Brucella canis Isolates from Canadian Dogs.
ABSTRACT Eleven Brucella canis isolates from Canadian dogs were characterized by dye and antibiotic sensitivity, phage susceptibility, urease and H(2)S production, CO(2) requirement, and reaction with monospecific A,M, and R anti-Brucella antiserum. The isolates could be separated into two distinct groups. One group had a sensitivity pattern similar to that seen with the American type strain RM666, while the other group had a pattern identical to that of a Mexican strain, Mex 51. Epidemiological studies supported contraction of infections in the United States and Mexico respectively. The characteristics of all isolates were stable after repeated subculture indicating that strain differences could serve as useful epidemiological markers and supporting division of the species into two biovars.
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ABSTRACT: Three procedures are commonly used for the serodiagnosis of B. canis infection: 1) The rapid slide agglutination test (RSAT), 2) the tube agglutination test (TAT), and 3) the modified 2-mercaptoethanol tube agglutination test (2ME-TAT). Hemocultures are always essential for diagnosis. The RSAT was developed to provide a presumptive diagnosis rapidly. It has been found accurate in identifying non-infected dogs; however false positive reactions are common due to shared determinants between the surface antigens of B. canis and certain other gram-negative bacteria. The RSAT has recently been modified to include brief reaction of test sera with 2ME (0.2M) prior to adding test antigen. The modification has improved specificity, but it has not eliminated false positive reactions. The TAT and 2ME-TAT are widely used. Although there is good agreement between tests, both suffer from lack of specificity but are valuable in kennels where B. canis has been identified by blood cultures. Agarose gel immunodiffusion (AGID) tests using extracted (SDC or hot PBS) cell wall antigenic complexes reveal precipitins in the sera of infected dogs usually 8 to 12 weeks postinfection (PI) that may persist 5 years. The antigenic complexes consist of at least 3 antigens; one (antigen '2R') appears to possess high B. canis specificity. Sera from noninfected dogs also may react nonspecifically in AGID tests that employ crude SDC or PBS antigenic extracts leading to 'false positive' interpretations. Cytoplasmic antigens gave up to four precipitin lines with sera from B. canis infected dogs. The antigens (protein or glycoprotein) were present in both S and R Brucella cells, but not in other gram-negative organisms examined. AGID tests that employed cytoplasmic antigens revealed precipitins against one or more (usually 2-3) antigens from PI months 4 through 64. In some dogs, precipitins were present 12 months after the bacteremia had ceased, a time when other tests were diagnostically insignificant, or equivocal. No 'false positive' field sera reached with the cytoplasmic antigens.Developments in biological standardization 02/1984; 56:371-83.
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ABSTRACT: In a serologic survey of stray and pet dog populations of Georgia, serums were screened for Brucella canis antibodies, using the slide agglutination test. If results were positive, B canis antibody titers were determined, using the standard tube agglutination test. The stray dogs had significantly (P less than 0.01) higher titers than did the pet dogs. The reactor rate was 58% higher for the slide agglutination test than for the tube agglutination test. The manufacturer's evaluation of the slide agglutination test was based on a comparison of the serologic results of that test with those of the tube agglutination test, using a comparative method that permitted the results to be interpreted as 99% agreement between the 2 tests. Reevaluation of the manufacturer's data by a different method indicated that the slide agglutination test is very accurate when the results are negative (99.7% specific) but less so when the results are positive (62.5% sensitive).Journal of the American Veterinary Medical Association 01/1977; 169(11):1214-6. · 1.72 Impact Factor
Brucella canis Isolates from Canadian Dogs
Lorry B. Forbes and James F. Pantekoek
Eleven Brucella canis isolates from Canadian dogs
were characterized by dye and antibiotic sensitivity,
CO2 requirement, and reaction with monospecific
A,M, and R anti-Brucella antiserum. The isolates
could be separated into two distinct groups. One group
had a sensitivity pattern similar to that seen with the
American type strain RM666, while the other group
had a pattern identical to that of a Mexican strain,
Mex 51. Epidemiological studies supported contrac-
tion of infections in the United States and Mexico
respectively. The characteristics of all isolates were
stable after repeated subculture indicating that strain
differences could serve
markers and supporting division of the species into two
urease and H2S production,
as useful epidemiological
Souches de Brucella canis, isoles chez des
chiens du Canada
Les auteurs ont determine les caracteristiques de
11 souches de Brucella canis, isolees chez des chiens
du Canada. Ils utiliserent a cette fin la sensibilite aux
colorants et aux antibiotiques, la susceptibilite aux
bacteriophages, la production d'urease et de H2S, les
besoins en CO2 et la reaction avec les antiserums
monospecifiques A, M et R contre Brucella.
reussirent a separer les isolats en deux
distincts. Le premier afficha un profil de sensibilite
semblable a celui de la souche RM666 du type ameri-
cain; quant au second, son profil de sensibilite s'appa-
rentait a celui de la souche mexicaine Mex 51. Une
etude epizootiologique permit de conclure que la con-
tamination se fit respectivement aux Etats-Unis et au
Mexique. Les caracteristiques de tous les isolats
cessives, indice que les differences entre les souches
pourraient servir de marqueurs epizootiologiques utiles
et qu'il convient de diviser l'espece en deux biotypes.
apres plusieurs cultures
Can Vet J 1988; 29: 149-152
116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3
(Forbes) and Reference Laboratories Branch, Animal Health
Division, Alberta Agriculture, 6909-116 Street, Edmonton,
Alberta T6H 4P2
Dr. Pantekoek: Animal Pathology Division, Halldon House,
2255 Carling Avenue, Ottawa, Ontario KIA 0Y9.
Brucella canis infection in the dog is often asymp-
tomatic. Clinical signs, when they occur, may be
limited to a generalized peripheral lymphadenopathy
with or without infertility. In the female, abortion
usually occurs late in gestation and is characterized by
fetal autolysis and a dark colored vaginal discharge
lasting from one to six weeks. Some bitches may whelp
stillborn or weak pups. Surviving pups have a gener-
alized lymphadenopathy and can carry infection to
maturity. The infected bitch continues to have normal
estrous cycles. Mature male dogs may develop orchitis
and epididymitis resulting in testicular enlargement.
In chronic infections, unilateral or bilateral testicular
atrophy is a more common sequel. Semen abnormali-
ties are evident by five weeks postinfection. Scrotal
enlargement due to the accumulation of fluid in the
tissues and scrotal dermatitis from licking and secon-
dary infection may occur. Less frequently observed
clinical aspects in the dog include discospondylitis,
multifocal pyogranulomatous dermatitis, and ocular
conditions such as corneal edema and endophthalmitis.
A comprehensive review of the clinical and epide-
miological aspects of canine brucellosis is available (1).
A variety of slide agglutination, tube agglutination,
and agar gel immunodiffusion tests have been described
for the diagnosis of B. canis in dogs (2,3,4,5,6).
Almost all have low specificity with false positive rates
ranging from about 10% to almost 60%. Recently
developed agar gel immunodiffusion procedures using
cytoplasmic protein antigens show improved sensitiv-
ity and specificity, and may lead to more reliable
routine tests. However, at present, the commonly used
serological tests for B. canis should be interpreted with
The definitive diagnostic procedure for canine
brucellosis is the isolation of B. canis. Tissues for
culture include blood, lymph nodes, spleen, liver,
kidney, bone marrow, testicular tissue, semen, urine,
vaginal discharges, milk, placenta, uterus, and aborted
fetuses. Hemoculture, though not always successful,
can be useful as the bacteremic phase in untreated dogs
may persist for up to five years (2).
The first reported isolates of B. canis were made
from Beagles in the northeastern United States in 1966
(7). The disease is now generally accepted as having
worldwide distribution (1). In Canada, clinical and
serological evidence indicates that B. canis occurs
sporadically or exists at very low levels (8,9,10). This
paper is, to our knowledge, the first published report
Can Vet J Volume 29, February 1988
Can Vet J Volume 29, February1988
of B. canis in Canadian dogs based on actual recovery
of the organism. We describe the characteristics of
eleven isolates of B. canis (from western Canada in
1982 and 1983) compared with the type strain RM666
and with Mex 51, a strain originating in Mexico.
Materials and Methods
Suspected field isolates of B. canis were
submitted for confirmation to the Animal Pathology
Laboratory, Agriculture Canada, in Saskatoon by the
Reference Laboratories Branch, Animal Health Divi-
BA28A, B and C, and isolates BA49B,C,E,F and G
originated from the same kennel and were received in
March of 1982. They came from five Poodles show-
ing clinical or serological evidence of B. canis infec-
tion. The organism was recovered at necropsy from
uterus, vagina, and pooled samples of spleen. Isolates
CII-T2, C12-T5 and C1I3-T4 were received in April
and May of 1983. They were from a seven-year-old
retriever with weight loss, lethargy, and
unilateral testicular swelling. The organism was ini-
tially recovered from urine and
kidney and pooled liver and spleen. All eleven isolates
in this study were obtained using routine bacteriological
procedures. Tissues were seared and sterile scissors
removed using either a sterile glass rod or a sterile
swab. Tissue inoculum and urine-soaked swabs were
streaked directly onto blood agar (5'Vo sheep's blood
in a trypticase soy agar base) and MacConkey agar,
both incubated aerobically at 350C, and blood agar
and chocolate agar, both incubated in
350C. Colonies were evident at two to three days under
CO2 and three to four days aerobically on both blood
Iowa. Strain Mex 51 was obtained from Dr. C. Rigby,
Animal Diseases Research Institute, Nepean, Ontario.
All eleven isolates were identified
as Brucella sp. by Gram's stain, lack of hemolysis,
nonfermentation of lactose, production of catalase,
and agglutination with antirough Brucella antiserum.
The isolates were then examined using accepted pro-
cedures (1 1) as well as an extended range of dye and
antibiotic concentrations, additional phage types and
sensitivity tests. Tests performed were as
follows: CO2 requirement; H2S production;
production; growth in the presence of thionin or basic
1:200,000), thionin blue (1:500,000 and
penicillin (1, 2.5, 5, 10, 15, 20 and 25 U/mL); meso-
and 2 mg/mL);
Firenze (Fi), S708, Weybridge (WVb), Berkeley2 (BK2),
rough (R), rough ovis (R/O), and rough canis (R/G)
Brucella A, M and R antisera.
lysis by Tbilisi (Tb),
Quality control consisted of monitoring
media, antisera, phages and incubator conditions using
the following cultures: B. abortus biovar 1, strain 544;
B. abortus biovar 2, strain 86/8/59; B. abortus biovar
4, strain 292; B. abortus strain 19; B. suis biovar 1,
strain 1330; B. suis biovar 4, strain 40; B. ovis strain
63/290; B. canis strain RM/666.
The submitted isolates did not require C02, produced
trace amounts of H2S, hydrolysed urea in 15 minutes,
grew in the presence of meso-erythritol and reacted
with anti-Brucella R antiserum but
Brucella A or M antisera on the tube agglutinationtest.
The results of the dye and antibiotic tests are sum-
marized in Table 1. All eight isolates from the Poodles
Can Vot J Volumo 29, Fobruary 19.t5
Can Vet J Volume 29, February1988
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had the same growth characteristics on dye and anti-
biotic media. They did not grow on either thionin or
basic fuchsin at a concentration of 1:25,000, and grew
poorly at 1:50,000. On thionin blue, there was reduced
growth at 1:1,000,000 and total inhibition at 1:500,000.
Similarly, on safranin-O, reduced growth was observed
at 1: 10,000 and total inhibition at 1:5,000. There was
reduced growth on penicillin plates at 2.5 U/mL, and
total inhibition at 5 U/mL. The type strain, RM666,
gave almost identical results. The control strain
Mex 51 and the other three isolates (Labrador retriever)
grew in the presence of all dye concentrations tested,
as well as on penicillin plates at concentrations of
10 U/mL. Reduced growth on higher concentrations
of penicillin was also observed with these strains. On
the basis of dye and antibiotic sensitivity tests,
Mex 51 and these three isolates appeared identical and
formed a group distinctly different from that seen with
RM666 and the other eight isolates.
Phages Tb [Routine Test Dilution (RTD) and
104RTD], Fi(RTD and 104RTD), S708(RTD),
Wb(RTD), and BK2(RTD) did not lyse any of the
isolates. Rough canis (RTD and 104RTD) lysed all
isolates. Phages R and R/O occasionally had a weak
lytic effect at 104 RTD but not at RTD. This
phenomenon was observed with all isolates except
The most frequently described strains of B. canis
resemble RM666, the type strain for the species. Char-
acteristics of this strain include resistance to thionin
(1:25,000) but sensitivity to basic fuchsin (1:50,000),
safranin-O (1:5,000), thionine blue (1:500,000), and
penicillin (5 U/mL) (11,12). Recently, several strains
of B. canis have been described that grow in the
presence of basic fuchsin (1:25,000) (13,14). A number
of these strains, including Mex 51, originated in
Mexico. Our examination of Mex 51 in parallel with
RM666 showed that Mex 51 consistently grew on con-
centrations of basic fuchsin, safranin-O, thionin blue,
and penicillin that were inhibitory to RM666. Without
exception, dye and antibiotic results for the Canadian
field isolates matched those of either RM666 or
Mex 51. The eight isolates from the kennel exhibited
the same characteristics as RM666 while the isolates
from the Labrador retriever were indistinguishable
from Mex 51.
The properties of RM666, Mex 51, and all field
strains remained unchanged after seven to ten sub-
cultures under laboratory conditions and storage at
-70°C in litmus milk. This stability indicates that
strain differences might be useful epidemiological
markers. Histories available with these 11 isolates
support this concept. Poodles from the kennel had
extensive contact with dogs from the United States,
where typical (RM666) strains are predominant. The
Labrador retriever spent approximately 4.5 months in
Mexico and the southwestern United States prior to
his return to Canada and the development of canine
Brucella canis is usually classified as H25 negative
(12,15). It will, however, slowly produce H25 after
several days of growth (16). In our system RM666,
Mex 51 and all field strains produced trace amounts
of H2S after five days.
The smooth phages Tb, Fi, S708, Wb, and BK2 did
not lyse any of the B. canis isolates, while R/C (RTD
and 104RTD) lysed all of them. These results are
expected for this group of phages. The frequency of
occurrence of the weak lytic effect produced by
R/0(104RTD) was twice that of R(104RTD). Varia-
tions in the specificity of the rough phages, particularly
the R/O group, are known to occur and may be
reflected in these observations (13).
All field and control strains of B. canis described
here grew on serum-dextrose-tryptose agar with added
meso-erythritol at 1 and 2 mg/mL. Brucellae that are
inhibited by erythritol can be detected on this medium
but actual erythritol utilization is difficult to assess.
The growth of RM666 in trypticase soy broth was not
enhanced by the addition of erythritol and it was con-
cluded that RM666 does not utilize this substrate (12).
Recent oxidative-metabolic studies have determined
that Mexican isolates can utilize erythritol and con-
firmed that RM666 cannot, leading to the conclusion
that differences in erythritol utilization and basic
fuchsin sensitivity are sufficient to warrant division of
the species into two biovars (14). The results obtained
here support this suggestion.
We wish to thank the following employees of the
Agriculture Canada, Animal Pathology Laboratory in
Saskatoon: Angie Chudy and Carla Berry for their
work in biotyping; Irene Bubyn for competent and
timely word processing and Dr. W.D.G. (Bill) Yates
for editorial comments. We also wish to thank
Dr. Charlotte Rigby, Animal Diseases Research Insti-
tute, Nepean for her constructive critique of this work
and Cindy Jenkins, Alberta Veterinary Laboratory,
for information on
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Can Vet J Volume 29, February 1988