Molecular Characterization and Analysis of Risk Factors Associated with Brucellosis in Central Indian and Meghalaya Population

Introduction

Abortus Melitensis Ovis Suis Brucella PCR (AMOS PCR) is a rapid screening and valuable multiplex molecular diagnostic tool that was developed at the National Animal Disease Control for molecular characterization of four important Brucella biovars namely Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis (Mohamed et al., 2013). Since its development, several investigators globally have investigated the utility of AMOS PCR for molecular typing to identify Brucella biovar from livestock (Gumaa et al., 2020; Kurmanov et al., 2022; Mohseni et al., 2017). However, only countable references exist in India for species typing of human brucellosis using AMOS PCR (Gumaa et al., 2020; Luna et al., 2016), which can be generally attributed to low or suboptimal culture sensitivity. To overcome this limitation, we explored a culture-independent method of species characterization, using DNA samples positive for genus-specific BCSP-31 PCR, which is a widely used molecular diagnostic tool for brucellosis diagnosis. In addition to molecular characterization, no studies exist in the Indian perspectives that have correlated clinical risk factors with species-specific disease outcomes.

In this study, we validated the utility of AMOS PCR for molecular characterization of Brucella species using human DNA samples positive for genus-specific BSCP 31 PCR from the central Indian and Meghalaya populations. The main rationale was to establish a culture-independent approach for the molecular characterization of Brucella species using DNA samples amplified directly from clinical samples. In addition, we also analyzed clinical and associated factors for brucellosis in these cohorts to correlate strain-specific disease outcomes in both populations.

Materials and Methods

Results

The overall positivity and distribution of four Brucella species in the study group are given in Table 1. Based on AMOS PCR, B. melitensis was found to be the predominant species in both study regions with the positivity of ∼58% (52/90) and 54.28% (34/70), respectively, in Central India and Meghalaya. B. melitensis, B. abortus was the second most abundant species associated with brucellosis with a positivity of 42.2% (38/90) and 38.57% (27/70), respectively, in Central India and Meghalaya. Although sporadic, brucellosis in Meghalayan cohorts was associated with B. suis and rare cases of B. ovis, which were found in 5 (7.14%) and 2 (2.85%) cases, respectively. State-wise stratification of the AMOS PCR cases in two different states indicated Chhattisgarh as having the highest cases of B. melitensis and B. abortus followed by Maharashtra and Madhya Pradesh in Central India (Table 2). In Meghalaya, East Khasi hills have higher B. melitensis cases, whereas B. abortus is highest in Ri Bhoi followed by B. suis and B. ovis (Table 3).

The correlation of associated risk factors for brucellosis with each of the four species in both regions highlighted diverse risk patterns, with exposure to animals and raw milk consumption being predominant risk factors associated with B. melitensis and B. abortus in Central India, as per statistically significant associations exposure in contrast to meat consumption, which was found to be the major mediating factor and a significant risk factor for all the Brucella strains in Meghalaya (with the exception of M. ovis) for human brucellosis (Table 4). Analysis of clinical manifestation among the four Brucella species, projected fever of unknown origin as a major symptom associated with B. melitensis, B. abortus, and B. suis in both study regions (p < 0.05), followed by myalgia (Table 5). Representative gel images of AMOS PCR for each of the four species on 1% agarose gel are provided in Supplementary Fig. S2.

Discussion

In this study, we evaluated the utility of AMOS PCR for the molecular characterization of Brucella species and analyzed the associated risk factors for brucellosis in Central Indian and Meghalaya population. To the best of our knowledge, this is the first comprehensive study that reports molecular characterization in addition to corroborating clinical risk factors of Brucella with identified serovars in both Indian regions. Our study highlighted B. melitensis as a dominant species in both populations followed by B. abortus. Both the Brucella biovars have been reported as dominant species in causing human brucellosis through contact with infected livestock and consumption of contaminated dairy products (Kolo et al., 2019; Ntivuguruzwa et al., 2022). Analysis of risk factors showed exposure to animals and consumption of raw milk as a major predisposing factor for brucellosis in Central India, which is in agreement with our previous epidemiological studies carried out in the region (Shukla et al., 2020).

As opposed to the Central Indian population, consumption of meat was found to be the major risk factor associated with causing brucellosis. Meghalaya is characterized by a subtropical climate according to altitude and mostly consists of extended periods of winter (Shukla et al., 2022). According to recent reports, the northeastern region of India is one of the highest producers of livestock and fish in the country, accounting for 4.09% of the country's total fish and meat production. Because of the cold temperature in the region, consumption of meat and fish is significantly higher compared with other parts of the country. Pork consumption in the region is particularly high, accounting for 58.6% of the total meat consumption, highlighting its importance in the diet of the people in the northeast.

Consumption of other types of meat in the region is also relatively high, at 29.39% of the country's total consumption, indicating a preference for unconventional meat in the region (Mahajan et al., 2015). Livestock rearing and fisheries are the most important agrarian activity in Meghalaya with meat as the major source of dietary protein. As per reports, ∼85% of the Meghalayan population consumes meat daily, which mainly includes pork and beef (Tripathi et al., 2019). The association of various Brucella serovars in Meghalaya therefore corroborates with the finding of our study, suggesting meat as a source of Brucella transmission in Meghalaya. In addition to occupational factors, we also analyzed clinical risk factors for Brucella in both regions and found undulant fever and arthralgia have major symptoms of brucellosis attributed to infection with B. melitensis and B. abortus. The above study highlights the importance of etiological diagnosis and its clinical correlation, which is needed for the identification of Brucella biovars that might vary in geographically distinct areas.

Conclusion

In lieu of cultures, DNA samples positive for BCSP-31 Brucella PCR can be effectively used for species identification and differentiation using AMOS PCR. Human brucellosis continues to be an arduous challenge for doctors in terms of its early and precise diagnosis. It poses a challenge because of its indefinite and vague clinical manifestations, delayed growth in blood cultures, and challenging serodiagnosis. This study emphasizes using AMOS PCR as a molecular characterization of Brucella species, which should be routinely undertaken to establish epidemiological important data for mitigation, advocacy, and antimicrobial stewardship for diagnosis of this neglected pathogen.