Improved husbandry to control an outbreak of rainbow trout fry syndrome caused by infection with Flavobacterium psychrophilum

Julie A. Bebak Freshwater Institute, 1098 Turner Rd, Shepherdstown, WV 25443

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Timothy J. Welch USDA National Center for Cool and Coldwater Aquaculture, 11861 Leetown Rd, Kearneysville, WV 25430

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Clifford E. Starliper US Geological Survey National Fish Health Research Laboratory, 11649 Leetown Rd, Kearneysville, WV 25430

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Ana M. Baya Maryland Department of Agriculture, 8077 Greenmead Dr, College Park, MD 20740

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Michael M. Garner Northwest ZooPath, 654 W Main St, Monroe, WA 98272.

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Abstract

Case Description—A cohort of 35,200, 13-week-old, female rainbow trout at a fish farm was evaluated because of a 2-week history of anorexia and lethargy and a mortality rate of approximately 100 fish/d.

Clinical Findings—Affected fish were lethargic and thin and had disequilibrium, bilateral exophthalmia, pale red gills and kidneys, red-tinged coelomic fluid, and pale brown livers. Some fish were differentially pigmented bilaterally. The presumptive diagnosis was bacterial or viral septicemia. The definitive diagnosis was rainbow trout fry syndrome caused by infection with Flavobacterium psychrophilum.

Treatment and Outcome—A strategy for controlling the outbreak based on reducing pathogen numbers in affected tanks and reducing pathogen spread among tanks was developed. The option of treating with antimicrobial-medicated feed was discussed with the farmer, but was declined. After changes were made, mortality rate declined quickly, with no more deaths within 10 days after the initial farm visit.

Clinical Relevance—Bacterial coldwater disease is the most common manifestation of infection with F psychrophilum in fingerling and adult rainbow trout. However, the organism can also cause rainbow trout fry syndrome. This condition should be included on a list of differential diagnoses for septicemia in hatchery-reared rainbow trout fry.

Abstract

Case Description—A cohort of 35,200, 13-week-old, female rainbow trout at a fish farm was evaluated because of a 2-week history of anorexia and lethargy and a mortality rate of approximately 100 fish/d.

Clinical Findings—Affected fish were lethargic and thin and had disequilibrium, bilateral exophthalmia, pale red gills and kidneys, red-tinged coelomic fluid, and pale brown livers. Some fish were differentially pigmented bilaterally. The presumptive diagnosis was bacterial or viral septicemia. The definitive diagnosis was rainbow trout fry syndrome caused by infection with Flavobacterium psychrophilum.

Treatment and Outcome—A strategy for controlling the outbreak based on reducing pathogen numbers in affected tanks and reducing pathogen spread among tanks was developed. The option of treating with antimicrobial-medicated feed was discussed with the farmer, but was declined. After changes were made, mortality rate declined quickly, with no more deaths within 10 days after the initial farm visit.

Clinical Relevance—Bacterial coldwater disease is the most common manifestation of infection with F psychrophilum in fingerling and adult rainbow trout. However, the organism can also cause rainbow trout fry syndrome. This condition should be included on a list of differential diagnoses for septicemia in hatchery-reared rainbow trout fry.

A West Virginia trout farmer reported a 2-week history of anorexia, lethargy, and death in a cohort of 13-week-old female rainbow trout fry (mean total body length, 7 cm) that were the offspring of neomales.1 Fish were reared in fresh water in flow-through, single-pass tanks. They were originally received as eyed eggs that were certified as being free from infectious pancreatic necrosis virus, infectious hematopoietic necrosis virus, and viral hemorrhagic septicemia virus. Before calling for assistance, the farmer had attempted treatment by adding formalin (concentration unknown) to affected tanks for 3 days, but no clinical improvement had been seen.

The population consisted of 35,200 fry with 2,200 fish/tank. The mortality rate was > 100 fish/d, or approximately 0.28% of the total population per day, and was noticeably higher than the typically observed mortality rate of < 5 fish/d for fish of this age at this farm. According to the farmer, the onset of the outbreak coincided with visiting students using equipment to measure water quality in the tank where the outbreak started.

Initially, fish stopped feeding in 1 tank, followed by fish in the 15 other tanks. The pattern of spread from tank to tank was consistent with the cleaning routine used by personnel as they moved from one tank to the next, using the same equipment.

Water quality variables were within reference limits for trout culture.2 Water temperature was 12°C, dissolved oxygen concentration was > 8 ppm, alkalinity was 150 mg/L as CaCO3, hardness was 75 mg/L as CaCO3, and pH was 7.0 to 7.2. Un-ionized ammonia-nitrogen and nitrite-nitrogen concentrations were not measured because tank turnover rate was at least 2 times/h.

In tanks with affected fish, approximately 5% to 10% of the fish appeared to be abnormal. Groups of fish were gathered in areas of the tanks with lower water velocity, which suggested that they were too weak to swim with their tank mates. Large numbers of fish were lethargic and thin. Many had bilateral exophthalmia. Some exhibited disequilibrium or were swimming in a spiral motion along the long axis of their bodies. Others were lying on the bottom of the tank with labored respiration and were differentially pigmented bilaterally, with normal color on one side and black on the other. A few fecal casts were seen. Twenty-five fish were euthanized by immersion in tricaine methane sulfonate (MS-222; 150 ppm).3

Four fish were necropsied. Gross pathologic examination revealed pale red gills and kidneys, an excessive amount of red-tinged, coelomic fluid, and pale brown livers. No external or internal parasites were detected. The clinical diagnosis was bacterial or viral septicemia with accompanying anemia.

A ventral midline incision was made in 4 fish, and the coelomic cavity was opened to allow access for fixative. Fish were preserved in neutral-buffered 10% formalin and submitted for histologic examination. Individual fish were embedded in paraffin and sectioned transversely at serial intervals. Sections were stained with H&E stain. Additional select sections were stained with Warthin-Starry silver stain and with Brown and Brenn tissue Gram stain. Histologic changes were consistent with bacterial septicemia, with lesions primarily involving the spleen, heart, and gills as well as bone and cartilage. The distribution of bone lesions was typical of infection with Flavobacterium psychrophilum.4 Lesions consisted of multiple foci of pyogranulomatous inflammation and necrosis involving cartilage and bone of the vertebrae, snout, and surrounding tissues. Small clusters of bacilli were detected in some areas of necrosis and inflammation in spleen and gill lesions and occasionally at the margins of necrotic cartilage. The organism stained with Warthin-Starry silver stain, but not with Brown and Brenn tissue Gram stain.

Samples of liver, spleen, and kidney were taken aseptically from 12 fish, and pooled samples (3 fish/ pool) were placed in tubes containing 5 mL of minimum essential medium with gentamicin (50 Mg/mL). Standard methods5 were used to isolate infectious pancreatic necrosis virus, infectious hematopoietic necrosis virus, and viral hemorrhagic septicemia virus from the samples, but results for all samples were negative.

Swabs of liver and kidney were aseptically obtained from 5 fish and submitted for bacteriologic culture. Three Flavobacterium-like isolates were obtained from 3 of the fish. On primary isolation on cytophaga agar, bacterial colonies were pale yellow and flat with slightly raised centers and weak spreading margins; colony morphology was consistent with the reported colony morphology for F psychrophilum.6 One of the isolates was confirmed to be F psychrophilum on the basis of results of standard biochemical testing.7–10 In particular, this isolate was positive for reduction of nitrate; production of oxidase, catalase, gelatinase, caseinase, elastase, and flexirubin-like pigments; and lysis of Escherichia coli cells. The isolate was negative for indole production, tyrosinase production, starch hydrolysis, xanthine degradation, and chondroitin lyase reduction. The capability of the isolate to use various sugars as a carbon source was determined by use of commercially available media.a It failed to assimilate any of a suite of 20 sugars, which is characteristic of F psychrophilum. All isolates were gram negative by conventional staining of smears from tissue and culture specimens. Cells were long, thin rods about 1 × 3 to 5μM. The isolate identified as F psychrophilum was compared with 10 Washington State reference isolates. Six of these were positive for tyrosinase production, whereas the isolate was negative. The isolate was positive for elastase production and reduced nitrate to nitrite, whereas none of the reference isolates were positive for either of these. Elastase production has been associated with body surface lesions caused by Aeromonas hydrophila,11 a pathogen of many cooland warm-water fish species. All 3 isolates grew on a 1:10 concentration of Mueller-Hinton agar, but none grew on MacConkey, brain heart infusion, tryptic soy, 5% sheep blood, or triple sugar iron agar or on the standard concentration of Mueller-Hinton agar. The test isolate and the reference strains grew to colonies 3 to 4 mm in diameter within 3 days at 6° and 16°C. The test isolate was the only one to grow at 24°C, albeit weakly in 4 days, and none of the isolates grew at 30°C.

The 3 isolates were verified to be strains of F psychrophilum by means of analysis of the 16S rDNA sequence. The 16S rDNA gene fragments were amplified by means of a PCR assay that incorporated primers Eubac27F and 1492R, as described.12 Amplification products were cloned with a commercial kit,b and DNA sequencing was performed on 3 clones from each of the strains with a commercial sequencerc (1,324 nucleotides sequenced). All 3 isolates had identical 16S rDNA sequences, and this sequence was identical to the 16S rDNA sequence for F psychrophilum strain CSF-259-93 (Genbank accession No. AY662494).

Because the farmer only used eggs certified as free from the most important viruses affecting trout and the groundwater supplying the facility was also considered free from these viruses, a viral etiology was considered to be less likely than a bacterial etiology. The farmer declined the option of treating with antimicrobial-medicated feed because, as a matter of policy, he avoided antimicrobial use whenever possible. The farmer also had a hazard analysis critical control point plan in place and indicated that his customers expected his product to be free from antimicrobials. For these reasons, a strategy for controlling the outbreak based on reducing pathogen concentrations in affected tanks and stopping pathogen spread from tank to tank was developed. The farmer was advised to remove all dying fish twice a day, work on tanks containing sick fish last, use separate equipment for each tank, use separate chlorine-containing (100 ppm) rinse buckets to rinse equipment, provide gloves or hand-washing facilities for fish handlers, and block off the area of the affected tanks with tape and cones. The farmer was also advised to transfer fish to tanks that were first cleaned with a surfactantd and then disinfected with chlorine (100 ppm) for 1 hour that was neutralized with sodium thiosulfate (740 ppm). Following these changes in management procedures, the mortality rate quickly decreased, and excess deaths stopped within 10 days.

To avoid future outbreaks, the farmer was advised to descale, clean, and disinfect the degassing tower and the interior of the water supply pipes, which could have provided reservoirs for the bacteria. He was also advised to restrict unauthorized and untrained people from access to the facility, tanks, and equipment.

Discussion

Flavobacterium psychrophilum is the cause of 2 common salmonid diseases: BCWD and RTFS. To the authors' knowledge, studies of risk factors for these 2 diseases have not been published. However, clinical signs and mortality rates during outbreaks of BCWD and RTFS have reportedly been related to fish size and water temperature, with higher mortality rates at water temperatures < 15°C. Bacterial coldwater disease, also known as peduncle disease and saddleback disease, usually occurs in fingerlings and larger fish. Typical clinical signs of BCWD include erosion or ulceration of the peduncle area ranging from a single cutaneous lesion to exposure of the vertebrae and spinal cord. Lesions cranial to the dorsal fin, on the mandible, and near the vent have also been reported. Exophthalmia, gill hemorrhage, and anemia may also occur.13

Other names for RTFS include fry mortality syndrome and fry anemia syndrome. Reported clinical signs of RTFS include lethargy; weakness; anorexia; bilateral exophthalmia; and pale red gills, kidneys, intestines, and liver, with an accompanying anemia. Disequilibrium and spiral swimming behavior have been associated with cranial and vertebral lesions.4,13

The reservoir of the bacteria that infected the fish described in the present report was unclear. The farmer reported that visiting students used equipment that might not have been properly disinfected to measure water quality in the tank where the outbreak started. However, vertical transmission is also a possibility.13,14 The farmer did report normal survival rates for the egg and sac fry stages, so vertical transmission is less likely, but the eggs could have arrived at the facility already infected with F psychrophilum.

Flavobacterium psychrophilum occurs naturally in soil and water. Therefore, the bacteria could have come from the facility water supply, with initial infection occurring at the egg or fry stage.15 Flavobacterium psychrophilum has been isolated from Koch rings used in a degassing tower for source water at an aquaculture facility.16 The pipes and degassing tower for the facility described in the present report had not been cleaned for many years, and the associated biofilm and mineral buildup could have provided a reservoir for the bacteria. Hence a recommendation was made to the farmer to clean and disinfect the degassing tower and the interior of the water supply pipes in an effort to reduce the risk of future outbreaks.

Husbandry and biosecurity changes recommended to the farmer were aimed at reducing the pathogen concentration in each tank, reducing transfer of the pathogen between tanks, and reducing pathogen numbers in bacterial biofilms on the tank walls. These changes likely contributed to the improved survival rate that was observed soon after they were implemented.

Findings for fish described in the present report suggest that RTFS should be included on a list of differential diagnoses for septicemia in rainbow trout fry, along with viral diseases such as infectious pancreatic necrosis and infectious hematopoietic necrosis.17 However, veterinarians should be aware that in many texts of fish disease, RTFS is not included with BCWD when infection with F psychrophilum is discussed.

ABBREVIATIONS

BCWD

Bacterial coldwater disease

RTFS

Rainbow trout fry syndrome

a.

OF (oxidation/fermentation) Basal Medium, Becton, Dickinson & Co, Sparks, Md.

b.

Topo TA cloning kit, Invitrogen, Carlsbad, Calif.

c.

ABI Prism 3700 DNA sequencer, Applied Biosystems, Foster City, Calif.

d.

Simple Green, Huntington Harbour, Calif.

References

  • 1

    Bye VJ, Lincoln RF. Commercial methods for the control of sexual maturation in rainbow trout (Salmo gairdneri R). Aquaculture 1986;57:299309.

    • Search Google Scholar
    • Export Citation
  • 2

    Piper RG, McElwain IB & Orme LE, et al. Fish hatchery management. Washington, DC: US Department of the Interior Fish and Wildlife Service, 1982.

  • 3

    2000 Report of the AVMA panel on euthanasia. J Am Vet Med Assoc 2001;218:669696.

  • 4

    Kent ML, Groff JM & Morrison JK, et al. Spiral swimming behavior due to cranial and vertebral lesions associated with Cytophaga psychrophila infections in salmonid fishes. Dis Aquat Organ 1989;6:1116.

    • Search Google Scholar
    • Export Citation
  • 5

    Diagnostic manual for aquatic animal diseases. 3rd ed. Paris: Office International des Epizooties, 2000.

  • 6

    Anacker RL, Ordal EJ. Studies on the myxobacterium Chondrococcus columnaris. I. Serological typing. J Bacteriol 1959;78:2532.

  • 7

    Bernardet J-F, Grimont PAD. Deoxyribonucleic acid relatedness and phenotypic characterization of Flexibacter columnaris sp. nov, nom. rev., Flexibacter psychrophilus sp. nov., nom. rev. and Flexibacter maritimus Wakabayashi, Hikida and Masumura 1986. Int J Syst Bacteriol 1989;39:346354.

    • Search Google Scholar
    • Export Citation
  • 8

    Bullock GL. Studies on selected myxobacteria pathogenic for fishes and on bacterial gill disease in hatchery-reared salmonids. In:US Bureau of Sport Fisheries and Wildlife technical paper No. 60. Washington, DC: US Fish & Wildlife Service, 1972.

    • Search Google Scholar
    • Export Citation
  • 9

    Cipriano RC, Schill WB & Teska JD, et al. Epizootiological study of bacterial cold-water disease in Pacific salmon and further characterization of the etiological agent, Flexibacter psychrophila. J Aquat Anim Health 1996;8:2836.

    • Search Google Scholar
    • Export Citation
  • 10

    Pacha RE. Characteristics of Cytophaga psychrophila (Borg) isolated during outbreaks of bacterial cold-water disease. Appl Microbiol 1968;16:97101.

    • Search Google Scholar
    • Export Citation
  • 11

    Hsu TC, Waltman WD, Shotts EB. Correlation of extracellular enzymatic activity and biochemical characteristics with regard to virulence of Aeromonas hydrophila. Dev Biol Stand 1981;49:101111.

    • Search Google Scholar
    • Export Citation
  • 12

    DeLong EF. Archaea in coastal marine environments. Proc Natl Acad Sci U S A 1992;89:56855689.

  • 13

    Nematollahi A, Decostere A & Pasmans F, et al. Flavobacterium psychrophilum infections in salmonid fish. J Fish Dis 2003;26:563574.

  • 14

    Cipriano RC. Intraovum infection caused by Flavobacterium psychrophilum among eggs from captive Atlantic salmon broodfish. J Aquat Anim Health 2005;17:275283.

    • Search Google Scholar
    • Export Citation
  • 15

    Rangdale RE, Richards RH, Alderman DJ. Colonisation of eyed rainbow trout ova with Flavobacterium psychrophilum leads to rainbow trout fry syndrome in fry. Bull Eur Assoc Fish Pathol 1997;17 (3/4):108111.

    • Search Google Scholar
    • Export Citation
  • 16

    Ryce EK, Zale AV. Bacterial coldwater disease in Westslope cutthroat trout: hatchery epidemiology and control. Final report to the Wild Fish Habitat Initiative. Bozeman, Mont: Montana Water Center, 2004.

    • Search Google Scholar
    • Export Citation
  • 17

    Wolf K. Fish viruses and fish viral diseases. Ithaca, NY: Cornell University Press, 1988.

  • 1

    Bye VJ, Lincoln RF. Commercial methods for the control of sexual maturation in rainbow trout (Salmo gairdneri R). Aquaculture 1986;57:299309.

    • Search Google Scholar
    • Export Citation
  • 2

    Piper RG, McElwain IB & Orme LE, et al. Fish hatchery management. Washington, DC: US Department of the Interior Fish and Wildlife Service, 1982.

  • 3

    2000 Report of the AVMA panel on euthanasia. J Am Vet Med Assoc 2001;218:669696.

  • 4

    Kent ML, Groff JM & Morrison JK, et al. Spiral swimming behavior due to cranial and vertebral lesions associated with Cytophaga psychrophila infections in salmonid fishes. Dis Aquat Organ 1989;6:1116.

    • Search Google Scholar
    • Export Citation
  • 5

    Diagnostic manual for aquatic animal diseases. 3rd ed. Paris: Office International des Epizooties, 2000.

  • 6

    Anacker RL, Ordal EJ. Studies on the myxobacterium Chondrococcus columnaris. I. Serological typing. J Bacteriol 1959;78:2532.

  • 7

    Bernardet J-F, Grimont PAD. Deoxyribonucleic acid relatedness and phenotypic characterization of Flexibacter columnaris sp. nov, nom. rev., Flexibacter psychrophilus sp. nov., nom. rev. and Flexibacter maritimus Wakabayashi, Hikida and Masumura 1986. Int J Syst Bacteriol 1989;39:346354.

    • Search Google Scholar
    • Export Citation
  • 8

    Bullock GL. Studies on selected myxobacteria pathogenic for fishes and on bacterial gill disease in hatchery-reared salmonids. In:US Bureau of Sport Fisheries and Wildlife technical paper No. 60. Washington, DC: US Fish & Wildlife Service, 1972.

    • Search Google Scholar
    • Export Citation
  • 9

    Cipriano RC, Schill WB & Teska JD, et al. Epizootiological study of bacterial cold-water disease in Pacific salmon and further characterization of the etiological agent, Flexibacter psychrophila. J Aquat Anim Health 1996;8:2836.

    • Search Google Scholar
    • Export Citation
  • 10

    Pacha RE. Characteristics of Cytophaga psychrophila (Borg) isolated during outbreaks of bacterial cold-water disease. Appl Microbiol 1968;16:97101.

    • Search Google Scholar
    • Export Citation
  • 11

    Hsu TC, Waltman WD, Shotts EB. Correlation of extracellular enzymatic activity and biochemical characteristics with regard to virulence of Aeromonas hydrophila. Dev Biol Stand 1981;49:101111.

    • Search Google Scholar
    • Export Citation
  • 12

    DeLong EF. Archaea in coastal marine environments. Proc Natl Acad Sci U S A 1992;89:56855689.

  • 13

    Nematollahi A, Decostere A & Pasmans F, et al. Flavobacterium psychrophilum infections in salmonid fish. J Fish Dis 2003;26:563574.

  • 14

    Cipriano RC. Intraovum infection caused by Flavobacterium psychrophilum among eggs from captive Atlantic salmon broodfish. J Aquat Anim Health 2005;17:275283.

    • Search Google Scholar
    • Export Citation
  • 15

    Rangdale RE, Richards RH, Alderman DJ. Colonisation of eyed rainbow trout ova with Flavobacterium psychrophilum leads to rainbow trout fry syndrome in fry. Bull Eur Assoc Fish Pathol 1997;17 (3/4):108111.

    • Search Google Scholar
    • Export Citation
  • 16

    Ryce EK, Zale AV. Bacterial coldwater disease in Westslope cutthroat trout: hatchery epidemiology and control. Final report to the Wild Fish Habitat Initiative. Bozeman, Mont: Montana Water Center, 2004.

    • Search Google Scholar
    • Export Citation
  • 17

    Wolf K. Fish viruses and fish viral diseases. Ithaca, NY: Cornell University Press, 1988.

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