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Strains of Aeromonas salmonicida ssp. salmonisalrnonicida ssp. masoucida, and several isolates not cida lacking pigment production were isolated from brown assigned to any of the valid subspecies, trout Salmo trutta m. lacustris and sea trout S. trutta m. trutta Recently a fourth subspecies, A. salmonicida ssp. cultivated in fresh water in south Finland. The bacteria isolated showed only minor deviations in biochen~ical charactersmithid, which readily produce the lstics compared to 2 strains of A. sillmonicjda ssp. salmoniclda pigment, was proposed by Austin et al. (1989). and the type strain of A. saln~onicjda ssp. salmoniclda (NCMB 1102). Several characters differed when compared to the type strain of A. salmonicida ssp. achromogenes (NCMB 1110). In challenge experiments, the strain tested was highly pathogenic to rainbow trout Oncorhynchus mykiss. Different forms of Aeromonas salmonicjda are frequently isolated from diseased salmonids as well as from non-salmonids (Wichardt 1983, Bohm et al. 1986, Wiklund 1990). Traditionally A. salmonicida has been divided into typical strains, that is, ssp. salmonicida which produce a brown water-soluble pigment and 'atypical' strains which do not produce or only very slowly produce the brown pigment. The 'atypical' strains include A. salmonicida ssp. achromogenes, A. According to 'Bergey's Manual of Systematic Bacteriology' (Popoff 1984) the production of brown pigment is one of the 9 key characteristics in separating ssp. salmonicida from 'atypical' strains (Table 1). The present paper, however, describes strains of Aerornonas salrnonicida ssp. salmonicida lacking pigment production. During routine examination of diseased farmed fish in our laboratory in 1991, non-pigmented variants of Aerornonas salmonicida were isolated from brown trout Salmo 'trutta m. lacustris and sea trout S. trutta m. trutta from 4 fish farms using fresh water. The fish farms, which were located in south Finland, were mainly raising smolts for stocking purposes. Table 1. Differential characteristics of subspecies of Aeromonas salmonicida and corresponding results of the non-pigmented strains. +: positlve reaction; -. negative reaction; 'NT: not tested Characteristic Present Aeromonas salmonicida strains ssp. ssp. ssp. ssp. salrnonicidaa achrornogenesd masoucidaa snithia Brown pigment + +/Indole production + + Esculin hydrolysis + + L-Arabinose utilization NT + Acid from sucrose + + +/Acid from mannitol + + +/Voges-Proskauer + Gas from glucose + NT Production of H2S + + "Popoff (1984); b ~ u s t i n et al. (1989) 0 Inter-Research 1993 220 Dis. aquat. Org. 15: 219-223, 1993 The total mortality of the affected fish stock in one of the farms was about 90%. Disease signs in the fish from this farm were more pronounced than in fish from the other farms: large necrotic lesions in the muscles, enlarged spleens, and hemorrhagic intestines. The total mortality in the other farms varied from 8 to 40 %. The disease signs of the fish from these farms included minor skin ulcers, enlarged spleens, and petechial hemorrhages in the perivisceral adipose tissue. The diseased fish in one of the farms were medicated with oxytetracycline. On 2 other farms the fish were treated with oxolinic acid. Mortality ceased during the medication period. Material and methods. Two to eleven fish from each farm (n = 4) were examined for bacterial infection in vlscerai organs. Samples irom kidney, i iver, dnci spleen from the dlseased fish were inoculated onto tryptic soy (TS) agar (Difco Laboratories; final NaCl concentration = I S % ) , supplemented with 5 % bovine blood. The agar plates were incubated at 20°C for 7 d. Biochemical tests were carried out on the isolates using methods described by MacFaddin (1983) or Cowan (1974) and the API 50 CHE diagnostic system (Bio Merieux, France). Because the development of brown pigment was previously shown to be dependent on tyrosine and phenylalanine (Griffin et al. 1953), TS agar supplemented either with tyrosine or phenylalanine (0.1 % final concentration) was used to detect the pigment production. As reference strains we included one pigmentforming strain (isolated concurrently with one of the non-pigmented strains), one strain of Aeromonas salmonicida ssp. salmonicida isolated from diseased farmed rainbow trout Oncorhynchus mykiss, and type strains of A. salmonicida ssp. salmonicida (NCMB 1 102) and A. salmonicida ssp. achromogenes (NCMB 1110). Virulence tests with one of the non-pigmented strains were performed by intraperitoneally injecting rainbow trout (5 fish per concentration) with 0.1 m1 sterile saline (0.9% NaC1) containing 1.3 X 107, 1.3 X 105, and 1.3 X 103 washed bacteria (CFU, colonyforming units). Control fish were injected with 0.1 m1 sterile saline. The infected fish were kept in recirculating, filtered tap water at 16 to 17OC for 2 wk. Samples of spleen, kidney, and liver from dead and surviving fish were examined bacteriologically. Results. Gram-negative, non-motile, cytochrome oxidase-positive, facultatively anaerobic, short rods were isolated from the examined fish. Pigmented (3 isolates out of 7) as well as non-pigmented (4 isolates out of 7) strains were isolated from diseased fish from one of the farms. The stralns isolated from the other 3 farms were all non-pigmented, and they were isolated from 67 to 100 % of the examined fish. In the biochemical tests, the strains lacking pigment production were almost identical to the 2 pigmentforming CO-isolates tested and they all showed only minor deviations (growth in 4 % NaCl and at 30°C) from the type strain of Aeromonas salmonicida ssp. salmonicida (NCMB 1102). Compared to the type strain of A. salmonicida ssp. achromogenes (NCMB 1110), several different characters (19 out of 92) were encountered (Table 2). However, the strain NCMB 11 10 gave a positive reaction in the VP test, although it should have been negative according to Popoff (1984). The strains tested did not produce pigment on TS agar supplemented with phenylalanine, but after 6 to 7 d of incubation a very weak pigment production was noted on TS agar supplemented with tyrosine. The Aeromonas salmonicida ssp. salmonicida refere~ce strains produced pigment on both agars. In the challenge test all fish injected with 1.3 X 107 and 1.3 X 10' bacteria died within 3 d, and 4 fish oul of 5 injected with 1.3 X 10"acteria died within 5 d. One fish survived for 2 ~ v k in the last-mentioned group. All fish in the control group were alive at the end of the experiment. The injected bacteria were re-isolated from visceral organs of all dead fish but not from the one surviving challenged fish. These isolates did not produce any pigment on TS agar. During 1992, the same type of disease occurred in 3 of the previously affected farms, but unfortunately no attempt to isolate bacteria was made. On the fourth farm there were no disease outbreaks during 1992. However, in 1992 non-pigmented strains of Aeromonas salmonicida ssp. salmonicida were isolated from diseased fish on 2 previously unaffected farms. One of the three of these non-pigmented variants tested did not produce gas from glucose and two of them produced acid from trehalose. In all other biochemical tests they were similar to the non-pigmented strains isolated in 1991. Discussion. To our knowledge there are very few reports of 'typical' Aeromonas salmonicida strains that do not produce a brown, water-soluble pigment when grown on agar containing tryptone (tyrosine or phenylalanine). Austin et al. (1989) reported one strain of A. salmonicida ssp. salmonicida out of 27 tested (4 %) not producing pigment. However, several characteristics of the group described (see Austin et al. 1989) did not match the type description of A. salmonicida ssp. salmonicida (Popoff 1984). Evelyn (1971) reported an aberrant strain of A. salmonicida that lost its ability to produce pigment after 2 yr of subcultivat~on. Also, Duff & Stewart (1933) noticed that isolates lost their ability to produce the brown pigment after prolonged subcultivation. Conversely, one strain recovered from an experimentally infected goldfish was reported not to produce brown pigment until it was subcultured 5 times. Wiklund et al.: Aerornonas lacking pigment production 221 Table 2. Morphological and biochemical characteristics of non-pigmented and pigmented Aerornonas salrnonicida ssp. salrnonicida strains and of type strains NCMB 1102 (A. salrnonicida ssp. salrnonic!da) and NCMB 11 10 (A. salmonicida ssp. achrornogenes). ASS: A. salrnonicida ssp. salrnonicida; ASA: A. salmonicida ssp. achrornogenes. +: positive reaction; -: negative reaction; (+): weak reaction; R: resistant; S: sensitive Non-pigmented Pigmented ASS ASS (n = 4)a (n = 2)" ASS AS A NCMB NCMB 1102 1110

Aeromonas salmonicida ssp. salmonicida lacking pigment production, isolated from farmed salmonids in Finland