Diagnosis of Brucella Infection

Sample for brucellosis

1. It is done on the serum of the patient.
   1. The serum is taken in the first week of illness and later in the 3 to 4 weeks.
2. Brucella can be cultured from the blood, sputum, bone marrow, CSF, tissue, lymph node, and urine.

Indications for brucellosis:

1. The patient with a fever of unknown origin.
2. The patient with a suspected history of contact with cattle.
3. The patient with signs and symptoms of Brucella infection.

Definition of Brucellosis

• Three members of the genus Brucella produce an uncommon febrile disease known as Brucellosis (Undulant fever).
• It is a chronic granulomatous intracellular infection due to small gram-negative aerobic coccobacilli seen in:
  1. Brucella melitensis = By goats and sheep.
  2. Brucella suis = By swine.
  3. Brucella abortus = By cattle.
  4. Brucella canis = By dogs.
• It spreads by infected animals or eating contaminated animal products.

Brucellosis (Undulant fever) is found as follows:

1. Brucella suis is pig-associated.
2. Brucella melitensis is goat-associated.
3. Brucella Canis is dog-associated.
4. Brucella abortus is cattle-associated.

Microbiology of Brucellosis:

1. The Brucella abortus, B.suis, B. melitensis, or B.canis cause brucellosis.
2. Brucella is a slow-growing aerobic gram-negative coccobacillus.
   1. Short, slender, pleomorphic, gram-negative bacilli (coccobacillus).
   2. Brucella is an oxidase-positive urease variable.
   3. Brucella is nonmotile.
   4. These are non-sporing.
3. Culture media:
   1. Enriched medium such as glucose serum or liver infusion broth or agar.
      1. Small transparent colonies develop after several days of incubation at 37 °C  in aerobic conditions.
      2. CO2 is needed for the growth of B. abortus.
4. There is growth in selective buffered charcoal yeast extract and Thayer-Martin medium.
5. Brucella Canis is very rare and is due to exposure to dogs.

Brucella bacteria

The characteristic feature of the Brucella organism:

Mode of spread:

1. It is by ingesting contaminated milk and milk products, especially goat milk.
2. Their milk is also contaminated when sheep, goats, cows, or camels are infected with these bacteria.
3. These names are based on infected animals.
4. Brucella is mostly spread by unpasteurized milk or raw dairy products.
5. Direct puncture of the skin of butchers and farmers. These bacteria can enter through the skin and mucous membranes.
   1. Brucella can spread through inhalation.
   2. Brucella can penetrate the skin, conjunctiva, lungs, and GI Tract.
6. Brucella also spread among meat industry workers, especially with swine (pigs).
7. Veterinarians and dairy farmers are also at risk.
8. Penetration is followed by the lymphatics spread.
   1. There is facultative intracellular growth in the macrophages.
   2. It also involves the organs.

Source of the spread of Brucella infection

Types of Brucella:

1. There are the following types of Brucella:
2. Brucella abortus source is from cattle and has moderate pathogenicity.
3. Brucella melitensis source is sheep, and these are highly pathogenic.
4. Brucella Canis source is from dogs and has moderate pathogenicity.
5. Brucella Sui’s source is pigs, and these are highly pathogenic.

Antigenic structure:

1. Three species share two antigens, A and M.
   1. B. melitensis contains an excess of M antigen.
   2. B. abortus and suis contain an excess of A  antigen.
2. Monospecific antisera can be prepared and used for identification.

Clinical Signs and Symptoms of Brucellosis:

1. This may be the acute or insidious onset of the symptoms.
2. The acute or insidious onset of signs and symptoms characterizes this multisystem disease.
3. There may be fever, chills, and night sweating.
   1. The fever peaks in the evening and slowly returns to normal by morning.
   2. Undulant fever because of a slow rise in temperature during the day, declining at night.
4. There is undue fatigue.
5. There is anorexia and weight loss.
6. There may be a headache and arthralgia.
   1. 25% of the patients develop single-joint arthralgia.
   2. There may be pain in muscles, joints, or back. Myalgia may be the first symptom.
7. Sometimes there is lymphadenopathy. But most lymph nodes are not enlarged.
8. 20% of the patients may show splenomegaly.
9. Some patients may develop pneumonia.
10. If not treated, then these patients may have:
    1. Arthritis.
    2. Recurrent fever.
    3. Swelling of the scrotal area and testes.
    4. Depression.
    5. Chronic fatigue.
    6. May develop endocarditis.
    7. Splenomegaly and hepatomegaly are common findings.
    8. Spondylitis is also common.
11. These symptoms may last months to years, but the outcome is not fatal.

Complications of brucellosis:

1. The patient may develop endocarditis.
2. There may be arthritis and osteomyelitis.
3. The infection of the testes gives rise to epididymal orchitis.
4. CNS involvement may lead to meningitis or encephalitis.
5. Inflammation of the liver and spleen leads to splenomegaly and hepatomegaly.
6. Relapses occur within the first year in about 10% of the cases.

Prevention of Brucellosis:

1. Please avoid the following:
   1. Use of unpasteurized milk and dairy products (cheese, ice cream).
   2. When handling animals, use preventive measures like gloves, etc.

Diagnosis of Brucella Infection:

1. WBC count is usually normal or decreased.
   1. There is mild anemia.
   2. There is relative lymphocytosis.
2. Liver function tests are abnormal.
3. B. melitensis infection is more severe, giving more raised AST, ALT, and LDH, and may give rise to thrombocytopenia.
4. By serological test detecting the Brucella antibodies.
5. A skin test with brucellergin is available, but a positive result only indicates exposure to the organism and does not prove active disease.

Culture of Brucellosis:

1. Blood culture. This is positive in 30% to 40% of the cases.
   1. Blood culture for B. abortus needs 10% CO2.
   2. Blood culture must be kept for at least four weeks.
   3. About 70% are positive.
2. Bone marrow.
   1. Bone marrow culture may be positive when blood culture and serologic tests are negative.
   2. Bone marrow culture has more positivity.
3. CSF.
4. Sputum.
5. Food specimen.
6. Biopsy and culture of affected tissue and organs.
7. By immunofluorescent demonstration of the organism in the clinical specimens.

Serology values:

1. Test results >1:80 are suspicious for brucellosis. The serologic tests become positive during the second to the third week of illness.
   1.  90% of the patients have a titer of ≥1:160.
   2. The rising titer is diagnostic.
   3. False-negative results are rare.
2. In chronic localized brucellosis, the titer may be negative or ≤1:200.
   1. These tests remain for a long time positive after the infection is cured.
3. EIA is the method of choice to detect specific IgM and IgG antibodies.
4. Failure of the decline in the titer indicates an incomplete cure.
5. With a high titer, multiple blood cultures are positive.
6. Normal serological values:
7. Source 1
   1. This is the detection of the presence of antibodies against the Brucella antigen.
   2. Borderline cases  1: 160
   3. Significant level = >1:160
   4. Highly suggestive of active infection =  > 1: 320.
   5. The rising titer is of diagnostic significance.
   6. In chronic localized brucellosis, the titer may be negative or <1:200.
8. False-negative results are rare.
9. False-positive serological tests are seen with tularemia or cholera, or cholera vaccination or after brucellin-skin test.
10. Source 2
    1. A rising titer ≥ 1:160 suggests infection, either past or present.
    2. A single titer of 1:160 or 1:320 may be suggestive if there are clinical S/S of brucellosis.
    3. It can prevent the prozone phenomenon by diluting the serum  ≥1: 1280.
11. Antibody levels decrease in 3 months or with antibiotics but persist low for years, especially in chronic infection.
    1. Antibody titer may last for 1 to 2 years after the recovery.

Brucella serology

Skin test:

1. Brucellergen (these are killed bacteria) are injected into the skin. After 24 to 48 hours, it shows a>5 mm reaction.
2. A definitive diagnosis needs the isolation of the organism by blood culture or the tissue sample.
3. Real-time PCR is more specific.

Treatment of brucellosis:

• These bacteria are sensitive to tetracycline combined with streptomycin. This combination is the choice of treatment.

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