Seroprevalence and molecular confirmation of brucellosis in sheep from Telangana with phylogenetic analysis of circulating Brucella sps.

Introduction

Brucellosis is one of the most important zoonotic bacterial diseases affecting livestock and humans worldwide. It is caused by members of the genus Brucella, which are Gramnegative, non-motile, non-spore-forming, facultative intracellular coccobacilli belonging to the family Brucellaceae (Leclercq et al., 2020) [25] . The disease affects a wide range of domestic animals, including cattle, sheep, goats, pigs, and wildlife species, resulting in significant reproductive and economic losses (Franc et al., 2018) [17] . Brucellosis remains a major public health concern, particularly in developing countries where livestock farming constitutes an important source of livelihood. In humans, the disease is characterized by intermittent or undulant fever, headache, weakness, excessive sweating, weight loss, joint pain, and other systemic manifestations (Pal and Jain, 1986; Addis, 2015; Pal, 2018) [30,4] . Human infection commonly occurs through the consumption of unpasteurized milk and milk products or through occupational exposure to infected animals and their , 2020) [7,45] . In India, Brucella melitensis biovar 1 is responsible for the majority of human brucellosis cases and contributes substantially to economic losses and public health burden (Kumara et al., 2015; Singh et al., 2018) [23,40] . India possesses one of the largest sheep populations in the world, estimated at 74.26 million, with Telangana accounting for approximately 25.67% of the national sheep population (GOI, 2023). Sheep farming plays a vital role in the socio-economic development of rural communities, particularly among small and marginal farmers. However, productivity in sheep production systems is constrained by several factors, including infectious diseases, among which brucellosis is of considerable importance. The disease is associated with abortions, infertility, retained placenta, reduced fertility, decreased lambing rates, and consequent economic losses.

The transmission of ovine brucellosis occurs primarily through ingestion of contaminated feed and water, inhalation of infected aerosols, and direct contact with aborted fetuses, placental tissues, and uterine discharges from infected animals (Radostits et al., 2007) [34] . Movement of infected animals between flocks and across geographical regions significantly contributes to disease dissemination (Bait et al., 2019) [11] . Pregnant animals are particularly susceptible because the presence of erythritol in the placenta promotes rapid multiplication of Brucella organisms, resulting in placentitis, endometritis, and abortion, especially during late gestation (Bait et al., 2019) [11] . Accurate diagnosis is essential for surveillance and control of brucellosis. Isolation and identification of Brucella organisms from tissues, milk, or vaginal secretions remain the definitive diagnostic method; however, culture techniques are labour-intensive, time-consuming, and pose a significant biosafety risk to laboratory personnel (Surucuoglu et al., 2009; Pérez-Sancho et al., 2013) [44,33] . Consequently, serological assays are widely employed for epidemiological investigations and herd-level screening.

Commonly used serological tests include the Rose Bengal Plate Test (RBPT), Enzyme-Linked Immunosorbent Assay (ELISA), and Lateral Flow Assay (LFA), owing to their simplicity, rapidity, and cost-effectiveness (Nielsen, 2002) [28] . [10,24,16,26,21,6] .

Sample size determination

The minimum sample size required to estimate the seroprevalence of brucellosis was calculated using the formula: n = Z²P (1-P)/d² (Charani et al., 2010 and Islam et al., 2021) [13,22] .

In this formula, Z represents 1.96, P represents the expected seroprevalence, and d denotes the desired precision set at 5%. An expected prevalence of 11.55% reported by Shome et al.

(2021) [39] was used for the calculation, yielding a minimum sample size of 158 animals. To improve the precision and reliability of the prevalence estimates and to compensate for potential sample loss or invalid test results, the sample size was increased to 245 sheep.

Collection and screening of blood samples from sheep

A total of 245 blood samples comprising 221 ewes and 24 rams were collected from the selected farms (Figur 1& Table 1). Blood samples (5 mL) were aseptically collected from the jugular vein using sterile vacutainer tubes containing clot activator. Samples were transported to the laboratory under refrigerated conditions and centrifuged for serum separation. The separated sera were stored at -20 °C until further analysis.

Serological screening by Rose Bengal Plate Test (RBPT)

All serum samples were screened for antibodies against Brucella spp. using the Rose Bengal Plate Test (RBPT) according to the method described by Alton and Jones (1967) [8] and Alton et al. (1988) [9] . The Rose Bengal antigen (Brucella abortus) was procured from the Institute of Animal Husbandry and Veterinary Biologicals (IAH&VB), Hebbal, Bengaluru.

Briefly, equal volumes (30 μL each) of serum and antigen were mixed thoroughly on a clean glass slide and observed for visible agglutination within 4 minutes. Samples exhibiting agglutination were considered positive, while those showing no agglutination were considered negative. Blood samples from RBPT-positive animals were subsequently subjected to molecular analysis (Figure 2).

Genomic DNA extraction

Genomic DNA was extracted from whole blood samples following a modified phenol-chloroform extraction protocol described by Leal-Klevezas et al. (1995) [24] . Briefly, erythrocytes were lysed using RBC lysis buffer followed by leukocyte lysis and protein digestion using Proteinase K. DNA was purified using phenol: chloroform alcohol (25:24:1), precipitated with isopropanol and sodium acetate, washed with 70% ethanol, and finally resuspended in TE buffer. Extracted DNA samples were stored at 4 °C until further use.

DNA quantification

The concentration and purity of extracted DNA were determined using a NanoDrop spectrophotometer. DNA quality was assessed by measuring absorbance at 260 and 280 nm. Samples with an A260/A280 ratio of approximately 1.8 were considered suitable for PCR amplification (Shirish, 2021) [38] .

PCR amplification of Brucella DNA Genus-specific PCR targeting the Brucella cell surface protein (BCSP31) gene was performed using F4 and R2 primers as described by Çiftci et al. (2017) [15] . PCR reactions were carried out in a final volume of 25 μL containing 2.5 μL of 10× PCR buffer, 2 μL MgCl₂ (25 mM), 0.5 μL dNTP mix (10 mM), 1 μL each of forward and reverse primers (10 pmol/μL), 0.6 μL Taq DNA polymerase (5 U/μL), 8 μL template DNA, and nuclease-free water to make up the final volume. Amplification was performed in a Prima-DUO® thermal cycler under the following conditions: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 90 s, and a final extension at 72 °C for 6 min.

Agarose gel electrophoresis

PCR amplicons were resolved by electrophoresis on 1% agarose gel prepared in 1× TBE buffer. Electrophoresis was carried out at 80-100 V for approximately 60 min. Following electrophoresis, gels were visualized under a UV transilluminator (Vilber BioPrint, France) and photographed. Samples producing a specific amplicon of 903 bp were considered positive for Brucella spp.

Sequencing and phylogenetic analysis

Representative PCR-positive amplicons were purified and commercially sequenced by Bioserve Biotechnologies (India) Pvt. Ltd., Hyderabad. Raw sequence data obtained in ABI format were edited and assembled using MEGA version 12.1 software. The resulting consensus sequences were subjected to Basic Local Alignment Search Tool (BLAST) analysis against sequences available in the NCBI GenBank database to determine sequence homology and confirm species identity. Phylogenetic relationships were inferred using reference sequences retrieved from GenBank.

Statistical analysis

The apparent seroprevalence of brucellosis was calculated as the proportion of RBPT-positive animals among the total animals tested. Descriptive statistics were used to summarize farm-level and animal-level data. Confidence intervals for prevalence estimates were calculated at the 95% confidence level.

Results

In the present study, Information on sheep farms established under government-subsidized sheep distribution schemes was obtained from the Department of Veterinary and Animal Husbandry, Telangana. A total of 13 sheep farms were identified, and baseline information regarding farm management practices, reproductive health status, and disease history was collected through farmer interviews, farm observations, and consultation with field veterinarians. Based on the presence of clinical signs and reproductive disorders suggestive of brucellosis, including abortion, retained placenta, stillbirths, infertility, and testicular swelling, 10 farms were considered suspected for brucellosis and selected for further investigation. From these farms, 245 sheep were randomly sampled for serological and molecular screening. Screening of suspected sheep for brucellosis Based on the sheep exhibiting reproductive disorders suggestive of brucellosis, including abortion, retained placenta, stillbirths, infertility, and testicular swelling, were selected for laboratory investigation. A total of 276 samples comprising 245 serum samples and 31 whole blood samples were collected from ten sheep farms located in different districts of Telangana. Serum samples were screened for Brucella-specific antibodies using the Rose Bengal Plate Test (RBPT), and whole blood samples from seropositive animals were subjected to molecular confirmation by PCR. Representative PCR-positive amplicons were further sequenced and analyzed for phylogenetic relationships.

Seroprevalence of brucellosis in sheep by RBPT

Out of 245 serum samples screened by RBPT, 31 samples were positive for Brucella antibodies (Figure 3), resulting in an overall seroprevalence of 12.65% (Figure 4). Farm-wise analysis revealed variation in seropositivity among the investigated flocks. The highest seroprevalence was recorded in Farm 5 (23.1%), followed by Farm 2 (16.7%), Farm 8 (14.8%), Farm 3 (14.3%), and Farm 6 (13.0%). The lowest prevalence was observed in Farm 9 (5.9%) and Farm 1 (6.7%) (Table 2). Sex-wise prevalence Among the 221 ewes tested, 27 animals were seropositive, corresponding to a prevalence of 12.2%. In contrast, 4 of the 24 rams tested were positive, yielding a prevalence of 16.7% (Table 3; Figure 5). Although seropositivity was numerically higher among rams, the association between sex and seroprevalence was not statistically significant (p > 0.01).

Age-wise prevalence

Age-wise analysis indicated that the highest seroprevalence was observed in sheep aged 1-2 years, where 19 of 129 animals (14.7%) tested positive. In the 2-4 years age group, 9 of 85 sheep (10.5%) were positive, while 3 of 31 sheep older than 4 years (9.6%) were seropositive (Table 4; Figure 6). Despite the higher prevalence observed in younger animals, no statistically significant association was detected between age and seropositivity (p > 0.01). Lambing-wise prevalence Among ewes, seropositivity varied according to lambing status. Sheep in their first lambing exhibited the highest prevalence, with 17 of 119 animals (14.2%) testing positive.

The prevalence decreased to 10.2% (8/78) among animals in their second lambing and to 8.3% (2/24) among ewes with more than two lambings (Table 5; Figure 7). However, these differences were not statistically significant (p > 0.01). Molecular detection of Brucella spp. by PCR All 31 RBPT-positive samples were subjected to genusspecific PCR using the F4/R2 primer pair targeting a 905 bp fragment of Brucella DNA. Before sample screening, PCR conditions were optimized using gradient PCR. Among the annealing temperatures evaluated (52-60 °C), 60 °C produced a distinct and specific amplification product of the expected size without non-specific bands and was therefore selected for subsequent analyses.

Of the 31 RBPT-positive blood samples examined, 5 (16.12%) yielded the expected 905 bp amplicon, confirming the presence of Brucella DNA (Figure 8). The remaining 26 samples (83.88%) were negative by PCR. Of the five PCRpositive animals, four were ewes and one was a ram (Table 6). Farm-wise distribution of PCR-positive cases indicated the presence of molecularly confirmed brucellosis in four farms. Two positive cases were detected in Farm 10, whereas Farms 2, 4, and 8 each contributed one positive case (Table 6).

Sequencing and phylogenetic analysis

One representative PCR-positive amplicon was subjected to nucleotide sequencing. The obtained sequence was edited and assembled using MEGA version 12.1.2 and subsequently analyzed using the BLAST algorithm available in the NCBI database. Sequence analysis confirmed homology with previously reported Brucella sequences, thereby validating the PCR results. Phylogenetic analysis demonstrated that the study isolate, designated VPHE/PVNRTGVU/2025, clustered within the Brucella lineage and exhibited close genetic relatedness to previously reported Brucella isolates from different geographical regions (Figure 9). The phylogenetic tree revealed distinct clustering patterns among B. melitensis and B. abortus isolates. The Indian isolates formed a separate branch with strong bootstrap support, indicating regional genetic divergence. The study isolates also showed phylogenetic affinity with strains reported from West Asian countries, including Syria, Turkey, Jordan, and Afghanistan, suggesting possible transboundary dissemination and genetic connectivity among circulating Brucella strains. The sequence generated in the present study contributes to the understanding of the molecular epidemiology and genetic diversity of Brucella circulating in sheep populations of Telangana. The observed prevalence suggests that brucellosis remains an important reproductive and zoonotic disease in small ruminants and may pose a potential risk to farmers, animal handlers, and veterinarians. The seroprevalence recorded in the present study is comparable with reports from Egypt (16.3%) (Selim et al., 2019) [37] , Qatar (16.7%) (Al-Hussain et al., 2024) [5] , Tunisia (16.1%) (Guesmi et al., 2023) [20] , Nepal (15%) (Gompo et al., 2021) [19] , and various regions of India where prevalence ranging from 8.4% to 15.5% has been reported in sheep using RBPT and ELISA-based assays ( [14,43,42] . However, the prevalence observed in the present study was higher than reports from Sudan, Ethiopia, Somalia, and Jammu & Kashmir, where prevalence ranged from 0.18% to 8.92% ( [1,45,12,27,32] . Conversely, lower prevalence than the present study has been reported from highly endemic areas of Egypt, Iraq, Andhra Pradesh, and Tamil Nadu, where prevalence exceeded 25% and reached up to 60% in some investigations (Abdeltwab et [2,3,35,41] . Variations in prevalence among different studies may be attributed to differences in geographical location, management practices, flock size, animal movement, climatic conditions, sample size, disease endemicity, and diagnostic methods employed. Studies utilizing ELISAbased assays often report higher prevalence because of their greater sensitivity compared with RBPT. Furthermore, investigations involving animals with abortion history or reproductive disorders generally yield higher prevalence than studies involving apparently healthy animals. Sex-wise analysis revealed a higher seropositivity in rams (16.7%) than in ewes (12.2%), although the difference was not statistically significant (p > 0.01). Similar observations have been reported by Selim et al. (2019) [37] , suggesting that sex may not be a major determinant of infection. The lack of statistical significance in the present study may also be influenced by the relatively small number of rams included compared with ewes. Age-wise analysis indicated the highest seroprevalence among sheep aged 1-2 years (14.7%), followed by animals aged 2-4 years (10.5%) and >4 years (9.6%). However, the differences among age groups were not statistically significant (p > 0.01). Although several studies have reported higher prevalence among older animals due to prolonged exposure and reproductive maturity, the present findings suggest that infection may occur irrespective of age in endemic flocks where continuous exposure to the pathogen exists.

Lambing-wise analysis demonstrated a higher prevalence among first-lambing ewes (14.2%) compared with secondlambing (10.2%) and multiparous animals (8.3%). However, no significant association was observed between lambing status and seropositivity. The relatively higher prevalence in first-lambing animals may be associated with physiological stress during the reproductive period, which could increase susceptibility to infection. The moderate seroprevalence observed in the present study may be associated with the large-scale movement of sheep under government-subsidized distribution programmes and the introduction of animals from neighbouring states. Such practices facilitate the dissemination of Brucella infection between flocks and may contribute to the persistence of the disease in the region. [15,38,36] . The relatively moderate detection rate may be attributed to intermittent bacteremia and the low concentration of bacterial DNA in peripheral blood, which can reduce PCR sensitivity.

Molecular Detection of Brucellosis by PCR

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