The detection of two antigenic groups among Renibacterium salmoninarum isolates

FEMSMicrobiologyLetters94 (1992) 105-110 © 1992Federationof EuropeanMicrobiologicalSocieties0378-1097/92/$05.00 Publishedby Elsevier

FEMSLE04933

The detection of two antigenic groups among Renibacterium samoninarum isolates Isabel Bandin, Ysabel Santos, Beatriz Magariflos, Juan L. Barja and Aiicia E. Toranzo Departamemode Microbiologiay Parasitolog[a, Facuhadde Biologia, Unit'ersidadde Santiago. Santiagode Compostela. Spabt

Received15October1991 Revisionreceived14March 1992 Accepted3 April 1992

Key words: Renibacterium salmoninamm; Protein; Antigenic diversity

1. SUMMARY

2. INTRODUCTION

The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium saimo,inamm revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected.

Bacterial kidney disease (BKD) caused by the Gram-positive bacterium Re, dbacterium salmoninature is considered a major obstacle in the culture of salmonids throughout the world [l]. Because of the slow-growing characteristics of the microorganism, serological methods are preferred over culture for the diagnosis of the diseasc [2-6]. Knowledge of the antigenic composition of7 R. salmoninarum is important for the development of vaccines and for reliable serological methods of detection. Getchelt et al. [7], reporting a heatstable cell surface antigen F common to seven R. salmoninarium isolates, claimed serological homogeneity within the species. However, in the present report, the analysis of the surface proteins and their immunogenic properties conducted in a group of 14 isolates of R. salmoninarum with different geographic origin and

Correspondence to: I. Bandin,Departamentode Microbiologia y Parasito[ogla,Facultad de Biologia,Universidadde Santiago, Santiagode Compostela,15706,Spain.

106 salmonid host indicates the existence of antigenic heterogeneity among the R. salmoninarum strains.

3. MATERIAL AND METHODS 3.1. Bacterial strains Fourteen strains of R, salmoninarum with different sources of isolation were used in this study (Table I). The isolates were confirmed to be R. salmoninarum, since all were non-motile, Grampositive rods that required cysteine for growth and reacted strongly with anti-R, satmoninarum serum in fluorescent antibody staining reactions (DFAT). The bacteria were grown on MuellerHinton agar (Difco) supplemented with 0.1% Lcysteine hydrochloride (Sigma), (MHA-C) at 15°C for 7 days and stored frozen at - 70°C in cysteine Table 1 Originof the isolatestested in this study Isolate number Origin MOC Sah'elinusfontinalis (Canada) 466 Oncorhynchuskitsuch (Canada) R1 Salmonidfish(Canada) K 57 Sabno solar (UK) K 76 Sahz,linusfonthzalis (Canada) K 84 Oncorhynchusmykiss (UK) MT 251 Salmo salar (UK) MT 409 Salrnosalar (UK) MT 417 Sabno salar (UK) MT 426 Salmo salar (UK) MT444 Sahno salar (UK) ATCC33209 Oncorhynchustschawytscha (USA) ATCC33739 Sah'elinusfontmalis (USA) NCMB1113 Sah'elinusJbntinalis

Source a J.G. Duly J.G. Duly R. Lallier B. Austin B. Austin B. Austin D.W. Bruno D.W. Bruno D.W. Bruno D.W. Bruno D.W. Bruno ATCC ATCC NCMB

a J.G. Daly, Departmentof Microbiology,Collegeof Biological Science,Universityof Guelph,Guelph,Ontario,Canada; R. Lallier,Facultd de M~decineVeterinaire,Universit6de Montreal, St. Hyacinthe,Quebec, Canada; B. Austin, Department of Brewingand BiologicalSciences,Heriot-Watt University, Edimburgh, U.K,; D.W. Bruno, SOAFD, Marine Laborary, Aberdeen, U.K., ATCC, American Type Culture Collection,gockville,MD, USA;NCMB,National Collectionof MarineBacteria, Aberdeen,U.K.

supplemented Mueller-Hinton broth (MHB-C) with 15% (v/v) glycerol. 3.2. Preparation of rabbit antiserum The rabbit antiserum against g salmoninarum ATCC 33209 was obtained as previously described [8], using formalin-killed bacteria as antigen injected intravenously. The collected serum was *~tored in aliquots frozen at -30°C until needed. Prior to immunization, rabbits were bled to obtain control serum. 3.3. Agglutination tes:s Slide agglutination tests were conducted according to the procedure of Toranzo et al. [9], suspending the bacteria in 0.2% saline to avoid autoagglutination observed with phosphate buffered saline (PBS). Quantitative agglutination tests were performed by using mierotitre plates with serial twofold dilutions of 50-/xl aliquots of the antiserum. The titre was recorded as the reciprocal of the highest dilution of the antiserum which gave positive agglutination after incubation overnight at 15°C. In all the assays bacterial suspensions in saline were used as controls. 3.4. Surface protein extraction Cell surface extracts from R. salmoninarum were prepared following the procedure of Crosa and Hodges [10]. Briefly, bacteria grown on MHA-C were scraped off the plates with saline solution (0.85% NaCI) and centrifuged at 7000 x g for 10 min (4°C). The cellular pellets were resuspended in 3 ml of 10 mM Tris[hydroxymethyl]amino-methane (Tris), pH 8 + 0.3% NaCI and disrupted by sonic treatment (Branson sonifier 250). After centrifugation at 10000 × g for 1 rain, the supernatant fluids were transferred to another tube and centrifuged again for 60 min at 20000 x g (4°C). The resultant pellets were dissolved in distilled water. These suspensions were frozen at -30°C until used. 3.5. EIectrophoresis Prior to electrophoresis the samples were boiled for 5 rain in buffer containing 65 mM

107 Tris-HCI (pH 6.8) 2% SDS, 10% (v/v) glycerol, 0.001% bromophenoi blue and 5% 2-mercaptoethanol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out overnight at constant current (20 mA) using 12.5% aerylamide in the separating gel and 3% in the stacking gel. The proteins were stained with Coomassie brilliant blue R-250 (Sigma) and the molecular masses determined by comparison with a mixture of molecules mass markers (Bio-Rad).

3.6. Western blotting After electrophoresis, proteins were electroblotted from the gel onto nitrocellulose paper. Transfer buffer consisted of 25 mM Tris, 192 mM glycine (pH 8.3) and 20% methanol. After transfer, cellulose membranes were blocked for 1 h with 3% gelatin in Tris buffered saline (TBS), pH 7.5, before immunostaining. Gelatin-blocked membranes were washed in TB5 plus 0.05% Tween 20 (TTBS) and then incubated for 1 h in control or immune rabbit serum diluted 1:1000 in TBS containing 1% gelatin After further washing in TI'BS, the membranes were incubated for 1 h with goat anti-rabbit lgG-alkaline phosphatase conjugate (Bio-Rad) diluted 1 : 3000. Bands were visualized by incubating membranes in 0.1 M carbonate buffer (pH 9.8) containing tetrazolium blue (0.3 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (0.15 mg/ml). Finally, the blots were rinsed in distilled water for approximately 3 min and air-dried.

4. RESULTS

4.1. Serological relationships among R. salmoninature isolates Slide agglutination tests using the antiserum raised against R. salmoninamm ATCC 33209 revealed that all the isolates analyzed shared antigens with the type strain. However, the quantitative agglutination assays performed to determine possible serological differences among the isolates indicated the existence of two separated groups (Table 2). The biggest group included 11

isolates with a high level of cross-agglutination with the reference strain (titres ranging from 1280 to 5120) the remaining three isolates displayed a weak reaction with the antisera assayed (titres < 10). Autoaggtutination was not observed in all cases.

4.2. SDS-PA GE profik.s Surface proteins obtained from 14 strains of R. saimoninan~m were subjected to SDS-PAGE electrophoresis. As can be observed (Fig. la), the protein profiles of 11 strains with different geographic origin and salmonid host were very similar, all showing a major common component of 57 kDa (Fig la, lanes D-N). However, three isolates K57, K76 and R1, showed a different pattern with a major protein that migrates to a position corresponding to a molecular weight of 30 kDa (Fig. la, lanes A, B and C). Other proteins of lower and higher molecular mass than the major component were also evident in all the samples. 4.3. Western blotting Protein preparations from Renibacterium isolates fraetionated by SDS-PAGE were tested by Western blotting with anti-R, salmoninantm ATCC 33209 and K57 rabbit sera.

Table 2 Agglutinationtitres of the R salmoninarumstrains Antigen Moc 466 R1 K57 K 76 K 84 MT 251 MT409 MT 417 MT 426 MT 444 ATCC33209 ATCC33739 NCMB i113

Antisera ATCC33209 2560 a 2560 <10 < 10 < 10 5120 2560 i 280 1280 1280 2560 5120 5120 256O

"~Numbers indicate the reciprocal of the highest dilution of the antiserumwhichgavepositiveagglutination

108

Similar to that observation in the SDS-PAGE, two different patterns were observed. When the immunoblot was performed using the antiserum produced against whole cells of R. salmoninarum ATCC 33209, numerous immunoreactive bands, including the 57 kDa protein, were detected

within the largest group obtained by electrophoresis (Fig. lb, lanes D-N) and only two components among the samples belonging to the minor group showed a positive reaction (Fig. lb, lanes A, B and C). One of these bands has a molecular size similar to the 57 kDa protein,

A BCDEFGHI J KLMN

92 57

64 45

30 24

A BODE F 6HI J KLBN

C ABC

DE

FGHI

J KLIRH

b 57

57

Fig. i, a. SDS-PAGE of membrane proteins from various R. salmoninan~m strains, b. Western blot of membrane protein preparations from R. salmoninarum isolates tested with rabbit serum anti-R, salmoninamm ATCC 33209. e. Western blot of membrane protein preparations from R. salmoninarum isolates tested with rabbit serum anti-R, salmoninarum K 57. Lanes: St, molecular mass standard; A, K 57; B, K 76; C, R 1; D, MT 251; E, MT 409; F, MT 417; G, MT 426; H, MT 444; !, K 84; J, 466; K, MOC; L, NCMB !113; M, ATCC 33209; N, ATCC 33739. Numbers indicate the molecular size in kilodalton.

1119

although we did not detect that component in the Coomassie blue stained gels. When the SDS-PAGE separated surface proteins were immunostained with anti-K57 serum a strong reaction was detected against compgnents from the minor group and only a weak reaction was observed against the major protein antigen (57 kDa) from the largest group (Fig. lc). No reaction was observed when the control (non-immune serum) was utilized.

5. DISCUSSION R. salmoninan+m isolates have been reported to be relatively homogeneous in a number of characteristics such as cell wall composition [11], cell surface hydrophobicity [12-14] and low extracelhdar enzymatic production [15]. In addition, they were described to be closely related serologieai!y [7,16-18]. How,:ver, although there are conflicting reports concerning the biochemical properties of R. salmoninarum [19,20], diversity has been described in this species [19]. In the present study we demonstrate the existence of antigenic heterogeneity among strains of R. salmonbtamm with different geographic origin. Slide agglutination tests showed that all R. sabnoninamm isolates reacted positively with the antiserum raised against the reference strain of this species. However, after titering the antisera a serological heterogeneity was evident with two distinct groups being detected (Table 2). In addition, the SDS-PAGE carried out with surface proteins preparations obtained from R. salmoninamm strains with wide geographical distibution and different salmonid host revealed the presence of two different patterns. The largest group with a major component of 57 kDa, possibly the antigen F described by Getehell et al. [7], included the type strain ATCC 33209 and another ten isolates from USA, Canada and UK. This analysis is in agreement with the differences observed in the quantitative agglutination tests. Furthermore, antigenic analysis performed by Western blotting indicates that only strains that shared the same protein pattern to R. salmoninamm ATCC 33209 showed a strong reaction

with the rabbit serum raised against whole cells of this strain. The three isolates that displayed a different protein profile in the SDS Coomassie stained gelr, reacted weakly in the immunoblot, with only two bands being detected weakly. Interestingly, one of the immunoreactive bands was found at a position similar to the 57 kDa antigen obsened in the strains belonging to the largest group. When the surface proteins were immunostained with anti-K57 serum the highest number of immunoreactive bands were detected among the strains that displayed the same protein profile as this isolate, and only a band corresponding to the 57 kDa protein was detected within the largest group. Since the instability of the 57 kDa protein has been previously reported [21], the results of the Western blot assays suggest that the profile observed in the minor group of R. salmonbmmm isolates could be the result of the degradation of the 57 kDa protein. Howe,,er, this protein seems to be so unstable in these particular strains growing 'in vitro', that surface proteins obtained from defrosted bacteria without passages in the laboratory present the constant and distinctive pattern reported in this study. In this patte, n, the native form of the antigen F (protein of 57 kDa) would be such a small amount that is only detectable by Western blot analysis (Fig. lb). An autologous proteinasc associated with the cell surface of some R. saimoninanon strains could be responsible for this degradation as suggested by Rockey et al. [221. The data presented in this work indicate the existence of serological diversity among R. salmonhlarunz. From the epidemiological point of view, it would be of interest to determine the ubiquity of the two antigenic patterns of R. sabnoninan~m, which can only be accomplished by testing a larger number of strains with different geographic origin and source of isolation.

ACKNOWLEDGEMENTS This work was supported in part by Grant PB87+i027 and MAR-91-1133-C02-01 from the

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Comisi6n Interministerial de Ciencia y Tecnologia. (CICYT), Spain. !. Band[n thanks the Ministerio de Educaci6n y Ciencia (MEC) for a Research Fellowship.

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