In this article we will discuss about Brucellae (Brucellosis):- 1. Morphology and Staining Brucellae 2. Cultural Characteristics of Brucellae 3. Pathogenicity and Clinical Features 4. Laboratory Diagnosis 5. Specimen 6. Antibody Levels 7. Detection of Animal Infection.
Contents:
- Morphology and Staining Brucellae
- Cultural Characteristics of Brucellae
- Pathogenicity and Clinical Features of Brucellae
- Laboratory Diagnosis of Brucellae
- Specimen of Brucellae
- Antibody Levels of Brucellae
- Detection of Animal Infection of Brucellae
1. Morphology and Staining Brucellae:
Brucellae are obligate, intracellular parasites, are Gram-negative coccobacillary forms, non-motile, non-sporing; some variants are capsulated.
2. Cultural Characteristics of Brucellae:
They are aerobic, grow at 37°C on Brucella agar, Albumin agar, Trypticase soy agar media. Only Br. abortus requires 5-10% CO2 Biochemically, carbohydrates are fermented without gas and acid. Some strains produce oxidases, catalase, H2S.
3. Pathogenicity and Clinical Features of Brucellae:
Human infection is commonly by ingestion of infected milk or by skin (contact) or by mucous membrane (droplets). Brucellae progress from the portal of entry via lymphatic’s, blood stream and are seeded into various organs (liver, spleen, bones); osteomyelitis, meningitis, cholecystitis, orchitis may occur.
Histologically, there is proliferation of mononuclear cells, exudation of fibrin, coagulation necrosis and peripheral fibrosis. The incubation period is 1-6 weeks. In acute stage, there is insidious onset with malaise, fever, weakness, ache, sweat; the fever rises in the afternoon, fails during night, is followed by drenching sweats.
There may be gastrointestinal or nervous symptoms. Spleen is palpable, hepatitis with jaundice. In chronic stage there is weakness, pain, low grade fever, nervousness.
4. Laboratory Diagnosis of Brucellae:
Blood, lymph nodes biopsy for culture and serum for serology are used. Blood can be cultured in diphasic trypticase soy agar medium in 10% CO2. Serology Tube agglutination test — 80 International units (IU) and above is positive and sensitive; 2-Mercaptoethanol test is not so sensitive; Skin test is unreliable.
Brucellosis is a systemic febrile disease with varied clinical manifestation. Clinical diagnosis is often difficult. Hence, Laboratory Diagnosis is of great necessity.
5. Specimen of Brucellae:
The most definite method of diagnosis is blood culture during acute phase of the disease. Urine, sputum, and milk may yield positive culture as the organisms are excreted intermittently in these specimens. In chronic stage, biopsied lymph node or bone marrow will become culturally positive.
(A) Blood culture can be done by Castaneda method using diphasic medium (both liquid—liver infusion broth; and solid medium—3% nutrient agar slope) in one Castaneda blood culture bottle. In this medium, the broth flows over the surface of agar slant when the bottle is tilted resulting in automatic subculture and incubated at 37°C under 5-10% CO2 for about 3-5 days to six weeks.
Blood culture becomes positive in 30-50 cases. Br. melitensis is more easily cultured than Br. abortus.
Identification:
Isolated organism is identified by dye test, biochemical tests, agglutination with mono-specific serum and lysis by bacteriophage.
(B) Serology:
When cultures are negative, serological tests are very useful in laboratory diagnosis. Antibodies against Br. melitensis and Br. abortus are detected. Ig M antibodies appear first and decrease in chronic stage, whereas Ig G appears later and persists longer. Ig M is mainly responsible for agglutination reaction and Ig G for complement fixation.
Incomplete (blocking) of Ig G and Ig A may prevent agglutination which can be detected by Coombs’ test.
(a) Direct agglutination test:
Diluted serum of patients is tested by slide or tube method against a brucella suspension. A titre of 80 IU or more is an indication of infection, latent active or past infection (Table): “Prozone phenomenon” (absence of agglutination in low dilution of serum due to high concentration of antibodies is common and hence a serial dilutes of serum is to be tested.
(b) Indirect agglutination test:
The agglutination can be prevented by blocking antibodies, therefore antibodies to brucella organisms can be detected by Coombs’ test. In this test, organisms are exposed to patient’s serum, incubated for half an hour and then centrifuged, washed and re-suspended in normal saline. The washed cells are treated with anti-human goblin which will cause agglutination.
(c) Screening test:
A loopful of patient’s serum is placed on a dried spot of stained brucella antigen. Antibody fixes the antigen (Castaneda strip test).
(d) Complement fixation test can detect blocking antibodies and is easier to perform than Coombs’ test.
(e) ELISA and RIA are valuable and very sensitive and can distinguish Ig M, Ig G and Ig A brucella specific antibodies and helpful to distinguish acute and chronic brucellosis.
Brucellin test (skin test) is an intradermal test carried out by using the extract of brucella for the detection of delayed hypersensitivity. An area of erythema and induration of 6 mm diameter within 24 hours indicates a positive reaction. It is not always reliable.
6. Antibody Levels of Brucellae:
1. Acute brucellosis:
Agglutination titres of 1,000 or more are seen. Both Ig M and Ig G are raised and antibody titres slowly decreased to low levels or zero with recovery.
2. Chronic brucellosis:
Ig G and Ig A are raised but not Ig M. Coombs’ test and CFT may be positive and direct agglutination test may become negative.
3. Healthy carriers:
In persons with occupational risk who separately come in contact with brucella organisms, the serological test for Ig G and Ig A becomes positive.
7. Detection of Animal Infection:
Direct agglutination (tube agglutination, plate agglutination test), Coombs’ test, CFT and skin test are useful in the diagnosis of brucellosis in animals. Milk and urine culture may give positive results. Milk Ring Test (MRT) can detect antibodies in milk.
Milk Ring Test:
It can detect brucella agglutinins in the milk of infected dairy cattle and is sensitive for testing the bulked milk supply from a herd:
1. One ml of pooled milk is taken in a 3 x 3/8 inch test tube.
2. A drop of concentrated suspension of Br. abortus or Br. melitensis stained with haematoxylin is added to the column of milk and mixed thoroughly by shaking.
3. The test tube with milk and stained bacterial suspension is incubated in a water bath at 37°C for 40-45 minutes till the cream rises.
Result:
In a positive test, the bacteria are agglutinated and rise with cream to form a blue line above the white skimmed milk, but if it is negative i.e. when brucella agglutinins are absent, the cream-line will be white and lies above a blue milk column.
Treatment, Prophylaxis:
Tetracycline or ampicillin is effective though combined treatment with streptomycin and tetracycline is more effective as brucellae are intracellular.
Prophylaxis:
Pasteurized milk should be used.