Brucella- zoonosis

1. BRUCELLA
Dr. V. S. Vatkar
Asso Prof
Microbiology Department

2. BRUCELLA
 Kingdom: BACtERiA
 PhyLUm: PRotEoBACtERiA
 CLAss:
ALPhA PRotEoBACtERiA
 oRdER: RhizoBiALEs
 FAmiLy: BRUCELLACEAE
 gEnUs: BRUCELLA

3. intRodUCtion
 Genus Brucella consists of
very small, aerobic ,non-motile, gram
negative coccobacilli, poorly grow in ordinary
media
Strict parasites of animals
i.e. goats, sheep, cattle, pigs, buffaloes, infection
transmitted to human by contact with infected
animal.

4. sPECiEs
1) Bruce (1887),isolated a small
microorganism from the spleen of
fatal cases in Malta.
This has been named Br.melitensis
2) Bang (1897),described
Br.abortus in Copenhagen,
the cause of contagious
abortion in cattle
3) Traum (1914), isolated Br.suis
from pigs, in USA
British Army physician
and microbiologist who
discovered Micrococcus
melitensis

5. AdditionAL sPECiEs
→Br.neotomae – isoltd.from desert wood rats in
USA
→Br.ovis – isoltd.from sheep in Australia
→Br.canis – isoltd.from dogs in USA

6. moRPhoLogy
 Gram negative coccobacilli
0.5 – 0.7 µ x 0.6 – 1.5 µ
motile
Non sporing
capsulated
Arranged singly or in short chains, non acid fast

7. CULtURAL
ChARACtERistiCs
 Strict aerobes
Optimum temp. 37°C ranging from 20°C -40°C
Optimum Ph is 6.6 to 7.4
 Can grow on ordinary media though growth is slow and
scanty
 Growth is improved by addition of serum, blood, glucose or
liver extract
 Br.abortus is capnophilic
 Br.suis is unaffected by CO2

8. CULtURAL ChARACtERistiCs
The media used are :
serum - dextrose agar
serum - potato – infusion agar
trypticase soy agar
tryptose agar
 The addition of bacitracin, cycloheximide, polymyxin B or
vancomycin make the media selective
 On solid media, colonies are small,moist,translucent and
glistening
 Mucoid,smooth and rough type of colonies appear,asso.
with change in antigenic structure & virulence

9. CULtURAL ChARACtERistiCs
 Brucellae grow on chorioallantoic
membrane of chick embryo & cause their
death in few days
 All grow intracellularly –
Br.melitensis in ectodermal cells
Br.abortus & Br.suis in cells of
mesodermal origin
Erythritol has a specially stimulating
effect on the growth of most strains of
Brucella

10. CULtiVAtion in PREsEnCE oF
dyEs
 The 3 species are differentiated on the
basis of their ability to grow in media
containing aniline dyes, like basic fuchsin
and thionin
→Br.melitensis is not inhibited by any of
the dyes
→Br.abortus is inhibited by thionin and not
by basic fuchsin
→Br.suis is inhibited by basic fuchsin and
not by thionin

11. BioChEmiCAL REACtions
 Catalase +
 Oxidase +
 Urease +
 Citrate –
 Indole –
 M.R. & V.P -
 Reduce nitrates to nitrites
 No carbohydrates are fermented though they
have oxidative capacity

12. RESISTANCE
Killed
 by heat at 60°C for 10 mins
 by pasteurization
 by 1% phenol in 15 mins
Survive
 in soil & manure for several weeks
 in refrigerated milk for 10 days, a month
in ice-cream & for months in butter
 Sensitive to direct sunlight and acid

13. ANTIGENIC STRUCTURE
 Two main somatic Antigens, A and M,are present
in varying amounts in the three major species
Antigen A is dominant in Br.abortus
Antigen M is dominant in Br.melitensis
Br.suis shows an intermediate antigenic pattern

14. ANTIGENIC STRUCTURE
The antigenic structure is more complex
Some strains behave biochemically as Br.abortus and some
serologically as Br.melitensis and vice-versa
A large no. of antigens have been identified by gel
ppt.technique
Antigenic cross reactions exist between –
 Vibrio cholera
 E.coli O:116 , O: 157
 Salmonella serotype N (O:30 antigen)
 Ps.maltophila
 F.tularensis
 Y.enterocolitica

15. BIOTYPES
 Br.abortus - 7 biotypes (1-9 )
 Br.melitensis - 3 biotypes
 Br.suis - 5 biotypes

16. PHAGE TYPING
 Several bacteriophages that lyse Brucella strains
have been isolated. All these phages are
serologically similar
 The Tblisi (Tb) reference phage is used
 At RTD and at 10000 RTD,it lyses Br.abortus
 Br.suis is lysed only at 10000 RTD
 Br.melitensis is not lysed at all

17. CLASSIFICATION
 Brucellae are classified on the basis of CO2
requirement,H2S production, sensitivity to aniline
dyes, agglutination by monospecific sera and by
phage lysis

19. PATHOGENICITY
 Br.melitensis is the most pathogenic
 Br.suis of intermediate pathogenicity
 Br.abortus is the least pathogenic
 Disease caused is Brucellosis
 Incubation period: 10-30 days but sometimes
prolonged.

20. PATHOGENICITY
 Mode of infection : Transmitted to Humans
1) By drinking contaminated or unpasteurised
raw milk or by ingestion of milk products from
infected animals
2) Direct contact with infected animal tissues –
butchers, dairy workers, farm workers &
veterinarians
3) Accidental ingestion,inhalation,injection and
mucosal or skin contamination may occur in
lab. workers

21. SOURCES OF BRUCELLA
INFECTION

22. PATHOGENICITY
 Types of infection :
1) Subclinical or latent infection - only serological
but no clinical evidence
2) Acute brucellosis – Known as undulant fever or
Malta fever. Mostly due to Br.melitensis & is
asso.with prolonged bacteraemia. Symptoms include
muscular & articular pains, nocturnal sweats,asthamatic
attacks,exhaustion,anorexia & constipation
3) Chronic brucellosis – Non-bacteraemic, symptoms
are related to a state of hypersensitivity. There is min.or
no fever at all but shows manifestations like sweating,
lassitude and joint pains

23. COURSE OF DISEASE
Bacilli
Lymphatic channels
Regional lymph nodes
Thoracic duct
Blood stream
Bacteraemia

24. PATHOGENICITY
 Primararily a disease of the RES
 May be demo. inside the phagocytic cells
 This accounts for their refractoriness to
chemotherapy & the coexistence of viable bacilli
with high levels of circulating antibodies

25. PATHOGENICITY
 Cell mediated immunity plays an imp. role in
recovery from brucellosis
 Brucella have a special predilection for placenta,
due to presence of Erythritol in the placenta,
which has a stimulating effect on brucella , in
culture

26. SPREAD OF BRUCELLAE IN THE
BODY

27. EPIDEMIOLOGY
 Brucellosis is worldwide in distribution
 Endemic in Mediterranean countries
 All Human infections in diff.parts of India
are due to Br.melitensis,acquired from
goats & sheep
 Serological survey in Haryana

28. LAB. DIAGNOSIS
 Varied clinical manifestations,
hence clinical diagnosis is very
difficult and sometimes
impossible
1) Culture
2) Serology &
3) Hypersensitivity tests
Specimens include :
Blood, bone marrow, liver ,
lymph node
Brucellae can occasionally be
isolated from
CSF,urine,sputum,breast
milk,vaginal discharge &
seminal fluid

29. LAB. DIAGNOSIS
 1) Blood culture :
Most definitive method for diagnosis
Blood collected during pyrexial phase.
5-10 ml blood is inoculated into 50-100 ml of
serum dextrose broth or trypticase soy broth or
liver infusion broth – incubated at 37°C ,in
presence of 5-10% CO2
Subcultures are made on solid media every 3-5
days, starting on the 4th
day
Growth is often delayed, so cultures should not
be declared negative in less than 6-8 weeks

30. LAB. DIAGNOSIS
 Castaneda’s method of
blood culture :
Contains both solid
(Trypticase soy agar)
and liquid (trypticase
soy broth) media in
the same bottle

31. LAB. DIAGNOSIS
 2) Serology :
Antibodies,IgG and IgM, appear in 7-10 days
after clinical infection. As the disease progresses,
IgM antibodies decline while IgG antibodies
persist or increase

32. LAB. DIAGNOSIS
a. Tube agglutination test :
Identifies mainly the IgM antibodies
Equal vol.of serial dilutions of patients serum
and the antigen (killed suspension of std.strain
of Br.abortus) are mixed & incubated at 37°C x
24 hrs
A single titre of more than 1:160 is evident of
brucella infection

33. LAB. DIAGNOSIS
 Several sources of error :
Sera often contains Blocking or Non-agglutinating
antibodies
Removed by heating the serum at 55°C for 30 mins or by
using 4% saline, as a diluent
Most imp. method for removing the blocking effect &
detecting the incomplete antibodies is ‘Antiglobulin test’ or
Coomb’s test
Several serum dilutions have to be tested as Prozone
phenomenon to high titres is frequent in brucellosis

34. LAB. DIAGNOSIS
b. Complement fixation test
( CFT) :
More useful in chronic
cases as it detects both
IgG and IgM antibodies
c. Castaneda strip test :
A surface fixation test
The antibody is able to
‘fix’ a coloured brucella
antigen, on a filter paper
strip, and prevent its
movement along the
paper

35. LAB. DIAGNOSIS
d. Indirect immunofluorescence :
Specific & a sensitive method for detecting
the antibodies
It may be positive even after the
agglutination test is negative
The procedure is too complex for its routine
use
e. ELISA & RIA :
Very sensitive & useful for screening of
antibodies & also for differentiation between
acute and chronic brucellosis
ELISA detects IgM and IgG separately

36. LAB. DIAGNOSIS
f. Huddleson’s opsonocytophagic test :
Based on the opsonic activity of brucella
antibodies but is not suitable for diagnostic
testing

37. LAB. DIAGNOSIS
 3) Hypersensitivity (Brucellin) test :
Delayed Type IV
An intradermal test using protein extract of the organisms
called Brucellin
Positive reaction is indicated by an erythema and
induration of 6 mm diameter, within 24 hrs.
Not useful for acute infections, as the test may remain
positive for years after acquiring infection
Brucellergen – is the antigen used for this test which is
commercially available

38. TESTS TO DETECT INFECTION IN
MILK
1) Rapid plate agglutination test
2) Rose Bengal card test
3) Milk ring test
4) Whey agglutination test

39.  MILK rING TEST :
Detects the Brucella antibodies in milk of infected dairy cattle
A sample of whole milk is mixed with a drop of stained brucella
antigen ,and incubated in water bath at 70°C for 50 mins
If antibodies are present in milk ,the bacilli are agglutinated and
rise with the cream to form a blue ring at the top, leaving the
milk unstained
If antibodies are absent ,no coloured ring is formed and the milk
remains blue

40. GENOME STrUCTUrE
 The genome of Brucella melitensis strain 16M is comprised
of 3,294,931 bp in two circular chromosomes. Chromosome
I has 2,117,144 bp and chromosome II has 1,177,787 bp;.
Genes encoding for DNA replication, protein synthesis,
core metabolism, and cell-wall biosynthesis can be found on
both chromosomes. It has been speculated that the second,
smaller chromosome might have evolved from a
megaplasmid
Brucella abortus has two circular chromosomes. The first
chromosome is 2,124,241 bp long with a G+C content of
57%.
The second chromosome is 1,162,204 with a 57% G+C
content. This species is able to cross the species barrier and
affects both livestock and humans. In livestock it causes
billions of dollars in losses due to miscarriages of cattle, in
humans it causes a undulant fever that has to be treated
with antibiotics. It is listed as a civilian, military, and
agricultural bioterrorism agent.

41. PrOPHYLAXIS
1. Persons handling the animals,
should use protective clothing &
gloves
2. Pasteurization of milk is done
3. Unimmunised infected animals
should be slaughtered
4. Vaccines :
Cattle are vaccinated with Live
attenuated Br.abortus strain 19
vaccine
Goats and sheep are vaccinated
with Br.melitensis Rev.1 vaccine or
killed H38 adjuvant vaccine
Br.abortus strain 19-BA – a more
attenuated variant of strain 19,has
been widely employed for Human
immunization in Russia, given
intradermally

42. TrEATMENT
 Tetracycline + streptomycin for 3 weeks
 WHO
Rifampicin 900 mg + Doxycycline 200 mg
single daily dose for 6 weeks