Introduction Morphology Culture characteristics Biochemical reactions Epidemiology and pathogenesis Lab diagnosis of human brucellosis Lab diagnosis of brucellosis in animals Treatment Prophylaxis Summary References

1. PRESENTED BY-
DEEPESH AGGARWAL

2. Lesson plan
• Introduction
• Morphology
• Culture characteristics
• Biochemical reactions
• Epidemiology and pathogenesis
• Lab diagnosis of human brucellosis
• Lab diagnosis of brucellosis in animals
• Treatment
• Prophylaxis
• Summary
• References

3. Introduction
• The genus Brucella consists of Gram –negative coccobacilli
• They are strict parasites of animals and may infect humans
• Brucellosis is zoonosis affecting goats, sheep, cattle, buffaloes, pigs
• Transmitted to human by contact with infected animal and their products
• David Bruse (1886) isolated small microorganism from spleen of fatal cases
in Malta
• This was named Brucella melitensis (Brucella after Bruce and melitensis
after Melita)

4. • Species which cause human infection are:
- Brucella melitensis
- Br. abortus
- Br. suis
Other species cause animal infection are-
Br. ovis
Br. neotomae
Br. canis

5. MORPHOLOGY
• GRAM NEGATIVE COCCOBACILLI
• SIZE 0.5-0.7 X 0.6-1.5 MICRO METER
• NON SPORING
• NON-CAPSULATED
• ARRANGED SINGLY OR IN SHORT CHAINS
• NON MOTILE

6. Culture characteristics
• Strict aerobe
• Br. abortus is capnophilic
• Optimum temperature is 37
.
C(range is 20-40
.
C)
• pH is 6.6-7.4
• Serum dextrose agar, serum potato infusion agar , typticase soy agar
employed
• Addition of bacitracin, cycloheimide, polymyxin B makes the media
selective

7. • Colonies are small, moist, translucent, glistening

8. Biochemical reactions
• Catalase positive
• Oxidase positive(except Br. neotomae and Br. ovis )
• O/F test – oxidative
• Urease positive
• Nitrate reduced to nitrites
• Citrate not utilized
• Indole is not produced
• MR-VP negative

9. Resistance
• In milk they are killed by pasteurization
• Holder method
• Flash method

10. Brucella bacteriophage
• Several bacteriophages have been isolated
• All are serologically similar
• The Tbilisi phage designated as reference phage

11. EPIDEMIOLOGY
• Brucella is a zoonotic disease
• All three species of Brucella are pathogenic to human being
• Br. melitensis is the most pathogenic species followed by Br. suis of
intermediate pathogenesis and Br. abortus is least pathogenic
• In India, Br. melitensis is predominate pathogen of human brucellosis
acquired from goat or sheep followed by Br. abortus of cattle region

12. Pathogenesis
• Primarily an intracellular pathogen affecting the reticuloendothelial
system
• Can be demonstrated inside phagocytic cells
• This account for refractoriness in chemotherapy and exist as viable
bacilli with high level circulating antibodies
• Immunity in brucellosis is cell mediated
• Activated macrophage kills can kill bacteria, which is the most
important mechanism in recovery in brucellosis

13. Cont..
• Brucella spread from initial site of infection through lymphatic
channels to lymph gland, where they multiply
• Spill over into bloodstream and disseminated throughout body
• They have predilection for placenta due to erythritol, which has
stimulating effect on brucellae in culture

14. Mode of infection:
• Brucella are transmitted to humans by-
• direct or indirect contact with infected animals.
- Direct contact with infected animal tissue dairy workers and
veterinarians are particularly at risk
• drinking contaminated raw milk or by ingestion of milk product from
infected animals
• Accidental ingestion, inhalation, injection, and mucosal or skin
contamination may occur in laboratory personnel

15. Types of infection
• Human may infect by three types-
• A) subclinical or latent infection- there is no clinical evidence of disease but is
detectable only by serological test
• B) Acute brucelosis- also known as undulant fever or Malta fever.
-Associated with bacteremia
-Incubation period is 2-3 weeks may extend to 6 months
- characterized by fever, chills, shivering, headache, bone and joint pain and mild
lymph node enlargement
• C) Chronic brucelosis- persist for 6 months, usually non-bactaeremic. Symptoms
are generally related to a state of hypersensitity

16. CHRONIC BRUCELOSIS IN HUMAN

17. LABORATORY DIAGNOSIS OF HUMAN
BRUCELLOSIS
Specimen
• Blood for culture- Blood culture is most definitive method of diagnosis of
acute brucellosis
• Can be isolated from bone marrow, liver, lymph nodes and occasionally
from CSF, urine, sputum, breast, milk, vaginal discharge and seminal fluid

18. Blood culture
1) CONVENTIONAL METHOD
• Blood is collected during pyerxial phase because chances of positive
blood culture are more during this period.
• 50-100ml of serum dextrose broth or trypticase soy broth and
incubated are 37.C aerobically in presence of 5-10% CO2.
• Subcultures are made on solid media every 3-5 days for 8 weeks, and
incubated at 37.C in presence of 5-10% co2
• The need for frequent subculture can be avoided by use of
Castaneda’s method of blood culture.

19. Cont..
• It contain both liquid and solid media in the same bottle.
• The blood is inoculated into the broth and bottle incubated at 37.C
• For subculture bottle is tilted so that broth flow over the surface of
solid agar slant
• It reduced the chances of contamination

21. 2)AUTOMATED METHOD-BacT/ALERT is rapid method for blood
culture and majority of samples become positive with in 5-6
days.

22. Identification of growth
• Colony morphology
• Biochemical tests
• Agglutination with monospecific sera
• Lysis by phage

23. Serology
• Serological methods are more useful as cultures are negative in high
percentage of cases:
1) Standard Agglutination Test
2) Compliment fixation test
3) ELISA
• IgM and IgG appear in 7-10 days after onset of illness
• Agglutination tests identifies mainly the IgM antibody
• IgA and IgG antibodies may act as blocking antibodies and may prevent
agglutination

24. 1) Standard agglutination test
• It is tube agglutination test in which equal volumes of serial dilution
of patient’s serum and standardized antigen (killed suspension of
standard strain of Br. abortus) are mixed and incubated at 37
.
C for 48
hrs.
• Agglutinins are detected in patient’s serum by IgM or IgG
• In some sera a blocking factor may interfere with agglutination at low
serum dilutions
• False-negative result due to this prozone phenomenon can be
avoided by testing a series of two fold dilution of serum from 1-in-20
1-in-640 in 0.4% phenol saline
• As a prozone phenomenon to high titers (upto 1/640) is very
frequent, several serum dilution should be tested
• The blocking effect of non-agglutinating antibodies can be removed
by heating serum at 55
.
C for 30 minutes or using 4% saline

25. • False positives in SAT- may be seen in cholera, Yersinia or immunization
2) Compliment fixation test
-useful in chronic cases as it detect IgG and IgG
3) ELISA
• It is sensitive and specific to distinguish acute and chronic brucellosis
• It can detect IgG and IgM
4) Mercaptoethonal agglutination test
• 2-Mercaptoethanol reduces the disulphide bond link IgM molecule to release
subunit
• It eliminated the titer of those agglutinins that remain reactive to presence of ME
• Procedure is same as SAT except serum dilutions are prepared in0.85% NaCl
Containing ME 0.05 mol/liter

26. • Intradermal test using protein extract of brucella (brucellin)
• Delayed hypersensitivity to brucella antigen
• Useful in chronic brucellosis
SKIN TEST

27. LAB DIAGNOSIS OF BRUCELLOSIS IN ANIMALS
• Immunofluorescence
• Culture
• Rapid card test
• Rose Bengal test
• Milk ring test
• Whey agglutuination test

28. Immunofluorescence
• It is specific and sensitive method for detection of antibodies and positive
when agglutination is negative
Culture
• Pooled milk samples may be tested for detection
Rose Bengal test
• It is a simple, rapid slide agglutination assay performed with stained Br.
abortus suspension

29. Whey Agglutination Test
• Quantitative serological test for detection of brucella infection in
animals
Milk Ring Test
• It detects brucella agglutinin in the milk of infected animals

30. Animal Inoculation
• Guinea pigs are injected with milk samples from the animals. The
animals are killed after six weeks and the serum is examined and
spleen is used for culture of Brucella

31. Prophylaxis
• Pasteurization of milk
• Elimination of infected animals in slaughter
• Vaccine have been developed for animals
• Br. abortus strain 19 vaccine is protective in cattle
• No suitable vaccine is available for human use

32. treatment
• Combination of doxycycline for 45 days with streptomycin IM for first
2 week in adults
• In children- cotrimoxazole with gentamycin or rifampicin

33. SUMMARY
• Brucella is gram negative coccobacilli, non-motile ,non-capsulated
aerobic bacteria
• Strict parasite of animals
• Br. melitensis, Br. abortus, Br. suis major species
• Man acquire infection by contact with infected animals and products
• Disease caused by Brucella known as brucellosis
• Media used for culture are serum dextrose agar, serum potato
infusion, trypticase soy agar