Academic journal article Iranian Journal of Public Health

Rocket and Two Dimensional Immunoelectrophoresis in Diagnosis of Caprine Brucellosis

Academic journal article Iranian Journal of Public Health

Rocket and Two Dimensional Immunoelectrophoresis in Diagnosis of Caprine Brucellosis

Article excerpt


Brucellosis is one of the most common zoonotic diseases that is endemic in rural areas of Mediter-ranean, Middle East and Latin American countries. This disease is caused by Brucella genus and many species of them are human pathogens (1). Brucel-losis can be transmitted from animals to humans in many ways: ingestion of infected meat or un-pasteurized dairy products, direct contact of bro-ken skin or mucous membrane with infected ani-mal tissues, and inhalation of infectious aero (2). Although, genitourinary complications such as epididymo-orchitis and prostatitis are seen in cases of human brucellosis, but, person to person trans-mission is still considered uncertain (3). Brucella organisms are Gram-negative facultative intracell-ular pathogens that may affect a range of different mammals, including man, cattle, sheep, goats, swine, rodents and marine mammals (4). Species infecting domestic livestock are B. melitensis (goats and sheep), B. suis (pigs), B. abortus (cattle and bi-son), B. ovis (sheep), and B. canis (dogs). In most host species, the disease primarily affects the repro- ductive system with a concomitant loss in produc-tivity of animals affected.

Ovine brucellosis is induced by B. melitensis along with B. ovis which can infect sheep, cattle, and sometimes humans (5). Infectious food-borne bru-cellosis usually result in humans when contami-nated or unpasteurized milk and cheese products are consumed (6). Therefore, control of brucellosis in animals, and thus prevention of human disease, depends on utilizing efficient diagnostic procedures. The diagnosis of brucellosis is mainly based on the detection of antibodies directed to the O-chain component of the lipopolysaccharide (LPS) antigen, expressed at the surface in Brucella species with smooth phenotype (4). Although the LPS antigens play the most important role in agglutination tests, other antigenic fractions of Brucella may be in-volved in other tests (7). Different methods are used for diagnosis and screening of animal and hu-man infected populations. The diagnosis of brucel-losis made by the isolation of Brucella species in blood cultures (BC) is successful in only 6-40 to 70-92% of cases (1, 8). Following Brucella infection, IgM appear within one week, reach a peak within 3 months and remain elevated for weeks to months. Moreover, IgG antibodies appear within 3 weeks of infection, they reach a peak within 6 to 8 weeks and they remain present, albeit at low levels, for months to years after the recovery of patients (9). Thus, laboratory diagnosis of brucellosis very often relies on detecting specific serum antibodies. Specific IgG antibodies can be detected by using of Coombs test, 2-mercaptoethanol (2-ME) slide agglutination test (SAT) and enzyme linked immunosorbent assay (ELISA) (10).

In this study, we tried to standardize two immunoe-lectrophoretic techniques, rocket and cross immu-noelectrophoresis, and compare the results obtained with those of BC, Rose Bengal, SAT, and 2-ME.

Materials and Methods

Serum samples

We used 20 sheep in two groups: Sera examined in this study were obtained, over a period of 125 days, from 15 female Mehraban sheep without any previous history of brucellosis vaccination which were infected with 1.5~108 CFU per sheep B. melitensis M16 (Razi Ins, Hesarak, Iran) subcutane-ously. Control sera were selected from other five healthy sheep without brucellosis who were not suffering from other bacteriological or parasitic diseases.

Cytosolic antigen preparation

Lyophilized B. melitensis M16 was cultured in Bru-cella broth (Sigma-Aldrich, Germany) at 37 cXC for 24 h. After recultivation of bacteria in blood agar and Brucella agar, the surface of agar was washed with normal saline, pH 7.2. The resulting suspen-sion was centrifuged at 3500 rpm for 30 min and then washed three times with normal saline pH 7.2. The washed bacterial cells and deionized wa-ter were mixed at a 1:1 ratio and disintegrated by sequential cold and hot extraction for 10 cycles of five min in liquid nitrogen of -196cXC and 10 min in a water bath of 60 cXC. …

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