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Molecular epidemiology of bacterial fish diseases: preliminary study with Vibrio anguillarum
Aquaculture, 107 (1992) 141-145 Elsevier Science Publishers B.V.. Amsterdam
141
Molecular epidemiology of bacterial fish diseases: preliminary study with Vibrio anguillarum Christian
P. Vivares’, Francine Grimontb, Felix Baudin-LaurencinC, Bruno Andrald and Patric A.D. Grimontb
‘Lab. Brologie Cornpar& da ProI~sIes (WA
CNRS no. 138). UnrversilP B/me FWICC
Pascal, Aubi>re,
ABSTRACT Vivares.C.P., Gtimont, F., Baudin-Laurencin, F.. Andral. B. and Grimm& P.A.D., 1992. Molecular epidemiology of bacteria: tirh diseases:preliminary study with Vibrio anguillarum. Aquacullure, lO7: 141-145.
six
A comparative study of 01 scrotype Vibrio angwIIarum strains from different areas and fishes was realized with three methods, of which two for the first time in fish pathology. Three plasmid proiiles were observed but all strains had the same rRNA gene restriction pattern.
INTRODUCTION
Among the bacterial pathogens of major importance in fish pathology, the vibrios produce diseases in estuarine and marine environments. The most commonly encountered Vibrio species pathogenic in fish is Vibrio anguilhum which is responsible for the majority of losses worldwide. Vibriosis is a stress-related disease and Y anguillarum constitutes a part of the natural microflora of the marine environment and fishes. Tanks, nets, and carrier and feral fishes can be sources of infection; thus an epidemiological study is necessary. The purpose of this preliminary work was to characterize, at the molecular level, different strains of K anguillarum isolated from fishes (rainbow trout, turbot and sea bass) of the Atlantic and Mediterranean coasts of France. A novel approach for differentiation and identification of bacterial species or strains is the comparison of patterns of restriction fragments hybridizing Correspondence la: Dr. C.P. Vivares, Laboraloire de Biologic Cornpar& CNRS no. 138), Universit6 Blake Pascal, 63117 Aubitre C&da, France.
0044-8486/92/$05.00
des Pmtistes (URA
0 1992 Elsevier Science Publishers B.V. All rights reserved.
142
C.P. “WARES ETAL.
with labelled rRNA (Grimont and Grimont, 1986). Since ribosomal ribonucleic acid (rRNA) genes are ubiquitous and contain highly conserved sequences, rRNA from a single species (F. coli) can be used as a probe to visualise restriction patterns in DNA from any species. rRNA gene restriction 1
2
3
4
-4.6
Fig. 1. Autoradiogram of a nylon membrane after transfer of HmdIII restriction fragments of four I’ifwio anguil/artw~ DNAs and hybridization with “zP-laheled E. coli 16+238 rRNA. Lane I, rRNA gene restriction pattern of V. mguillarutn 10482: lane 2, 10466: lane 3. 10283; lane 4, ATCC 19264.
MOLEC”LAR
EPlDEYlOlBCY
OF BAcrERI4L
143
FlSH DISEASE*
TABLE 1 EcoRl restnction endunuclease cleavage patterns ofplarmid DNA ofsix slrainr of Vibrio angurlhrum (kb)
10283
10466
1 zs!l 1l.lnJ
12.50 11.00
9.40
9.40
7.4n
7.50
4.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
d.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
12.50 11.00
V62
408
10724
12.50 I I .oo
12.50 II.00 I Dal
12.50 11.00
8.00 7.50
8.00 7.50
4.50 4.30
4.50 4.30
4.10 3.20 3.00 2.80 2.10 2.00 1.55
4.10 3.20 3.00 2.80 2.10 2.OD 1.55
7.40 4.70 4.50
7.40 4.70 4.50
4.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
4.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
patterns can be species-, subspecies-, or type-specific depending on the degree of intraspecies genomic heterogeneity. Another approach was a study of relatio~tships among plasmid and phenotypes (Wiik el al., 1989). A tinal method was to use biochemical nutritional tests with 99 pure carbon sources and comparison with reference species (Grimont et al., 1988). MATERIAL
AND METHODS
Strains of Vibrio anguilkvum from LPAA-CNEVA 29263 Plouzane, France and ATCC 19264 were studied. Three types, isolated from different fishes, were characterized with API System 20E to show the differences in the assimilation of amygdaline and melibiose: (I ) strain nos. 10482 and V62 (amy negative and me1 negative, from Dicenlrarchus 1abru.xL.); (2) strain nos. 10466 and 10283 (amy positive and me1 negative, from Scophthalmus maximur (L.)); (3) strain nos. 408 and 10724 (a;ny positive and me1 positive from respectively, Oncorhynchus mykiss Richardson and Scophthalmus max. imus (L.)). The method followed for rRNA gene restriction patterns is described in Grimont and Cirtmont ( 1986). To determine plasmid profiles, a rapid method (Birnboim and Doly, 1979) was used for extraction of plasmid DNA which was cleaved by restriction endonucleases EcoRI and Hinrz!III (Amersham ). For biochemical nutritional tests 99 pure carbon sources listed
C.P. “IVARESETAL.
144 TABLE
2
HindIll restriction hum (kb)
endonuclease
10283
10466
cleavage patterns of plasmid DNA of six strains of Mbrio angurl-
10482
V62
13.50
13.50
408
10724
9.00
IO.00 9.00
8.70
8.70
7.00 6.50
7.00 6.50
7.00 6.50
7.00 6.50
4.10 3.90 3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 1.40 1.30
4.10
4.10
4.10
7.10 7.00 6.50 4.80 4.30 4.20 4.10
3.90
3.90
3.90
3.90
3.90
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 I .40 1.30
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 1.40 I .30
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 I .4cl 1.30
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 I .80 1.70 I .40 1.30
3.80 3.30 2.50 2.35 2.30 2.20
7.00 6.50
4.10
2.10 2.00 I.80 I.70 1.40 1.30
in Grimont et al. ( 1988) were used in a minimal medium, provided by API System and supplemented with I6 growth factors; galleries were incubated at 2OC and examined for unambiguous turbidity after 2 and 4 days. Nutritional data were entered in an Apple IIE compatible computer (API System) and compared with nutritional data stored in a data base (P.A.D. Grimont). RESULTS
AND
DISCUSSION
rRNA gene restriction patterns obtained with Hi&III digested DNAs of K anguihrum are shown in Fig. 1. The three 01 serotype lCanguihrum (nos. 10482, 10466 and 10283) had identical patterns with 15 fragments. Strain ATCC 19264 (02 serotype) showed a similar RNA pattern with 1I common fragments. Plasmid profiles are shown in Tables 1 and 2. With EcoRl restriction endonuclease, some fragments are present and common to both strains: a 9.4-
MOLECULAR
EP,OEM,OLOGY
OF BAcTERlAL
FlSH DISEASES
145
kb band in strains IO283 and 10466; a 4.7-kb band in strains 10482 and V62; and 4.3-, 7.5 and 8.0-kb bands in strains 408 and 10724. Similar results were obtained with HinmII restriction endonuclease: a 8.7-kb band in strains 10283 and 10466: a 13.5kb band in strains 10462 and V62: and a 9.0-kb band in strains 408 and 10724. In nutritional tests, comparison with a reference type of V. angdlr;rum ( ATCC 19264, serotype 02: D-trehalose positive, D-gluconate positive, myoinositol negative, cellobiose and arabinose positive) showed different types: ( 1) all strains are D-trehalose negative, (2) strain 408 is D-gluconate negative, (3) strain 10724 is myo-inositol positive, (4) strains 10482 and V62 are cellobiose and arabinose negative. The comparative study of different strains isolated from diseased fish shows different types of V. anguillarum. Three methods were used (two for the first time in fish pathology) on strains first characterized with a rapid method (API 20 E) by which three types were found. The plasmid profiles agree with these preliminary observations; it is possible that some corresponding metabolic genes are localized on plasmids. The other methods demonstrated the common characters in six different strains. Thus, an epidemiological study may be realized with genomic system.
REFERENCES
Birnboim,H.C.and Daly, J., 1979. A rapid alkaline
extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res., 7: 1513-l 523. Grimont, F. and Grimont, P.A.D., 1986. R&osonal ribonusleic acid gene restriction patterns aspotentialtaxonomic tools. Ann. Inst. Pasteur/Microbial.. 137B: 165-175. Grimont. P.A.D.. Jackson, T.A., Ageron, E. and Noonan, ML, 1988. Serrariaenromophilasp. nav. associated with amber disease irl :he Yew Zealand grass grub Cowlytra relandica. Int. J. Syst. Bacterial., 38: l-6. Wiik. R.. HoiF. LA., Andersen, K. and Daae, FL., 1989. Relationships between p!ssmids and phenotypes of presumptive strains of Vibrio angullarum isolated from different fish species. Appl. Environ. Microbial., 55: 826-831.
141
Molecular epidemiology of bacterial fish diseases: preliminary study with Vibrio anguillarum Christian
P. Vivares’, Francine Grimontb, Felix Baudin-LaurencinC, Bruno Andrald and Patric A.D. Grimontb
‘Lab. Brologie Cornpar& da ProI~sIes (WA
CNRS no. 138). UnrversilP B/me FWICC
Pascal, Aubi>re,
ABSTRACT Vivares.C.P., Gtimont, F., Baudin-Laurencin, F.. Andral. B. and Grimm& P.A.D., 1992. Molecular epidemiology of bacteria: tirh diseases:preliminary study with Vibrio anguillarum. Aquacullure, lO7: 141-145.
six
A comparative study of 01 scrotype Vibrio angwIIarum strains from different areas and fishes was realized with three methods, of which two for the first time in fish pathology. Three plasmid proiiles were observed but all strains had the same rRNA gene restriction pattern.
INTRODUCTION
Among the bacterial pathogens of major importance in fish pathology, the vibrios produce diseases in estuarine and marine environments. The most commonly encountered Vibrio species pathogenic in fish is Vibrio anguilhum which is responsible for the majority of losses worldwide. Vibriosis is a stress-related disease and Y anguillarum constitutes a part of the natural microflora of the marine environment and fishes. Tanks, nets, and carrier and feral fishes can be sources of infection; thus an epidemiological study is necessary. The purpose of this preliminary work was to characterize, at the molecular level, different strains of K anguillarum isolated from fishes (rainbow trout, turbot and sea bass) of the Atlantic and Mediterranean coasts of France. A novel approach for differentiation and identification of bacterial species or strains is the comparison of patterns of restriction fragments hybridizing Correspondence la: Dr. C.P. Vivares, Laboraloire de Biologic Cornpar& CNRS no. 138), Universit6 Blake Pascal, 63117 Aubitre C&da, France.
0044-8486/92/$05.00
des Pmtistes (URA
0 1992 Elsevier Science Publishers B.V. All rights reserved.
142
C.P. “WARES ETAL.
with labelled rRNA (Grimont and Grimont, 1986). Since ribosomal ribonucleic acid (rRNA) genes are ubiquitous and contain highly conserved sequences, rRNA from a single species (F. coli) can be used as a probe to visualise restriction patterns in DNA from any species. rRNA gene restriction 1
2
3
4
-4.6
Fig. 1. Autoradiogram of a nylon membrane after transfer of HmdIII restriction fragments of four I’ifwio anguil/artw~ DNAs and hybridization with “zP-laheled E. coli 16+238 rRNA. Lane I, rRNA gene restriction pattern of V. mguillarutn 10482: lane 2, 10466: lane 3. 10283; lane 4, ATCC 19264.
MOLEC”LAR
EPlDEYlOlBCY
OF BAcrERI4L
143
FlSH DISEASE*
TABLE 1 EcoRl restnction endunuclease cleavage patterns ofplarmid DNA ofsix slrainr of Vibrio angurlhrum (kb)
10283
10466
1 zs!l 1l.lnJ
12.50 11.00
9.40
9.40
7.4n
7.50
4.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
d.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
12.50 11.00
V62
408
10724
12.50 I I .oo
12.50 II.00 I Dal
12.50 11.00
8.00 7.50
8.00 7.50
4.50 4.30
4.50 4.30
4.10 3.20 3.00 2.80 2.10 2.00 1.55
4.10 3.20 3.00 2.80 2.10 2.OD 1.55
7.40 4.70 4.50
7.40 4.70 4.50
4.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
4.20 4.10 3.20 3.00 2.80 2.10 2.00 1.55
patterns can be species-, subspecies-, or type-specific depending on the degree of intraspecies genomic heterogeneity. Another approach was a study of relatio~tships among plasmid and phenotypes (Wiik el al., 1989). A tinal method was to use biochemical nutritional tests with 99 pure carbon sources and comparison with reference species (Grimont et al., 1988). MATERIAL
AND METHODS
Strains of Vibrio anguilkvum from LPAA-CNEVA 29263 Plouzane, France and ATCC 19264 were studied. Three types, isolated from different fishes, were characterized with API System 20E to show the differences in the assimilation of amygdaline and melibiose: (I ) strain nos. 10482 and V62 (amy negative and me1 negative, from Dicenlrarchus 1abru.xL.); (2) strain nos. 10466 and 10283 (amy positive and me1 negative, from Scophthalmus maximur (L.)); (3) strain nos. 408 and 10724 (a;ny positive and me1 positive from respectively, Oncorhynchus mykiss Richardson and Scophthalmus max. imus (L.)). The method followed for rRNA gene restriction patterns is described in Grimont and Cirtmont ( 1986). To determine plasmid profiles, a rapid method (Birnboim and Doly, 1979) was used for extraction of plasmid DNA which was cleaved by restriction endonucleases EcoRI and Hinrz!III (Amersham ). For biochemical nutritional tests 99 pure carbon sources listed
C.P. “IVARESETAL.
144 TABLE
2
HindIll restriction hum (kb)
endonuclease
10283
10466
cleavage patterns of plasmid DNA of six strains of Mbrio angurl-
10482
V62
13.50
13.50
408
10724
9.00
IO.00 9.00
8.70
8.70
7.00 6.50
7.00 6.50
7.00 6.50
7.00 6.50
4.10 3.90 3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 1.40 1.30
4.10
4.10
4.10
7.10 7.00 6.50 4.80 4.30 4.20 4.10
3.90
3.90
3.90
3.90
3.90
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 I .40 1.30
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 1.40 I .30
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 1.80 1.70 I .4cl 1.30
3.80 3.30 2.50 2.35 2.30 2.20 2.10 2.00 I .80 1.70 I .40 1.30
3.80 3.30 2.50 2.35 2.30 2.20
7.00 6.50
4.10
2.10 2.00 I.80 I.70 1.40 1.30
in Grimont et al. ( 1988) were used in a minimal medium, provided by API System and supplemented with I6 growth factors; galleries were incubated at 2OC and examined for unambiguous turbidity after 2 and 4 days. Nutritional data were entered in an Apple IIE compatible computer (API System) and compared with nutritional data stored in a data base (P.A.D. Grimont). RESULTS
AND
DISCUSSION
rRNA gene restriction patterns obtained with Hi&III digested DNAs of K anguihrum are shown in Fig. 1. The three 01 serotype lCanguihrum (nos. 10482, 10466 and 10283) had identical patterns with 15 fragments. Strain ATCC 19264 (02 serotype) showed a similar RNA pattern with 1I common fragments. Plasmid profiles are shown in Tables 1 and 2. With EcoRl restriction endonuclease, some fragments are present and common to both strains: a 9.4-
MOLECULAR
EP,OEM,OLOGY
OF BAcTERlAL
FlSH DISEASES
145
kb band in strains IO283 and 10466; a 4.7-kb band in strains 10482 and V62; and 4.3-, 7.5 and 8.0-kb bands in strains 408 and 10724. Similar results were obtained with HinmII restriction endonuclease: a 8.7-kb band in strains 10283 and 10466: a 13.5kb band in strains 10462 and V62: and a 9.0-kb band in strains 408 and 10724. In nutritional tests, comparison with a reference type of V. angdlr;rum ( ATCC 19264, serotype 02: D-trehalose positive, D-gluconate positive, myoinositol negative, cellobiose and arabinose positive) showed different types: ( 1) all strains are D-trehalose negative, (2) strain 408 is D-gluconate negative, (3) strain 10724 is myo-inositol positive, (4) strains 10482 and V62 are cellobiose and arabinose negative. The comparative study of different strains isolated from diseased fish shows different types of V. anguillarum. Three methods were used (two for the first time in fish pathology) on strains first characterized with a rapid method (API 20 E) by which three types were found. The plasmid profiles agree with these preliminary observations; it is possible that some corresponding metabolic genes are localized on plasmids. The other methods demonstrated the common characters in six different strains. Thus, an epidemiological study may be realized with genomic system.
REFERENCES
Birnboim,H.C.and Daly, J., 1979. A rapid alkaline
extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res., 7: 1513-l 523. Grimont, F. and Grimont, P.A.D., 1986. R&osonal ribonusleic acid gene restriction patterns aspotentialtaxonomic tools. Ann. Inst. Pasteur/Microbial.. 137B: 165-175. Grimont. P.A.D.. Jackson, T.A., Ageron, E. and Noonan, ML, 1988. Serrariaenromophilasp. nav. associated with amber disease irl :he Yew Zealand grass grub Cowlytra relandica. Int. J. Syst. Bacterial., 38: l-6. Wiik. R.. HoiF. LA., Andersen, K. and Daae, FL., 1989. Relationships between p!ssmids and phenotypes of presumptive strains of Vibrio angullarum isolated from different fish species. Appl. Environ. Microbial., 55: 826-831.