Evaluation of serological assays for the detection of bovine brucellosis and optimization of an in-house rose Bengal antigen

Introduction

Brucellosis is widely regarded as one of the world's most important zoonotic diseases, affecting livestock productivity and human health. In India, it remains highly endemic, with reported prevalence rates in livestock ranging from 6.5% to 16.4% [1,2,3,4,5] . The disease in animals is primarily characterized by reproductive disorders, including abortions and stillbirths [6] . The serological diagnosis of bovine brucellosis is usually performed using a stepwise approach that combines screening with confirmatory assays. Bacterial culture is the gold standard but is time-consuming and requires high-biosafety laboratory conditions. Consequently, serodiagnosis remains the cornerstone of surveillance. Commonly used methods include the Rose Bengal test (RBT), standard tube agglutination test (STAT), and indirect enzyme-linked immunosorbent assay (i-ELISA) [7,8] . This study aimed to develop and optimize a cost-effective in-house RBT antigen and comprehensively evaluate its performance alongside established conventional and ELISA-based serological assays to improve disease surveillance in bovine herds.

Materials and Methods

Specimens:

A total of 236 blood samples (Table 2.1) were collected from cattle with a history of abortion from farms in the Pune and Mumbai regions. Serum was separated and stored at -20°C for serological testing. Standard reference strains (B. melitensis 16M, Rev1, B. abortus S99, S19, 544) were acquired from IVRI, Izatnagar.

Development of In-house Rapid Assay (RBT):

The reference strain Brucella abortus S99 was maintained on Brucella Agar Medium, harvested using phenol saline, and inactivated at 65°C for 60 min. Cells were stained with 1% Rose Bengal dye and suspended in a buffered diluent. The final packed cell volume (PCV) was adjusted to 8%, and the pH was standardized to 3.65 ± 0.05 [9] .

Serological Techniques:

• RBPT: Serum was tested using both in-house and commercial antigens. Equal volumes (25-30 µl) of serum and antigen were mixed and observed for agglutination after 4 min. • STAT: Serum was serially diluted in carbol saline, mixed with B. abortus antigen, and incubated at 37°C for 24h. Titers >80 IU/ml were considered positive. • ELISA: Commercial i-ELISA and c-ELISA kits were used according to the manufacturer's instructions. A commercially available Brucella antibody detection diagnostic kit and antigen were obtained from Texas (USA) and Uttar Pradesh (India). The Brucella-Ab C-ELISA kit was obtained from INDICAL BIOSCIENCE GmbH, (Leipzig, Germany). For c-ELISA, the percent inhibition (PI) of the samples was calculated, with values > 30% considered positive.

Statistical Analysis:

Diagnostic parameters (sensitivity, specificity, PPV, and NPV) were calculated using c-ELISA as the reference test (Table 2.2). Agreement was evaluated using Cohen's kappa coefficient.

Comparative Evaluation:

Using c-ELISA as the reference, i-ELISA demonstrated the highest sensitivity (87.05%) and specificity (96.91%) with almost perfect agreement (kappa = 0.820). The RBT demonstrated moderate sensitivity (74.29%) and specificity (82.29%). STAT exhibited the lowest sensitivity (69.01%) but the highest absolute specificity (98.94%) (Table 3.5)

Discussion

The Rose Bengal Plate Test (RBPT) is widely used as a primary rapid screening tool due to its simplicity [11] . Our optimized in-house RBT antigen demonstrated identical diagnostic performance to commercial kits. While some studies report higher positivity with local antigens due to circulating strain differences [12,13,14] . Our findings confirm comparable efficacy between standardized in-house and commercial preparations [15] . However, an elevated falsenegative rate (31.30%) in RBT indicates chronic cases may be missed if RBT is used in isolation. STAT primarily detects early agglutinating antibodies (IgM and IgG) [16] , achieving exceptionally high specificity (98.94%). However, its limited sensitivity (69.01%) reflects its inability to detect nonagglutinating antibodies dominating chronic stages [17,18,19] . ELISA-based methods overcome these limitations, demonstrating superior capacity to detect both antibody types, yielding a high sensitivity (87.05%) and perfect agreement with the c-ELISA reference [20,21,2] .

Conclusion

An in-house RBT antigen was successfully optimized from B. abortus S99, demonstrating field-level performance comparable to commercial antigens. While conventional tests like RBT and STAT remain practical and cost-effective for screening, their diagnostic sensitivity is inherently limited during chronic infection. Therefore, combining these rapid screening tools with highly sensitive ELISA methods is recommended to ensure robust case detection in bovine brucellosis control programs.

Declarations

Acknowledgement

AcknowledgmentsThe authors thank the Associate Dean, Mumbai Veterinary College, Mumbai, India for providing the necessary facilities for this study. The authors are also grateful to the advisory committee members for their technical support during the research.

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