Identification and Biotyping Of Brucella spp
Authors;
J.A.Stack and A.P.MacMillan
FAO/WHO Collaborating Centre for Reference and Research on Brucellosis
Central Veterinary Laboratory
New Haw
Addlestone
Surrey
Kt15 3NB
United Kingdom
Introduction
Brucella is named after Sir David Bruce who, in 1886, first isolated the organism
from the spleen of a soldier at post-mortem with what was then called Malta fever.
The genus Brucella currently contains six nomen species : B. melitensis, B. abortus,
B. canis, B. ovis, B. suis and B. neotomae, which vary in their ability to infect host
animals.
B. abortus primarily infects cattle but is transmitted to buffaloes, camels, deer, dogs,
horses, sheep and man.
B. melitensis causes a highly contagious disease in sheep and goats although cattle can
be infected. It is the most important species in human infection.
B. suis covers a wider host range than most other Brucella species.This species has 5
biovars, biovars 1 and 3 infect swine primarily, biovar 2 causes infection in European
wild hares, biovar 4 is responsible for infection in reindeer and wild caribou and
Biovar 5 was isolated from rodents in the USSR. All biovars can be transmitted to
man with the possible exception of biovar 2.
B. canis causes epididymo-orchitis in the male dog and abortion and metritis in the
bitch. It has not been reported in other animal species except man.
B. ovis is responsible for epididymitis in rams and occasionally abortion in ewes, but
does not infect other animals or man. Goats are susceptible to the disease by
experimental infection.
B. neotomae is only known to infect the desert wood rat under natural conditions, and
no other cases have been reported.
Brucella spp. constitute a high risk to the worker and brucellosis is one of the most
easily acquired laboratory infections therefore work should only be carried out under
containment level 3 (biosafety 3 ) conditions. The degree of risk varies due to the
virulence of the organism (B. melitensis and B. suis are most dangerous for man) and
with the number of bacteria in the material being processed. Aborted and infected
1
placental material may contain up to 1013 bacteria per gram. Lymph nodes and milk
present less risk , as does blood. Milk should be considered a source of human
infection. The organism may be recovered from amniotic fluid, vaginal discharge,
lymph nodes, mammary glands, uterus, testes, semen, milk, blood, fetal membranes,
spleen and urine from infected animals. Brucella produce generalised infections with
a bacteremic phase followed by localisation in the reproductive organs and
reticuloendothelial system. Infection in the pregnant animal will cause fetal and
placental infection which will often lead to abortion. Most Brucella strains are slow
growing fastidious organisms on primary isolation and grow poorly on nutrient media
unless supplemented with 5-10% serum or blood. Serum Dextrose Agar (SDA) is the
recommended media to support all strains, but it is usually after at least 48 hours
before colonies appear on primary isolation. At this stage they are just visible, being
approx. 0.5- 1.0mm diameter.
Where contaminating organisms are a possibility, selective media should be used e.g.
SDA supplemented with antibiotics. If the concentration of organisms is likely to be
low, e.g. in blood, milk or serum, enrichment media is recommended using the
method of Casteneda.
Preliminary Examination
a.
Make heat-fixed smears of representative colonies and stain by Hucker’s
modification of Gram’s stain.. Examine microscopically under the oil
immersion objective. Brucella organisms will appear as Gram-negative cocci,
cocco-bacilli or short rods. If the organisms are vibrios, spirillae, large rods,
occur in long chains or are Gram-positive, Brucella can be excluded.
b.
If required, make a further examination of the culture for bipolar staining,
spores, capsules, flagella and acid-fast staining.. These should give negative
results in the case of Brucella isolates. Experience is required in performing
and interpreting these staining reactions.
c.
Mix a loopful of a suspension of culture in distilled water with a loopful of 0.1
% w/v acriflavine on a clean glass slide and examine for agglutination. If no
auto-agglutination occurs mix a loopful of culture suspension with a loopful of
unabsorbed antiserum to smooth Brucella on another slide and agitate gently
for 30 seconds. If no agglutination occurs, the culture is unlikely to be
Brucella . If agglutination is observed, it is a strong indication that the culture
is Brucella. Mix a loopful of culture suspension with a loopful of rough
Brucella antiserum and examine after rotating gently for 30 seconds. The
absence of agglutination should exclude rough Brucella. A negative control
should be set up using Brucella negative serum.
d.
If the isolate gives a positive reaction with the appropriate Brucella antiserum,
sub-culture into two tubes of Albimi brucella both using a heavy inoculum.
Incubate one of these at 37ºC in air + 10% v/v CO2 and the other at 22ºC in a
similar atmosphere (a candle jar of anaerobic jar filled with the appropriate gas
mixture may be used if CO2 incubator is not available). After 24 - 48 hours
2
incubation examine both preparations for motile organisms by the hanging
drop method. Positive motility excludes Brucella.
e.
When setting up the cultures for d, also sub-culture onto SDA, sheep blood
agar and MacConkey agar plates. Incubate these at 37ºC in air + 10 % v/v
CO2. Replicate SDA plates should also be incubated at 37ºC in an anaerobic
jar in an atmosphere of CO2 + H2 or H2 alone, and aerobically at room
temperature (18 - 2ºC). Inspect the plates daily.
Brucella strains will not grow under strictly anaerobic conditions nor will they
produce visible colonies in 24 - 48 hours when incubated aerobically at room
temperature.
3
After 48 - 72 hours incubation at 37ºC in air + 10 % v/v CO2, smooth Brucella
isolates will grow on SDA to produce circular, convex colonies, 1-3 mm in
diameter, with a smooth glistening surface. The colonies are a transparent
honey colour in transmitted light and have a bluish-white translucent
appearance in reflected light. Rough Brucella isolates produce colonies of
similar size and shape but of a more opaque off-white colour and often with a
rather granular surface.
On blood agar growth is slower than on SDA, with the production of nonhaemolytic greyish-white glistening colonies 0.5 - 1 mm in diameter after 48 72 hours incubation.
Little or no growth is produced by most Brucella strains on MacConkey agar,
even after five days incubation at 37ºC.
The production of rapidly growing, haemolytic or lactose fermenting colonies
on the appropriate medium excludes Brucella.
f.
Test cultures grown on blood-free medium for catalase by transferring about
0.5 ml of a heavy suspension of the bacteria into a test tube plugged with
cotton wool and adding about 1 ml or 3 % w/v hydrogen peroxide by means of
a Pasteur pipette inserted through the plug. Vigorous gas formation indicates a
positive reaction. A negative reaction will exclude Brucella.
The cytochrome C oxidase test may also be done at this stage by smearing a
loopful of culture grown on blood-free medium across a piece of filter paper
impregnated with one per cent w/v tetramethyl p-phenylenediamine solution.
The development of a purple colour within ten seconds indicates a positive
reaction. Most Brucella strains will give a positive result but B. neotomae and
B. ovis give negative reactions.
g.
Inoculate the cultures into:
ii
Koser’s citrate medium,
iii
nutrient broth,
iv
O-nitrophenyl -D-galactoside broth
v
nitrate broth (0.1 % w/v KNO3 in nutrient broth),
vi
glucose-peptone water,
vii
gelatin stabs.
4
Incubate (i) and (ii) for 48 hours, (vii) for up to 14 days and the others for five
days at 37ºC. After incubation, add two drops of 0.04 % w/v methyl red to i.
A red colour indicates a positive methyl red reaction, a yellow colour indicates
a negative reaction. After completing the methyl red test, add 0.6 ml of 5 %
w/v ethanolic -naphthol solution and 0.2 ml of 40 % w/v KOH, mix well and
examine after 15 minutes and one hour. A positive reaction is indicated by a
red colour (Voges-Proskauer reaction). Examine (ii). for signs of turbidity, the
absence of this indicates a negative result.
Test (iii). for indole production by adding 0.5 ml of Kovacs reagent (5 % w/v
p-dimethylaminobenzaldehyde in 25 % v/v HC1 in iso-amyl alcohol) and
shaking for one minute. A positive result is indicated by the development of a
red colour in the lower layer.
Examine (iv). for the development of a yellow colour. If this is absent the
result is recorded as negative.
Test (v). for nitrate reduction by adding 1 ml of 0.8 % w/v sulphanilic acid in 5
mol/litre acetic acid followed by 1 ml of 0.5 % w/v -napthylamine in 5
mol/litre acetic acid. A positive reaction is indicated by a red colour which
develops within five minutes. Negative reactions should be checked by adding
a few mg of zinc powder and allowing the mixture to stand for ten minutes.
The development of a red colour indicates a negative nitrate reduction of
nitrate to ammonia or nitrogen.
In (vi). the absence of acid or gas production in glucose-peptone water is taken
as a negative result.
The gelatin stabs from (vii). should be examined daily after chilling to 4ºC.
The absence of liquefaction after 14 days incubation is taken as a negative
reaction.
All Brucella cultures will give negative results to the glucose fermentation, methyl
red, Voges-Proskauer, indole, citrate utilisation and gelatin liquefaction tests. The
only exception to this is B. neotomae which may ferment glucose. All Brucella
cultures, except B. ovis, will give positive results to the nitrate reduction test.
These tests will usually permit an organism to be identified or dismissed as a Brucella
strain in the majority of cases.
5
Methods for the Identification of Brucella
The genus and the six species of Brucella currently recognised are defined as follows:
Brucella Meyer & Shaw (1920)
Small, non-motile, non-sporing, Gram-negative cocci, coccobacilli or short rods, 0.5 0.7 µM by 0.5 - 1.5 µM, occurring singly, in pairs or short chains. Aerobic, no growth
under strictly anaerobic conditions. Carboxyphilic. Metabolism is mainly oxidative,
usually showing little fermentative action on carbohydrates in conventional media.
Multiple amino acids, thiamin, biotin and nicotinamide are required. Growth of many
strains is improved by calcium pantothenate and meso-erthritol. Haemin (X factor)
and nicotinamide adenine dinucleotide (V factor) are not required. Catalase positive.
Usually oxidase positive. Usually reduce nitrates. Produce H2S and hydrolse urea to
a variable extent. Do not produce indole. Do not liquefy gelatin or lyse blood. Do
not produce acetyl-methyl carbinol (Voges-Proskauer test). Do not utilise citrate.
Give a negative methyl red reaction. Do not release O-nitrophenol from Onitrophenol--D-galacotoside. Do not change litmus milk or may render it alkaline.
Optimum growth temperature 37C, range 20-40 C. Optimum pH for growth
between pH 6.6 and 7.4. Contain cytochrome C. Guanine + cytosine content. of DNA
is 55 - 58 moles per cent. Show greater than 90 per cent homology in DNA
hybridisation tests. Possess characteristic internal antigens. Intracellular parasites
producing characteristic infections in animals transmissible to man.
The Biovars within species are differentiated on the basis of specific combinations of
characteristics. These include phage lysis, CO2 requirement, H2S production,
serological specificity, tolerance to dyes and in the case of B. suis strains, oxidative
metabolic profiles with standard substrates. The current biotyping scheme is
summarised in Table 1.
Brucella abortus (Schmidt & Weis 1901) Meyer & Shaw 1920
Catalase and oxidase positive. Usually require supplementary CO2 for growth,
especially on primary isolation. Usually produce H2S from sulphur-contianing amino
acids or proteins. Usually hydrolyse urea but some strains may not. Usually grow in
the presence of basic fuchsin, methyl violet, pyronin and safranin O but not thionin, at
standard concentrations. Reduce nitrates to nitrites and may also reduce nitrites.
Smooth strains may have A, M or A and M surface antigens reactive in tests with
monospecific antisera, depending upon biovar. Oxidise L-alanine, D-alanine, Lasparagine, L-glutamic acid, D-galactose, D-glucose, D-ribose, and meso-erythritol.
Do not oxidise D-xylose, L-arginine, DL-citrulline, DL-ornithine. or L-lysine.
Cultures in the smooth or smooth-intermediate phase are lysed by brucella-phages of
the Tbilisi (Tb), Weybridge (Wb), Firenze (Fz), M51-S708, Berkeley (Bk), MC/75
and D groups at routine test dilution (RTD) and 104 RTD. Non-smooth cultures are
lysed by brucella-phage R at RTD.
6
Seven biovars are currently recognised.
Type Strain
544
86/8/59
Tulya
292
B3196
870
C68
(Reference strain for biovar 1)
(Reference strain for biovar 2)
(Reference strain for biovar 3)
(Reference strain for biovar 4)
(Reference strain for biovar 5)
(Reference strain for biovar 6)
(Reference strain for biovar 9)
7
Brucella melitenis (Hughes 1893) Meyer & Shaw 1920
Catalase and oxidase positive. Do not require supplementary CO2 for growth. Do not
produce H2S, or no more than a trace, when grown on recommended media. Usually
hydrolyse urea but some strains may not. Usually grow in the presence of basic,
fuchsin, thionin, methyl violet, pyronin and thionin blue at the standard
concentrations. Reduce nitrates to nitrites and may also reduce nitrites. Smooth
cultures may have the A, M or both A and M surface antigens reactive in tests with
monospecific sera. Oxidise D-glucose, meso-erythritol, L-alanine, D-alanine, Lasparagine and L-glutamic acid. Do not oxidise L-arabinose, or L-lysine. Cultures in
the smooth phase are usually susceptible to lysis by Bk phage at RTD and 104 RTD,
but are not lysed by brucella-phages of the Tb, Wb, Fz, M51-S708, MC/75, D or R
groups at RTD or 104 RTD. Non-smooth cultures are resistant to lysis by all of these
phages.
Type Strain
16M (Reference strain for biovar 1)
63/9 (Reference strain for biovar 2)
Ether (Reference strain for biovar 3)
Brucella suis Huddleson 1929
Catalase and oxidase positive. Supplementary CO2 is not required for growth.
Produce large amounts of H2S (biovar 1) or none at all (other biovars). Usually
hydrolyse urea rapidly. Usually grow in the presence of thionin but most strains are
inhibited by basic fuchsin, methyl violet, pyronin, safranin O and malachite green at
standard concentrations. Smooth cultures have the A surface antigen predominant
except for those of biovar 4 which react equally with antisera monospecific for the A
and M Suface antigens. Usually oxidise D-ribose, D-glucose, meso-erythritol, Dxylose, L-arginine, DL-citrulline, DL-ornithine and L-lysine. Oxidation of Lasparagine, L-glutamic acid, L-arabinose and D-galactose varies with the biovar. Do
not oxidise L-alanine or D-alanine. Smooth or smooth-intermediate cultures are lysed
by brucella-phages of the Wb, M51-S708, Bk, MC/75 and D groups at RTD and 104
RTD. Phages of the Fz group produce partial lysis at RTD, those of the Tb group do
not produce lysis at RTD but are lytic at 104 RTD. Non-smooth cultures are not lysed
by any of these phages or by phage R at RTD or 104 RTD.
Five biovars are currently recognised.
Type Strain
1330
Thomsen
686
40
513
(Reference strain for biovar 1
(Reference strain for biovar 2)
(Reference strain for biovar 3)
(Reference strain for biovar 4)
(Reference strain for biovar 5
8
Brucella neotomae Stoenner & Lackman 1957
Catalase positive, oxidase negative. Do not require supplementary CO2 for growth.
Usually produce H2S in large amounts and hydrolyse urea rapidly. Do not grow in the
presence of basic fuchsin even at 1/150 000, nor in the presence of safranin O at
1/10000 or thionin blue at 1/150 000, but will grow in the presence of thionin at 1/150
000. Reduce nitrates to nitrites. In smooth cultures the A surface antigen is
predominant. Oxidise L-asparagine, L-glutamic acid, L-arabinose, D-galactose, Dglucose, meso-erythritol and D-xylose. Do not oxidise L-alanine, D-alanine, Larginine, DL-citrulline, DL-ornithine or L-lysine. Oxidation of D-ribose may be
variable. In peptone media, ferment D-glucose, D-galactose, L-arabinsoe and Dxylose. Smooth or smooth-intermediate cultures are lysed by brucella-phages of the
Wb, M51-S708, Fz, Bk, MC/75 and Dgroups at RTD. Phages of the Tb group produce
partial lysis with few very small plaques at RTD, and complete lysis at 104 RTD.
Rough or mucoid cultures are not lysed by these phages or brucella-phage R, at RTD
or 104 RTD. No biovars are recognised.
Type strain 5K33.
Brucella ovis Buddle 1956
Catalase positive, oxidase negative. Require supplementary CO2 for growth. Does
not produce H 2S or hydrolyse urea. Inhibited by methyl violet but usually grows in
the presence of basic fuchsin and thionin at standard concentrations. Does not
produce nitrite from nitrate. A true smooth phase does not occur. Although the
colonial morphology may resemble the smooth form cultures are always in the rough
phase on primary isolation. Rough (R) - specific surface antigens cross-reacting with
other non-smooth brucellae are predominant. Oxidise L-alanine, D-alanine, Lasparagine, D-asparagine, L-glutamic acid, DL-serine and adonitol. Do not oxidise Larabinose, D-galactose, D-glucose, D-ribose, meso-erythritol, D-xylose, L-arginine,
DL-citrulline, DL-ornithine or L-lysine. The cultures are not lysed by brucella-phages
of the Tb, Wb, M51-S708, Fz, Bk, MC/75, D or R groups at RTD or 104 RTD but are
lysed by phage R/O at RTD and 104 RTD.. No biovars are recognised.
Type strain 63/290.
Brucella canis Carmichael & Bruner 1968
Catalase positive, oxidase positive. Does not require supplementary CO2 for growth.
Do not produce H2S. Hydrolyse urea very rapidly. Reduce nitrates to nitrites.
Growth is inhibited by basic fuchsin but not by thionin at the standard concentrations.
A smooth phase is not known, cultures are always in the rough or mucoid phase on
primary isolation. Form a mucoid sediment in liquid media. Rough-(R)-specific
surface antigens cross-reacting with other brucellae are predominant. Oxidise Dribose, D-glucose, L-arginine, DL-citrulline, DL-ornithine, L-lysine. Oxidation of
meso-erythritol is variable. Do not oxidise L-alanine, D-alanine, L-asparagine,
L-glutamic acid, L-arabinose, D-galactose. They are not lysed by brucella-phages of
the Tb, M51-S708, Wb, Fz, Bk, MC/75, D, R, or R/O groups at RTD or 104 RTD.
9
No biovars are recognised.
Type strain, RM6/66.
Other species
A number of Brucella isolates have been described whose properties do not closely
agree with the descriptions for recognised species. The status of most of these strains
has not been finally decided and it is possible that some or all of them will be found
eventually to correspond to atypical cultures of existing species or biovars.
Recently, isolations of previously unidentified species of Brucella have been reported
from seals, cetateans and an otter from Scotland and the coast around northern
England and from a bottle-nosed dolphin from California. The characterisation of
these strains cannot be assigned to nomen species of the genus Brucella .These
findings have raised questions concerning exposure, prevalence of infection,
distribution and possible pathogenicity and zoonotic potential. A serological survey
was carried out to investigate the range of marine mammal species which may have
been exposed to the organism .
10
Table 1: CHARACTERS USED IN THE DIFFERENTIATION OF
BRUCELLA SPECIES AND BIOVARS
Species
Biotype
CO2
req’t
H 2S
prod’n
Growth on
media
containing
thionin*
fuchsin*
Agglutination
with
monospecific
antisera
A
M R
Lysis by phage†
at RTD
Tb
Wb
Bk
Fz
1
2
3**
4
5
6**
9
(+)‡
(+)
(+)
(+)
-
+
+
+
+
(-)‡
+
+
+
+
+
+
+
+***
+
+
+
+
+
+
+
-
+
+
-
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
1
-
+
+
-****
+
-
-
L
L
PL
2
-
-
+
-
+
-
-
L
L
PL
3
-
-
+
-
+
-
-
L
L
PL
4
-
-
+
(-)
+
+
-
L
L
PL
5
-
-
+
-
-
+
-
N
L
N
L
N
L
N
L
N
L
L
L
PL
1
-
-
+
+
-
+
-
NL
L
2
-
-
+
+
+
-
-
NL
L
3
-
-
+
+
+
+
-
N
L
N
L
N
L
NL
L
N
L
N
L
N
L
B.ovis
+
-
+
(+)
-
-
+
N
L
NL
N
L
N
L
B. canis
-
-
+
-
-
-
+
N
L
NL
N
L
N
L
L
L
L
B.abortus
B. suis
B. melitensis
B. neotomae
-
+
-
-
+
-
-
N
L
or
PL
L = Confluent lysis
*
†
‡
**
PL = Partial lysis
NL = No lysis
Concentration = 1/50 000 w/v
Phage R will lyse non-smooth Brucella abortus at RTD
Phage R/O will also lyse B. ovis at RTD
(+) = Most strains positive (-) = Most strains negative
For more certain differentiation of B. abortus Type 3 and Type 6,
thionin at 1/25 000 (w/v) is used in addition. Type 3 = + ,
Type 6 = - .
11
***
****
Some strains of this biovar are inhibited by basic fuchsin
Some isolates may be resistant to basic fuchsin, pyronin and safranin O
Brucella Typing Procedure
Once an isolate has been identified as a Brucella culture, it may then be subjected to
further tests in order to identify its species and biovar. In some cases it may be
necessary to determine if the isolate has the characteristics of one of the live vaccine
strains of B. abortus or B. melitensis.
In all case, when typing organisms suspected to be Brucella, it is essential to include
in the test at least the Brucella reference strains B. abortus 544, B .melitensis 16M and
B. suis 1330 as a check on media and methods. If the strains undergoing
characterisation are non-smooth and/or are thought to belong to one of the other
Brucella species, then at least one strain typical of these should be included in the test.
It is recommended that the procedure is carried out at a specialist reference centre to
further identify the species and biovar level.
Tests Include :
 Dissociation
Brucella cultures growing in vitro may undergo changes in colonial morphology
which are accompanied by alterations in antigenic structure, phage susceptibility and
virulence. This process is termed dissociation and is particularly likely to occur when
smooth strains are grown in static liquid culture. The variants produced may range
from grossly aberrant mucoid or rough forms to others which are transitional between
the extreme stages; intermediate or smooth-intermediate forms. A variety of methods
are available for detecting dissociation:
a)
b)
c)
d)
Direct observation using obliquely transmitted light
Staining of colonies with crystal violet
Agglutination in acriflavine
Agglutination by antiserum to rough Brucella
B. ovis and B. canis are naturally rough on primary isolation.

Growth requiring CO2
Some biovars of B. abortus and B. ovis require CO2 for growth

Production of hydrogen sulphide
Lead acetate strips are used to identify the production of H2S during growth. For
example, B. suis biovar 1 produces large amounts but B. melitensis is negative.
12

Urease Test
In general, B. canis, B. neotomae and B. suis will give a strong urease reaction which
turns Christensen’s medium and magenta colour in less than 15 minutes. B. ovis does
not hydrolyse urea, neither do some strains of B. abortus, including B. abortus 544 the
type strain for biovar 1 .
Growth on dye plates
Growth is measured on SDA plates containing basic fuchsin and thionin. All biovars
of B. melitensis grow in the presence of fuchsin and thionin althuogh recently, atypical
B.melitensis biovar 1 strains which are resistant to these dyes have been reported. All
biovars of B. abortus, except biovar 2 grow in the presence of fuchsin.


Agglutination reactions
Monospecific autisera to smooth and rough forms of Brucella are used.

Phage sensitivity
Currently used for culture identification are Tb, Wb, Fz, Bk and R phages. These are
propagated at CVL and titrated against appropriate host Brucella strains. Lysis of
strains by phage forms a distinct and integral part of the routine identification
procedure.

Oxidative metabolism
In addition, the tetrazolium reduction assay can be performed to support the above
classical biotyping methods if required.

Identification of vaccine strains
Using the above classical techniques, Strain 19 cannot be distinguished from other
CO2 independent strains of B. abortus biovar 1. Additional tests can be performed
using thionin blue and meso-erythritol dyes in conjunction with growth in the presence
of penicillin to aid differentiation.
Also, B. melitensis strain Rev 1 cannot be differentiated from virulent isolates of B.
melitensis biovar 1 using routine techniques. Supported tests include using penicillin
and streptomycin to discriminate between the vaccine strain and field isolates.
13