonicida and one strain of V anguillarum. A decrease in antimicrobial activity was observed as the incubation temperature was lowered from 15 to 4°C; however, no significant statistical difference between the measured values was demonstrated.     

Cell-Surface-Associated Properties of Fish Pathogenic Bacteria

Abstract

Pathogenicity assays showed that 33 of 42 potentially pathogenic strains of bacteria tested were virulent to rainbow trout Oncorhynchus mykiss. Regardless of their degree of virulence to fish, strains of motile Aeromonas, A. salmonicida, and Vibrio anguillarum were moderately hydrophobic. Only 46 and 25°10 of the strains were able to hemagglutinate human and trout erythrocytes, respectively. Hydrophobicity and hemagglutination were practically absent in isolates of Yersinia ruckeri. A notable number of the strains positively adhered to salmonid (51%) and nonsalmonid (55%) fish cells. Whereas the treatment of the bacteria with proteinase K or trypsin did not decrease the hydrophobicity of the isolates, within motile Aeromonas and A. salmonicida species, strains with both protease-sensitive and -resistant hemagglutinating and adhesive abilities occurred. The effects of heat and sugars on hemagglutinating and hydrophobic properties varied within all bacterial groups. Although treatment of strains with D-mannose or L-fucose had distinct effects on adhesiveness according to the bacterial species and the cell system used, none of the heat-treated (80°C for 15 min) bacteria lost their capacity to adhere to cultured fish cells. The results showed that there was no direct relationship between any of the cell surface properties analyzed and the degree of virulence of the strains.     

Journal of Aquatic Animal Health: Guide for Authors 2014

Abstract

Brook trout Salvelinus fontinalis were treated with single 60-min static baths of 250 mg formalin/L, 3% NaCl, and 15 mg Chloramine-T/L to evaluate the efficacy of these compounds against external infections of Aeromonas salmonicida. Prevalence of A. salmonicida was significantly lower in brook trout treated with Chloramine-T than among those treated with formalin or salt. Further laboratory tests substantiated the therapeutic value of a single treatment of ChloramineT (15 mg/L) against A. salmonicida. In two experiments, viable counts of A. salmonicida in mucus did not vary among replicate groups of treated brook trout, but the counts for treated fish were significantly (P < 0.05) lower than those for untreated controls. In vitro tube dilution assays indicated that mean minimum inhibitory concentrations of Chloramine-T for 10 isolates of A. salmonicida were 9.0 mg/L for 1 h and 2.25 mg/L, for 24 h. In field trials at the White River National Fish Hatchery (Bethel, Vermont), the pathogen was detected principally as an external infection of juvenile Atlantic salmon Salmo solar maintained in two culture ponds. In one pond, the bacterium accounted for 100% of the total distribution of tnicroflora isolated from mucus. Seven days after treatment with Chloramine-T, A. sahnonicida accounted for 11% of the total bacterial counts identified from these fish. In the second pond, A. salmonicida composed 3% of the counts of bacteria isolated from the mucus of fish before treatment but was not isolated after treatment.     

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.     

Physiological and Immunological Effects of Adjuvanted Aeromonas salmonicida Vaccines on Juvenile Rainbow Trout

Abstract

The effects of injectable vaccines against Aeromonas salmonicida on oxygen consumption, growth, kidney lysozyme activity, and anti-A. salmonicida plasma antibody titers of juvenile rainbow trout Oncorhynchus mykiss were examined. The vaccines were A. salmonicida bacterin only, bacterin adjuvanted with levamisole, bacterin in emulsified oil, microencapsulated bacterin, microencapsulated bacterin with muramyl dipeptide, microencapsulated bacterin with β-1,3-glucan, and microencapsulated bacterin with Vibrio anguillarum lipopolysaccharide (LPS). The greatest and broadest ranges of responses were caused by the microencapsulated bacterin with V. anguillarum LPS. Oxygen consumption rates and specific growth rates were significantly higher over the course of 1 month among fish treated with the LPS vaccine. These fish also maintained a higher anti-A. salmonicida plasma antibody titer and kidney lysozyme activity for a substantially longer period than fish receiving the other treatments.     

Occurrence and Distribution of Aeromonas salmonicida Infections on Swedish Fish Farms, 1951–1987

Abstract

The occurrence of typical and atypical Aeromonas salmonicida infections on Swedish fish farms was surveyed, and the methods for controlling the disease are described here. This review is based on data for a period of 36 years from fish health control studies conducted by the National Veterinary Institute and the Salmon Research Institute. The epizootiology and routes of transmission of the pathogens are described, and policy and methods for disease control are discussed. The distribution of atypical A. salmonicida infections was entirely different from that of typical infections. There was also circumstantial evidence of different routes of transmission for typical and atypical A. salmonicida infections. Although subclinical carriers of atypical A. salmonicida occurred among Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss on fish farms, acute disease outbreaks were most frequent among Arctic char Salvelinus alpinus and brown trout Salmo trutta. On the other hand, acute outbreaks of typical A. salmonicida infections were frequent among Atlantic salmon and rainbow trout.     

Prevention of Ulcer Disease in Goldfish by Means of Vaccination

Abstract

A vaccine comprising cells of Aeromonas bestiarum grown in tryptic soy broth and atypical A. salmonicida cells produced in iron-limited and iron-supplemented media protected goldfish Carassius auratus when administered by immersion (dosage ≈ 5 × 10⁷ cells/mL for 60 s) followed after 28 d by an oral booster (dosage = 5 × 10⁷ cells/g of feed), which was fed for 7 d so that each fish received about 1 g of vaccine-containing feed. After challenge by intramuscular injection of a virulent culture of atypical A. salmonicida, the relative percent survival (RPS) was more than 90%. The approach was more successful than using a commercial furunculosis vaccine with or without supplementation with A. bestiarum or atypical A. salmonicida cells. Moreover, a smooth derivative of the virulent rough culture of atypical A. salmonicida was less effective as a vaccine candidate, yielding an RPS of only 65%. Low antibody titers of 1:39–1:396 were found in the vaccinated fish. The vaccinated fish had a significantly higher proportion of dead head kidney macrophages (10.9 ± 3.5%; P = 0.0149) than did the controls (6.8 ± 3.1%). However, differences in the number of erythrocytes and leukocytes, the level of phagocytic and lysozyme activities, and the proportion of lymphocytes, monocytes, and polymorphonuclear cells were not statistically significant between the two groups.     

Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis

Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.     

Fast detection of Vibrio species potentially pathogenic for mollusc

Vibrio tasmaniensis, Vibrio splendidus and Vibrio neptunius species were distributed worldwide and associated with aquaculture and have been reported as the cause of diseases in aquatic organisms. Polyphasic analyses for bacterial identification are not feasible for routine diagnostic because of the time involved. The aim of this study is to design three PCR primer sets that can assist with fast detection of these species. They were designed from the 16S ribosomal RNA gene, and PCR conditions were found. Each PCR test successfully identified all the tested strains of each target species. The combined specificity of V. tasmaniensis and V. splendidus primer sets offered the best coverage (86%) in terms of separating target organisms from other related species. The primer set of V. tasmaniensis showed a lower sensitivity limit (500 fg of DNA) than the V. splendidus set (1 pg) and both sets gave positive amplification using homogenized tissues from inoculated clams, with 10² and 10⁴ cfu/g of clam, respectively. The primer set of V. neptunius was highly specific, showing only cross-reaction with V. parahaemolyticus species from 44 tested species. Its sensitivity limit was 100 pg of DNA. A small number of biochemical tests were proposed concurrently with the PCR to differentiate the cross-reacting bacteria. The time of detection of the three tested species was reduced and the further affected animals can be diagnosed in a rapid fraction of time. The detection of virulent strains of V. tasmaniensis pointed to the risk of mollusc culture outbreaks.     

Monoclonal Antibody-Based Analysis of the Renibacterium salmoninarum p57 Protein in Spawning Chinook and Coho Salmon

Abstract

Renibacterium salmoninarum, causative agent of bacterial kidney disease of salmonid fish, produces large amounts of soluble proteins during infection and broth culture. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies was developed for the precise quantification of p57, a major component of these proteins. Kidney, spleen, blood, and reproductive fluids of adult Pacific salmon Oncorhynchus spp. were examined by means of the assay. Kidney and spleen harbored the highest concentrations of this antigen. In the populations of returning salmon tested, 5–25% of the fish had p57 concentrations above a baseline level of 3 ng antigenig tissue, and antigen concentrations as high as 200 μg/g tissue were detected in kidneys of individual fish. The ELISA was compared to direct fluorescent antibody analysis, in which rabbit anti-R. salmoninarum antiserum was used to identify infected fish. There was 99% agreement (199 of 201 examined fish) between the two methods. Western blot analysis was used to demonstrate that antigenic proteins present in infected fish were similar to those seen from antigen prepared from broth culture of R. salmoninarum, although less degradation of p57 appears to occur in vivo.     

Host factors associated with the detection of Aeromonas salmonicida and Yersinia ruckeri in Ontario, Canada government fish hatcheries

This paper presents an epidemiological investigation of Ontario Ministry of Natural Resources Fish Health Laboratory data from 1981 to 1997, to determine whether fish species and age were associated with lot-level detection of Aeromonas salmonicida and Yersinia ruckeri in hatchery fish. In stepwise logistic regression, the species brook trout and back-cross (lake trout crossed with the hybrid “splake”) were more likely to test A. salmonicida-positive compared to all other species reared in the hatcheries. Similarly, the species brook trout was significantly more likely to test Y. ruckeri-positive compared to all other species. For both pathogens, the 1–5-month age group was associated significantly with detection. These findings suggest that purposive sampling of higher-risk fish lots could increase the likelihood of detecting both study pathogens.     

Search for an antigenic relationship between aeromonads and Brucella abortus

A. hydrophila, A. punctata subsp. vaviae, A. salmonicida, and Plesiomonas shigelloides strains did not serologically react with three strains of Brucella abortus when tested by serum agglutination, immunodiffusion, and immunoelectrophoresis.     

Nature of Aeromonas salmonicida Carriage on Asymptomatic Rainbow Trout Maintained in a Culture System with Recirculating Water and Fluidized Sand Biofilters

Abstract

An asymptomatic carrier population of rainbow trout Oncorhynchus mykiss was examined for Aeromonas salmonicida by primary dilution counts on Coomassie Brilliant Blue agar and also by streaking on bacteriological media after a 24–48-h pre-enrichment in tryptic soy broth. The pathogen was detected by primary dilution plate counts in 1 spleen, 15 gill, and 19 mucus samples of the 100 trout examined. Aerornonas samonicida was detected only after 48-h preenrichment in the one spleen that had already tested positive via primary dilution counts. The pathogen was not detected in kidney, liver, and intestinal samples. The occurrence of the pathogen in mucus and gills suggests a predominantly external nature of asymptomatic carriage of A. salmonicida within this population of fish. Repeated examination of fluidized biofilters and tank water showed that A. salmonicida did not become established in the recirculation system.     

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.     

Occurrence of Aeromonas salmonicida,Renibacterium salmoninarum,and Infectious Pancreatic Necrosis Virus in Ontario Salmonid Populations

Abstract

The results of samples collected from private and government fish farms and wild and feral fish populations in Ontario from 1981 to 1995 were synthesized to obtain prevalence estimates in salmonids at both the fish and site levels for three pathogens. Renibacterium salmoninarum and Aeromonas salmonicida were both detected on at least one site for every year investigated. Ontario Ministry of Natural Resources (OMNR) culture stations had the highest percentages of sites with infected fish for R. salmoninarum. Natural water bodies had the highest percentages of sites with infected fish for A. salmonicida. Infectious pancreatic necrosis virus (IPNV) was only detected sporadically on some commercial farms and never in OMNR hatcheries or in wild or feral fish. Although screening for any virus that would yield cytopathological effect was carried out during all the years surveyed, no virus other than IPNV was isolated. The low prevalence and “source-specific” presence of IPNV in Ontario demonstrates the necessity of representative sampling for the detection of rare pathogens. It was estimated that, overall, less than 1% of all fish in the sampled populations were infected with each of the three pathogens for almost every year studied. The importance of summarizing pathogen-testing data and the possible implications on disease control policy planning and assessment are discussed.     

Isolation and Classification of a Nocardia species from Diseased Giant Gourami Osphronemus goramy

Abstract

An enzootic caused by bacteria occurred among cultured giant gourami in two freshwater fish farms in Kagoshima, Japan, in 1982. Typical external characteristics of the diseased fish were sloughed scales, exophthalmia, and opaque eyes. Internally, numerous creamy white nodules were found in the gills, spleen, kidney, and liver and a lesser number in the bladder and stomach. Nocardia sp. was isolated from spleen and kidney tissues of all diseased fish examined. To clarify the taxonomic position of the isolates, 164 tests were performed, and results were compared to reference strains of six species of Nocardia and one species of Rhodococcus. The isolates formed a homogenous group in a single taxon at a similarity of 98.7% based on an average linkage analysis and were clearly separated from the reference strains. The isolated strains were more similar to Nocardia kampachi than to the other representative species. However, 12 distinct differences were found between the isolates and N. kampachi, including growth on nutrient agar, sensitivity to 250 and 500 μg hydroxylamine/mL and 0.01% phenol, tolerance of 0.05% salicylate and 0.1% picric acid in brain-heart infusion medium, hydrolysis of starch, nicotinamidase reaction, and acid from fructose, sucrose, and dextrin. Accordingly, the isolates from the diseased giant gourami were classified as a new species of Nocardia.     

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.     

Development of Similar Broth Microdilution Methods to Determine the Antimicrobial Susceptibility of Flavobacterium columnare and F. psychrophilum

Flavobacterium columnare and F. psychrophilum are major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnare and F. psychrophilum to facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnare and 72–96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller–Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim–sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria.

Received June 24, 2015; accepted October 2, 2015.     

Effect of Nanophyetus salmincola and Bacterial Co-Infection on Mortality of Juvenile Chinook Salmon

The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon Oncorhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmincola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations.

Received February 24, 2015; accepted September 7, 2015     

In Vitro Study of Adherence,Invasion, and Persistence of Streptococcus iniae in Fibroblastic-Like Fish Cell Line SAF-1

Abstract

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Gentamicin protection assays were used to investigate the ability of different S. iniae strains to invade and adhere to fibroblastic-like fish cell line SAF-1. All strains tested were detected intracellularly using both techniques, with variable internalization degrees between strains. The experiments carried out at 4°C demonstrated that active cell metabolism is necessary for bacterial internalization. Intracellular bacteria were detected for up to 3 d with a round morphology and were stained with 4′,6-diamidino-2-phenylindole (DAPI), indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that S. iniae is capable of adhering, entering, and surviving within fibroblastic cells, which may be important for the persistence and establishment of a carrier state.

Received July 8, 2011; accepted February 12, 2012