Seroprevalence of caprine brucellosis in Karnataka

Aim: To study the seroprevalence of caprine bruellosis in Karnataka and compare the relative sensitivity and specificity among the different serological tests used. Materials and Methods: A total of 252 serum samples were collected from the goats of Karnataka and subjected to 5 different serological tests, i.e., Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (STAT), 2-mercaptoethanol test (2MET), Indirect ELISA (I-ELISA) and Dot-ELISA to detect the Brucella antibodies. Results: Test-wise, the seroprevalence in goats was 5.15% by RBPT, 6.34% by STAT, 1.98% by 2-MET, 9.52% by I-ELISA and 7.14% by Dot-ELISA. The prevalence of brucellosis was found to be highest among goats of northeast Karnataka followed by northwest Karnataka, central Karnataka and south Karnataka. I-ELISA detected maximum number of positive samples. Conclusions: The study used five serological tests to determine the apparent seroprevalence of caprine brucellosis in Karnataka. Taking I-ELISA as reference, the tests revealed the relative sensitivity values in the following order: DotELISA>STAT>RBPT>2-MET.


Introduction
as compared to B. abortus [3].In India, goats have been reported to account for the transmission of brucellosis to Brucellosis is a major bacterial zoonosis of maximum number of human cases and also caprine global, economic and public health significance, which brucellosis is shown to be endemic [5].poses a serious threat to the livestock economy.Brucellosis Brucellosis in goats has been reported from has shown a wide prevalence among livestock populavarious parts of the country including Karnataka [6].In tion all over the world.Caprine brucellosis due to Karnataka, goat husbandry is extensively practiced.Brucella melitensis which is widespread in India is a Based on the available literature with respect to major cause of abortion in goats and also accounts for prevalence of brucellosis in Karnataka, though caprine large number of human brucellosis cases [1,2], the brucellosis was found in discrete parts of the state, no source of infection for man being occupational individual study encompassing all the four regions of exposure during handling of unconfirmed cases of Karnataka is attempted yet.abortions and consumption of goat milk containing B.
Thus the present study aimed to apply multiple melitensis [3].The most incontrovertible method of tests like RBPT, STAT, MET, I-ELISA and Dot-ELISA diagnosis for brucellosis is by isolation and to determine the apparent prevalence of caprine brucellosis identification [4] of the organism though it has some in Karnataka and compare the relative sensitivities and limitations like low sensitivity, health risk to laboratory specificities among the different tests applied.personnel, time of sample collection and type of sample collected.As a result, recourse is taken in  overnight.The solution was then centrifuged at 10,000g for 15 min at 4°C and the supernatant was decanted.
At the end of incubation, the plates were washed thrice Another 80 ml of DW was added to the pellet, which with PBS-T as before and 100 µl of working dilution of was then stirred for 1 h and centrifuged as before.The anti-species conjugate (Bangalore Genei) tagged with two supernatant were pooled, filtered through HRPO (1:8000 for goat) was dispensed in each well membrane filter (0.3mm), and 50-100mg each of and the plates were incubated at 37°C for 1 h.The ribonuclease, deoxyribonuclease and proteinase K were plates were washed thrice with PBS-T as earlier.After added.This mixture was incubated for 18 h at 20°C.It last wash, 100 µl of substrate solution containing 6 mg was re-precipitated with methanol and resuspended as orthophenyl-diamine (OPD) (Sigma) and 4µl of H O 2 2 above in 2 ml of DW.The solution was dialyzed (30 %) in 10 ml of substrate buffer (Citrate buffer; pHextensively against DW until free of phenol.The 4.5) was dispensed in each well.Plates were kept in resultant antigen was lyophilized, weighed and resusdark for 15 min for color development.After 15 min, pended in DW to give 1mg LPS/ml.This was finally the reaction was stopped by adding 50 µl of 3M H SO 2 4 freeze dried in 1 ml volume and stored at 4°C for future and absorbance was measured at 492 nm in an ELISA use.The LPS so obtained by above method was further reader (MICROSCAN MS5605A9).In each microtitre dissolved at the concentration of 1mg/3ml in sterilized plate, strong positive, moderate positive, negative and DW, aliquoted in 200 µl volume and stored at -20°C.conjugate controls were included .The optical This was treated as the stock solution.For testing, the density (OD) of strong positive control (absorbance LPS stock solution was thawed and vortexed.A 10 µl of value between 1.000 and 1.500) was used to calculate stock LPS was dispensed into the 10 ml coating buffer the percent positivity (PP) value for test samples using (carbonate/bicarbonate buffer; pH-9.6), vortexed and the following equation: PP = (absorbance of test 100 µl per well was dispensed in flat bottom microtitre sample/absorbance of strong positive control) × 100.plates (Nunc).The plates were then incubated at 4ºC Serum sample having PP value > 62 in goats was taken overnight.Next day, the plates were washed thrice as positive.A cut-off value of 62 was determined based using the phosphate buffered saline (PBS; 0.01M; pHon the results of the analysis of the mean and standard 7.4) containing 0.05 % Tween-20 (PBS-T).Before the deviation (SD) of the total negative population in the final wash, 1:200 dilution of each test serum sample study.The mean of the test values from uninfected and control serum was prepared.After final wash, 100 animals + 2 SD was used as the rationale in deciding µl of each diluted serum was dispensed in the microtitre the cut-off for the I-ELISA [11] .plates in duplicate wells and incubated for 1 h at 37°C.The test was performed as described by Dot-ELISA.I-ELISA detected maximum number of Sharma et al. [12].LPS was dissolved at the concensera, i.e., 24 (9.52%) as positive (Table -1).Interestingly, tration of 1mg/1ml in sterilized DW and was stored atthere were 5 (1.98%) sera samples positive only to I-20°C.This was considered as stock solution.For test ELISA and negative in rest of tests, while 5 (1.98%) proper, the LPS stock solution was thawed and samples were positive to all the 5 serological tests vortexed.The optimal concentration of LPS antigen .It can also be seen from table that 8 (3.17%) was determined by checker-board titration fixed to sera samples were found positive to RBPT, STAT, I-62.5ng/µl and accordingly 1 µl of this antigen was ELISA and Dot-ELISA but negative to 2-MET.coated onto the centre of the nitrocellulose membrane Further, 2 (0.79%) samples were positive only to I-(NCM) strips.The strips were allowed to dry at 37°C ELISA and Dot-ELISA.The regional prevalence of for 2 hours.To block the unbound sites in the NCM, the brucellosis was found to be highest among goats of strips were incubated with 5% skim milk powder at 37° northeast Karnataka and lowest in south Karnataka C for 2 h.The blocked NCM strips were rinsed in PBS-( Table-4).I-ELISA detected most number of Brucella T for four times, dried and later, the strips were positive goats in all the different geographical regions incubated in the serum samples (1:200) at the 37°C for of Karnataka with highest prevalence in northeast 45 min.After incubation, NCM strips were washed Karnataka (10.90%) followed by northwest Karnataka with PBS-T for four times.Further, the NCM strips (10.00%), central Karnataka (9.09%) and south Karnataka were incubated with anti-species conjugate tagged (5.00%).Overall, the prevalence of brucellosis in goats with HRPO (1: 2000) for 45 min at 37°C.The plates was lowest in central Karnataka based on all the tests were washed four times with PBS-T.After last wash, employed.Taking I-ELISA as reference, the tests the NCM strips were dipped in substrate solution revealed the relative sensitivity values in order of Dotcontaining 6 mg diaminobenzidine (DAB) (Sigma) and ELISA>STAT>RBPT>2-MET.The Dot-ELISA emerged 4µl H O (30 %) in 10 ml of substrate buffer (Citrate as being more sensitive than RBPT, STAT and 2-MET 2 2 buffer; pH-4.5) for 5 min.The reaction was terminated with a relative sensitivity of 75%.The 2-MET showed by washing NCM strips with distilled water.The NCM least relative sensitivity of 20.82%.The relative sensistrips were air dried and reaction was observed for the tivity of RBPT was 54.16% and that of STAT was development of a brown spot (Figure -6).Appearance 66.66%.It is also interesting to note that all the four of brown spot was taken as positive.
tests showed 100 percent specificity.

Discussion
Of the 252 goat sera samples, 13 (5.15%)were The most incontrovertible diagnosis of brucellosis positive to RBPT, 16 (6.34%)to STAT, 5 to 2-MET is made by bacteriological isolation which has draw-(1.98%),24 (9.52%) to I-ELISA and 18 (7.14%) to backs like low sensitivity, time consuming and (DW) and heated to 66°C.An equal volume of phenol Veterinary Research Institute (IVRI), Izatnagar.The (90% v/v) in DW, also heated to 66°C, was added and RBPT, STAT and 2-MET were performed as described the solution was stirred continuously for 20 min.It was by Alton et al. [8].Appearance of agglutination within then cooled to 4°C and centrifuged at 12,000g for 20 4 min of mixing of antigen and serum was considered min at 4°C.The phenol phase (bottom layer) was as positive while absence of agglutination was recovered and filtered through Whatman #1 to which recorded as a negative result for RBPT (Figure-2).For three volumes of chilled methanol reagent was added.STAT (Figure-3) and 2-MET (Figure-4), the samples It was mixed thoroughly and left to precipi-tate at 4°C showing > 50% agglutination at a dilution of > 1:20 for 2 h.The precipitate was recovered by centrifugation (40 I.U.) were considered positive.at 12,000g at 4°C and resuspended in the 80 ml of DW I-ELISA: The ELISA [9] was performed using smooth and centrifuged at 6,000g for 20 min.The pellet was lipopolysaccharide (S-LPS) extracted from B. abortus resuspended in 80ml of DW and stirred at 4°C S 99.S-LPS was extracted from heat-killed cells of B.

For RBPT, STAT and 2-MET: The colored antigen for
RBPT the extraction, 5g of lyophilized cells of B. abortus and B. abortus plain antigen for STAT and 2-MET were strain 99 was suspended in 170ml of distilled water procured from Biological Products Division, Indian

Table - 1
: Prevalence rates by different serological tests

Table - 3
: Relative sensitivity and specificity with I-ELISA as reference

Table - 4
: Seroprevalence of brucellosis among goats (n=252) in different regions of Karnataka