Characterization of the Causal Agents of Bacterial Kidney Disease

B.AUSTIW

Introduction
Bacterial kidney disease (BKD) is a severe debilitating disease of salmonid fish, particularly Oncorhynchus spp. and Sa/mo spp., with incidences in North America (Ordal and 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 it has been referred to as a coryneform or Corynebacterium (Bullock et aI., 1974; Vladik et aI., 1974; Sanders et aI., 1978), on account of the distinctive micro-morphology of small gram-positive, paired cocco-bacilli. However, the genus Corynebacterium contains many asporogenous gram-positive rods, and extensive efforts have been made to re-define the genus. Thus, Corynebacterium should be restricted sensu stricto to near neighbours of the human pathogen, C. diphtheriae, i.e., club-shaped cells with evidence of snapping division (Cowan, 1974; Minnikin et aI., 1978). Hence, the majority of BKD 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, it has been the purpose of this study to characterize these micro-organisms in depth.

Materials and Methods

Source of Strains Twenty-one strains, each considered to be a causal agent of BKD were received from several sources (Fig. 1). Four fresh UK isolates, 1/79,2/79, 10/79, and 11/79, were obtained from swabs of kidney removed aseptically from diseased rainbow trout (Sa/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, England

W. Ahne (ed.), Fish Diseases Springer-Verlag Berlin Heidelberg 1980

148
Percentage similarity

B. Austin

50

r---~--_r----r---,_--~

60

70

80

90

100

Strain No. 4/79 U.S.A.

8/79
10/79 11/79 12/79 5/79 9/79 33/77 47/77 2/79 7/79

Canada
England

England
Japan U.S.A. NCTC 5224 Scotland Canada England Canada

CorynebacterIUm pyogenes

PHENON 1

6/79
36/77

Canada
Canada

43/77

Canada

34/77
35/77

scotland
Canada U.K.

39/77 41/77 40/77

Canada
U.K.

PHENON 2

42/77
38/77 44/77
45/77

Canada
Canada Canada
U.S.A.

1/79 3/79 32/77

England U.S.A. France

50

60

70

90

100

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

Characterization of the Strains

Each strain was examined for over 100 unit characters, as currently used in extensive taxonomic investigations (Colwell and Weibe, 1970), using modified Mueller-Hinton agar 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 incubation at 16C for 28 days, when results were reduced to a unit character state, and the data examined by the procedures of numerical taxonomy (Sneath and Sokal, 1973).

Electron Microscopy Cells 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 determined using a Jeol CX100 transmission electron microscope.

Characterization of the Causal Agents of Bacterial Kidney Disease

149

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

Serology
Washed cells were suspended in sterile 0.9% (w/v) saline to ca. 1011 cells/ml and their agglutination reactions were determined using doubling dilutions of antisera to strains 33/77,36/77,39/77,41/77, and 2/79, prepared in female New Zealand white rabbits (McCarthy and Rawle, 1975).

Results and Discussion

On the basis of overall similarity, the strains separated into 2 numerically dominant groups, phena 1 and 2, and 7 single member clusters (Fig. 1). Phenon 1 contained the type strain of Corynebacterium pyogenes (NCTC 5224). Characteristics of the Strains A summary of the characteristics of phena 1 and 2 has been given in Table I. With the exception of strains 6/79 and 7/79, which comprised large gram-positive rods, all isolates appeared morphologically similar when gram-stained smears and wet preparations were examined by light microscopy. However, subtle-variation in cell size and shape were 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, occurring singly (Fig. 3) or, rarely, in pairs. Moreover, distinctions between phena extended beyond micro-morphology insofar as the 6 strains of phenon 1 were extremely fastidious in their growth requirements, taking 28 days at 16C to produce colonies on enriched Mueller-Hinton agar. In contrast, members of phenon 2 grew readily on a wide assortment of standard bacteriological media, including nutrient agar (Oxoid) and tryptone soya agar (Oxoid). Thus, from a comparison of the phenotypic characteristics, it would appear that the micro-organisms considered as the causal agents ofBKD, represent a diverse spectrum of bacterial taxa. Nevertheless, serology data summarised in 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 is apparent that there are some shared antigens among the isolates. Taxonomic Considerations The presence of the type culture of Corynebacterium pyogenes (NCTC 5224) with isolates clustered in phenon 1 has, on first appearances, provided much information

150

B. Austin

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

Characteristic Biochemistry: Aesculin degradation Arginine dihydrolase Casein hydrolysis Catalase Methyl-red test Phosphatase Tween 40 hydrolysis Growth in: 2.5% (w/v) NaCl Enriched CO 2 Anaerobiosis Crystal violet Malachite green Potassium tellurite Growth at: 4C 16C 22C Sensitivity to: Ampicillin Cephaloridine Cloramphenicol Erythromycin Tetracycline a Weakly positive Phenon 1 Phenon 2

+ + +
(+)a

+ + + + + + + + +

+ +

+ + +

+ + +
+ +

+ + + +

2
Fig. 2. Negatively stained cell representative of the isolates clustered in phenon 1. Bar equals Illm

Characterization of the Causal Agents of Bacterial Kidney Disease

lSI

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

on the taxonomic status of this group of micro-organisms. Moreover, there was very close similarity between the description of C. pyogenes (Cummins et aI., 1974) and the characteristics of phenon 1, except for the inability of the fish isolates to grow at 37C. Unfortunately, C. pyogenes has been considered distinct from other Corynebacterium spp. (Jones, 1975) to such an extent that it has been proposed to accommodate the species in another, as yet unspecified, genus (Harrington, 1966; Minnikin et al., 1978). The relationship of this taxon to the lactic acid bacteria, notably Streptococcus 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, the classification of phenon 1 must await improvements in the taxonomy of C. pyogenes.
Table 2. Titres obtained by agglutination of representative BKD strains against antisera Bacterial isolate { 4/79 8/79 { 34/77 36/77 1/79 Antisera to: 36/77 1/64 1/64 1/32 1/1,024 1/128 1/8 1/2 1/512

39/77 1/64 1/64 1/512 1/128 1/2,048 1/8 1/16 1/8

41/77 1/128 1/128 1/128 1/1,024 1/1,024 1/4 1/32 1/2 1/32

33/77 1/1,024 1/1,024 1/8 1/4 1/8 1/2 1/16

2/79 1/2,048 1/2,048 1/8 1/4 1/4 1/8 1/128 1/16

Phenon 1 Phenon 2

Miscellaneous

{ 32/71 2/79
3/79 6/79

152

B. Austin

Similarly, the taxonomic status of phenon 2 is unclear. With Corynebacterium restricted sensu stricto to near neighbours of C. diphtheriae, clearly phenon 2 belongs in another genus. Lactobacillus may be an appropriate home for these organisms, insofar as 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 to 53.4 mole% (Rogosa, 1974), this genus is in dire need of taxonomic revision. It is noteworthy that Lactobacillus was recognised previously as pathogenic to fish, causing "pseudokidney" 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, notably Lactobacillus and Streptococcus. However, it is disquieting that 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 be confusing several chronically related diseases, or even isolating erroneous strains on laboratory media. Nevertheless, it is apparent that more work is necessary to clear the myths enshrouding BKD. The reference to proprietary products in this paper should not be construed as an official endorsement of these products, nor is any criticism implied of similar products which have not been mentioned.

References
Bullock GL, Stuckey HM, Chen PK (1974) Corynebacterial kidney disease of salrnonids: growth and serological studies on the causative bacterium. Appl icrobiol 28:811-814 Colwell RR, Weibe WJ (1970) "Core" characteristics for use in classifying aerobic heterotrophic bacteria by numerical taxonomy. Bull Ga Acad Sci 28: 165-185 Cowan ST (1974) Cowan and Steel's manual for the identification of medical bacteria. 2nd edn. Cambridge University Press, Cambridge, pp 238 Cummins CS, Lelliott RA, Rogosa M (1974) Genus 1. Corynebacterium Lehmann and Newmann 1896,350. In: Buchanan RE, Gibbons NE (eds) Bergey's manual of determinative bacteriology, 8 th edn. Williams and Wilkins Co, Baltimore, pp 602 -617 Evelyn TPT (1977) An improved growth medium for the kidney disease bacterium and some notes on using the medium. Bull Off Int Epiz 87:511-513 Fijan N (1977) Corynebacteriosis (Dee disease, kidney disease) of salrnonids in Yugoslavia. Bull Off Int Epiz 87:509 Halici G, Istanbulloglu E, Arda M (1977) An outbreak of bacterial kidney disease in fish farming station of Bayinder Dam and its treatment (in Turkish). J Fac Vet Med Univ Istanbul 3 :22-27 Harrington BJ (1966) Numerical taxonomic study of some corynebacteria and related organisms. J Gen MicrobioI45:31-40 Jones D (1975) A numerical taxonomic study of coryneform and related bacteria. J Gen Microbiol 87:52-96 Kimura T (1978) Bacterial kidney disease of salrnonids. Fish Patho116:43 -52 McCarthy D, Rawle CT (1975) The rapid serological diagnosis of fish furunculosis caused by "smoth" and "rough" strains of Aeromonas salmonicida. J Gen Microbiol86: 185 -187 Minnikin DE, Goodfellow M, Collins MD (1978) Lipid composition in the classification and identification of coryneform and related taxa. In: Bousfield IJ, Calleby AG (eds) Coryneform bacteria. Academic Press, London New York, pp 85-160

Characterization of the Causal Agents of Bacterial Kidney Disease

153

Ordal EJ, Earp BJ (1956) Cultivation and transmission of etiological agent of kidney disease in salmonid fishes. Proc Soc Exp BioI Med 92:85 -88 Rosoga M (1974) Genus 1. Lactobacillus Beijerinck 1902, 212, Nom cons Opin 38, Jud Comm 1971, 104. In: Buchanan RE, Gibbons NE (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: 191 Sanders JE, Pilcher KS, Fryer JL (1978) Relation of water temperature to bacterial kidney disease in coho salmon (Oncorhynchus kisutchj. sockeye salmon (0. nerkaj. and steelhead trout (Saimogairdnerij. J Fish Res Board Can 35:8-11 Smith IW (1964) The occurrence and pathology of Dee disease. Freshwater Salmon Fish Res 34:1-12 Sneath PHA, Sokal RR (1973) Numerical taxonomy. The principles and practice of numerical classification. WH Freeman and Co, San Francisco, pp 573 Vladik P, Vitovec J, Cervinka S (1974) The taxonomy of Gram-positive immobile diplobacilli isolated from necrotizing nephroses in American char and rainbow trout. Vet Med 19:233-238 Whittenbury R (1964) Hydrogen-peroxide formation and catalase activity in the lactic acid bacteria. J Gen Microbiol 35: 13-26