Apr 27, 1981 - observed cross-reactions between some A. hydrophila and A. sobria .... Interestingly, cross-reactions ..... Erwing, W., R. Hugh, and J. Johnson.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1981, p. 56-60 0099-2240/81/070056-05$02.00/0
Vol. 42, No. 1
Serogrouping of Motile Aeromonas Species Isolated from Healthy and Moribund Fish D. LEBLANC, K. R. MITTAL, G. OLIVIER, AND R. LALLIER* Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University ofMontreal, St. Hyacinthe, Quebec J2S 7C6, Canada
Received 9 March 1981/Accepted 27 April 1981
A total of 195 strains of motile Aeromonas isolated from fish were characterized Aeromonas hydrophila and Aeromonas sobria. In view of the frequency of isolation and the importance of motile Aeromonas species as fish pathogens, a serological classification of these organisms was attempted. Antisera were prepared in rabbits against formalinized whole cell suspensions and against boiled cells of 12 different isolates. Seventy-six strains could be grouped by tube agglutination with whole cells as antigen and anti-whole cell antiserum. However, only 39 strains were typable with anti-O serum. Differentiation was made between heat-stable antigens and heat-labile antigens which did not block the 0 agglutination reaction. The same heat-labile antigen could be associated with different heat-stable particulate antigens, and a relationship was observed between the heat-stable particulate antigens and the virulence of A. hydrophila for fish. In addition to these two types of antigen, motile Aeromonas possessed heat-stable soluble antigens which could be detected by indirect hemagglutination. One strain seemed to possess various heat-stable soluble antigens; so far, however, it does not appear to be feasible to use these antigens for serology. Finally, we also observed cross-reactions between some A. hydrophila and A. sobria strains. as
gens (4, 5). Preliminary data on serotyping suggested a possibility for grouping the motile Aeromonas strains (3, 6, 12). The objective of this work was to characterize the antigenic structure of motile Aeromonas strains with a view to classifying them into serogroups. (Preliminary reports of this work were presented at the meeting of the American Society for Microbiology in Miami, Fla., 11-17 May 1980.) MATERIALS AND METHODS
Motile Aeromonas strains are regularly isolated from the environment and are also considered to be normal inhabitants of the fish intestinal tract (7, 16). They are also recognized as fish pathogens, and some could also be pathogenic for humans and other mammals (2). Schubert (14) classified such motile strains as Aeromonas hydrophila and Aeromonas punctata. Another group, working with A. hydrophila and Aeromonas sobria, proposed a new classification (13). Shaw and Hodder found that the core lipopolysaccharides of A. hydrophila strains were different from those of A. sobria (15). A higher percentage of deoxyribonucleic acid-deoxyribonucleic acid hybridization was observed among A. hydrophila isolates as compared to that observed between A. hydrophila and A. sobria (9). Boulanger et al. (1) reported that A. hydrophila could be isolated from cutaneous lesions and kidneys of diseased fish, whereas both A. hydrophila and A. sobria could be recovered from the intestine of normal fish. We also observed that A. hydrophila strains are more hemolytic, dermonecrotic, and virulent for fish than A. sobria (8; G. Oliver, R. Lallier, and S. Lariviere, Can. J. Microbiol., in press). Motile Aeromonas strains contain thermostable 0, thermolabile K, and flagellar H anti-
Strains. A total of 195 strains isolated from healthy and moribund fish were identified as A. hydrophila and A. sobria as already described (1). Antiserum preparation. Bacteria were grown on tryptic soy agar (Difco) slants plus 0.1% glucose for 18 h at room temperature. Cells were suspended in 0.85% saline. For the 0 antigen, cells were boiled at 100°C for 1.5 h, washed three times in saline, and then adjusted to an optical density of 1.0 at 540 nm. For the whole-cell (WC) antigen, the strains were grown in brain heart infusion broth (Difco) for 18 h at room temperature. Formalin was added to obtain a final concentration of 1%, and the optical density at 540 nm was adjusted to 0.3. Antisera were prepared in rabbits against 0 and WC antigens of A. hydrophila and A. sobria strains. Rabbits were injected intravenously twice a week for 3 weeks with increasing volumes (0.25, 0.50, 1.00, 2.00, 3.00, and 3.00 ml) of antigen, and they were bled 7 days after the last injection. 56
VOL. 42, 1981
MOTILE AEROMONAS SPECIES FROM FISH
57
Slide agglutination tests. The test strains were added, and the tubes were incubated at room temperpicked from an 18-h-old colony on 5% blood-tryptic ature for 4 h before reading. The highest dilution of soy agar plates and mixed thoroughly with a drop of serum giving complete agglutination was taken as the a 1:5 diluted rabbit anti-WC serum. The reactions end titer. Controls consisted of sensitized and unsenwere observed for 1 to 2 min. sitized sheep erythrocytes mixed with known positive Tube agglutination test. For each strain, 0 and and negative sera. WC antigen preparations were tested against different anti-O and anti-WC sera. Twofold dilutions of serum RESULTS in 0.2-ml volumes were made in 0.85% saline. An equal Before the serotyping of the 195 isolates was volume of antigen standardized to an optical density of 0.4 at 540 nm was added. The tubes were then attempted, the specificity of the different sera incubated for 18 h at 370C, followed by further incu- was tested by tube agglutination with the 12 strains used for immunization. In all cases except bation for 6 h at 40C before reading. Immunoelectrophoresis. The 18-h brain heart one (A. sobria strain 9), each strain was aggluinfusion broth (5 ml) cultures were centrifuged, and tinated only with either anti-O or anti-WC hothe cells were suspended in 5 ml of 0.85% saline. The mologous serum (Tables 1 and 2). Boiled and cells were boiled for 1.5 h, sonicated, and centrifuged cell preparations of the 183 other (8,000 x g, 10 min), and the resulting supernatants fornalinized isolates were then tested by tube agglutination were used as soluble antigens. For immunoelectrophoresis, 20 pl of antigen was added to each well of a 1% using the 12 original antisera. Almost twice as many strains could be grouped by using the WC agarose gel in 0.05 M barbital buffer (pH 8.0), and current was applied for 60 min at 25 mV/cm. The anti- antigen preparation with anti-WC serum than O sera were then added to the trough, and immunoby using the 0 antigen with anti-O or anti-WC diffusion was carried out at room temperature over- serum (Table 3). Interestingly, cross-reactions night. were observed between A. hydrophila and A. Passive hemagglutination test. The passive sobria strains. In fact, 17 of 67 A. sobria isolates hemagglutination test was performed as described by reacted with one of the anti-A. hydrophila sera. Mittal and Ingram (10). A total of 57 strain of Aero- We also determined whether slide agglutination monas were selected randomly and grown in 4 ml of brain heart infusion broth for 24 h at room tempera- could be used as an aid for preliminary screening ture. Cell sediments obtained after centrifugation were when using an anti-WC serum. Among strains suspended in 2 ml of 0.85% saline. After boiling for 1 positive by slide agglutination, some were negah in a water bath, the tubes were centrifuged at 3,000 tive in tube testing; however, all the isolates x g for 10 min, and the supernatant was used as the negative by slide testing were also negative in heat-stable soluble extract. A volume of 0.2 ml of tube tests. packed sheep erythrocytes was mixed with 2 ml of Figure 1 shows the typical immunoelectrophoheat extract and incubated at 370C for 1 h. The sheep resis pattern obtained with three reference erythrocytes were washed three times in 0.85% saline strains against their homologous anti-O sera; no and suspended to 0.5%. The rabbit antisera were heat inactivated at 560C for 30 min and absorbed with 10% reaction was observed with heterologous serum. sheep erythrocytes at room temperature for 1 h. Serial Furthermore, four strains in each group, based twofold dilutions of the sera, starting at 1:100, were on tube agglutination, produced a pattern simimade in 0.85% saline in agglutination tubes. The same lar to that of the typical strain in immunoelecvolume of sensitized sheep erythrocyte suspension was trophoresis. TABLE 1. Tube agglutination using antiserum prepared against boiled cells and homologous strains as antigen Titera of anti-O serum from strain:
Antigen
LL1 TF2 75 Ba8 9 3 64
Sab-20 Po-238 Po-218 Po-208 ATCC
75 -
Ba8
9
3
64
Sab-20
Po-238
Po-218
Po-208
ATCC
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1,280
-
-
-
1,280
-
-
1,280
-
LL1
TF2
10,240 -
640
-
-
1,280
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
640
-
-
-
-
-
-
-
-
10,240
-
-
-
-
-
-
-
-
1,2805,120
10,240-
-
-
-
1,280 a Reciprocal titer of the last dilution showing a positive agglutination. -, No reaction, or reaction at a titer lower than 1:20. -
-
-
-
-
-
-
-
-
-
-
58
APPL. ENVIRON. MICROBIOL.
LEBLANC ET AL.
We have previously reported that the virulence of A. hydrophila strains varies from strain to strain (8, 11). Table 4 summarizes the virulence of tested strains in relation to their serogroup. Strains reacting with the anti-O serum from strain LL1 were virulent, and strains reacting with strain TF2 anti-O serum were weakly virulent for fish, whereas the typable strains or strains reacting with the anti-O sera other than anti-LL1 and anti-TF2 were nonvirulent for fish. Furthermore, strains reacting with the LL1 antiWC or the TF2 anti-WC sera, but not with their corresponding anti-O sera, were not virulent for fish. Heat-stable soluble extracts of different strains were used for indirect hemmagglutination against the 12 different anti-O sera. It appeared that strains belonging to one 0 group possessed more than one heat-stable soluble determinant. In addition, it also appeared that strains which did not belong to a particular 0 group could possess some determinants present'
in the representative strains of the group (Table
5). DISCUSSION The data presented in this paper suggest that not all motile Aeromonas strains are homogeneous and that they can be separated serologically into various groups. We found that motile Aeromonas strains contained at least three types of antigens: heat-labile surface antigens and two types of heat-stable antigens. The heat-labile surface antigens could be identified when using formalinized whole cells as the antigen and anti-WC sera. A total of 76 strains are now classified in 12 different groups based on their heat-labile antigens. De Meuron and Peduzzi (4) reported that two of seven strains of A. hydrophila possessed thennolabile K antigens which partly inhibit the 0 reaction in tube but not in slide agglutination testing. However, our results suggest that the presence of thermolabile antigens on the cell surface does not
TABLE 2. Tube agglutination using WC antiserum and homologous strains as antigen Titera of anti-WC serum from strain: Antigen
LL1 TF2 75 Ba8 9 3 64
Sab-20 Po-238 Po-218 Po-208 ATCC
LL1 320
TF2
75
Ba8
9
3
64
Sab-20
Po-238
Po-218
Po-208
-
-
-
-
-
-
-
-
-
-
-
-
5,120
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
320
-
-
-
-
-
-
-
-
-
320
-
-
-
-
-
-
320 160
ATCC
-
-
-
-
-
10,240
-
-
-
-
-
-
-
-
-
-
-
-
1,280
-
-
-
-
-
-
-
-
-
-
-
-
2,560
-
-
-
-
-
-
5,120
-
-
-
-
-
-
-
-
-
-
-
-
640
-
-
-
-
-
-
-
-
-
-
320
-
-
-
-
-
-
-
-
-
-
-
-
-
1,280
a Reciprocal titer of the last dilution showing a positive agglutination. -, No reaction, or reaction at a titer lower than 1:20.
TABLE 3. Serogrouping of 195 Aeromonas strains by using different antiserum preparations No. of strains reactive' with antiserum: A. hydrophila serotype: A. sobria Antigen prepn Antigen prepn TF2 75 LL1 strain 9 Ba8
~~~~~~~~strain
Boiled cells A. hydrophila A. sobria
O WC
O WC
O
WC
O
WC
O
WC
9 1
10 4
2 1
2 1
1 0
1 0
1 3
1 3
9 1
10 4
Total no.
of strams stran ~~~~~~~~~~~~~~~~~of No. of Other strainsb
7 0
grouped wihatserum:
O
WC
30 9
40 9
nontypable
strains 98 58
WC A. hydrophila 9 10 10 15 2 13 1 10 1 1 7 30 56 72 A. sobria 1 3 4 7 1 3 0 4 3 3 0 9 20 47 a Number of strains which gave a titer higher than 1:40. b Seven other strains were used to prepare the anti-O and anti-WC sera. These sera reacted only against their homologous strains.
MOTILE AEROMONAS SPECIES FROM FISH
VOL. 42, 1981
block the detection of the 0 antigen. In fact, the formalinizd WC preparations of 39 strains reacted with the anti-O sera as well as with the anti-WC sera. On one hand, we found that 48% of the strains serotyped by the heat-labile antigens were negative with the corresponding anti0 serum. On the other hand, all of the strains showing a positive response to an anti-O serum were also positive with the corresponding antiWC serum. Based on these observations it is suggested that the same heat-labile antigen could be associated with different somatic heatstable 0 antigens; however, this does not exclude the possibility that one strain may possess more than one heat-stable or heat-labile antigen. The feasibility of slide agglutination was also evaluated. It appeared that strains showing no reaction in the slide agglutination test can be considered as nontypable; strains showing a pos-
59
itive reaction should be further tested by tube agglutination for final serogrouping since some strains that were positive by the slide test were negative in the tube test. From a practical point of view, it is suggested that slide agglutination, using anti-WC serum, should be used for a rapid and preliminary screening. With the positive strains, tube agglutination should then be done using a formnlinized WC preparation as the antigen and anti-WC and anti-O sera. However, if anti-O serum is not available the serotyping could be done using two antigen preparations (WC and boiled cells) and anti-WC serum. In addition to the heat-stable antigen which still associated with the cells after boiling, other heat-stable determinants could be dissociated from the cells after boiling. These antigens could be detected by indirect hemagglutination test. It appeared that one strain possessed more than TABLE 5. Serogrouping of strains, based on their soluble heat-stable antigens, by indirect hemagglutination
LL-I
Antigenb
0 group' TF-2I
FIG. 1. Immunoelectrophoresis patterns obtained with the representative strains. Cells were boiled for 1.5 h, sonicated, and centrifuged. The resulting supernatants were used as antigens. A 20-ILI volume of antigen was added to wells of 1% agarose gel in 0.05 M barbital buffer (pH 8.0), and current was applied for 60 min at 25 mV/cm. Anti-0 sera were then added to the trough, and immunodiffusion was carried out at room temperature overnight.
TABLE 4. Relation between
LL1 NT NT TF2
LL1; P01; L4
NT TF2 TF2 NT 75
Sab-7; 143
aAs
Antisera producing tination hemagglu-
146
P-79-149 TF2; 66; 73 80
Ba5 P-79-41
LL1; 75; p-79-3c; p-79-64c LL1; 75; P-79-3; P-79-64 LL1; P-79-3; Ba8 TF2; Sab-20; P-79-3; SauIc TF2; Sab-20; P-79-3; SauI TF2; P-79-3; SauI TF2; Sab-20; SauI
Sab-20; Saul
75 determined by tube agglutination. NT, Not 75
typable. b Erythrocytes coated with heat-stable soluble antigen of the indicated strain(s). c Antisera were also produced against these strains in addition to the 12 standard strains.
irnmunoelectrophoresis pattern (based on 0 antigen), serogrouping by tube agglutination, and virulence for rainbow trout A. sobria
A. hydrophila
Serogroupa
OLL1WCLL1 O?WCLL1 OTF2WCTF2
0?WCTF2
No. of strains
IEb
9 1 10 5
3/3 0/1 7/7
0/2 1/1
Virulence in fish'
1045 (7) 107 (1) 105-5 (5) 107 (2) 107 (1)
No. of strains 1
Virulence in
IEb 0/1
fishc 107 (1)
2
_d
4 3
1/1
107 (2)
0/1 0/1 0/2
-
1 2 075WC75 2 11 0/5 O?WC75 107 (5) 54 107 (4) 90 Others 0 = OXWCS Strains reacting with sera raised against the and WC preparations of the indicated strains. O?WCX = Strains reacting only with the indicated anti-WC serum. b Number of strains with the immunoelectrophoretic (IE) pattern of the group/number of strains tested. 'The 50% lethal dose (number of organisms) of the strains; parentheses indicate number of strains tested. d -, Not done. a
60
LEBLANC ET AL.
one heat-stable soluble antigen, since by indirect hemagglutination one preparation reacted with various antisera. Of 57 isolates tested by this method, 14 could be grouped into seven groups, but at this time the relationship between the indirect hemagglutination and tube agglutination results is unclear. In fact, strains of a particular 0 group as determined by tube agglutination belonged to different indirect hemagglutination groups, and some strains which were nontypable by tube agglutination were classified in the same indirect hemagglutination group. An interesting relationship seems to exist between the serogroup and the virulence of motile Aeromonas strains for fish. All of the virulent Aeromonas strains tested belonged to one particular 0 serogroup (LL1). Less virulent strains belonged to another serogroup (TF2), and the nonvirulent strains belonged to different groups. It appears that this relationship is associated with heat-stable somatic antigens rather than with heat-labile antigens. We are also aware that strains of other serogroups could be found virulent for fish. For this reason, it is suggested that preliminary differentiation between virulence and nonvirulence should be based on some other cell surface characteristics, as we have already reported, such as the fact that only virulent strains precipitate after boiling and are resistant to the bactericidal effect of normal serum, whereas nonvirulent strains are not (11). ACKNOWLEDGMENTS We gratefully acknowledge the financial support of Le Conseil des Recherches et Services Agricoles du Quebec, grant 77-664, and the support of Ministere de l'Education, Gouvernement du Quebec, grant EQ-1164. G.O. held a studentship under the former grant. It is a pleasure to acknowledge the valuable assistance provided by Francine Bernard.
LITERATURE CITED 1. Boulanger, Y., R. Lallier, and G. Cousineau. 1977. Isolation of enterotoxigenic Aeromonas from fish. Can. J. Microbiol. 23:1161-1164.
APPL. ENVIRON. MICROBIOL. 2. Davis, W. A., J. G. Kane, and V. F. Garagusi. 1978.
Human Aeromonas infection. Medicine 57:267-277. 3. De Figuredo, J., and J. A. Plumb. 1977. Virulence of different isolates of Aeromonas hydrophila in channel catfish. Aquaculture 11:349-354. 4. De Meuron, P.-A., and R. Peduzzi. 1979. Caracterisation de souches du genre Aeromonas isolees chez des poissons d'eau douce et quelques reptiles. Zentralbl. Veterinaermed. Reihe B 26:153-167. 5. Erwing, W., R. Hugh, and J. Johnson. 1961. Studies of the Aeromonas group. U.S. Dept. of Health, Education and Welfare, Center for Disease Control, Atlanta. 6. Fliermans, C. B., and T. C. Hazen. 1980. Immunoflorescence of Aeromonas hydrophila as measured by fluorescence photometric microscopy. Can. J. Microbiol. 26:161-168. 7. Hazen, T. C., C. B. Fliermans, R. P. Hirsch, and G. W. Esch. 1978. Prevalence and distribution of Aeromonas hydrophila in the United States. Appl. Environ. Microbiol. 33:114-122. 8. Lallier. R., Y. Boulanger, and G. Olivier. 1980. Difference in virulence of Aeromonas hydrophila and Aeromonas sobria in rainbow trout. Prog. Fish Culturist 42: 199-200. 9. MaclInnes, J. I., T. J. Trust, and J. H. Crosa. 1979. Deoxyribonucleic acid relationships among members of the genus Aeromonas. Can. J. Microbiol. 25:579-586. 10. Mittal, K. R., and D. G. Ingram. 1975. Factors involved in bactericidal activity of sheep serum. Am. J. Vet. Res.
36:1183-1187. 11. Mittal, K. R., G. Lalonde, D. Leblanc, G. Olivier, and R. Lallier. 1980. Aeromonas hydrophila in rainbow trout: relation between virulence and surface characteristics. Can. J. Microbiol. 26:1501-1503. 12. Paterson, W. D., D. Douez, and D. Desautels. 1980. Relationships between selected strains of typical and atypical Aeromonas salmonicida, Aeromonas hydrophila and Haemophiluspiscium. Can. J. Microbiol. 26: 588-598. 13. Popoff, M., and M. Veron. 1976. A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group. J. Gen. Microbiol. 94:11-12. 14. Schubert, R. H. W. 1974. Genus 11. Aeromonas, p. 345348. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore. 15. Shaw, R. H., and H. J. Hodder. 1978. Lipopolysaccharides of motile aeromonads: core oligosaccharide analysis as an aid to taxonomic classification. Can. J. Microbiol. 24:864-868. 16. Trust, T. S., and R. A. H. Sparrow. 1974. The bacterial flora in the alimentary tract of freshwater salmonid fishes. Can. J. Microbiol. 20:1219-1228.
