25 characterization of the causal agents

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Characterization of the Causal gentsof Bacterial Kidney iseaseB.AUSTIW

IntroductionBacterial kidney disease (BKD) is a severe debilitating disease of salmonid fish, particularly Oncorhynchus spp. and Sa/mo spp., with incidences in North America (Ordaland Earp, 1956; Bullock et aI. 1974), Japan (Kimura, 1978), and Europe, particularly France (Evelyn, 1977), Great Britain (Smith, 1964), Turkey (Halici et aI., 1977),and Yugoslavia (Fijan, 1977). The aetiological agent has been misclassified, although ithas been referred to as a coryneform or Corynebacterium (Bullock et aI., 1974; Vladiket aI., 1974; Sanders et aI., 1978), on account of the distinctive micro-morphology ofsmall gram-positive, paired cocco-bacilli. However, the genus Corynebacterium contains many asporogenous gram-positive rods, and extensive efforts have been made tore-define the genus. Thus, Corynebacterium should be restricted sensu stricto to nearneighbours of the human pathogen, C diphtheriae, i.e., club-shaped cells with evidenceof snapping division (Cowan, 1974; Minnikin et aI. 1978). Hence, the majority ofBKD isolates do not conform to the true definition of Corynebacterium.

Diagnosis of BKD has resulted usually from histological examination of kidney,and the isolation of slow-growing gram-positive, rod-shaped bacteria on enriched media(Ordal and Earp, 1956), no further effort being made to identify the pathogen. Consequently, t has been the purpose of this study to characterize these micro-organisms indepth.

Materials and MethodsSource of StrainsTwenty-one strains, each considered to be a causal agent of BKD were received fromseveral sources (Fig. 1). Four fresh UK isolates, 1/79 2/79 10/79, and 11/79, wereobtained from swabs of kidney removed aseptically from diseased rainbow troutSa/mo gairdnerij and inoculated on to plates of Mueller-Hinton agar (Oxoid) supplemented with 0.1 (w/v) L-cysteine hydrochloride and 20 (v/v) fetal bovine serum.Plates were examined after 28 days incubation at 16C when isolates were removed,and re-streaked three times on fresh media to ensure purity. All strains were maintained on modified Mueller-Hinton agar at 4C.1 Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fish Diseases

Laboratory, Weymouth, Dorset DT 4 BUB EnglandW. Ahne (ed.),Fish Diseases

Springer-Verlag Berlin Heidelberg 1980


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148Percentage similarity

50 60 70 80 90 100 Strain No.r - - - ~ - - _ r - - - - r - - - _ - - ~4/79 U S A8/79 Canada

10/79 EnglandPH NONorynebacterIUm pyog n s11/79 England

12/79 Japan5/79 U S A9/79 NCTC 5224

33/77 Scotland47/77 Canada

2/79 England7/79 Canada6/79 Canada

36/77 Canada43/77 Canada

34/77 scotland35/77 Canada39/77 U K41/77 Canada40/77 U K PH NON 242/77 Canada38/77 Canada44/77 Canada45/77 U S A1/79 England3/79 U S A

32/77 France

50 60 70 90 100

Fig. 1. A simplified dendogram based on the SJ coefficient and single-linkage method ofclustering, showing relationships between the strains

Characterization of the Strains

B. Austin

Each strain was examined for over 100 unit characters, as currently used in extensivetaxonomic investigations (Colwell and Weibe, 1970), using modified Mueller-Hintonagar or media supplemented with 0.1 (w/v) L-cysteine hydrochloride and 5 (v/v)fetal bovine serum as the basal medium. Tests were recorded after prolonged incubationat 16C for 28 days, when results were reduced to a unit character state, and the dataexamined by the procedures of numerical taxonomy (Sneath and Sokal, 1973).

Electron MicroscopyCells grown on basal medium were suspended in 0.9 (w/v) saline and negatively stained with 2 (w/v) aqueous uranyl acetate. The micro-morphology was determinedusing a Jeol CX100 transmission electron microscope.


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Characterization of the Causal Agents of Bacterial Kidney Disease 149

Guanine Plus Cytosine Content of DNADespite numerous attempts, purified DNA could not be isolated from cells of 34/77,39/77,5/79 or 8/79.

SerologyWashed cells were suspended in sterile 0.9 (w/v) saline to ca. 1 11 cells/ml and theiragglutination reactions were determined using doubling dilutions of antisera to strains33/77,36/77,39/77,41/77, and 2/79, prepared in female New Zealand white rabbits(McCarthy and Rawle, 1975).

Results and DiscussionOn the basis of overall similarity, the strains separated into 2 numerically dominantgroups, phena 1 and 2, and 7 single member clusters (Fig. 1). Phenon 1 contained thetype strain of orynebacterium pyogenes (NCTC 5224).Characteristics of the StrainsA summary of the characteristics of phena 1 and 2 has been given in Table I. With theexception of strains 6/79 and 7/79, which comprised large gram-positive rods, all isolates appeared morphologically similar when gram-stained smears and wet preparationswere examined by light microscopy. However, subtle-variation in cell size and shapewere revealed by high-resolution transmission electron microscopy. Thus strain 5/79,a representative of phenon 1, consisted of diplo-cocco-bacilli, 2.0xl 8 /Jm in size(Fig. 2), whereas strain 34/77 of phenon 2 comprised rods, 3 .0xl.0 /Jm, occurringsingly (Fig. 3 or, rarely, in pairs. Moreover, distinctions between phena extendedbeyond micro-morphology insofar as the 6 strains of phenon 1 were extremely fastidiousin their growth requirements, taking 28 days at 16C to produce colonies on enrichedMueller-Hinton agar. In contrast, members of phenon 2 grew readily on a wide assortment of standard bacteriological media, including nutr ient agar (Oxoid) and tryptonesoya agar (Oxoid). Thus, from a comparison of the phenotypic characteristics, itwould appear that the micro-organisms considered as the causal agents ofBKD, represent a diverse spectrum of bacterial taxa. Nevertheless, serology data summarisedin Table 2 show that there was cross-agglutination between antisera and strains representative of phena 1 and 2, and some of the uncIustered isolates. Therefore, it isapparent that there are some shared antigens among the isolates.

Taxonomic ConsiderationsThe presence of the type culture of orynebacterium pyogenes (NCTC 5224) withisolates clustered in phenon 1 has, on first appearances, provided much information


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150Table 1 Summary of the characteristics of the isolates clustered in phena 1 and 2Characteristic

Biochemistry:Aesculin degradationArginine dihydrolaseCasein hydrolysisCatalaseMethyl-red testPhosphataseTween 40 hydrolysis

Growth in:2.5 (w/v) NaClEnriched CO 2AnaerobiosisCrystal violetMalachite greenPotassium tellurite

Growth at:4C16C22C

Sensitivity to:AmpicillinCephaloridineCloramphenicolErythromycinTetracycline

a Weakly positive

2

Phenon 1

B Austin

Phenon 2

+)a

Fig 2 Negatively stained cell representative of the isolates clustered in phenon 1. ar equals l lm


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Characterization of the Causal Agents of Bacterial Kidney Disease lS

3Fig 3. Transmission electron micrograph of cell representative of isolate from phenon 2. Barequals Ij.tm

on the taxonomic status of this group of micro-organisms. Moreover, there was veryclose similarity between the description of C pyogenes Cummins et aI., 1974) and thecharacteristics of phenon 1 except for the inability of the fish isolates to grow at37C Unfortunately, C pyogenes has been considered distinct from other Corynebac-terium spp. Jones, 1975) to such an extent that it has been proposed to accommodatethe species in another, as yet unspecified, genus Harrington, 1966; Minnikin et al.,1978). The relationship of this taxon to the lactic acid bacteria, notably tr pto-coccus and Lactobacillus has been indicated Whittenbury, 1964; Cummins et al.,1974), and needs clarification. However, it is noteworthy that the electron micrograph of 5/79 Fig. 2) is indicative of the spherical cells of Streptococcus. Thus, theclassification of phenon 1 must await improvements in the taxonomy ofC pyogenes.

Table 2. Titres obtained by agglutination of representative BKD strains against antiseraBacterial Antisera to:isolate 36/77 39/77 41/77 33/77 2/79

{ 4/79 1/64 1/64 1/128 1/1,024 1/2,048Phenon 1 8/79 1/64 1/64 1/128 1/1,024 1/2,048{ 34/77 1/32 1/512 1/128 1/8Phenon 2 36/77 1/1,024 1/128 1/1,024 1/8 1/41/79 1/128 1/2,048 1/1,024 1/4 1/4

{ 32/711/8 1/8 1/4 1/8 1/8

Miscell- 2/79 1/32 1/128aneous 3/79 1/2 1/16 1/2 1/26/79 1/512 1/8 1/32 1/16 1/16


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152 B. AustinSimilarly, the taxonomic status of phenon 2 is unclear. With Corynebacterium re-

stricted s nsu stricto to near neighbours of C. diphtheriae clearly phenon 2 belongs inanother genus. Lactobacillus may be an appropriate home for these organisms, insofaras they meet the genus description of gram-positive, non-motile, catalase-negative, fermentative rods with complex organic nutritional requirements (Rogosa, 1974). However, with the guanine plus cytosine content of DNA occupying a range of 34.7 to53.4 mole% (Rogosa, 1974), this genus is in dire need of taxonomic revision. t is noteworthy that Lactobacillus was recognised previously as pathogenic to fish, causingpseudokidney disease (Ross and Toth, 1974), although the characteristics of this

micro-organism differ considerably from those of phenon 2.Therefore, the strains examined in this study have been shown to be related to the

lactic acid bacteria, notablyLactobacillus and Streptococcus. However, it is disquietingthat such a wide diversity of bacterial taxa should be associated with one disease.Perhaps the vague description of BKD is at fault. Thus inexperienced workers may beconfusing several chronically related diseases, or even isolating erroneous strains onlaboratory media. Nevertheless, it is apparent that more work is necessary to clear themyths enshrouding BKD.

The reference to proprietary products in this paper should not be construed as anofficial endorsement of these products, nor is any criticism implied of similar productswhich have not been mentioned.

eferencesBullock GL, Stuckey HM Chen PK (1974) Corynebacterial kidney disease of salrnonids: growthand serological studies on the causative bacterium. Appl icrobiol 28:811 814Colwell RR, Weibe WJ (1970) Core characteristics for use in classifying aerobic heterotrophicbacteria by numerical taxonomy. Bull Ga Acad Sci 28: 165 185Cowan ST (1974) Cowan and Steel's manual for the identification of medical bacteria. 2nd edn.Cambridge University Press, Cambridge, pp 238Cummins CS Lelliott RA, Rogosa M (1974) Genus 1 Corynebacterium Lehmann and Newmann1896,350. In: Buchanan RE, Gibbons NE (eds) Bergey's manual of determinative bacteriology,8 th edn. Williams and Wilkins Co, Baltimore, pp 602 617Evelyn TPT (1977) An improved growth medium for the kidney disease bacterium and some notes

on using the medium. Bull Off Int Epiz 87:511 513Fijan N (1977) Corynebacteriosis (Dee disease, kidney disease) of salrnonids in Yugoslavia. Bull OffInt Epiz 87:509Halici G Istanbulloglu E, Arda M (1977) An outbreak of bacterial kidney disease in fish farmingstation of Bayinder Dam and its treatment (in Turkish). J Fac Vet Med Univ Istanbul 3:22 27Harrington BJ (1966) Numerical taxonomic study of some corynebacteria and related organisms.J Gen MicrobioI45:31-40Jones D (1975) A numerical taxonomic study of coryneform and related bacteria. J Gen Microbiol87:52 96Kimura T (1978) Bacterial kidney disease of salrnonids. Fish Patho116:43 52

McCarthy D Rawle CT (1975) The rapid serological diagnosis of fish furunculosis caused bysmoth and rough strains of Aeromonas salmonicida. J Gen Microbiol86: 185 187Minnikin DE, Goodfellow M Collins MD (1978) Lipid composition in the classification and iden

tification of coryneform and related taxa. In: Bousfield IJ, Calleby AG (eds) Coryneform bacteria. Academic Press, London New York, pp 85 160


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Characterization of the Causal Agents of Bacterial Kidney Disease 153Ordal EJ, Earp BJ (1956) Cultivation and transmission of etiological agent of kidney disease in sal-

monid fishes. Proc Soc Exp BioI Med 92:85 88Rosoga M (1974) Genus 1 Lactobacillus Beijerinck 1902, 212, Nom cons Opin 38, Jud Comm

1971, 104. In: Buchanan RE, GibbonsNE (eds) Bergey s manual of determinative bacteriology,8th edn. Williams and Wilkins Co, Baltimore, pp 576 593

Ross AJ, Toth RJ (1974) Lactobacillus - a new fish pathogen. Prog Fish Cult 36: 191Sanders JE Pilcher KS, Fryer JL (1978) Relation of water temperature to bacterial kidney diseasein coho salmon Oncorhynchus kisutchj. sockeye salmon 0. nerkaj. and steelhead troutSaimogairdnerij. J Fish Res Board Can 35:8 11Smith IW (1964) The occurrence and pathology of Dee disease. Freshwater Salmon Fish Res

34:1 12Sneath PHA, Sokal RR (1973) Numerical taxonomy. The principles and practice of numerical

classification. WH Freeman and Co, San Francisco, pp 573Vladik P Vitovec J, Cervinka S (1974) The taxonomy of Gram-positive immobile diplobacilli iso

lated from necrotizing nephroses in American char and rainbow trout. Vet Med 19:233 238Whittenbury R (1964) Hydrogen-peroxide formation and catalase activity in the lactic acid bacteria. J Gen Microbiol 35: 13 26

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