Brucellosis is a common zoonotic disease caused by Brucella spp., which are Gram-negative, non-motile, non-spore-forming, slow-growing, and facultative intracellular bacteria (Di Bonaventura et al., 2021). Brucellosis, also known as undulant fever or Malta fever, was first reported in the 1850s in Malta from a patient who was said to have acquired the infection through consumption of infected goat’s milk (Hull and Schumaker, 2018). In 1886, David Bruce first isolated and identified Brucella melitensis from the spleens of four soldiers as the causative agent of the disease (Aleixo et al., 1999). Currently, 12 Brucella species have been identified, including six classical species and six novel species (Bricker et al., 2000). Among, four strains exhibit pathogenicity in humans and animals, B. melitensis is the most infectious, the other strains are Brucella suis and Brucella abortus and more recently human cases being infected with Brucella cetaceae have been reported (Gorvel, 2008; Orsini et al., 2022). Direct or indirect contact with infected animals and consumption of unpasteurized dairy products are the main transmission routes of brucellosis to humans, and people may also be infected by inhalation of contaminated dust or aerosols (Al Dahouk et al., 2003; Khalafalla, 2023). Domestic animals and wildlife, including goats, sheep, cattle, deer, and camels, are natural hosts of Brucella spp (Liu et al., 2020). The persistent worldwide prevalence of human brucellosis has caused serious public health concerns and vast economic losses in the farming industry (Singh et al., 2018).

Human brucellosis is prevalent worldwide, and an evidence-based report suggests that the global incidence of human brucellosis is 2.1 million new cases every year, with the highest incidence of human brucellosis in the Middle East and Central Asia (Laine et al., 2023). Furthermore, human brucellosis has become a serious public health challenge, and the cases reported in all 31 provinces in mainland China and the affected regions have expanded from northern to southern, including Jiangsu province (Lai et al., 2017). During the years 2006-2021, 1,347 brucellosis cases were reported in Jiangsu Province, with an average annual incidence of 0.1036 per 100,000 individuals (Zhang et al., 2023). Four cluster cases of brucellosis from one family were reported in Shuyang County, Jiangsu Province, China, and isolates from the four patients were indistinguishable by MLVA profiling, displaying a unique type in Jiangsu Province (Tan et al., 2015). Whole-genome sequencing single-nucleotide polymorphism (WGS-SNP) analysis is a widely used genotyping tool for identifying the transmission pattern and phylogenetic inferences of Brucella strains (Janowicz et al., 2018; Xue et al., 2023). And, cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection (Abdel-Glil et al., 2022). In this study, we performed WGS-SNP and cgMLST analyses on Brucella melitensis strains isolated from human patients in Jiangsu Province to elucidate their transmission patterns and phylogenetic relationships, thereby providing critical insights for developing targeted surveillance and control strategies.

Human brucellosis is a re-emerging disease in Jiangsu province. Sporadic cases were reported after 2006, and the number of reported cases has significantly increased since 2011 (Zhang et al., 2023). However, the species/biovars, origin, and transmission patterns of circulating Brucella strains remain unclear. Bacteriology, bio-typing, and WGS-SNP analyses were applied to uncover the original and transmission patterns of circulating Brucella strains. Our investigation showed that B. meitensis strains were prevalent in Jiangsu province and neighboring regions, including Anhui and Shandong provinces, dominated by B. melitensis bv. 3. Previous studies have shown that human brucellosis has changed significantly over the past decades, and B. melitensis strains have become a dominant circulating species in China, and it has expanded from north to south regions (Zhu et al., 2020). An outbreak caused by B. melitensis at Jinchi Biotechnological Company engaged in collecting and disposing of kitchen waste from catering units in Lianyungang has been reported (Zhang et al., 2020). Brucellosis has become an important public health issue in Anhui Province, serum and milk samples obtained from goats in different regions of Anhui province were tested, the investigation frequency of brucellosis using RBPT, SAT, MRT, and PCR methods was 3.9% (n=7), 4.45% (n=8), 11.67% (n=7), and 86.67% (n=156), respectively (Rahman et al., 2019). These data show that ruminant brucellosis has been a serious public health concern in Jiangsu and Anhui provinces.

MLST analysis showed that all strains belonged to ST8, which is the dominant ST in the northern region (Sun et al., 2016). ST8 is the prevalent sequence type and the transmission of osteoarthritis-associated B. melitensis among different geographical areas in Inner Mongolia (Zhu et al., 2024). In Guangxi province, the population structure of Brucella strains had changed considerably; ST17 and ST21, two previously predominant populations, appeared to have been replaced by the recently emerging ST8 group (Liu et al., 2019). A previous report demonstrated that the cause of an outbreak was the plentiful influx of unchecked sheep from the northern part of China, and the employees in the process of sheep slaughtering or trading lacked effective prevention programs (Xiang et al., 2014).

WGS-SNP analysis revealed that 89 strains were divided into five SNP clades and 17 SNP genotypes (STs); each clade formed a ladder topology, with strains lacking apparent territory ownership, implying that cross-region cases occurred due to common sources of infection. Whole-genome SNP analysis showed that four B. melitensis strains were closely related to strains from China’s northern provinces, and the source of infection was partly human brucellosis in this province, which may have been from these regions (Li et al., 2020). In Shaanxi, multiple cross-county brucellosis outbreak events are driven by multiple indigenous circulating B. melitensis lineages (An et al., 2024).

The global phylogeny analysis showed that strains displayed a high genetic homogeneity with strains from multiple northern provinces, implying that multiple B. melitensis strain lineages were imported due to animal trade that co-drove the human brucellosis epidemic in Jiangsu. According to their territorial affiliation between the five clade strains, the absence of a clear differentiation suggests that strains continuously expanded and spread in Jiangsu province and neighboring regions. Similarly, 110 Chinese animal B. melitensis strains were grouped into five clusters, reflecting the existence of multiple lineages, and Chinese lineages were more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq (Sun et al., 2017). The active transfer and trade of animals (sheep and goats) between these regions is a reasonable explanation. A relevant survey showed that this province mainly breeds and raises its own cattle and sheep, the imported sheep come from Inner Mongolia, Shandong, Anhui and other provinces (Xu and Wang, 2018). The lack of geographic clustering among Jiangsu strains provides strong evidence for frequent cross-regional livestock movements, which significantly compromises local disease control efforts. The observed high genetic concordance with northern Chinese strains particularly highlights the need for an integrated One Health approach to effectively manage transmission risks at the human-animal interface. Key interventions should include: (1) implementing mandatory testing and quarantine protocols for livestock imported from endemic regions, (2) establishing real-time genomic surveillance systems to accurately trace outbreak origins, and (3) enhancing farmer education programs to mitigate high-risk behaviors such as direct contact with infected animals and consumption of unpasteurized dairy products (Liu et al., 2023). Finally, to effectively curb the southward spread of brucellosis, it is essential to strengthen inter-sectoral collaboration between agriculture and public health authorities while enhancing inspection and quarantine measures for introduced livestock.

At present, bacteriology and WGS-SNP have been applied to uncover the origin and transmission of Brucella strains, underscoring the fact that B. melitensis strains have become a severe public health issue in Jiangsu province and neighboring regions. Due to livestock trade and transfer, multiple B. melitensis lineages eventually trigger a local human brucellosis epidemic and spread. This investigation provides a vital cluse for devising and exerting a tailor control measure; however, further genomic epidemiological analysis on a regional and national scale will provide more detailed information to understand the transmission pattern of B. melitensis from north to south.