RBPT and IS711 PCR-Based detection of bovine brucellosis in Tripura, India

Introduction

Brucellosis is one of the most widespread bacterial zoonotic diseases worldwide and remains endemic in India, posing a significant threat to both animal and public health. Brucellosis has been eradicated in many developed countriesEurope, Australia, Canada, Israel, Japan, and New Zealand (Geering et al., 1995) [6] but remains endemic in Africa, the Mediterranean, the Middle East, parts of Asia, and Latin America (Refai, 2002) [19] . Brucellosis is caused by bacteria of the genus Brucella, which are small, non-motile, aerobic, facultative intracellular, Gram-negative coccobacilli. Brucella ability to survive and multiply within host cells and enables persistent infection and evasion of both innate and adaptive immunity (Fichi, 2003) [5] . Several earlier study showed that brucellosis is associated with substantial economic losses in the livestock sector due to reproductive disorders, including abortion, infertility, retained placenta, reduced milk production, and restrictions on the trade of animals and animal products. In India, brucellosis is estimated to cause a median annual economic loss of approximately US$3.4 billion (Singh et al., 2015) [20] , highlighting its considerable impact on livestock productivity and the national economy. Brucellosis is recognized as one of the most important occupational infectious diseases in humans, particularly affecting those working in slaughterhouses, as butchers, and as veterinarians. Furthermore, the clinical signs of brucellosis can be highly variable, ranging from non-specific, flu-like symptoms (in the acute form) to undulant fever, arthritis, orchitis, and epididymitis. (Hassan et al., 2012) [8] Transmission of Brucella occurs through both direct and indirect routes, including horizontal and vertical transmission in bovines. Major sources of infection include aborted foetuses, placental membranes, uterine discharges, In domesticated animals, the clinical manifestations of brucellosis mainly associated with reproductive pathology. In females, it responsible for the metritis, mastitis, repeat breeding, third-trimester abortion, retained placenta, and reduced milk yield and in males, it manifests through epididymitis, orchitis, and infertility (Radostits et al., 2000) [18] The variety of possible transmission routes, combined with its zoonotic nature, makes brucellosis one of the most dreaded and contagious diseases among all zoonotic and reproductive failure diseases in animals. Infected animals shed the organism through milk, urine, and semen. The gravid uterus of infected pregnant animals harbours large numbers of the organism, making reproductive materials particularly important in disease transmission. The definitive diagnosis of brucellosis relies on the isolation and identification of Brucella spp (Alton et al., 1988) [1] . Bacterial culture remains the gold standard for diagnosis (Qureshi et al.,2024) [17]. Samples testing sero-positive on screening assays such as the Rose Bengal Plate Test (RBPT) should be confirmed using the Complement Fixation Test (CFT), the method recommended by the OIE (World Organisation for Animal Health) to support disease eradication efforts (OIE) [15] However, it is labour-intensive, time-consuming, and requires stringent biosafety measures because of the zoonotic nature of the pathogen. Consequently, serological tests have gained widespread acceptance (Nazir et al.,2025) [13]. as practical, economical, and rapid tools for the diagnosis and surveillance of brucellosis in both animals and humans. In recent years, nucleic acid-based detection methods have emerged as valuable alternatives for the diagnosis of brucellosis. Molecular techniques offer high sensitivity and specificity while eliminating the need to handle viable organisms, thereby reducing laboratory-associated risks. Several genetic targets have been employed for the detection and characterization of Brucella spp., including bcsp31 (Ingle et al.,2023) [9] , outer membrane protein (omp) genes (Gupta et al., 2007) [7] , and the insertion sequence IS711 (Paul et al., 2020) [16] .A recent study on the diagnosis and differentiation of vaccine strains of Brucella confirmed that a single-tube duplex PCR assay is useful for the early and rapid detection and differentiation of B. abortus vaccine strain S19 from other Brucella spp. in both milk and clinical specimens (Das et al., 2019) [3]. Considering the importance of rapid and accurate diagnosis, the present study was undertaken to detect bovine brucellosis using the Rose Bengal Plate Test (RBPT) as a serological screening method and polymerase chain reaction (PCR) as a molecular diagnostic tool for the detection of Brucella spp. in clinical samples.

Materials and Methods

Place of study

The present study was carried out in the Departments of Veterinary Microbiology and Veterinary Pathology, College of Veterinary Sciences and Animal Husbandry, R.K. Nagar, West Tripura, during the period 2024-2025.

Collection of clinical samples

A total of 45 clinical samples, including blood, vaginal discharge, aborted foetal organs, and placenta, were randomly collected from cattle with a history of abortion, from cattle farms in and around Agartala as well as from various organized farms in the West district of Tripura. Based on prior information regarding abortion history, the animals were kept under continuous monitoring, and prior to sample collection, all animals were serologically tested by the Rose Bengal Test (RBT) and confirmed to be seropositive for brucellosis.

Rose Bengal plate test (RBPT)

The coloured antigen required for the RBPT test was purchased from the Indian Veterinary Research Institute, Division of Biological Products, Izzatnagar. The serum samples were processed for the detection of brucella antibodies by RBPT as per WOAH, 2009 [22] .

PCR assays Bacterial DNA extraction

Genomic DNA was extracted from Brucella broth cultures using the boiling method. Briefly, culture aliquots were transferred to microcentrifuge tubes, boiled for 5 min, and immediately snap-chilled on ice (Didugu et al., 2015) [4] . The lysate was then centrifuged at 10,000 × g for 5 min, and the resulting supernatant was used as the template DNA for downstream PCR applications.

Primer design

Oligonucleotide primers targeting the IS711 insertion sequence of Brucella spp. were custom-synthesized by Eurofins Genomics India Pvt. Ltd. The primer sequences, lengths, and expected amplicon size are presented in Table 1.

Screening of clinical samples

Genomic DNA extracted from the Brucella abortus S19 vaccine strain was used as the positive control, while nuclease-free water (NFW) was used as the negative control. Forty-five clinical samples comprising of blood, vaginal discharge, aborted foetal organs and placenta, were randomly collected from cattle with a history of abortion, from west district of Tripura were screened by the optimized IS711PCR assay. . The ability of PCR to detect infection at an early stage is particularly important for preventing disease transmission and implementing timely control measures. In the present study, it has been observed that, two samples were positive by RBPT but negative by PCR. This discrepancy may be attributed to the persistence of antibodies following previous exposure or recovery from infection, resulting in positive serological reactions despite the absence of detectable bacterial DNA. This outcome is in complete in agreement with the earlier study done by Muñoz, et al (2005) [12] and study showed that, falsepositive RBPT reactions may occur because of crossreactivity with other Gram-negative bacteria possessing similar smooth lipopolysaccharide antigens, such as Yersinia enterocolitica O:9. Variations in bacterial load, sample quality, DNA extraction efficiency, and the intermittent presence of the organism in clinical specimens may also contribute to negative PCR results in serologically positive animals. The IS711-PCR assay employed in the present study successfully amplified the expected 498 bp product in the positive control (Brucella abortus S19) and in PCR-positive clinical samples, confirming the presence of Brucella DNA. The IS711 insertion sequence is widely recognized as a highly specific molecular target because it is present in multiple copies within the Brucella genome, thereby enhancing the sensitivity of detection. The successful amplification of the target fragment validates the reliability and specificity of the assay for molecular diagnosis of bovine brucellosis.

Results

Conclusion

This study demonstrates that PCR offers superior sensitivity over RBPT for detecting Brucella abortus infection in bovine clinical samples. Nonetheless, RBPT retains considerable value as a rapid, cost-effective, and fieldfriendly screening tool for large-scale epidemiological surveillance. Integrating serological and molecular diagnostic approaches can substantially enhance detection accuracy and enable earlier identification of infected animalsan approach that is especially critical for the surveillance and control of bovine brucellosis in Tripura and other North-Eastern states of India, where livestock rearing forms a vital component of the rural economy. The continued detection of B. abortus in bovine clinical samples from Tripura confirms the ongoing circulation of the pathogen in the region, underscoring the need for intensified surveillance, routine screening programmes, and robust biosecurity measures to curb transmission both among animals and from animals to humans.

Acknowledgement

AcknowledgementThe authors are highly thankful to the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India, New Delhi-110016, for providing the necessary funds and facilities to carry out this investigation under the externally funded research project CRG/2021/007338.

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