Molecular diversity and growth features of Flavobacterium columnare strains isolated in Finland.

Columnaris disease caused by Flavobacterium columnare is a problem in fish farming worldwide. During the last 15 yr, outbreaks have started to emerge in Finland. Flavobacterium columnare Type Strain NCIMB 2248T and 30 Finnish F. columnare isolates were studied using analysis of 16S rDNA by restriction-fragment length polymorphism (16S RFLP), length heterogeneity analysis of polymerase chain reaction (LH-PCR) products, automated ribosomal intergenic spacer analysis (ARISA), and 16S rDNA sequence analysis. All isolates fell into RFLP Genomovar I and had the same length in the LH-PCR analysis. Based on ARISA, 8 genetically different strains were selected for further analyses. The growth of these strains under different temperatures, NaCl concentrations, and pH values was tested. The Finnish F. columnare strains did not grow at NaCl concentrations >0.1% or at pH values < or = 6.5, and they were susceptible to several antimicrobial agents, but not to Polymyxin B or neomycin. These findings may aid in development of methods for disease management at fish farms.

Aeromonas species in fish, fish-eggs, shrimp and freshwater.

Aeromonas spp. are common contaminants of fish and seafood. They also are ubiquitous in the water environment. Aeromonas spp. were identified in 27 (93%) of 29 fish, in 17 (100%) fish-egg, in two (16%) of 12 shrimp samples and in 23 (100%) freshwater samples. In total, 117 Aeromonas strains were isolated from 69 positive samples, several samples having had two or three Aeromonas species. Included in this were also 26 mesophilic Aeromonas strains isolated in association with the study on fish diseases. The distribution of the species into 13 known hybridization groups (HGs) were studied by phenotypic and molecular methods. Ribopattern analysis of SmaI digested DNA was used for the identification of HGs. The predominant HG in fish, fish-eggs and freshwater samples was A. hydrophila HG 3 because 63% (22/37), 28% (16/57) or 80% (16/20) of the strains, respectively, were in HG 3. A. hydrophila HG 2 was also common in fresh fish samples but was not identified in fish-egg samples. HG 7 was common in fish samples studied for fish diseases and in freshwater samples. Strains which were not allotted to any HGs were common (19 of 143 strains). A. hydrophila HG 1, A. caviae HG 4, A. veronii subspecies sobria or subspecies veronii HG 8/10 known to be associated with human diarrhea were uncommon in all samples. The three strains isolated from frozen shrimp during two suspected food-borne outbreaks were A. hydrophila HG 2 and HG 3.

Molecular and phenotypic methods for the characterization of atypical Aeromonas salmonicida.

Atypical Aeromonas salmonicida form a taxonomically diverse group among the psychrophilic A. salmonicida. Characteristics of 53 atypical A. salmonicida strains originating from Finland, Denmark, Norway and Sweden were studied using 60 phenotypic tests. Ribopattern analysis and plasmid profiles were used as genetic methods. The production of brown pigment on the furunculosis agar containing L-tyrosine divided the atypical oxidase-positive strains into two groups: pigment-producing (n = 35) and achromogenic (n = 16). PstI differentiated all the atypical A. salmonicida from A. salmonicida subsp. salmonicida. Combined ribopatterns ClaI/PstI divided pigment producing atypical strains into four major groups B/B, G/G, G/T, F/F. Most of the achromogenic oxidase-positive strains belonged to two major groups H/H or U/U. Cluster analysis of ribopatterns and plasmid profile analysis also supported the division of atypical oxidase-positive A. salmonicida into pigment-producing and achromogenic groups. The oxidase-negative strains formed a distinct group which differed from oxidase-positive atypical A. salmonicida type biochemically and in terms of ribopatterns. Our results also support the use of ribopattern analysis as a valid method to study the epidemiology of infections caused by atypical A. salmonicida on fish farms.

Serological survey and epidemiological investigation of maedi-visna in sheep in Finland.

A survey for antibodies to maedi-visna virus (MV) in the Finnish sheep surveillance flocks was conducted in 1994. Examination of a total of 12931 serum samples from animals over 1 year of age from 545 flocks (81% of all flocks) revealed eight seropositive flocks and the subsequent epidemiological investigation yielded one additional seropositive flock, indicating a low prevalence of 1.6%. The infection was very probably imported from Sweden in 1981, but it was not detected until the survey was conducted 13 years later. The entire primary infection flock was slaughtered in 1995. 77% of the sheep were seropositive but the animals were clinically healthy and only one (5%) of the contact flocks of the primary infection flock had contracted the infection. This secondary infection flock, 77% of which was seropositive, was slaughtered in 1994; however, animals in this flock had respiratory problems and the lungs of three sheep showed typical MV lesions. Seven (24%) of its contact flocks had contracted the infection and these each had one or two seropositive animals except for one flock which had seven (18%) seropositive animals. The results show that the initial spread of MV can be insidious and wide before infection is revealed in surveys or any clinical cases are encountered.

Preventing the spread of maedi-visna in sheep through a voluntary control programme in Finland.

The sheep disease maedi-visna (MV) was introduced into Finland in 1981 and had spread to eight flocks in the southwestern part of the country when first detected in a survey in 1994. Six more seropositive flocks were subsequently traced, bringing the total to 14. MV has a notifiable disease status in Finland that provides for official restrictive measures to which all infected herds are subject. These measures are withdrawn once the seropositive animals and their progeny are culled and the flock has showed negative signs in the test done twice, or after total culling. A voluntary control programme was initiated in January 1995 to extend official control efforts. The programme furnishes a guideline for culling, restrictions on contacts, and a timetable for testing the flock to attain MV-free status. Seven flocks of the 14 were slaughtered either immediately or after a period under restrictive measures. One flock finished sheep production after four years under restrictive measures. Selective culling and repeated testing was attempted with the other six flocks, three of which attained MV-free status. One flock finished sheep production after two years in the control programme, the other two dropped out of the programme when the restrictive measures were withdrawn. It was concluded that the control programme was salient in eradicating MV from Finland and that serological monitoring of the situation must be continuous.

The prevalence of toxoplasma antibodies in swine sera in Finland.

A total of 1847 swine sera obtained from the 10 largest abattoirs slaughtering swine in Finland were examined by ELISA for toxoplasma antibodies. The sample represented 0.64% of the total number of swine slaughtered in these abattoirs over a period of 2 months. The prevalence of toxoplasma antibodies in swine sera was 2.5%.

Evaluation of ELISA for the detection of Toxoplasma antibodies in swine sera.

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from greater than 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving less than 10 EIU were negative in the other tests, and all those with greater than 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10-70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.

Bovine mastitis in Finland in 1988 and 1995--changes in prevalence and antimicrobial resistance.

Two surveys were carried out (during 1988 and 1995) to estimate the prevalence of bovine mastitis in Finland. In 1988, 17,111 quarter milk samples were obtained from 4495 cows, and in 1995 the corresponding figures were 10,410 and 2648. Antimicrobial susceptibility of mastitis pathogens was studied. Prevalence of mastitis on cow basis decreased from 47.8% in 1988 to 37.8% in 1995. Staphylococci was the largest group of pathogens isolated. The proportion of Staphylococcus aureus decreased and that of coagulase-negative staphylococci (CNS) increased. The proportion of strains resistant to at least one antibacterial drug increased with regard to S. aureus from 36.9% in 1988, to 63.6% in 1995 and with CNS from 26.6% to 49.7%. Most of the increase in antibacterial resistance was due to a higher number of beta-lactamase producing strains. Multiresistance also increased, but it was proportional to the overall increase in resistance. All the predominant mastitis streptococci were susceptible to beta-lactams tested.

Atypical growth of Renibacterium salmoninarum in subclinical infections.

Two growth types of Renibacterium salmoninarum were isolated from subclinically infected rainbow trout, one producing the smooth colonies typical of R. salmoninarum and the other forming a thin film on the surface of the agar with no separate colonies. The atypical growth was present on kidney disease medium agar in primary cultures of the kidney but not on selective kidney disease medium (SKDM). Fluorescent antibody staining of the fresh isolate and polymerase chain reaction amplification were the most reliable techniques to identify the atypical growth of R. salmoninarum. The condition was reversible, with growth reverting from atypical to the smooth colony form in experimentally infected rainbow trout and under laboratory conditions. There was no mortality, or any clinical signs of bacterial kidney disease (BKD) in the fish challenged with the atypical growth, although small numbers of smooth colonies of R. salmoninarum were isolated from 8% of these fish. The atypical growth reported here may explain some of the failures of culture, when SKDM agar alone is used for the detection of BKD in subclinically infected fish.

Genetic diversity of atypical Aeromonas salmonicida studied by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) pattern analysis with XbaI restriction enzyme was used to study the genetic heterogeneity of 88 atypical Aeromonas salmonicida strains which were earlier or during this study characterized phenotypically, by ribotyping (ClaI/PstI) and by plasmid profile analysis. The strains of certain'ribotypes were also analysed by digestion with SpeI. The strains represented different geographic locations: Finland (72 strains), Iceland (5 strains), Norway (5 strains), Sweden (4 strains) and Denmark (2 strains), and they were from 17 fish species during 1981 97. Thirty-one PFGE genotypes found among these strains correlated well with the ribotypes, and in most cases PFGE pattern analysis subdivided ribotypes into several PFGE genotypes, and further within a PFGE genotype into subtypes. XbaI and SpeeI digests produced concordant results. In most cases, PFGE patterns of strains with the same ribotype shared many fragments, suggesting genetic relatedness. PFGE patterns of most Norwegian and Icelandic strains isolated during an approximately 10-year period had the same ribotype and their PFGE patterns shared most fragments, suggesting close genetic relatedness. Moreover, atypical strains of ribotypes B/B and H/H isolated from the same Finnish fish farms had closely related patterns suggesting genetic stability and persistence of these genotypes. Genotype 29 of Achromogenic strains was strongly associated with disease of Finnish arctic char and grayling. PFGE was shown to be a distinguishing method to study the genetic heterogeneity of atypical A. salmonicida. epidemiology of these infections.

Phenotypic and molecular characteristics of Aeromonas salmonicida subsp. salmonicida isolated in southern and northern Finland.

Aeromonas salmonicida subsp. salmonicida causes outbreaks of furunculosis in salmonid fish. Furunculosis was first detected in Finland in 1986 on fish farms located on the Finnish coast of the Bothnian Bay. Molecular methods, SDS-PAGE, ribotyping, randomly amplified polymorphic DNA (RAPD) and plasmid profile analysis as well as phenotypic characteristics (biochemical characteristics, maximum growth temperature, pigment and elastase production) were used both for typing the strains and to study the possible routes of transmission of the organism to Finland and the spread of infection within Finland from 1986 to 1993. Ribopattern analysis of chromosomal DNA digested with SmaI, BglI, PstI and ClaI of 28 Finnish strains and eight foreign strains (Denmark, Sweden, Norway or Canada) showed that all Finnish strains and the Swedish strain originating from the Swedish coast of the Bothnian Bay had identical ribopatterns. All other foreign strains had distinct, unique ribotypes except for the second Swedish strain studied, the ribotype of which was identical with that of one Danish strain. RAPD typing, based on the results of two arbitrary primers, yielded 15 types for the Finnish strains. Except for both Danish strains, which had the RAPD type which was identical with that of one Finnish strain, the foreign strains had RAPD patterns differing from those of the Finnish strains. Plasmid profile typing and RAPD profile typing did not correlate. Ribotyping with four different enzymes proved to be the most sensitive method for studying genetically homogeneous Aer. salmonicida subsp. salmonicida. Ribopattern analysis showed that the infection which first started in 1984/1985 on the Swedish coast of the Bothnian Bay may have been transmitted to Finland where the first outbreaks occurred in 1986. The strains infecting Finnish fish farms were very homogeneous, with most differences seen, for example, in maximum growth temperature, plasmid profiles and the RAPD profiles of the strains.

Molecular genetic characterization of the Fennoscandian cervid strain, a new genotypic group (G10) of Echinococcus granulosus.

The northern biotype of Echinococcus granulosus occurs in North America and northern Eurasia in life-cycles involving cervids. Previously, cervid isolates of E. granulosus from North America have been characterized using molecular genetic techniques as the G8 genotype. In this study, 5 isolates of E. granulosus were collected from 4 reindeer and 1 moose in north-eastern Finland. DNA sequences within regions of mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NI)I) genes and the internal transcribed spacer 1 (ITS-1) fragment of the ribosomal DNA were analysed. The mitochondrial nucleotide sequences were identical in all isolates, but high sequence variation was found in the ITS-1 region. Mitochondrial and nuclear sequences of the Finnish cervid E. granulosus and the camel strain (G6) of E. granulosus resembled closely each other. According to phylogenetic analyses, the Finnish isolates have close relationships also with the pig (G7) and cattle (G5) strains. Although some similarities were found with the previously published North American cervid strain (G8), particularly in the NDI sequence and some of the ITS-1 clones, the Finnish E. granulosus form represents a distinct, previously undescribed genotype of E. granulosus. The novel genotype is hereby named as the Fennoscandian cervid strain (G10).

Trichinella spiralis in wild animals, cats, mice, rats and farmed fur animals in Finland.

A total of 1 399 samples of wild animals, cats, mice, rats and fur animals were examined for Trichinella larvae during the period 1.1.1982-30.6.1984. Samples were obtained both from the problem area, were Trichinella larvae had been found in pigs, and for comparison from the rest of Finland. The frequency of the infection in wild carnivores, badgers, pine martens, raccoon dogs, foxes and wild mink was significantly higher in the problem area than in the rest of Finland.

Campylobacter hyointestinalis subsp. hyointestinalis, a common Campylobacter species in reindeer.

AIMS: To study the prevalence of Campylobacter spp. in the faecal material of reindeer, and to identify the isolates by means of a polyphasic approach. In addition, to study the genetic diversity of Camp. hyointestinalis subsp. hyointestinalis reindeer isolates by pulsed-field gel electrophoresis (PFGE). METHODS AND RESULTS: The material, collected during the slaughter period in autumn 1998, comprised 399 faecal contents from the reindeer (Rangifer tarandus), a semi-domesticated, meat-producing ruminant of northern Finland. These samples came from 16 herds in the areas of eight reindeer slaughterhouses. Samples were cultured by methods suitable for isolation of fastidious Campylobacter species. Of all samples, 6% (24/399) were Campylobacter-positive. Phenotypic characteristics, SDS-PAGE protein patterns, dot blot DNA-DNA hybridization, 23S rDNA restriction fragment polymorphism analysis and PFGE identified the isolates as Camp. hyointestinalis subsp. hyointestinalis. CONCLUSIONS: Campylobacter hyointestinalis subsp. hyointestinalis was the only Campylobacter species isolated from reindeer in this study. The isolates showed high genomic diversity in PFGE with the restriction enzymes SmaI and KpnI. SIGNIFICANCE AND IMPACT OF THE STUDY: PFGE analysis is a useful subtyping method for epidemiological studies. Contaminated reindeer meat can be a source for human infections.