Titers of Anti-Brucella Antibodies by Enzyme Linked Immunosorbant Assay in Vaccinated and Unvaccinated Brucellosis Infected Cattle

Brucellosis is an important re-emerging zoonotic disease caused by Brucella organisms. In the absence of a Differentiation of Infected from Vaccinated Animal (DIVA) assay for bovine Brucellosis, it becomes difficult to assess whether the anti-Brucella antibody response in an animal is due to vaccination or infection. We compared the anti-Brucella antibody titers of naturally Brucellosis affected unvaccinated cows, previously vaccinated infected cows, normal healthy vaccinated cows and healthy unvaccinated calves. The titers of anti-Brucella antibodies were estimated by indirect ELISA. The mean titer (log10) was found to be 1.518 ± 0.005 in case of naturally Brucellosis affected cattle which had been vaccinated during calf hood. The mean titer in case of naturally infected cattle which had never been vaccinated was 1.5441 ± 0.005. The mean titer in healthy unaffected cattle vaccinated during calf hood was 1.504 ± 0.002 and that of unvaccinated healthy calves was 0.560 ± 0.016. It was interesting to find that the antibody titers in naturally affected cattle which had never been vaccinated were very significantly (p < 0.01) higher than those of Brucellosis affected cows which had been vaccinated during calf hood. The titer in vaccinated infected cattle was very significantly (p < 0.01) higher than that of uninfected vaccinated cows.


Introduction
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 [1] in females and orchitis and epididymitis in males.The organisms are excreted in semen, uterine discharges, and milk [2].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 [3].

Materials and Methods
Permission of the Institutional Animal Ethics Committee (IAEC) was obtained and IAEC guidelines were followed throughout the study.

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.

Rose Bengal Plate Test (RBPT)
RBPT was carried out on sera as per the standard method [5].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.

Enzyme Linked Immunosorbant Assay (ELISA)
Serum samples of cattle were tested by ELISA [6] using Ingezim Brucella Bovina 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.

Statistical Analysis of Data
Data pertaining to serum antibody titers was statistically analyzed by ANOVA.

Results and Discussion
The antibody titers estimated by ELISA are presented in Tables 1-4; Figure 1.
The mean antibody titer (log 10 ) 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)   In a study [7] 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 How to cite this paper: Raj, S., Saxena, H.M. and Singh, S.T. (2017) Titers of Anti-Brucella Antibodies by Enzyme Linked Immunosorbant Assay in Vaccinated and Unvaccinated Brucellosis Infected Cattle.Open Journal of Veterinary Medicine, 7, 131-137.https://doi.org/10.4236/ojvm.2017.710013S. Raj et al.DOI: 10.4236/ojvm.2017.710013132 Open Journal of Veterinary Medicine

2. 0
Brucellosis serum ELISA test kit (Ingenasa).ELISA was carried out as per the S. Raj et al.DOI: 10.4236/ojvm.2017.710013133 Open Journal of Veterinary Medicine 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).

Figure 1 .
Figure 1.Antibody titers in infected and vaccinated cattle by ELISA.

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.