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Comparative evaluation of PCR and ELISA for diagnosing bovine brucellosis.


Brucellosis is an important zoonotic disease causing huge economic losses to the dairy farmers and is of public health relevance. It is a major cause of reproductive losses, abortions, placentitis, epididymitis and orchitis in animals. The disease is worldwide in distribution and is more common in countries with poor animal and public health programs (Capasso, 2002). It remains an uncontrolled problem in regions of high endemicity such as Africa, the Mediterranean, Middle East, parts of Asia and Latin America (Refai 2002). It is endemic in India and is prevalent in all states of the country including Punjab (Aulakh et al., 2008). Brucellosis is caused by the small, non-motile, non-spore forming, Gram negative facultative intracellular coccobacilli of genus Brucella. The clinical diagnosis is complicated by variable incubation period and absence of apparent clinical signs, except abortion. The current serodiagnostic methods like RBPT, STAT and CFT have their own limitations and are not fool proof. In order to control and eradicate brucellosis from humans and livestock it is essential to establish an accurate method for diagnosis of brucellosis.

While culture and isolation of Brucella spp. is regarded as the gold standard test for laboratory diagnosis of brucellosis, its sensitivity is low because the Brucella are fastidious micro-organisms that can easily be overgrown by other contaminating bacteria. More importantly, the procedure is associated with high risk of infection to laboratory personnel (Alton et al., 1988). Therefore, serological tests are often relied upon for the diagnosis of brucellosis.

The classical Rose Bengal Plate Test (RBPT) is often used as a rapid screening test for the diagnosis of brucellosis (Oomen and Waghela, 1974, Ruiz-Mesa et al., 2005). The sensitivity of RBPT is high but the specificity can be disappointingly low (Barroso-Garcia et al 2002 and Kiel and Khan, 1987). As a result, the positive predictive value of the test is low and a positive test result thus requires confirmation by a more specific test (Smits and Kadri, 2005). The RBPT could sometimes give a false positive result because of [S.sub.19] vaccination or of false positive serological reactions (OIE, 2009). Also, Gram negative bacteria such as Yersinia enterocolitica, Vibrio cholera, Campylobacter fetus, Bordetella bronchiseptica and Salmonella spp. may cross react with smooth Brucella spp. (Corbel et al., 1984).

Materials and Methods Collection of blood samples

Blood samples from 200 cattle and buffaloes were collected from Veterinary clinics, dairy farms and gaushalas, in and around Ludhiana, Punjab. All the animals were of age more than two years. About 10ml of blood was collected aseptically from the jugular vein of the animal. Serum was collected by centrifuging the clotted blood at 3000 rpm for 15minutes. ELISA and PCR were applied on all the serum samples.

Enzyme--Linked Immunosorbent Assay (ELISA)

A commercial kit--The IBL--America Brucella IgG Antibody ELISA kit (a) was used. The kit has been designed for the detection of anti-Brucella antibodies of IgG class in serum and plasma. The test was performed according to the instructions provided in the kit manual. The test serum samples were diluted 1:101 with ready-to-use sample diluent. Wells of microtiter plate were prepared for standards, controls and samples in duplicate as well as for a substrate blank. 100[micro]l each of the diluted serum samples were pipetted into the wells. Ready-to-use standards and controls were pipetted in the respective wells. One well was left empty as a substrate blank.

The plate was covered and incubated at room temperature for 60 mins. After the incubation, the wells were emptied by dumping the contents. Washing was carried out by pipetting 300 [micro]l of diluted washing solution into each well followed by discarding the contents. This procedure of washing was repeated three times, after which the rest of the washing buffer was removed by gently tapping the plate over a tissue paper. About 100 pl of ready-to-use conjugate was then pipetted into each well except the substrate blank. The plate was then covered with a plate cover and incubated at room temperature for 30 mins. After the incubation, the wells were emptied by discarding the contents. Washing was carried out three times, after which the washing buffer was removed by gently tapping the plate over a tissue paper. The ready-to-use substrate (100 [micro]l) was pipetted into each well. This time the substrate blank was also pipetted. The plate was covered and incubated at room temperature for 20 mins. To terminate the substrate reaction, 100 [micro]l of ready-to-use stop solution was pipetted into each well. The substrate blank was also pipetted. After thorough mixing and wiping the bottom of the plate, reading of the absorption was recorded with a spectrophotometer at 450 nm within one hour of the completion of the test.

Polymerase Chain Reaction (PCR)

PCR is considered as a sensitive, specific and rapid test for detecting a variety of pathogens. Using serum as a clinical sample for PCR is a new approach (Zerva et al., 2001).

The composition of buffer was as follows:

1x TBE--Working Buffer

10 x TBE: 10ml; DW: To make the volume up to 100ml.

Lysis Buffer

The lysis buffer contained 100mM KCl, 20mM Tris HCl (pH8.3), 5mM Mg[Cl.sub.2] 0.2mg of gelatin per ml and 0.9% polysorbate 20. 1g of agarose was dissolved in 100 ml of working buffer (1 X TBE) and Ethidium Bromide was added at 0.5 [micro]g/ml of 1 X TBE buffer.

Extraction of genomic DNA

For PCR, DNA was isolated from serum samples using the method described by Yamakami et al. (1996). 100 [micro]l serum sample was mixed with 100 [micro]l lysis buffer. Proteinase K was added to a final concentration of 60[micro]g/ml and the mixture was incubated for 60 mins at 55[degrees]C. Proteinase K was inactivated by heating the mixture to 95[degrees]C for 10 min followed by centrifugation at 12000 g for 10 min at 4[degrees]C. The supernatant was collected in a fresh centrifuge tube to which 0.1 volume of sodium acetate (3M) and 0.6 volume of isopropanol were added. The contents were mixed gently and kept on ice for 1 hour and then centrifuged at 8000 g for 10 min. the pellet was washed with 70% alcohol twice and dried at 37[degrees]C. Finally the pellet was suspended in 20-40[micro]l of Tris EDTA buffer and stored at -20[degrees]C till further use.

Primer sequences

The PCR assay was carried out using Brucella genus specific F4/R2 primers (Romero et al 1995). These primers are derived from 16S rRNA of B. abortus. The details of primers used are given in the Table 1.

The PCR reactions were carried out in a Gradient Thermal Cycler (Sensoquest, Germany) in a 25pl reaction mixture (Table 2) with the cycling conditions described in Table 3.

Agarose gels premixed with ethidium bromide were examined under UV rays using the Gel Documentation System (AlphaInnotech) to visualize the amplified products. The duration of the UV exposure was optimized and photographs were taken. The images were normalized with the AlphaImager software for better visibility. The amplicon size and concentrations were determined by comparing with the standard molecular ladder (GeneRuler 100 bp plus, Fermentas) which was run along with the samples.

Results and Discussion

Serological evidences are suggestive of high endemicity of brucellosis in India. Because of the costs, difficulty of performance, and lack of sensitivity of culture procedures, other methods of diagnosis are employed. In the present study, PCR was compared to ELISA for the diagnosis of bovine Brucellosis.

ELISA is considered to be a specific and sensitive test for Brucellosis (Radostits et al., 2000). All the 200 samples were subjected to ELISA using a commercial kit. Out of the 200 samples, 75 were detected as positive (Table 4).

PCR was carried out on all the 200 serum samples. However, only three samples showed a band of size 905bp, typical of Brucella spp (Fig. 1). Other samples failed to show positive reaction by PCR.

Keid et al. (2010) carried out a comparative study between blood PCR assay and serum PCR assay, where sensitivity of blood PCR was found to be 97.14% and that of serum PCR was 25.71%. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had a low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood PCR. In another study carried out by Takele et al. (2009), a comparison between PCR assay with serum and whole blood samples for detection and differentiation of Brucella melitensis was done. They suggested that though use of serum-PCR may lead to assay simplification and shorten turnaround time, the optimal clinical specimen for this test was not serum but whole blood, which leads to maximum assay sensitivity. Hafez et al. (2011) recommended the use of the blood PCR assay for accurate diagnosis of ovine brucellosis especially in the early stage of infection, which is difficult to achieve by the applied serological tests. The present study indicates that serum PCR is not a suitable method for screening animals for Brucellosis. Serum is not the appropriate material for carrying out the confirmatory PCR.


Alton, G.G., Jones, L.M., Angus, R.D. and Verger, J.M. (1988). Techniques for the brucellosis laboratory. INRA Paris.

Aulakh, H.K., Patil, P.K., Sharma, S., Kumar, H., Mahajan, V. and Sandhu, K.S. (2008). A study on the epidemiology of bovine Brucellosis in Punjab (India) using

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Capasso, L. (2002). Bacteria in two millenia-old cheese, and related epizoonosis in Roman populations. J. Infect 45: 122-27.

Corbel, M.J., Stuart, F.A and Brewer, R.A. (1984). Observations on serological cross-reactions between smooth Brucella species and organisms of other genera. Develop Biolo Standard 56: 341-48.

Hafez, S.M.A, Khaled, A., Razik, A.L., Hany, M., Hassan, L. and Ibrahim, G. (2011). Comparative diagnosis of ovine brucellosis using single step blood-PCR with old and new serological tools. African J. Microbiol. Res. 5: 3976-80.

Keid, L.B., Soares, R.M., Vasconcellos, S.A., Salgado, V.R., Megid, J. and Richtzenhain, L.J. (2010). Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis. Vet. Rec. 167:96-99.

Kiel, F.W. and Khan, M.Y. (1987). Analysis of 506 consecutive positive serologic tests for brucellosis in Saudi Arabia. J Clincal Microbiol. 25: 1384-87.

OIE. (2009). Bovine Brucellosis, Section 2.4.3. Manual of the Diagnostic Tests and Vaccines for Terrestrial Animals, vol. 1., 5th edition Office International Des Epizooties, Paris, France, pp. 624-59.

Oomen, L.J. and Waghela, S. (1974). The Rose Bengal Plate Test in human brucellosis. Tropical and Geographical Medicine 26: 300-02.

Radostits, O.M., Gay, C.C., Blood, D.C. and Hinchcliff, K.W. (2000). Diseases caused by Brucella spp. A Textbook of the Disease of Cattle, Sheep, Pigs, Goats and Horses. 9th Edn., London, Harcourt Publishers Limited. pp. 881-90.

Refai, M. (2002). Incidence and control of brucellosis in the Near East region. Vet. Microbiol. 90: 81-110.

Romero, C., Gamazo, C., Pardo, M. and Lopez-Goni, I. (1995). Specific Detection of Brucella DNA by PCR. J. Clin. Microbiol. 33: 615-17.

Ruiz-Mesa, J.D., Gonzalez, J.S., Reguera, J.M., Martin, L., Palmero, S.L. and Colmenero, J.D. (2005). Rose Bengal Test: diagnostic yield and use for the rapid diagnosis of human brucellosis in emergency departments in endemic areas. Clin. Microbiol. Infec. 11: 221-25.

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Takele, Belay, Y., Bejo, Khairani, S., Bahaman, Rani, A., Omar and Rahman, A. (2009). Comparison of PCR assay with serum and whole blood samples of experimental trials for detection and differentiation of Brucella melitensis. J. Anim. Vet. Advan. 8:1637-40.

Yamakami, Y., Hashimoto, A., Tokimatsu, I. and Nasu, M. (1996). PCR detection of DNA specific for Aspergillus species in serum of patients with invasive aspergillosis. J. Clin. Microbiol. 34: 2464-68.

Zerva, I., Bourantas, K., Mitka, S., Kansouzidou, A. and Legakis, N.J. (2001). Serum is the preferred clinical specimen for the diagnosis of human brucellosis by PCR. J. Clin. Microbiol. 39: 1661-64.

Shubhada Chothe, H.M. Saxena (1) and Deepti Chachra

Department of Veterinary Microbiology

College of Veterinary Science

Guru Angad Dev Veterinary and Animal Sciences University

Ludhiana--141004 (Punjab)

(1.) Corresponding Author


(a) - Brand of Immuno-Biological Laboratories, America

Table 1: Primer sequences used for
PCR amplification

Primer                      Sequence    Reference
                            Size of

F4        5'-TCGAGCGCCC                   Romero
          GCAAGGGG-3'        905bp        et al.
R2        5'-AACCATAGTG

Table 2: Brucella PCR reaction mixture

PCR components     Amount ([micro]l)

PCR Master Mix            12.5
Forward Primer            0.75
Reverse Primer            0.75
DNA Sample                 10
Distilled Water            1
Total Volume               25

Table 3: Brucella PCR programme used

Stage    Step               Temperature     Duration     No. of
                            ([degrees]C)                  cycles

I        Initial                 94           5 min         1
II       Denaturation            94          45 secs        35
         Annealing               53          45 secs
         Extension               72           5 min
III      Final Extension         72           5 min         1

Table 4: Analysis of Sera by ELISA and Serum PCR

ELISA Positive Samples           ELISA Negative Samples
(Animal Number)                  (Animal Number)

BE, BI, BS, BT, 2426, 2452,      J, BD, BJ, BK, BM, BO, BQ,
2467, 2490, 2554, 2567, 2581,    23(20/4/11), 34(20/4/11),
101(15/9/11), 104(15/9/11),      42(20/4/11), 51(20/4/11),
19(20/4/11), 32(20/4/11),        53(20/4/11), 54(20/4/11),
86(T 5/10/11), 87(T 5/10/11),    69(20/4/11), 72(20/4/11),
89(T 5/10/11), CD, CE, CH,       73(20/4/11),76(20/4/11),
CS, CX, DQ, DU, [T.sub.2],       79(20/4/11),80(20/4/11),
[T.sub.2062] (25/11/11),         83(20/4/11), 100(20/4/11),
H, I, Q, U, W, X, Y, Z, AH,      [T.sub.1], [T.sub.3], [T.sub.4],
AK, AM, AN, AO, AQ, AR, AS,      [T.sub.6], [T.sub.8], 2(1/3/11),
AU, AV, AW, AX, BW, BX, BZ,      6(1/3/11), 4(1/3/11),
CR, CT, CZ, DB, DC, DG, DJ,      1(1/3/11), 11(1/3/11),
DO, DP, 2489, 2574, 2582,        13(1/3/11) *, 19(1/3/11) *,
25(20/4/11), 88(T5/10/11),       20(1/3/11), 23(1/3/11) *,
80(T 5/10/11), 2413,             21(T 5/10/11), 76(T 5/10/11),
[T.sub.29] (25/11/11),           79(T 5/10/11), 82(T 5/10/11),
[T.sub.77] (25/11/11),           92(T 5/10/11), 5(13/10/11)
[T.sub.78] (25/11/11),           10(13/10/11), 11(13/10/11), A, B,
[T.sub.84] (25/11/11),           E, F, G, AT, BA, BB, BF, BH, BR,
[T.sub.86] (25/11/11), 2218,     CM, CN, CY, DD, DL, DN, DR,
2308, 2362, 2417.                12(13/10/11), [T.sub.81] (25/11/11),
                                 [T.sub.85] (25/11/11), K, L, M, N,
                                 O, P, R, S, T, V, AA, AB, AC, AD,
                                 AE, AF, AG, AI, AJ, AL, AP, AY, AZ,
                                 BC, BG, BL, BP, BY, CB, CI, CK, CV,
                                 16(20/4/11), 66(20/4/11),
                                 68(20/4/11), 70(20/4/11),
                                 71(20/4/11), 74(20/4/11),
                                 77(20/4/11), 78(20/4/11),
                                 81(20/4/11), 82(20/4/11),
                                 [T.sub.53] (1/3/11), 9(1/3/11),
                                 3 (13/10/11), 2379, 102(15/9/11),
                                 7(20/4/11), T7, 6(13/10/11),
                                 7(13/10/11), T 83 (25/11/11),
                                 T2061 (25/11/11), 29(15/9/11),
                                 40(15/9/11), 103(15/9/11),
                                 13(20/4/11, 15(20/4/11),
                                 17(20/4/11), 20(20/4/11).

* Only sera from animal numbers 13(1/3/11), 19(1/3/11) and
23(1/3/11) were positive by serum PCR.
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Title Annotation:Research Article
Author:Chothe, Shubhada; Saxena, H.M.; Chachra, Deepti
Publication:Intas Polivet
Article Type:Report
Geographic Code:9INDI
Date:Jan 1, 2013
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