Classification of the genus Brucella : the current position(*)

Rev. sci. tech. Off. int. Epiz., 1982, 1 (1), 281-289. Classification of the genus Brucella : the current position(*) by M.J. CORBEL and W.J. BRIIMLEY MORGAN(**) The present system of taxonomy for the genus Brucella is based on recommendations made by the Sub-Committee on Taxonomy of Brucella of the International Committee on Bacteriological Nomenclature (1963) (1) and subsequently extended in later reports (1967) (2), (1971) (3). This scheme was devised to eliminate problems which arose in the identification of the original species, B. melitensis, B. abortus and B. suis, when these were typed by the conventional procedures involving examination of CO 2 requirement, H 2 S production, dye sensitivity and agglutination reactions with monospecific antisera. The introduction of tests for sensitivity to lysis by phage and measurement of oxidative metabolism with selected substrates resolved these problems and enabled a system of species identification to be achieved which was consistent with the epidemiological evidence. Currently phage lysis and oxidative metabolism tests are used as the primary means of identification at the species level and the conventional tests are used for the differentiation of the biotypes. Attention has also been given to criteria to be applied for recognition at the genus level. These are included among recommendations made on-behalf of the Sub-Committee on Taxonomy of the genus Brucella of the International Committee on Systematic Bacteriology (4). Identification is achieved in the first instance by examination of morphological, cultural, metabolic and serological properties. Confirmatory evidence may be provided in the case of atypical isolates by examination of DNA purine-pyrimidine base composition (5), DNA nucleotide sequence (5, 6), electrophoretic separation patterns produced by phenol-acetic acid-water-soluble proteins (7), absorption spectrum of cytochrome a and c bands (8) and demonstration of the presence of intracel- (*) Document WHO/BRUC/81.370. (**) FAO/WHO Collaborating Centre for Reference and Research on Brucellosis, Central Veterinary Laboratory, Weybridge, Surrey, England.

- 282 - lular antigens shared with Brucella reference (9, 10) strains. Gas-liquid chromatography of the fatty acid methyl esters is also a useful supplementary procedure as the structural fatty acids of Brucella strains produce a characteristic elution profile (11). The phage lysis pattern and oxidative metabolic profile are used for species identification, but the phage tests are also useful for confirmation of genus identity as they are specific for Brucella. The conventional tests, including examination of CO 2 requirement, dye sensitivity, H 2 S production, urease activity, and agglutination reactions with monospecific antisera, are of supplementary value for species identification and are also used to differentiate the biotypes of the major species, B. abortus, B. melitensis and B. suis. The genus Brucella and its species have been defined as follows : Small non-motile gram-negative cocci, coccobacilli or short rods with straight or slightly convex sides and rounded ends 0.5-0.7 µm wide by 0.6-1.5µm long. Arranged singly, less commonly in pairs, short chains or small clusters. Do not produce capsules, spores or flagella. Do not usually show bipolar staining. Not acid-fast but may resist decolorization by weak acids or alkalis as in the Macchiavello or modified Köster staining procedures. Aerobic, metabolism respiratory. Many strains require supplementary (5-10%) CO2 for growth especially on primary isolation. Chemo-organotrophic with complete nutritional requirements, including multiple amino acids, thiamin, nicotinamide, biotin and magnesium. Some strains require serum or other colloid for growth but haemin (X factor) and nicotinamide adenine dinucleotide (V factor) are not required. Catalase positive and usually oxidase positive but B. neotomae, B. ovis and some strains of B. abortus are oxidase negative. Do not ferment carbohydrates in conventional media except for B. neotomae. Oxidize various amino acids and carbohydrates. Usually reduce nitrates to nitrites but B. ovis does not. Urea is hydrolysed to a variable extent. Production of H 2 S varies between species and biotype. Citrate is not utilized as sole carbon source. Indole is not produced. The methyl red test and Voges-Proskauer reactions are negative. Gelatin is not liquefied and erythrocytes are not lysed. Litmus milk is either unchanged or rendered alkaline. Temperature range 20-40 C; optimum -37 C. Optimum ph 6.6-7.4. Optimum osmotic pressure 2-6 atmospheres (0.05-0.15 molar NaCl). Branched electron transport system includes cytochromes a, a3, b, c and O. Susceptibility to lysis by genus-specific phages varies between species, structural fatty acids produce characteristic elution profile on gas-liquid chromatography as methyl esters. Possess common structural proteins soluble in phenol-acetic acid-water which produce genus-specific patterns on disc electrophoresis. Major surface

- 283 - antigens differ between smooth and non-smooth strains but some intracellular antigens are common to all strains and are genus specific. The guanine + cytosine content of the DNA ranges from 56 to 58 moles per cent (buoyant density). DNA polynucleotide sequencies show 90% homology in hybridization studies. Facultative intracellular parasites producing characteristic infections in a wide range of animals. TYPE SPECIES 1. BRUCELLA MELITENSIS CO 2 independent. Produces no H 2 S, or no more than a trace, on peptone media. Usually grows in the presence of basic fuchsin and thionin. Usually hydrolyses urea. Smooth strains may react with M, A or A and M monospecific antisera depending upon biotype. Not lysed by phages Tb, Fi, or Wb at RTD or 104 RTD. Smooth cultures are lysed by phage Bk 2 at RTD and 10* RTD. Oxidizes L-alanine, L-asparagine, L-glutamic acid, D-glucose, and i arginine, DL-citrulline, DL-ornithine or L-lysine. Usually pathogenic for sheep and goats but may infect cattle and man. FAO/WHO neotype and biotype reference strains : NCTC B. melitensis 16M ( biotype 1 ) 10094 B. melitensis 63/9 (biotype 2) 10508 B. melitensis Ether (biotype 3) 10509 ATCC 23456 23457 23458 2. BRUCELLA ABORTUS Usually requires supplementary (5%) CO 2 for growth, especially on primary isolation. Usually hydrolyses urea and produces moderate amounts of H 2 S but some strains may not. Usually grows in the presence of basic fuchsin, some biotypes will also grow in the presence of thionin and some are inhibited by both dyes. Smooth strains may have A, M or A and M surface antigens reactive in tests with monospecific antisera depending upon biotype. Cultures in the smooth or smooth-intermediate phase are lysed by phages Tb, Fi, Wb and Bk 2 at RTD. Non-smooth cultures are lysed by phage R/C at RTD. Oxidizes L-alanine, L-asparagine, L-glutamic acid, L-arabinose, D- galactose, D-glucose, D-ribose and i-erythritol. Does not oxidize D-xylose, L- arginine, DL-citrulline, DL-ornithine or L-lysine. Usually pathogenic for cattle, causing abortion; can also infect other species including sheep, goats, camels, yaks, buffaloes, horses, dogs and man.

- 284 - FAO/WHO neotype and biotype reference strains : NCTC ATCC B. abortus 544 (biotype 1) 10093 23448 B. abortus 86/8/59 (biotype 2) 10501 23449 B. abortus Tulya (biotype 3) 10502 23450 B. abortus 292 (biotype 4) 10503 23451 B. abortus B3196 (biotype 5) 10504 23452 B. abortus 870 (biotype 6) 10505 23453 B. abortus 63/75 (biotype 7) 10506 23454 B. abortus C68 (biotype 9) 10507 23455 B. abortus biotype 8 is no longer recognized. 3. BRUCELLA SUIS C0 2 independent. Hydrolyses urea rapidly. Produces large amounts of H 2 S or none at all depending upon biotype. Grows in the presence of thionin and usually inhibited by basic fuchsin but some strains grow on both dyes. Smooth strains are usually reactive with A monospecific serum but some strains may react with M or A and M monospecific antisera depending upon biotype. Smooth strains are not lysed by phage Tb at RTD but are lysed at 10 4 RTD and are partially lysed by phage Fi and lysed by phages Wb and Bk 2 at RTD. Oxidize D-ribose, D-glucose, i-erythritol, D-xylose, L-arginine, DLcitrulline and DL-ornithine. Do not usually oxidize L-alanine or L-asparagine. Oxidation of L-lysine, L-glutamic acid, L-arabinose and D-galactose varies with biotype. Usually pathogenic for pigs except for biotype 4 which is usually pathogenic for reindeer. May also infect other species including hares, rodents, dogs and man. FAO/WHO neotype and biotype reference strains : NCTC ATCC B. suis 1333 (biotype 1) 10316 23444 B. suis Thomsen (biotype 2) 10510 23445 B. suis 686 (biotype 3) 10511 23446 B. suis 40 (biotype 4) 11364 23447 4. BRUCELLA NEOTOMAE CO 2 independent. Produces H 2 S. Hydrolyses urea rapidly. Does not grow in the presence of basic fuchsin but will grow in the presence of thionin (at 1:150,000). Smooth strains have the A surface antigen reactive in tests with monospecific antisera. Smooth strains are partially lysed by phage Tb at RTD and completely lysed at 10 4 RTD. They are also lysed by phages Fi, Wb and Bk 2 at RTD. May produce acid from D-glucose, D-galactose, L-arabinose, and D-xylose in peptone media. Oxidize L-asparagine, L-glutamic acid, L- arabinose, D-galactose, D-glucose, i-erythritol and D-xylose. Do not oxidize

- 285 - L-alanine, L-arg nine, DL-c trulline, DL-orn th ne or L-lys ne. Oxidation of D- ribose is variable. Occurs in the desert wood rat of the western United States of America (Neotoma lepida Thomas). Natural infections unknown in other species. FAO/WHO reference type strain : B. neotomae 5K33 NCTC 10084 ATCC 23459 No biotypes are recognized. 5. BRUCELLA OVIS Requires supplementary (5-10%) C0 2 for growth. H 2 S is not produced. Usually does not hydrolyse urea but some strains may show weak activity after seven days. Grows in the presence of basic fuchsin and thionin. Does not reduce nitrate. A smooth phase does not occur, cultures are always in the rough phase on primary isolation. Does not react with A and M monospecific antisera but is agglutinated by R antiserum. Cross-reacts with B. canis and other non-smooth brucellae. Not lysed by phages Tb, Fi, Wb or Bk 2 at any concentration. Lysed by phage R/C at RTD. Oxidizes L-alanine, L-asparagine and L-glutamic acid. Does not oxidize L-arabinose, D-galactose, D-glucose, D-ribose, i-erythritol, D-xylose, L-arginine, DL-citrulline, DL-ornithine or L- lysine. Adonitol is oxidized and this is useful for identification, as B. ovis and B. neotomae are the only species consistently active on this substrate. Pathogenic for sheep causing epididymitis in rams and abortion in ewes. Natural infections are unknown in other species. FAO/WHO reference type strain : B. ovis 63/290 NCTC 10512 ATCC 25840 No biotypes are recognized. 6. BRUCELLA CAN/S CO 2 independent. Hydrolyses urea rapidly. Does not produce H 2 S. Usually reduces nitrates but some strains may not. Usually grows on thionin but not on basic fuchsin. Cultures are always in the rough or mucoid phase on primary isolation. Does not react with monospecific antisera for A and M antigens but reacts with antiserum to R antigen. Cross-reacts serologically with B. ovis and other non-smooth brucellae. Not lysed by phages Tb, Fi, Wb or Bk 2 at any concentration. Lysed by phage R/C at RTD. Oxidizes D-ribose, D- glucose, L-arginine, DL-citrulline, DL-ornithine and L-lysine. Does not oxidize L-alanine, L-asparagine, L-glutamic acid, L-arabinose, D-galactose or D- xylose. Oxidation of /-erythritol is variable. Pathogenic for dogs causing epididymo-orchitis in the male and abortion and metritis in the female. May be transmitted to man.

- 286 - FAO/WHO reference type strain : B. canis RM 6/66 NCTC 10854 ATCC 23365 No biotypes are recognized. TYPING OF BRUCELLA Because of their infectivity for man, Brucella cultures should be examined by adequately trained staff provided with the facilities necessary to prevent accidental infections. Specific recommendations have been made for the safety precautions to be taken with Brucella strains (10). In most cases Brucella cultures isolated during the course of diagnostic procedures or surveys can be readily identified as members of the genus on the basis of microscopic appearance of smears stained with Gram's stain, colonial morphology on serum dextrose agar or similar medium, agglutination with antiserum to smooth Brucella species if in the smooth colonial phase or with antiserum to a rough Brucella strain if non-smooth, lysis by brucella phages. Subsequent identification at the species and biotype level is then readily achieved by the use of the conventional tests. These procedures are simple and can be performed by any laboratory equipped for general bacteriology and having the necessary safety facilities. In contrast, oxidative metabolism tests are not suitable for the routine identification of cultures. They require expensive apparatus and specially trained staff, are hazardous and time-consuming to perform and need expert interpretation. Their performance is best left to reference laboratories with experience in these methods. Simpler methods for the semi-quantitative determination of oxidative metabolic activity using thin-layer chromatography have been described (6, 7). These are safer and easier to perform than manometric techniques and require only simple apparatus. They do not always produce results entirely in agreement with those obtained by manometric methods however, as unlike the latter they are influenced by non-oxidative degradation of substrates. Nevertheless, the overall pattern of substrate utilization demonstrated by thin-layer chromatography is usually adequate for species identification. Oxidative metabolic tests are invaluable for the identification of atypical cultures, for example, the rare phage-resistant smooth isolate or more commonly, non-smooth variants of the normally smooth species. The invariably non-smooth species, B. ovis and B. canis, are now amenable to identification by phage typing and conventional tests. It is recommended that national brucellosis centres should carefully examine cultures isolated in their own country by the phage and conventional tests. Those that differ from established biotypes or that have been isolated from unusual hosts should be preserved by freeze-drying or vacuum-drying as soon as possible after isolation. When a number of similar cultures has been isolated and the type appears to be of epidemiological significance or of

- 287 - Preferred host J Cattle Sheep, goats Swine Swine, hares Swine Reindeer Murine and cricetine rodents Desert wood rat Sheep Dogs loiuqiaja-/ + + + + + + + + 1 + 1 aujsa -"i 1 1 + 1 + + + 1 1 + TABLE 1. Classification of the genus Brucella into species. Oxidation of substrates phage at RTD au!lji ujo-- a 1 1 + + + + + i i + aujnnjuo-iq 1 1 + + + + + i i + auju ßje-i 1 1 + + + + + i i + aso Áx-Q +l 1 + + + + + i i i asoon ß-Q + + + + + + + + 1 + asoqu-q asoioe eß-a + I + + + + + + 1 1 + + 1 +1+1 1 1 1 + 1 + 1 asouiqejb--] + i + + 1 1 1 + 1 + 1 P OB OjLUBin ß--] + + 1 +1 +1 +1 + + + + au!ßejedsb-- + + 1 +1 1 1 + + + i au UB B-"l + + + M +1 i i +1 +i +i R/C o/y i t i i 1 i 1 I I _l, _ l I 1 I 1 1 I -J m Z >. ir j ' 1 1 1. 1 NL = No lysis PL = Partial lysis L = Lysis + = QO 2N>50 - = QO 2N<50 ± = Q0 2 N variable Lysis Wb -O 1- Brucella species -1 2-1 J - 1-1 - 1 -J 2 2, 1 1 1 1 "râ" T CMCO "O ~<ñ.to CD QJ % 55 s s ê 1 3 S î 1.c 0 "S.<2 -S3 o O -SS 5 -ScS 5 c c b S c S eu fc co <o 3 cocj Qá CQCQ Oj OQOQ OQ (a) Smooth strains

- 288 2. Classification of Brucella biotypes. TABLE Agglutination with monospecific antisera Growth on media containing' 3 ) basic fuchsin thionin H 2 S production C0 2 requirement Biotype Species DC I I I I I I I I I I I I I I I I I + + I I I + + I + + + I + + I I + + I I I < + + + I I + + I I + + + + + + I + I I + I + + + + + I +++T++Ii i i i 5 1 + 1 + + + + + + + + + + + + - + + + + + + I - J - + + I I I + I I I I + I 1 s,. Ä,. + i. l i l i I I I 1 1 1 1 1 1 + 1 <-cng )<tflr>^í )rva5 <- CM co *-CM00'*tg 73 c B. abortus B. melitensis B. suis o B. neotomae B. ovis B. canis (a) Concentration = 1/50,000 w/v. (b) (+) = most strains positive; ( ) = most strains negative. (c) For more certain differentiation of biotypes 3 and 6 thionin at 1/25,000 w/v is used; type 3 = positive, type 6 = negative. (d) Growth will occur in the presence of thionin at 1/150,000 w/v.

- 289 - unusual interest, an FAO/WHO reference centre should be contacted with regard to the regulations to be observed for the transport of cultures across national boundaries. Full information on preliminary typing results, source and history should accompany the cultures. The differential characteristics of the species and biotypes of the genus Brucella are given in Tables 1 and 2. REFERENCES 1. STABLEFORTH A.W. and JONES L.M. Int. Bull. bact. Nomencl., 1963, 13, 145-158. 2. JONES L.M. Int. J. system. Bact, 1967, 12, 371-375. 3. JONES L.M. and WUNDT W. Int. J. system. Bact., 1971, 21, 126-128. 4. CORBEL M.J. and MORGAN W.J.B. Int. J. system. Bact., 1975, 25, 83-89. 5. HOYER B.H. and MCCULLOUGH N.B. J. Bact., 1968a, 95, 444-448. 6. HOYER B.H. and MCCULLOUGH N.B. - J. Bact., 19686, 96, 1783-1790. 7. MORRIS J.A. - J. gen. Microbiol., 1973, 76, 231-237. 8. DRANOVSKAYA E.A. and KUSHNAREV V.M. Zh. Mikrobiol. (Mosk.), 1968,12, 3-5. 9. DIAZ Ft., JONES L.M. and WILSON J.B. J. Bact., 1968, 95, 618-624. 10. CORBEL M.J. et al. Techniques in the identification and classification of Bruce/la species. In : SKINNER F.A. and LOVELOCK D.W., Eds., «Identification methods for microbiologists», 2nd ed., The Society for Applied Microbiology Technical Series No. 14, London, Academic Press, 1979. 11. TANAKA S. et al. Ann. Sclavo, 1977, 19, 67-82. 12. BALKE E., WEBER A. and FRONK B. - Zbl. Bakt., I Abt. Orig., 1977, A 237, 523-529. 13. CORBEL M.J., GILL K.P.W, and THOMAS E.L. Methods for the identification of Brucella. Ministry of Agriculture, Fisheries and Food, Pinner, England, 1978.