I "eterinao, Microbiology, 27 ( 1991 ) 397-401 Elsevier Science Publishers B.V., A m s t e r d a m

397

Short Communication Biochemical typing of Actinobacillus

pleuropneumoniae Marc Sirois and Robert Higgins* Ddpartment de Pathologie et Microbiologie, Facult~ de Mddecine V~;tOrinaire, Universitd de Montrdal. Saint-Hyacinthe, QuObec, J2S 7C6 Canada (Accepted 19 November 1990)

ABSTRACT Sirois, M. and Higgins, R., 1991. Biochemical typing ofActinobacillus pleuropneumoniae, l'et. Microbiol., 27: 397-401. A study was conducted to evaluate the possibility of using biochemical differences among strains of a given serotype ofActinobacillus pleuropneumoniae as epidemiological markers, to rapidly identify the source of infection in herds affected with swine pleuropneumonia. Out of 38 different biochemical and physiological tests performed on a total of 67 strains belonging to serotypes 1 and 5 ofA. pleuropneumoniae, three fermentation tests, glycerol, lactose and raffinose, allowed the classification of serotype 1 strains into 6 phenotypic groups and serotype 5 strains into 4 of these groups. Groups II and llI were exclusively composed of serotype 1 strains, whereas the majority of strains in groups I and IV belonged to serotypes 1 and 5 respectively, the latter comprising almost all the serotype 5 studied.

INTRODUCTION

Actinobacillus pleuropneumoniae is presently the most severe respiratory pathogen in swine. The prevalence of swine pleuropneumonia is always very important in intensive swine operations and is responsible for important economic losses in this industry. This causative agent is usually identified with tests such as production of hemolysis on blood agar, positive CAMP reaction, need of N A D (nicotinamide adenine dinucleotide) factor for growth, urea degradation (Biberstein et al., 1977) and serotyping, which allows to distinguish 12 different serotypes (Nielsen, 1986). Serotyping is a useful phenotypic marker for epidemiological studies of A. pleuropneumoniae especially when the serotype of a particular isolate is uncommon. Most of isolates in Canada belong to serotypes 1 and 5, and in many feeder operations, piglets from different sources are mixed together and sus*Author for correspondence.

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ceptible animals arc contaminated by infected animals. 1he purposct el" this work was to determine, by the use of biochemical a n d / o r physiological reactions, whether phenotypic variations were present within each of these two serotypes and to establish a biochemical typing scheme to determine cpidcmiological markers that could be employed by any diagnostic laboratory to help identifying the source of infection in swine herds. MATERIALS A N D METH()I)S

A total of 65 field isolates of,4. pleuropneumoniae were collected at the clinical bacteriology laboratory of the Facult6 de M6decine V6t6rinaire of the Universit6 de Montr6al, from acute cases of swine pleuropneumonia. Among these isolates, 45 belonged to serotype 1 of A. pleuropneumoniae and 20 belonged to serotype 5. In addition, reference strains 4074 and K17 belonging respectively to serotypes 1 and 5 and kindly supplied by Dr K.R. Mittal of the Facult6 de M6decine V6t6rinaire of the Universit6 de Montr6al. were tested concurrently to the field isolates. All 67 strains were submitted to a total of 38 different tests. The need for NAD was tested using a commercial strip of V factor ( BBL, Cockeysville, M D ) placed after the streaking of the strain on a Mueller-Hinton agar plate (Difco, Detroit, MI ). The ability to synthetize porphyrins from delta-aminolevulinic acid was tested according to Kilian (1974). The CAMP reaction was tested on Trypticase Soy agar ( Difco ) with 5% sheep blood, with the use of a streak of a/3-toxigenic Staphylococcus aureus strains (Kilian, 1976b ). Catalase production was tested with a drop of 30% hydrogen peroxide on a glass slide and oxidase activity was determined by using the API (API system, St-Laurent, Qc) reagent and the manufacturer's specifications. Determination of the production of urease,/3-galactosidase, lysine and ornithine decarboxylases, arginine dihydrolase and reduction of nitrates were performed with the specifications of the Difco Manual (tenth edition): production of indole was tested in Tryptic Soy broth without dextrose (Difco), as r e c o m m e n d e d by McFaddin (1980). Production of acid from carbohydrates was carried out according to Kilian (1976b) with some modifications A total of 25 fermentation tests were conducted in Phenol Red Broth Base (Difco, Detroit, MI) with 1% of the carbohydrate and 10 p g / m l of N A D grade III-C (Sigma, Philadelphia). Carbohydrates tested were: N-acetyl-D-glucosamine, adonitol, arabinose, cellobiose, dulcitol, galactose, glucose, glycerol, inositol, inulin, lactose, levulose, maltose, mannitol, mannose, melibiose, melizitose, raffinose, rhamnose, ribose, saccharose, salicin, sorbitoi, trehalose and xylose. For all preceeding tests except when otherwise mentioned, inocula were prepared by culturing each strain on PPLO-Yeast enriched agar plates ( Difco ). Plates were incubated at 37 °C overnight and colonies were harvested with 10

BIOCHEMICAL TYPING OFACTINOB.4CILLUSPLEL'ROPNEUMONIAE

399

ml of a sterile saline (0.85% w/v ) solution to obtain a dense bacterial suspension; 0.1 ml was used to inoculate the tests. All tests were carried out in duplicate. RESULTS

Of the 38 tests performed in this study, the following 17 reactions gave positive results with all the 67 strains of A. pleuropneumoniae: V-factor dependancy (need for N A D ) , porphyrins synthesis, nitrates reduction, production offl-galactosidase and urease, CAMP reaction and hemolysis production of acid from N-acetyl-D-glucosamine, galactose, glucose, levulose, maltose, mannitol, mannose, saccharose, ribose and xylose. A total of 17 reactions were uniformly negative and they are catalase, indole, lysine and ornithine decarboxylase, arginine dihydrolase, absence of production of acid from adonitol, arabinose, cellobiose, dulcitol, inositol, inulin, melibiose, melizitose, rhamnose, salicin, sorbitol, and trehalose. The oxidase test was discarded because of variable results with a particular strain. Finally, three tests gave variable results between strains and appeared to respond to the objective of this work; they are production of acid from glycerol, lactose and raffinose. As shown in Table 1, the 46 strains belonging to serotype 1 were classified into six phenotypic groups, while the 21 strains belonging to serotype 5 were divided into four of the preceeding groups. An important number of serotype 1 strains (43%) were in group 1 and were glycerol and lactose negative, and raffinose positive. Most serotype 5 strains (80%) were in group IV and were glycerol negative, and lactose and raffinose positive. Phenotypic group II TABLE 1

S u m m a r y o f the b i o c h e m i c a l v a r i a t i o n s

ofActinobacilluspleuropneumoniaeserotypes

Proposed phenotypic groups

Serotype

No. of isolates

1

1 5

20 2

-

II

1

11

--

Ill

1

5

IV

1 5

V VI

1 a n d 5 isolates

B i o c h e m i c a l tests glycerol

lactose

raffinose

m

m

+

-

+

5 17

-

4-

4-

1 5

4 1

+

4-

4-

1 5

1 1

+

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(glycerol, lactose and rail]nose negative) was exclusively composed of serotype 1 strains, as well as group Ill (glycerol and raffinose positive, and lactose negative) which included 5 serotype 1 strains. I)IS('tISSION

The majority of the 38 tests performed on 67 serotypes 1 and 5 A. pleuropneurnoniae strains did not show any discriminatory capacity and this suggests, in accordance with other authors (Kilian, 1976a; Kilian et al., 1978, Pohl et al., 1983 ), a high degree of homogeneity among members of this species. The catalase test, which was constantly negative in our study, had previously been reported as variable (Kilian, 1976a, Kilian et al., 1978; O'Reilley et al., 1984). This could be explained by the fact that 30% hydrogen peroxide was used in the present work. The N-acetyI-D-glucosamine fermentation test, reported for the first time with this microorganism, did not appear to be useful in showing phenotypic variations. The oxidase test was known to present variations (Kilian, 1976a; Kilian et al., 1978) but with the API reagent in our hands, variations with a particular strain were detected and it was not considered as a reliable test in this study. Previous studies had shown variations with lactose and raffinose (Biberstein, 1977, Kilian et al., 1978), but based on a study with 43 isolates, glycerol has been reported invariably negative (Kilian et al., 1978 ). The name "phenotypic group" is used here because of the fact that the ,4. pleuropneumoniae species already comprises 2 biovars (Pohl et al., 1983) includes V-factor dependant strains and biovar 2 represents V-factor independant species (Bertschinger and Seifert, 1978 ). It is possible that our phenotypic groups represents sub-biovars of V-factor dependant strains. The repartition of the 46 isolates of serotypes 1 into 6 phenotypic groups in comparison to the concentration of the 21 serotype 5 isolates into 1 particular phenotypic group may be related to the lower n u m b e r of serotype 5 strains studied. Interestingly, the majority of serotype I isolates do not belong to the same phenotypic group in which belong nearly all the serotype 5 isolates, showing homogeneity divergence between the 2 main serotypes responsible for swine mortality in Canada. Despite the fact that the n u m b e r of strains could have been more important in order to definitively confirm the validity of this biotyping system, observation of differential fermentation reactions allowed establishment of 6 phenotypic groups within serotype 1 and 5 that led us propose a biochemical typing scheme based on the production of acid from 3 carbohydrates namely glycerol, lactose and raffinose. This further phenotypical characterization, along with serotyping, could be useful for epidemiological studies that are essential in efforts to eradicate the disease. For example, the system could demonstrate that pigs infected with a

BIOCHEMICAL TYPING OF ACTINOBACILLUS PLEUROPNEUMONIAE

401

g i v e n s t r a i n h a v e b e e n i n f e c t e d b y pigs f r o m a h e r d in w h i c h t h e s a m e s t r a i n belonging to the same serotype and to the same phenotypic group has been isolated. ACKNOWLEDGEMENTS T h i s w o r k w a s p a r t o f a s t u d y s u p p o r t e d b y t h e C o n s e i l des R e c h e r c h e s en P 6 c h e s et A g r o - A l i m e n t a i r e d u Q u 6 b e c , a n d t h e M i n i s t 6 r e de l ' E n s e i g n e m e n t S u p 6 r i e u r et de la S c i e n c e d u Q u 6 b e c .

REFERENCES Bertschinger, H.U. and Seifert, P., 1987. Isolation of a Pasteurella haemolytica-like organism from porcine necrotic pleuropneumonia, Abstract M 19, Proc. 5th Int. Pig Vet. Soc. World Congress. Zagreb, Yugoslavia. Biberstein, E.L., Gunnarsson, A. and Hurvell, B., 1977. Cultural and biochemical criteria for the identification ofHaemophilus spp. from swine. Am. J. Vet. Res., 38:7-11. Kilian, M., 1974. A rapid method for the differentiation of Haemophilus strains: the porphyrin test. Acta Path. Microbiol. Scand. Sect., B, 82: 835-842. Kilian, M., 1976a. A taxonomix study of the genus Haemophilus, with the proposal of a new species, J. Gen. Microbiol., 93: 9-62. Kilian, M., 1976b. The haemolytic activity of Haemophilus species. Acta Path. Microbiol. Scand. Sect., B, 84: 339-341. Kilian, M., Nicolet, J. and Biberstein, E.L., 1978. Biochemical and serological characterization of Haemophilus pleuropneumoniae (Matthews and Pattison, 1961) Shope 1964 and proposal ofa neotype strain. Int. J. Syst. Bacteriol., 28: 20-26. McFaddin, J.F., 1980. Biochemical tests for identification of medical bacteria. Williams & Wilkins, Baltimore, Maryland, 2nd Edn. pp. 173-183. Nielsen, R., 1986. Serological characterization of Actinobacilluspleuropneumoniae strains and proposal of a new serotype: serotype 12. Acta Vet. Scand., 27:453-455. O'Reilley, T.S., Rosendal, S. and Niven, N.F., 1984. Porcine haemophili and actinobacilli: characterization by means of API test strips and possible taxonomic implications. Can. J. Microbiol., 30: 1229-1238. Pohl, S., Bertschinger, H.U., Frederiksen, W. and Mannheim, W., 1983. Transfer of Haemophilus pleuropneumoniae and the Pasteurella haemolytica-like organism causing porcine necrotic pleuropneumoniae to the genus Actinobacillus (Actinobacilluspleuropneumoniae comb. nov. ) on the basis of phenotypic and deoxyribonucleic acid relatedness. Int. J. Syst. Bacteriol, 33: 510-514.

Biochemical typing of Actinobacillus pleuropneumoniae.

A study was conducted to evaluate the possibility of using biochemical differences among strains of a given serotype of Actinobacillus pleuropneumonia...
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