Brucellosis is endemic in India and is prevalent in all parts of the country. It causes heavy economic losses to the industry through delayed conception, late-term abortions, and retention of placenta  in females and orchitis and epididymitis in males. The organisms are excreted in semen, uterine discharges, and milk  . The occurrence of the disease varies from 10% in marginal herds to 50% in organized farms.
The Brucella abortus strain 19 vaccine against bovine Brucellosis has been found to be very useful under most conditions but has some undesirable traits also  . Although vaccination with S19 is effective in preventing Brucellosis in cattle and buffaloes, it is not uncommon to find adult cattle vaccinated in calf hood with S19 vaccine, getting naturally infected with Brucellosis  . Although there is vast information on various aspects of bovine Brucellosis in the available literature, there is hardly any systematic study on the comparison of antibody levels between infected and vaccinated animals. We therefore compared the antibody titers of different categories of animals by ELISA to investigate if titers alone could indicate whether the animal is infected or vaccinated. ELISA has been claimed to be more sensitive followed by RBPT and STAT when applied to cattle sera. The present study offers valuable insight which could help in devising appropriate control strategies for this dreaded zoonosis.
2. Materials and Methods
Permission of the Institutional Animal Ethics Committee (IAEC) was obtained and IAEC guidelines were followed throughout the study.
2.1. Collection of Serum
Blood samples were collected from naturally infected clinical cases of Brucellosis in cattle which had never been vaccinated (6) identified in villages in and around Ludhiana district, from naturally infected animals vaccinated during calfhood (21), normal healthy (uninfected) vaccinated cattle (6) and healthy unvaccinated calves (6) of a dairy farm in Haibowal area, Ludhiana. Sera were separated from clotted blood and stored at −20˚C till further use for studying the antibody response of the animals.
2.2. Rose Bengal Plate Test (RBPT)
RBPT was carried out on sera as per the standard method  . Equal volumes (25 μl each) of RBPT colored antigen (Punjab Veterinary Vaccine Institute, Ludhiana) and test serum were mixed on a clean glass slide. The slide was observed till 3 min. for formation of clumps. Formation of clumps (agglutinate) indicated a positive reaction while the absence of clear clumps was considered as a negative reaction.
2.3. Enzyme Linked Immunosorbant Assay (ELISA)
Serum samples of cattle were tested by ELISA  using Ingezim Brucella Bovina 2.0 Brucellosis serum ELISA test kit (Ingenasa). ELISA was carried out as per the manufacturer’s instructions accompanying the kit described below.
a) 90 μl of diluent was added in each well of the plate except the ones destined as controls. 10 μl of diluted individual sample (1/10) was added to the remainder wells of the plate.
b) 100 μl of the controls provided in the kit were added. Plate was incubated for 1 hour at room temperature (RT, 20˚C - 25˚C).
c) The plate was washed three times with 300 μl of washing solution.
d) 100 μl of the conjugate was added to each well and plate was incubated for 30 minutes at room temperature. The plate was washed three times with 300 μl washing solution.
e) 100 μl of substrate was added to each well and plate was kept in dark for 10 min at RT.
f) 100 μl of the stop solution was added to each well.
g) Optical density (O.D.) of each well with spectrophotometer was done at 450 nm within 5 minutes after the addition of stop solution.
The test was valid when: O.D. value for positive control serum was ≥ 1.0, and was considered negative when the OD of the control serum was ≤ 0.2.
Cut off = OD450 nm positive Control × 0.4 = 40% positivity
2.4. Statistical Analysis of Data
Data pertaining to serum antibody titers was statistically analyzed by ANOVA.
3. Results and Discussion
The antibody titers estimated by ELISA are presented in Tables 1-4; Figure 1. The mean antibody titer (log10) in case of Brucellosis affected cattle which had been vaccinated during calf hood was found to be 1.518 ± 0.005 (Table 1). The mean titer in case of naturally infected cattle which had never been vaccinated was 1.5441 ± 0.005 (Table 2). In case of healthy unaffected cattle vaccinated during calf hood the mean titer was 1.504 ± 0.002 (Table 3) and in unvaccinated healthy calves it was 0.560 ± 0.016 (Table 4). It was interesting to find that in naturally affected cattle which had never been vaccinated previously the antibody titers were very significantly (p < 0.01) higher than those of Brucellosis affected cattle which had been vaccinated during calf hood and normal healthy vaccinated cattle unaffected by the disease. The mean titer in Brucellosis affected cattle which had been vaccinated during calf hood was very significantly (p < 0.01) higher than that of normal healthy vaccinated cattle unaffected by the disease.
The differences between mean titers of vaccinated infected and unvaccinated infected, vaccinated infected versus vaccinated healthy, vaccinated infected versus unvaccinated healthy, unvaccinated infected versus vaccinated healthy, unvaccinated infected versus unvaccinated healthy and vaccinated healthy versus unvaccinated healthy were very significant (p < 0.01).
The titers in unvaccinated infected cattle were very significantly (p < 0.01)
Table 1. Titers of anti-Brucella antibody by ELISA in infected cattle vaccinated in calfhood.
Table 2. Anti-Brucella antibody titers by ELISA in unvaccinated infected cattle.
Table 3. Anti-Brucella antibody titers by ELISA in healthy vaccinated cattle.
Table 4. Anti-Brucella antibody titers by ELISA in unvaccinated healthy calves.
Figure 1. Antibody titers in infected and vaccinated cattle by ELISA.
higher than those of infected vaccinated, healthy vaccinated and healthy unvaccinated animals. The titers in vaccinated infected cattle were very significantly (p < 0.01) higher than those of healthy vaccinated and healthy unvaccinated animals.
In a study  the performance of an ELISA for detection of total antibodies to Brucella spp. was compared with that of the Rose Bengal Plate Test, standard tube agglutination test and Coombs test in the diagnosis of Brucellosis. ELISA was the most sensitive test (97%), showing greater specificity (96%) and good predictive positive and negative values (98% and 94% respectively). ELISA was the only positive test in 6% of patients in whom Brucellosis had been confirmed by culture. ELISA has been claimed to be more sensitive followed by RBPT and STAT when applied to cattle sera  .
Assessment of immunological data from Brucella infected cattle can be helpful in charting the disease process, diagnosis and prognosis and may help in understanding the pathophysiology of disease. It was reported  that naturally affected animals have very high titers of agglutinating antibodies than the vaccinated animals.
Numerous outer and inner membrane, cytoplasmic, and periplasmic protein antigens of Brucella have been characterized, yet the antigen that dominates the antibody response is Lipopolysaccharide (LPS). Some antigens are recognized by the immune system during infection and are potentially useful in diagnostic tests.
The role of humoral immunity against intracellular bacterial infections is limited and may not be protective. Antibody mediated opsonization by immunoglobulins (IgM, IgG1, IgG2a and IgG3) enhances phagocytic uptake of bacteria, limiting the level of initial infection with Brucella but has little effect on intracellular course of infection   . The L7/L12 ribosomal proteins are important in stimulating cell-mediated responses  .
Since the infected animals in the first group were the ones who had already been vaccinated during calf hood, the infection in these animals may suggest that the vaccine was unable to induce adequate protective levels of antibody. Secondly, the heightened antibody response after infection in vaccinated animals may indicate a secondary immune response to Brucella antigens against which the lymphocytes were primed by calf hood vaccination.
The anti-Brucella antibody titers by ELISA in naturally affected unvaccinated cattle were very significantly higher than those of Brucellosis affected vaccinated cattle and normal healthy vaccinated cattle unaffected by the disease.
Conflict of Interest
The authors declare that there is no conflict of interest.
The study was funded by a grant under RKVY scheme (PI: Prof. H M Saxena).
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