Vol. 30, No. 9
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1992, p. 2432-2434 0095-1137/92/092432-03$02.00/0
Biochemical and Antigenic Properties of Streptococcus bovis Isolated from Pigeons PETER DE HERDT,1* FREDDY HAESEBROUCK,l LUC A. DEVRIESE,' AND RIK DUCATELLE2 Laboratory of Veterinary Bacteriology' and Department of Poultry Diseases,2 Faculty of Veterinary Medicine, University of Gent, Casinoplein 24, B-9000 Gent, Belgium Received 6 April 1992/Accepted 23 June 1992
Biochemical and serological properties of 60 strains of Streptococcus bovis isolated from healthy pigeons and from pigeons that died from S. bovis septicemia were determined. On the basis of the hemolysis of bovine erythrocytes, the production of polysaccharides on saccharose-containing media, and the fermentation of mannitol, inulin, trehalose, and L-arabinose, the isolates were classified in five biotypes and two subbiotypes. Slide agglutination and microagglutination tests using monospecific rabbit antisera allowed the classification of all isolates in five serotypes.
Streptococcus bovis is generally regarded as a harmless inhabitant of the gastrointestinal tract in numerous animal species. Recently, however, this bacterium has been described as an important cause of septicemia in pigeons (4, 5). The main symptoms include inability to fly, lameness, inappetentia, emaciation, polyuria, and production of green, slimy droppings, and it may result in sudden death in pigeons of all ages. Unpublished observations in our laboratories showed that a large number of healthy pigeons are carriers of S. bovis in the gastrointestinal tract. It was the aim of the present study to investigate whether strains isolated from carriers have the same biochemical and antigenic characteristics as strains obtained from the organs of pigeons that died from S. bovis septicemia. Furthermore, in this article a proposal for biochemical and serological classification is given.
MATERIALS AND METHODS Strains. Sixty strains of S. bovis were used in this study. Twenty-three of these isolates were obtained from organ lesions of pigeons that died from S. bovis septicemia. The other strains were isolated from crop or cloaca samples of healthy pigeons, all from different lofts. All strains were isolated on Slanetz and Bartley agar (Oxoid, Basingstoke, England) and identified according to the method of Devriese et al. (5). The strains were stored at -70°C until used in the biochemical and serological tests. All media used in this study were incubated at 37°C in an atmosphere that was enriched by 5% CO2. Biochemical tests. For biochemical tests, strains were grown overnight on Columbia agar (GIBCO, Paisley, Scotland) with 5% bovine blood. Acid production from L-arabinose, inulin, mannitol, lactose, raffinose, ribose, sorbitol, and xylose was examined in phenol red (Merck, Darmstadt, Germany). Hemolysis was observed on Columbia agar with 5% bovine blood. The production of polysaccharide was determined by the growth of slimy colonies on 5% saccharose-supplemented Columbia agar plates. Results were evaluated after 24, 48, and 72 h of incubation. Immune sera. Antisera to 12 strains were prepared by *
Corresponding author.
immunizing rabbits with 6-h cultures prepared in ToddHewitt broth (Oxoid) with 5% horse serum (GIBCO). The bacteria were washed once in phosphate-buffered saline solution (PBS) and resuspended in PBS containing 0.5% formaldehyde. After 24 h, the bacteria were washed with PBS and adjusted to a concentration of approximately 1010 bacteria per ml. Three milliliters of a bacterin, consisting of an equal volume of the bacterial suspension and Freund complete adjuvant (Difco, Detroit, Mich.), was injected into the footpads and subcutaneously at four sites as described by Nicolet (10). Three weeks later, four intravenous injections were performed at 3-day intervals with 0.3, 0.5, 0.7, and 0.9 ml, respectively, of a suspension containing 109 inactivated bacteria per ml. The sera were collected 1 week after the last injection, inactivated at 56°C for 20 min, and stored at -20°C. Serological tests. For serological tests, strains were cultivated at 37°C for 6 h in Todd-Hewitt broth with 5% horse serum. Cultures were harvested, washed once in PBS, and resuspended in PBS containing 0.5% formaldehyde. After 24 h, the bacteria were washed with PBS and adjusted to a concentration of approximately 1010 bacteria per ml. Antigenic properties of the strains were studied by slide agglutination and microagglutination tests. In the slide agglutination tests, 50 IlI of the bacterial suspensions and 50 ptl of 10-fold-diluted rabbit antisera were mixed on a microscope slide. The slide was rocked and tilted slowly. Reactions which became visible within 2 min were recorded as positive. Microagglutination tests were performed in 96-well U-microtiter plates (Greiner Labortechnik, Nurtingen, Germany). The optimal antigen concentration was determined by making twofold dilutions of the bacterial suspensions with 50-,Il droppers and diluters. The highest antigen dilution in which a clearly visible button was formed was used in the agglutination tests. In these tests, twofold serial dilutions were made from 1/40-prediluted sera with 50-pl multichannel pipettes. To each serum dilution, 50 pul of the optimal antigen dilution was added. Antigen controls (50 pl of PBS with 50 ,ul of antigen) were included in each test. After shaking, the plates were incubated for 24 h at 37°C. Agglutination titers were expressed as the reciprocal of the highest serum dilution that showed agglutination. Adsorption tests. Microagglutination tests and slide agglutination tests were also performed with antisera adsorbed 2432
VOL. 30, 1992
S.
Biotype
Fermentation of: Mannitol Inulin Trehalose L-Arabinose
Polysaccharide productiona Alpha-hemolysis
2b
3
4
5
-
-
+ + -
+
+ + +
+
-
+ +
-
+
+
-
-
-
+ -
1
2a
+
+ -
Vb (+) -
+
2433
TABLE 2. Cross agglutination tests with reference strains of pigeon S. bovis serotypes and reference hyperimmune sera
TABLE 1. Biochemical classification of S. bovis from pigeons Biochemical character
BOVIS IN PIGEONS
+ +
a On saccharose-containing media.
bVariable.
with heterologous and homologous serotypes. Suspensions containing 5 x 10'° formaldehyde-inactivated bacteria were centrifuged at 12,000 x g for 2 min. The supernatant was discarded, and the pellet was used for the adsorption of the antisera. For heterologous adsorption, pellets of bacteria belonging to the heterologous serotypes were suspended in 2 ml of 1/40-diluted antiserum. For homologous adsorption, 2 ml of the 1/40-diluted antisera was adsorbed to pellets of bacteria belonging to the homologous serotype. The suspensions were incubated for 5 h at 37°C and shaken every hour. The adsorbed antisera were obtained after centrifugation at 12,000 x g for 2 min. Statistical analysis. Student's t test (6) was used to determine the significance of differences in prevalence of the S. bovis serotype-biotype groups isolated from organs of pigeons that died from septicemia versus those obtained from the intestinal tract of healthy pigeons. A significance level of 0.05 was used. RESULTS Biochemical tests. Lactose and raffinose were fermented by all strains of S. bovis. None of the isolates produced acid from ribose, sorbitol, or xylose. Reaction patterns obtained with the other carbohydrates, the presence or absence of hemolysis of bovine erythrocytes, and the production of polysaccharides on saccharose-containing media allowed classification of S. bovis in five biotypes (Table 1). In about 95% of the biotype 1 strains, slime formation on saccharosecontaining media was evident. In the remaining 5%, polysaccharide production was very slight or absent. Biotype 2 strains were divided in two subbiotypes, based on their ability to produce acid from trehalose. Serological tests. The slide agglutination and microagglutination tests allowed us to classify all isolates in five serotypes. Strains STR 357, STR 329, STR 598, STR 675, and STR 669 were used as reference strains for the serotypes 1, 2, 3, 4, and 5, respectively. Sera prepared against these strains were used as reference sera. Titers obtained with reference strains and nonadsorbed reference sera in microagglutination tests are given in Table 2. After adsorption with heterologous serotypes, titers against the homologous serotype remained unchanged or were 1 dilution lower whereas titers against heterologous serotypes were lower than 40. After adsorption with the homologous serotypes, titers against all serotypes were lower than 40. The slide agglutination test with nonadsorbed sera gave clear results for 53 of the 60 isolates. Seven isolates reacted with more than one nonadsorbed reference serum sample. Slide agglutination and microagglutination tests with ad-
Antiserum titer' against serotype (type strain"):
Antigen
serotype 1 (STR 357) 2 (STR 329) 3 (STR 598) 4 (STR 675) 5 (STR 669) 1 2 3 4 5
1,280 80
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1992, p. 2432-2434 0095-1137/92/092432-03$02.00/0
Biochemical and Antigenic Properties of Streptococcus bovis Isolated from Pigeons PETER DE HERDT,1* FREDDY HAESEBROUCK,l LUC A. DEVRIESE,' AND RIK DUCATELLE2 Laboratory of Veterinary Bacteriology' and Department of Poultry Diseases,2 Faculty of Veterinary Medicine, University of Gent, Casinoplein 24, B-9000 Gent, Belgium Received 6 April 1992/Accepted 23 June 1992
Biochemical and serological properties of 60 strains of Streptococcus bovis isolated from healthy pigeons and from pigeons that died from S. bovis septicemia were determined. On the basis of the hemolysis of bovine erythrocytes, the production of polysaccharides on saccharose-containing media, and the fermentation of mannitol, inulin, trehalose, and L-arabinose, the isolates were classified in five biotypes and two subbiotypes. Slide agglutination and microagglutination tests using monospecific rabbit antisera allowed the classification of all isolates in five serotypes.
Streptococcus bovis is generally regarded as a harmless inhabitant of the gastrointestinal tract in numerous animal species. Recently, however, this bacterium has been described as an important cause of septicemia in pigeons (4, 5). The main symptoms include inability to fly, lameness, inappetentia, emaciation, polyuria, and production of green, slimy droppings, and it may result in sudden death in pigeons of all ages. Unpublished observations in our laboratories showed that a large number of healthy pigeons are carriers of S. bovis in the gastrointestinal tract. It was the aim of the present study to investigate whether strains isolated from carriers have the same biochemical and antigenic characteristics as strains obtained from the organs of pigeons that died from S. bovis septicemia. Furthermore, in this article a proposal for biochemical and serological classification is given.
MATERIALS AND METHODS Strains. Sixty strains of S. bovis were used in this study. Twenty-three of these isolates were obtained from organ lesions of pigeons that died from S. bovis septicemia. The other strains were isolated from crop or cloaca samples of healthy pigeons, all from different lofts. All strains were isolated on Slanetz and Bartley agar (Oxoid, Basingstoke, England) and identified according to the method of Devriese et al. (5). The strains were stored at -70°C until used in the biochemical and serological tests. All media used in this study were incubated at 37°C in an atmosphere that was enriched by 5% CO2. Biochemical tests. For biochemical tests, strains were grown overnight on Columbia agar (GIBCO, Paisley, Scotland) with 5% bovine blood. Acid production from L-arabinose, inulin, mannitol, lactose, raffinose, ribose, sorbitol, and xylose was examined in phenol red (Merck, Darmstadt, Germany). Hemolysis was observed on Columbia agar with 5% bovine blood. The production of polysaccharide was determined by the growth of slimy colonies on 5% saccharose-supplemented Columbia agar plates. Results were evaluated after 24, 48, and 72 h of incubation. Immune sera. Antisera to 12 strains were prepared by *
Corresponding author.
immunizing rabbits with 6-h cultures prepared in ToddHewitt broth (Oxoid) with 5% horse serum (GIBCO). The bacteria were washed once in phosphate-buffered saline solution (PBS) and resuspended in PBS containing 0.5% formaldehyde. After 24 h, the bacteria were washed with PBS and adjusted to a concentration of approximately 1010 bacteria per ml. Three milliliters of a bacterin, consisting of an equal volume of the bacterial suspension and Freund complete adjuvant (Difco, Detroit, Mich.), was injected into the footpads and subcutaneously at four sites as described by Nicolet (10). Three weeks later, four intravenous injections were performed at 3-day intervals with 0.3, 0.5, 0.7, and 0.9 ml, respectively, of a suspension containing 109 inactivated bacteria per ml. The sera were collected 1 week after the last injection, inactivated at 56°C for 20 min, and stored at -20°C. Serological tests. For serological tests, strains were cultivated at 37°C for 6 h in Todd-Hewitt broth with 5% horse serum. Cultures were harvested, washed once in PBS, and resuspended in PBS containing 0.5% formaldehyde. After 24 h, the bacteria were washed with PBS and adjusted to a concentration of approximately 1010 bacteria per ml. Antigenic properties of the strains were studied by slide agglutination and microagglutination tests. In the slide agglutination tests, 50 IlI of the bacterial suspensions and 50 ptl of 10-fold-diluted rabbit antisera were mixed on a microscope slide. The slide was rocked and tilted slowly. Reactions which became visible within 2 min were recorded as positive. Microagglutination tests were performed in 96-well U-microtiter plates (Greiner Labortechnik, Nurtingen, Germany). The optimal antigen concentration was determined by making twofold dilutions of the bacterial suspensions with 50-,Il droppers and diluters. The highest antigen dilution in which a clearly visible button was formed was used in the agglutination tests. In these tests, twofold serial dilutions were made from 1/40-prediluted sera with 50-pl multichannel pipettes. To each serum dilution, 50 pul of the optimal antigen dilution was added. Antigen controls (50 pl of PBS with 50 ,ul of antigen) were included in each test. After shaking, the plates were incubated for 24 h at 37°C. Agglutination titers were expressed as the reciprocal of the highest serum dilution that showed agglutination. Adsorption tests. Microagglutination tests and slide agglutination tests were also performed with antisera adsorbed 2432
VOL. 30, 1992
S.
Biotype
Fermentation of: Mannitol Inulin Trehalose L-Arabinose
Polysaccharide productiona Alpha-hemolysis
2b
3
4
5
-
-
+ + -
+
+ + +
+
-
+ +
-
+
+
-
-
-
+ -
1
2a
+
+ -
Vb (+) -
+
2433
TABLE 2. Cross agglutination tests with reference strains of pigeon S. bovis serotypes and reference hyperimmune sera
TABLE 1. Biochemical classification of S. bovis from pigeons Biochemical character
BOVIS IN PIGEONS
+ +
a On saccharose-containing media.
bVariable.
with heterologous and homologous serotypes. Suspensions containing 5 x 10'° formaldehyde-inactivated bacteria were centrifuged at 12,000 x g for 2 min. The supernatant was discarded, and the pellet was used for the adsorption of the antisera. For heterologous adsorption, pellets of bacteria belonging to the heterologous serotypes were suspended in 2 ml of 1/40-diluted antiserum. For homologous adsorption, 2 ml of the 1/40-diluted antisera was adsorbed to pellets of bacteria belonging to the homologous serotype. The suspensions were incubated for 5 h at 37°C and shaken every hour. The adsorbed antisera were obtained after centrifugation at 12,000 x g for 2 min. Statistical analysis. Student's t test (6) was used to determine the significance of differences in prevalence of the S. bovis serotype-biotype groups isolated from organs of pigeons that died from septicemia versus those obtained from the intestinal tract of healthy pigeons. A significance level of 0.05 was used. RESULTS Biochemical tests. Lactose and raffinose were fermented by all strains of S. bovis. None of the isolates produced acid from ribose, sorbitol, or xylose. Reaction patterns obtained with the other carbohydrates, the presence or absence of hemolysis of bovine erythrocytes, and the production of polysaccharides on saccharose-containing media allowed classification of S. bovis in five biotypes (Table 1). In about 95% of the biotype 1 strains, slime formation on saccharosecontaining media was evident. In the remaining 5%, polysaccharide production was very slight or absent. Biotype 2 strains were divided in two subbiotypes, based on their ability to produce acid from trehalose. Serological tests. The slide agglutination and microagglutination tests allowed us to classify all isolates in five serotypes. Strains STR 357, STR 329, STR 598, STR 675, and STR 669 were used as reference strains for the serotypes 1, 2, 3, 4, and 5, respectively. Sera prepared against these strains were used as reference sera. Titers obtained with reference strains and nonadsorbed reference sera in microagglutination tests are given in Table 2. After adsorption with heterologous serotypes, titers against the homologous serotype remained unchanged or were 1 dilution lower whereas titers against heterologous serotypes were lower than 40. After adsorption with the homologous serotypes, titers against all serotypes were lower than 40. The slide agglutination test with nonadsorbed sera gave clear results for 53 of the 60 isolates. Seven isolates reacted with more than one nonadsorbed reference serum sample. Slide agglutination and microagglutination tests with ad-
Antiserum titer' against serotype (type strain"):
Antigen
serotype 1 (STR 357) 2 (STR 329) 3 (STR 598) 4 (STR 675) 5 (STR 669) 1 2 3 4 5
1,280 80
