Brucella

for undergraduate and post graduate students
Pyrexia of unknown origin

1. Dr. Abhijeet Mane

2. Introduction and History Genus Brucella consists of very small, non motile, aerobic, GNCB Zoonosis. Primarily affecting goats, sheep, cattle, buffaloes, pigs Also known as Mediterranean fever, Malta fever, undulant fever Named after British Doctor, David Bruce Species causing human infections – melitensis , abortus , suis

3. David Bruce

4. Morphology Coccobacilli or short rods, 0.5-0.7 x 0.6-1.5um in size, arranged singly or short chains Maybe mistaken for cocci Non motile, non capsulated, non sporing Gram negative and non acid fast

5. Cultural characteristics Strict aerobes B.abortus is capnophilic , 5-10% CO2 Optimum temp. – 37 deg C, pH – 6.6-7.4 Simple media – growth is slow Serum dextrose agar, serum potato infusion agar, trypticase soy agar, tryptose agar

6. Selective – addition of bacitracin, polymyxin , cycloheximide Liquid media – growth uniform Solid media – colonies small, moist, translucent, glistening. Mucoid , smooth and rough types of colonies appear Erythritol – stimulating effect

7. Biochemical reactions Carbohydrates not fermented Catalase positive, oxidase positive

8. Urease positive

9. Nitrates reduced to nitrites Citrate not utilized Indole not produced MR and VP negative

10. Resistance Killed by Pasteurization Remain viable in refrigerated milk for 10 days, 1 month – ice cream, 4 months – butter, many weeks - meat

11. Antigenic structure Somatic antigen determinants – A and M Present in various amounts in 3 major species Antigenic cross reaction exist between brucellae and vibrio cholerae

12. Classification Classified into different species based on CO2 requirement, H2S production, sensitivity to dyes, agglutination by monospecific sera, phage lysis B.melitensis B.abortus B.suis

13. Bacteriophage Tblisi (Tb) phage designated as reference phage Routine test dilution lyses only B.abortus B.suis – lysed at 10000 RTD B.melitensis – not lysed at all

14. Pathogenicity All 3 major species of Brucellae are pathogenic to human beings B.melitensis – most pathogenic Primarily intracellular pathogen affecting RE system LPS component of cell wall - virulence factor Organisms from infected animal enter human body (thru wound, conjunctiva, inhalation, ingestion)

15. Incubation Period :– 10 – 30 days Organisms spread from site of infection thru lymphatic channels to local lymph glands Then spill over in blood stream Disseminated through out the body Predilection for placenta, due to erythritol , stimulates brucellae Fever, sweats and extreme fatigue - in 2-4 weeks

16. Human infection maybe of 3 types: Latent infection with serological but no clinical evidence Acute or sub acute brucellosis Chronic brucellosis

17. Acute brucellosis Mostly due to B.melitensis Known as undulant fever, maybe misleading Associated with prolonged bacteremia and irregular fever Symptoms – muscular pains, asthamatic attacks, nocturnal drenching sweats, exhaustion, anorexia, constipation, nervous irritability, chills Complications – articular, osseous, visceral or neurological

18. Chronic Brucellosis Maybe non bacteremic, low grade infection Symptoms generally related to state of hypersensitivity in the patient CF – sweating, lassitude, joint pain, with minimal or no pyrexia Illness lasts for years

19. Immunity Mainly CMI Activated macrophages can kill bacteria Th1 type T helper cell response and CMI required TNF alpha and gamma & IL 1 and 12 important mediators of protective response Tissue reaction - granuloma formation with EC, giant cells, lymphocytes and plasma cells Granulomas heal with fibrosis and sometimes calcify

20. Epidemiology Human infection acquired from animals Goats, sheep, cattle, buffalo Modes of infection: ingestion, contact, inhalation or accidental inoculation Most important vehicle: raw milk Ingestion; Milk products, meat and raw vegetables or water supplies contaminated by faeces or urine of infected animals

21. Contact infection: veterinarians, butchers, animals handlers Inhalation: of dried material of animal origin such as dust from wool Most human infections in various parts of India due to B.melitensis

22. Laboratory diagnosis Blood culture : Most definitive method for diagnosis Blood inoculated into bottle of trypticase soy broth or Brucella broth in a biphasic medium Castaneda method Cultures not be declared negative in less than 6-8 weeks Automated cultures become positive in 5-6 days

23. Castaneda method: Minimizes materials and manipulation Reduces chances of contamination Reduces risk of infection to laboratory workers Blood cultures positive in only 30-50% cases Bone marrow yield higher remain positive long after blood culture is negative

24. Other samples: Lymph nodes, CSF, urine, abscesses Occasionally, sputum, breast milk, vaginal discharges and seminal fluid

25. Serological methods Standard agglutination test (SAT) Tube agglutination test Performed most often Significant titre: 160 or more Most patients with acute brucellosis develop titres of 640 or more by 3-4 weeks

26. Sources of error Presence of blocking or non agglutinating antibodies Prozone phenomenon to high titres (Upto 1/640) is very common in brucellosis Hence several serum dilutions need to be tested False positive: cholera, tularemia, Yersinia infection Express agglutinin titres in IU

27. Both IgM and IgG antibodies appear in 7-10 days Agglutination test mainly identifies IgM Rise in titre is diagnostic

28. Complement fixation test More useful in chronic cases as it detects IgG also ELISA Sensitive and specific Can detect IgM and IgG separately Rapid methods: dipstick and rose Bengal card test

29. Delayed hypersensitivity-type skin tests With brucella antigens Detection in milk and infected animals: Microscopy of pathological specimens Immunofluorescence

30. Rapid methods Milk ring test Whole milk + stained brucella antigen (a conc. Suspension of killed B .abortus stained with hematoxylin) Incubated in water bath at 70 deg. C x 40-50 mins Positive: bacilli agglutinated, rise with cream to form blue ring at top , milk unstained Rose B engal card test and Rapid plate agglutination tests – screening infected herds

31. Prophylaxis Majority infections by ingestion of contaminated milk Checking dairy animals for brucellosis Pasteurization of milk B.abortus strain 19 vaccine protective in cattle No suitable vaccine for human use

32. Vaccination

33. Treatment Combination of Doxycycline for 45 days with i.m. Streptomycin daily for 2 weeks For children, cotrimoxazole with Rifampicin or Gentamycin