Comparative evaluation of three serological tests for the detection of Brucella antibodies from infected cattle herds

ABSTRACT

Three serological methods used in the diagnosis of bovine brucellosis were conducted to assess the diagnostic sensitivity of the tests in brucellosis-infected herds. A total of 487 unvaccinated bovine serum samples collected from infected herds in the southeast region of Turkey having history of abortion with a known bacteriological status for the last three years were tested for anti-Brucella antibodies detection using the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and indirect enzyme-linked immunosorbent assay (i-ELISA). A protein A/G conjugate was used in i-ELISA in order to reduce background reactivity and to use the test in multispecies if required. The serum samples were found as positive in 74.3%, 83.7% and 81.3% when we employed the CFT, i-ELISA and RBPT, respectively. i-ELISA could be used as a single test or combined with the RBPT for serologic diagnosis for brucellosis in infected herds.

KEYWORDS:

• Brucella
• ELISA
• serology
• complement fixation test

1. Introduction

Brucellosis is a common zoonotic disease that has important veterinary and public health concerns and economical impact. The control and eradication of brucellosis is primarily based on the serological testing of animals (Nielsen 2002; OIE 2016). However, the isolation of Brucella spp. from cattle with positive serology is the only definitive diagnosis of the disease. Sera from different stages of infection may not give positive results and there is currently no single serological test to detect all stages of infection. For this reason, a combination of screening and confirmatory test is generally used in order to detect infection status more accurately (Nielsen 2002; McGiven et al. 2003).

Although several serological tests can be used for the diagnosis of bovine brucellosis, the Rose Bengal plate test (RBPT) and the complement fixation test (CFT) are the official tests currently used for serologic diagnosis of the disease. RBPT is a simple plate agglutination test mainly used as a screening test in cattle. The low pH of the antigen prevents partial agglutination by IgM and then reduces non-specific reactions. CFT has been regarded as a specific confirmatory test especially because it detects almost only IgG1 (Nielsen 2002). But CFT is technically a complex test, in which various different test reagents should be titrated against each other and all control reagents should be included in each set of the test. Anticomplementary activity needs heat inactivated serum samples and prozone phenomenon are the other limitations. Besides, it requires good laboratory facilities and well-trained personnel to accurately perform the test and to maintain its reagents. CFT is also a prescribed test for international trade recommended by the World Organisation for Animal Health (OIE 2016). So there was a need for simpler, sensitive and specific serological tests. A large number of indirect enzyme-linked immunosorbent assays (i-ELISAs) with numerous modifications have been reported in the literature. i-ELISA has been shown to be a highly sensitive and suitable test for large-scale screening for bovine brucellosis. Different i-ELISA techniques were the results of different antigens used and conjugates chosen for the test (Sutherland et al. 1986; Wright et al. 1990; Jacques et al. 1998; Garcia-Bocanegra et al. 2014; O’Grady et al. 2014).

The present study attempted to evaluate and compare three serological tests for anti-Brucella antibodies of cattle from infected herds.

2. Materials and methods

2.1. Samples

A total of 487 bovine sera were collected from 27 unvaccinated infected herds where Brucella abortus had bacteriologically been demonstrated in the southeast region of Turkey between April 2013 and February 2016. Ten millilitre of whole blood in a tube without anticoagulant was collected from each animal by jugular venipuncture. Collected blood samples were transported to the laboratory in cold chain. Samples were centrifuged at 3000 rpm for 10 min and then samples were stored at −20°C until analysed.

2.2. Conventional serological tests

The RBPT and CFT were performed according to the procedures recommended by the OIE (2016).

2.3. Rose Bengal plate test

Equal volumes of antigen and serum samples were mixed on a clean slide by a stirring stick. The slide was manually rotated for 4 min and examined for any degree of agglutination. When noticed, The RBPT was considered as positive.

2.4. Complement fixation test

All reagents of CFT were obtained commercially (Virion-Serion, 1297, Würzburg, Germany). The assay was carried out in a microtitre format by cold fixation with two units of complement. Test sera and positive (OIEISS) and negative sera were diluted twofold in Veronal Buffer (VB) starting from 1:5 dilution. Diluted serum samples (1:5) were inactivated at 58°C for 50 min. The minimum haemolytic dose (MHD) was estimated for each test set-up using 2% sensitized sheep red blood cell (SRBC) in VB. Two MHD units were used throughout the test. The end point titre was taken as the first well showing approximately 50% lysis of SRBC. Serum dilutions of 1:5 or higher giving a titre equivalent to 20 international CFT unit (ICFTU)/ml or more were considered as positive for the CFT.

2.5. Indirect enzyme-linked immunosorbent assay

Antigen preparation from B. abortus S99 was carried out in a biosafety level 3 laboratory. Ninety-six-well polystyrene microplates (Nunc 69620, USA) were coated with 100 µl B. abortus S99 SLPS (smooth lipopolysaccharide) antigen at a dilution of 1:6000 in 0.05 M carbonate buffer, pH 9.6 for 18–24 h at 4°C. All the wells were rinsed five times with the washing solution (0.1 mM disodium hydrogen orthophosphate, 0.01% Tween 20, PBS/T). The control and test sera were diluted 1:100 in the diluting buffer (PBS, 0.05% Tween 20, 0.1% phenol red, pH 7.3) and 100 µl diluted serum samples were applied to respective wells in duplicate. The plates were then incubated for 30 min at room temperature (RT) on a rotary shaker. After five washes in PBS/T, protein A/G horseradish peroxidase conjugate (Thermo Scientific, 32490, IL, 61101, USA) was added – 100 µl/well – and incubated for 1 h at RT. Finally, after five washes in PBS/T, 100 µl of chromogenic substrate (10 mg OPD (o-Phenylenediamine) tablet, (P-8287, Sigma, St. Louis, MO, USA) in 25 ml of 0.05 M phosphate-citrate buffer pH 5.5, with 10 µl 30% H₂O₂) per well was added. The plates were shaken continuously on an orbital shaker for 15 min prior to reading at OD₄₅₀ nm in a microplate reader (VERSAmax 3.13/B2573). The cut-off level was determined by adding three standard deviations to the mean of the OD of negative controls. Any samples with ODs greater or equal to this value were considered positive.

3. Results

This study focused on the results of an i-ELISA in comparison with those obtained in conventional tests. Of the 487 bovine sera, 362 (74.3%), 408 (83.7%) and 396 (81.3%) were found to be positive by the CFT, i-ELISA and RBPT, respectively. In our study, i-ELISA gave the highest number of positive results followed by the RBPT. The lowest positivity rate was obtained by the CFT (Table 1). i-ELISA detected anti-Brucella antibodies in 15 serum samples which were found negative by the RBPT. On the other hand, three of the serum samples which were found as positive by the RBPT were found negative by i-ELISA. Seven of the bovine serum samples found positive by both i-ELISA and the CFT were found negative by the RBPT (Table 2).

Table 1. Serological test results of the serum samples from infected cattle herds.

Total serum number	Serological tests
	RBPT		CFT		i-ELISA
	Positive %	Negative %	Positive %	Negative %	Positive %	Negative %
487	396 (81.3)	91 (18.7)	362(74.3)	125 (25.7)	408(83.7)	79 (16.3)

Table 2. Comparison of serological test results.

Number of serum samples	RBPT	CFT	i-ELISA
355	+	+	+
79	−	−	−
38	+	−	+
8	−	−	+
7	−	+	+
3	+	−	−
Total	396	362	408

4. Discussion

Bovine brucellosis is an economically important zoonotic infection resulting in abortions, stillbirths and infertility and continues to remain endemic in many countries, including Turkey. Brucellosis also constitutes a serious public health threat to consumers of milk and milk-products (İyisan et al. 2000; Çelebi et al. 2016; OIE 2016). The diagnosis of the disease is largely based on serological testing although the bacterial identification is always ‘gold standard’.

By comparing the overall results of these three serological tests for brucellosis in the southeast region of Turkey, it can be seen that the CFT gave the lowest positive percentage (74.3%), whereas i-ELISA showed the highest positivity rate (83.7%) (Table 1). In many countries, the RBPT is used as a screening test, followed by the CFT as a confirmatory test to detect brucellosis in an infected animal (Glynn & Lynn 2008). The present study showed a higher sensitivity of i-ELISA and the RBPT compared with those recorded in the literature (Erganis et al. 2002; Mailles et al. 2008).

In the northeastern part of Turkey, a total of 626 serum samples obtained from 27 infected cattle herds were tested by i-ELISA and the CFT. Out of 626 test samples, 221(35.30%) and 247 (39.45%) were found positive by the RBPT and i-ELISA, respectively (Şahin et al. 2008). O’Grady et al. (2014) tested 248 bovine sera in Ireland and they found 38.7% and 16.1% positivity rates by i-ELISA and the RBPT, respectively. In another study conducted in 36 sheep flocks with history of abortion in Elazığ and its surrounding area, 500 serum samples were tested by i-ELISA, RBPT and CFT using B. abortus S99 as antigen and positivity rates were 20.6%, 11% and 17.8%, respectively (Öngör et al. 2001). These findings are in agreement with our findings that brucellosis is endemic in our country and the RBPT and i-ELISA detect more positive samples than CFT does.

Successful control programmes based on the use of simple tests of high sensitivity, such as the RBT, followed by confirmatory serological tests of high specificity, such as the CFT. In this study, i-ELISA detected 15 serum samples as positive while these were found negative by the RBPT. This could be due to prozoning which is not rarely seen in acidified antigens like the RBPT (Nielsen 2002). On the other hand, three of the serum samples which were found as positive by the RBPT were found negative by i-ELISA. This might result from cross-reacting antibodies in the RBPT. Seven of the bovine serum samples found positive by both i-ELISA and the CFT were found negative by the RBPT. This could be explained by lower sensitivity of the RBPT compared to i-ELISA.

Several researchers reported that i-ELISA is more sensitive than conventional serological tests (Nielsen 2002; Ferreira et al. 2003; OIE 2016). Jacques et al. (1998) evaluated the efficacy of i-ELISA in comparison with conventional serological tests on sera from infected ewes. i-ELISA was shown to be a good screening test to be used alone or in combination with the RBPT. Out of 487 samples, 355 serum samples were found positive by all three tests. There was no single serum sample found positive by the CFT while negative to i-ELISA (Table 2). This could be an expected finding since such cases are rare because of relatively low sensitivity of the CFT compared to i-ELISA (MacMillan 1990; Mailles et al. 2008; OIE 2016). The 38 serum samples were positive to both the RBPT and i-ELISA although they were negative by the CFT. This might represent early infection or result from some cross-reacting antibodies.

In conclusion, i-ELISA was found the most sensitive test in our study. Turkey has been currently trying to reduce the prevalence rate of brucellosis by mass vaccination of livestock and therefore is not close to the final stage of eradication where the specificity of the test is of paramount importance. Therefore, i-ELISA could be used as a screening test whether or not combined with the RBPT for serologic diagnosis for brucellosis.

Disclosure statement

No potential conflict of interest was reported by the authors.