Brucella

Dr. Neha Sharma

Introduction

  • Brucella is a genus of Gram-negative, facultative intracellular coccobacilli.

  • Brucella species are the causative agents of brucellosis, also known as undulant fever, Malta fever, or Mediterranean fever.

  • Brucellosis is a major zoonotic disease, transmitted from animals to humans.

  • Humans acquire infection through:

    • Consumption of unpasteurized milk and dairy products

    • Direct contact with infected animals or animal products

    • Inhalation of infectious aerosols (laboratory exposure)

  • Common pathogenic species include:

    • Brucella melitensis

    • Brucella abortus

    • Brucella suis

    • Brucella canis

  • Brucella organisms have the ability to survive and multiply within macrophages, contributing to chronic infection.

  • The disease affects multiple systems, particularly:

    • Musculoskeletal

    • Reticuloendothelial

    • Genitourinary

    • Nervous system

  • Laboratory diagnosis relies on:

    • Culture

    • Serological tests

    • Molecular methods

  • Brucellosis is an important occupational hazard for veterinarians, farmers, abattoir workers, and laboratory personnel.

  • Control strategies focus on animal vaccination, food safety, surveillance, and public health education.

 

General Character

Genus: Brucella

Family

  • Brucellaceae

Species and Primary Hosts

  1. Brucella abortus

    • Primarily affects cattle

    • Causes abortion in animals and brucellosis in humans

  2. Brucella melitensis ⭐

    • Primarily affects goats and sheep

    • Most virulent species for humans

    • Common cause of severe human brucellosis

  3. Brucella suis

    • Primarily affects pigs

    • Associated with chronic and suppurative infections in humans

  4. Brucella canis

    • Affects dogs

    • Causes relatively milder disease in humans

Morphology and Staining Characteristics

Gram Staining

  • Gram-negative bacteria

  • Appear pink due to:

    • Thin peptidoglycan layer

    • Presence of outer membrane with LPS

Shape

  • Small coccobacilli

  • Short, plump rod-shaped organisms

Arrangement

  • Occur:

    • Singly

    • In small clusters

  • Do not form chains

Oxygen Requirements 

  • Obligate aerobes

  • Some strains (especially B. abortus) require 5–10% CO₂ for primary isolation

 Key concept:

  • Brucella species are facultative intracellular organisms,
    NOT facultative anaerobes

 

Morphology

1. Size

  • Very small bacteria

  • Measure approximately 0.5–0.7 µm × 0.6–1.5 µm

2. Shape

  • Coccobacilli

  • Short, plump rod-shaped organisms

  • Appear intermediate between cocci and bacilli

3. Gram Reaction

  • Gram-negative

  • Appear pink on Gram staining due to:

    • Thin peptidoglycan layer

    • Presence of outer membrane with LPS

  • Often poorly stained with routine Gram stain

4. Arrangement

  • Occur:

    • Singly

    • Occasionally in small clusters

  • Do not form chains or palisades

5. Motility

  • Non-motile

  • Lack flagella

6. Capsule

  • Non-capsulated

  • No true polysaccharide capsule

7. Spores

  • Non-sporing

8. Pleomorphism

  • Minimal pleomorphism

  • Fairly uniform morphology

9. Special Staining Characteristics

  • Weakly acid-fast

  • Demonstrated by:

    • Modified Ziehl–Neelsen stain (1% sulfuric acid)

  • Stamp’s modification:

    • Organisms stain red against a blue background

    • Commonly used in veterinary microbiology

 

Cultural Characteristics

Growth Requirements

  • Fastidious, slow-growing organisms

  • Obligate aerobes

  • Optimal temperature: 35–37 °C

  • Optimal pH: 6.6–7.4

  • Some strains (especially B. abortus) require 5–10% CO₂ for primary isolation

  • Visible growth usually appears after 3–7 days, may take up to 2–3 weeks

Culture Media Used

A. Blood Agar

  • Enriched medium

  • Supports growth of most Brucella species

  • Colonies appear after 48–72 hours or later

B. Chocolate Agar

  • Better recovery than blood agar

  • Commonly used for routine isolation

C. Brucella Agar (Selective Medium)

  • Enriched with:

    • Blood or serum

    • Vitamins

  • Selective agents:

    • Polymyxin B

    • Bacitracin

    • Nystatin

    • Vancomycin

  • Used for specimens with mixed flora

D. Castaneda Biphasic Medium ⭐

  • Contains:

    • Solid agar phase + liquid broth phase

  • Medium of choice for blood culture

  • Minimizes risk of laboratory contamination and aerosol exposure

Colony Morphology

On Blood / Chocolate Agar

  • Small (1–2 mm), round, smooth, convex colonies

  • Translucent, glistening

  • Non-hemolytic

  • “Honey-drop / dew-drop” appearance

Colony Types (Antigenic Phases)

Colony Type Species Characteristics
Smooth (S) B. abortus, B. melitensis, B. suis Smooth LPS, virulent
Rough (R) B. canis Rough LPS, less virulent

Growth on Other Media

  • MacConkey agar:

    • Usually no growth

    • If growth occurs → non-lactose fermenting colonies

  • Low nutrient media: Poor or no growth

 

Biochemical Reactions

A. Oxidase Test

  • Positive in all Brucella species

B. Catalase Test

  • Positive in all species

  • Helps survival within macrophages by neutralizing hydrogen peroxide

C. Urease Test 

  • Strongly positive

  • Rapid urease activity (within minutes to a few hours)

  • One of the most characteristic features of Brucella

D. Nitrate Reduction Test

  • Positive in most Brucella species

  • Helps in differentiation from other Gram-negative coccobacilli

E. Indole Test

  • Negative

F. Citrate Utilization

  • Negative

G. Hydrogen Sulfide (H₂S) Production 

Species H₂S Production
B. abortus Positive
B. suis Positive
B. melitensis Negative
B. canis Negative

 

Pathogenicity

Source of Infection

  • Infected animals:

    • Cattle (B. abortus)

    • Goats & sheep (B. melitensis)

    • Pigs (B. suis)

    • Dogs (B. canis)

  • Animal products:

    • Unpasteurized milk, cheese

    • Meat, placenta, aborted fetuses

Mode of Transmission

  • Ingestion: Unpasteurized dairy products (most common)

  • Direct contact: Through skin abrasions or mucous membranes

  • Inhalation: Aerosols (laboratory-acquired infection)

  • Rarely: Vertical transmission, blood transfusion

Portal of Entry

  • Gastrointestinal tract

  • Respiratory tract

  • Skin and conjunctiva

Virulence Factors

(Brucella lacks classic exotoxins)

A. Intracellular Survival Mechanisms 

  • Smooth LPS:

    • Poorly endotoxic

    • Inhibits phagolysosome fusion

  • Type IV secretion system (VirB)

    • Enables intracellular replication

  • Superoxide dismutase & catalase

    • Protect against oxidative killing

  • Urease

    • Aids survival in acidic environments

Pathogenesis 

  1. Entry into the host via ingestion, inhalation, or contact

  2. Phagocytosis by macrophages and dendritic cells

  3. Survival and multiplication within phagosomes

  4. Spread via lymphatics and bloodstream

  5. Localization in reticuloendothelial organs:

    • Liver

    • Spleen

    • Bone marrow

    • Lymph nodes

  6. Formation of granulomas

  7. Persistence leads to chronic infection

Host Immune Response

  • Cell-mediated immunity is crucial

  • Th1 response (IFN-γ) activates macrophages

  • Antibodies:

    • Useful for diagnosis

    • Limited role in protection

 

Laboratory Diagnosis

Specimen Collection 

A. Common Specimens

  • Blood (most important)

  • Bone marrow (highest yield)

  • CSF (neurobrucellosis)

  • Synovial fluid

  • Tissue biopsy (lymph node, liver)

B. Safety Note

  • Highly infectious organism

  • All specimens handled with BSL-3 precautions

Direct Microscopy

  • Gram stain:

    • Small Gram-negative coccobacilli

    • Low sensitivity

  • Not reliable for routine diagnosis

Culture Methods

A. Blood Culture

  • Castaneda biphasic medium – medium of choice

  • Incubation:

    • 35–37 °C

    • Aerobic

    • With 5–10% CO₂ (especially B. abortus)

  • Duration:

    • Up to 4–6 weeks

  • Subcultures done weekly

B. Bone Marrow Culture

  • Most sensitive culture method

  • Useful in:

    • Chronic cases

    • Prior antibiotic therapy

Automated Blood Culture Systems

  • BACTEC / BacT-ALERT

  • Faster detection (within 5–7 days)

  • Improved sensitivity and safety

Identification of Isolates

  • Colony morphology on blood/chocolate agar

  • Biochemical tests:

    • Oxidase positive

    • Catalase positive

    • Rapid urease positive

  • H₂S production and dye sensitivity (reference labs)

Serological Tests

A. Standard Agglutination Test 

  • Detects anti-Brucella antibodies

  • Diagnostic titer:

    • ≥1:160 (single sample)

    • Fourfold rise in paired sera

  • Limitation:

    • Cannot differentiate acute vs chronic

    • Prozone phenomenon

B. Rose Bengal Plate Test (RBPT)

  • Rapid screening test

  • High sensitivity

  • Used for:

    • Mass screening

    • Field surveys

C. 2-Mercaptoethanol (2-ME) Test

  • Detects IgG antibodies

  • Helps distinguish:

    • Active vs chronic infection

    • Past exposure

D. ELISA

  • Detects IgM, IgG, IgA

  • High sensitivity and specificity

  • Useful in:

    • Early diagnosis

    • Chronic and complicated cases

Molecular Methods

PCR

  • Detects Brucella DNA

  • Highly sensitive and specific

  • Useful in:

    • Early disease

    • Relapse

    • Culture-negative cases

Hematological Findings

  • Anemia

  • Leukopenia or leukocytosis

  • Raised ESR

  • Mild thrombocytopenia

 

Antibiotic Resistance

Natural Susceptibility Pattern

  • Generally susceptible to:

    • Tetracyclines (Doxycycline)

    • Rifampicin

    • Aminoglycosides (Streptomycin, Gentamicin)

    • Fluoroquinolones

    • TMP–SMX

Intrinsic Resistance

  • Brucella species are intrinsically resistant to:

    • β-lactam antibiotics (Penicillins, Cephalosporins)

    • Macrolides

  • Reasons:

    • Poor intracellular penetration

    • Low affinity for penicillin-binding proteins

    • β-lactamase production (low-level)

Acquired Resistance

  • True acquired resistance is uncommon

  • Occurs mainly due to:

    • Inadequate therapy

    • Poor compliance

    • Monotherapy

  • Reported resistance:

    • Rifampicin resistance (mutations in rpoB)

    • Fluoroquinolone resistance (rare)

Recommended Combination Therapy (WHO)

Clinical Situation Regimen
Uncomplicated brucellosis Doxycycline + Rifampicin (6 weeks)
Severe disease Doxycycline + Streptomycin
Neurobrucellosis Doxycycline + Rifampicin + TMP-SMX
Pregnancy / Children Rifampicin + TMP-SMX

Antibiotic Susceptibility Testing

  • Not routinely performed

  • Done in:

    • Treatment failure

    • Relapse

    • Epidemiological studies

  • Methods:

    • MIC determination

    • Molecular detection of resistance genes

 

Prevention

Control in Animals 

A. Animal Vaccination

  • Live attenuated vaccines used in animals:
    • B. abortus → Strain S19 and RB51
    • B. melitensis → Rev-1
  • Vaccination reduces:
    • Animal disease
    • Human transmission

B. Test and Slaughter Policy

  • Serological screening of livestock
  • Elimination of infected animals
  • Effective in non-endemic regions

Food Safety Measures

  • Pasteurization of milk
  • Avoid consumption of:
    • Raw milk
    • Unpasteurized cheese and dairy products
  • Proper cooking of meat

Occupational Protection

High-Risk Groups

  • Farmers
  • Veterinarians
  • Abattoir workers
  • Laboratory personnel

Preventive Measures

  • Use of:
    • Gloves
    • Masks
    • Protective clothing
  • Proper handling and disposal of animal products
  • Education and training

Laboratory Safety 

  • Brucella is a common laboratory-acquired infection
  • Requires:
    • Biosafety Level-3 (BSL-3) practices
  • Avoid aerosol generation
  • Use biosafety cabinets

Human Vaccination

  • ❌ No effective human vaccine available
  • Past trials discontinued due to:
    • Toxicity
    • Poor efficacy

Chemoprophylaxis

  • Not routinely recommended
  • May be considered after:
    • High-risk laboratory exposure
  • Drugs used:
    • Doxycycline + Rifampicin (short course)

Surveillance and Public Health Measures

  • Notification of cases
  • Monitoring of animal and human disease
  • Control of animal movement
  • Health education in endemic areas
  • Live attenuated vaccines used in animals:
    • B. abortus → Strain S19 and RB51
    • B. melitensis → Rev-1
  • Vaccination reduces:
    • Animal disease
    • Human transmission

 

MCQs

1. Brucella species belong to which family?

A. Enterobacteriaceae
B. Brucellaceae
C. Pasteurellaceae
D. Neisseriaceae

✅ Answer: B

2. Morphology of Brucella is best described as:

A. Gram-positive bacilli
B. Gram-negative cocci
C. Gram-negative coccobacilli
D. Acid-fast bacilli

✅ Answer: C

3. Most virulent Brucella species for humans is:

A. B. abortus
B. B. suis
C. B. canis
D. B. melitensis

✅ Answer: D

4. Brucella organisms are:

A. Obligate intracellular anaerobes
B. Facultative intracellular aerobes
C. Facultative anaerobes
D. Obligate anaerobes

✅ Answer: B

5. Brucella species are best described as:

A. Highly invasive extracellular bacteria
B. Toxin-producing pathogens
C. Facultative intracellular pathogens
D. Obligate intracellular pathogens

✅ Answer: C

6. Most common mode of transmission of brucellosis is:

A. Vector borne
B. Inhalation
C. Direct contact only
D. Ingestion of unpasteurized dairy products

✅ Answer: D

7. Brucella is weakly acid-fast due to:

A. Mycolic acid
B. Lipoarabinomannan
C. Cell wall lipid content
D. Capsule

✅ Answer: C

8. Best specimen for diagnosis of brucellosis is:

A. Urine
B. Blood
C. Throat swab
D. Stool

✅ Answer: B

9. Highest yield culture specimen in brucellosis is:

A. Blood
B. CSF
C. Bone marrow
D. Synovial fluid

✅ Answer: C

10. Culture medium of choice for blood culture in brucellosis is:

A. MacConkey agar
B. Blood agar
C. Lowenstein-Jensen medium
D. Castaneda biphasic medium

✅ Answer: D

11. Brucella species require which condition for optimal growth?

A. Anaerobic
B. Microaerophilic
C. Aerobic
D. Alkaline

✅ Answer: C

12. Which Brucella species requires CO₂ for primary isolation?

A. B. melitensis
B. B. abortus
C. B. suis
D. B. canis

✅ Answer: B

13. Brucella colonies on blood agar are:

A. Large, hemolytic
B. Mucoid and pigmented
C. Small, smooth, non-hemolytic
D. Dry and wrinkled

✅ Answer: C

14. Most characteristic biochemical test for Brucella is:

A. Oxidase negativity
B. Slow urease reaction
C. Rapid urease positivity
D. Sugar fermentation

✅ Answer: C

15. Brucella species are oxidase:

A. Negative
B. Weakly positive
C. Variable
D. Positive

✅ Answer: D

16. H₂S production is seen in:

A. B. melitensis
B. B. canis
C. B. abortus
D. All species

✅ Answer: C

17. Brucella does NOT ferment:

A. Glucose
B. Lactose
C. Sucrose
D. All of the above

✅ Answer: D

18. Main virulence factor of Brucella is:

A. Exotoxin
B. Capsule
C. Intracellular survival ability
D. Hemolysin

✅ Answer: C

19. Brucella survives intracellularly by inhibiting:

A. Phagocytosis
B. Complement activation
C. Phagolysosome fusion
D. Antibody production

✅ Answer: C

20. Type of immunity important in brucellosis is:

A. Humoral immunity
B. Complement mediated
C. Cell-mediated immunity
D. Innate immunity only

✅ Answer: C

21. Characteristic fever pattern in brucellosis is:

A. Continuous fever
B. Step-ladder fever
C. Undulating fever
D. Relapsing fever

✅ Answer: C

22. Most common system involved in brucellosis is:

A. Respiratory
B. Musculoskeletal
C. Gastrointestinal
D. Skin

✅ Answer: B

23. Most common cause of death in brucellosis is:

A. Neurobrucellosis
B. Hepatitis
C. Endocarditis
D. Septic shock

✅ Answer: C

24. Screening test for brucellosis is:

A. ELISA
B. PCR
C. Rose Bengal test
D. SAT

✅ Answer: C

25. Diagnostic titer in Standard Agglutination Test is:

A. ≥1:40
B. ≥1:80
C. ≥1:160
D. ≥1:320

✅ Answer: C

26. 2-Mercaptoethanol test detects mainly:

A. IgM
B. IgA
C. IgE
D. IgG

✅ Answer: D

27. Best test for chronic brucellosis is:

A. Blood culture
B. Rose Bengal test
C. ELISA
D. Weil-Felix test

✅ Answer: C

28. Gold standard for diagnosis of brucellosis is:

A. Serology
B. Culture
C. PCR
D. ELISA

✅ Answer: B

29. Brucella is a common cause of:

A. Hospital-acquired infection
B. Laboratory-acquired infection
C. Vector-borne disease
D. Nosocomial pneumonia

✅ Answer: B

30. Biosafety level required for Brucella handling is:

A. BSL-1
B. BSL-2
C. BSL-3
D. BSL-4

✅ Answer: C

31. Drug of choice for brucellosis is:

A. Penicillin
B. Doxycycline
C. Ciprofloxacin
D. Azithromycin

✅ Answer: B

32. Recommended treatment for uncomplicated brucellosis is:

A. Doxycycline alone
B. Rifampicin alone
C. Doxycycline + Rifampicin
D. TMP-SMX alone

✅ Answer: C

33. Duration of treatment in brucellosis is usually:

A. 1 week
B. 2 weeks
C. 3 weeks
D. 6 weeks

✅ Answer: D

34. Brucella shows intrinsic resistance to:

A. Tetracyclines
B. Rifampicin
C. Aminoglycosides
D. β-lactam antibiotics

✅ Answer: D

35. Relapse in brucellosis occurs due to:

A. Toxin production
B. Antigenic variation
C. Intracellular persistence
D. Spore formation

✅ Answer: C

36. Human vaccine against brucellosis is:

A. Available
B. Under trial
C. Live attenuated
D. Not available

✅ Answer: D

37. Animal vaccine used against B. abortus is:

A. Rev-1
B. RB51
C. BCG
D. S19 only

✅ Answer: B

38. Vaccine used against B. melitensis is:

A. S19
B. RB51
C. Rev-1
D. BCG

✅ Answer: C

39. Most effective preventive measure for brucellosis is:

A. Antibiotic prophylaxis
B. Human vaccination
C. Animal vaccination
D. Isolation of patients

✅ Answer: C

40. Pasteurization prevents brucellosis by:

A. Killing spores
B. Killing vegetative bacteria
C. Neutralizing toxins
D. Preventing fermentation

✅ Answer: B

41. Brucella LPS is best described as:

A. Highly endotoxic
B. Weakly endotoxic
C. Neurotoxic
D. Cytolytic

✅ Answer: B

42. Brucella canis shows which colony type?

A. Smooth
B. Mucoid
C. Rough
D. Hemolytic

✅ Answer: C

43. Brucella infection mainly involves which system?

A. Reticuloendothelial system
B. Respiratory system
C. Gastrointestinal tract
D. Skin

✅ Answer: A

44. Brucella species are non-motile due to absence of:

A. Pili
B. Capsule
C. Flagella
D. Fimbriae

✅ Answer: C

45. Which test differentiates IgM from IgG antibodies?

A. Rose Bengal test
B. SAT
C. 2-ME test
D. PCR

✅ Answer: C

46. Most common occupational group affected by brucellosis is:

A. Teachers
B. Healthcare workers
C. Veterinarians
D. Office workers

✅ Answer: C

47. Brucellosis is also known as:

A. Enteric fever
B. Malta fever
C. Dengue fever
D. Q fever

✅ Answer: B

48. Brucella species are killed by:

A. Cold storage
B. Pasteurization
C. Salting
D. Freezing

✅ Answer: B

49. Reason for prolonged therapy in brucellosis is:

A. Spore formation
B. Toxin production
C. Intracellular survival
D. Capsule formation

✅ Answer: C

50. Humans act as which type of host in brucellosis?

A. Definitive host
B. Intermediate host
C. Reservoir host
D. Dead-end host

✅ Answer: D