Human brucellosis, caused by Brucella spp., is a global public health concern (Pappas et al., 2006; Di Bonaventura et al., 2021). This disease exerts negative effects on human health and the animal farming industry. Brucella strains can result in severe clinical manifestations in the population, including fever, sweat, and fatigue, along with substantial economic losses in animal farming due to factors such as abortion and decreased milk productivity (Bagheri Nejad et al., 2020; Jamil et al., 2021). Although some developed countries have nearly eliminated this disease, the majority of low-income countries remain vulnerable to this zoonotic threat, including Africa, Latin America, Asia, and China (Gwida et al., 2010; Zhu et al., 2020). Since the 2000s, the incidence of human brucellosis has been continuously increasing and is distributed across all mainland regions in China, including the Ningxia Hui Autonomous Region (Ningxia) (Lai et al., 2017). Ningxia has a history of being an endemic area for brucellosis. The first survey of human brucellosis was conducted from 1958 to 1962, involving the sampling of 18,698 people, among whom 3,185 tested positive, resulting in a positivity rate of 17.03%. At that time, immunization had not yet been widely promoted, leading to the infection rate gradually increasing. Subsequently, a second brucellosis survey conducted from 1974 to 1980 revealed a positivity rate of 4.34% (68,609/1580,082). Following the implementation of mass ruminant immunization, the human brucellosis epidemic has been relatively stable, with 76.19% of the counties in the region reaching the national standard for stable brucellosis control (Deqiu et al., 2002).

Although vaccination and test-slaughter program animals, and surveillance and intervention plan in human population were exerted continuously. Since 2008, human brucellosis has steadily increased, with the current incidence surpassing historical levels. Conducting timely epidemiological analysis of resurging brucellosis areas is vital to enhance disease surveillance and improve intervention and treatment outcomes (Franco et al., 2007; Yagupsky et al., 2019; Matle et al., 2021). Additionally, pathogen surveillance is necessary for the formulation of targeted control measures and to provide a source for molecular epidemiological investigations (Laine et al., 2022). Therefore, the aim of this study was to illustrate the epidemiology of human brucellosis and the molecular association of Brucella strains in Ningxia to provide valuable insights for the development of targeted measures to halt its spread. To achieve this, we employed epidemiological analysis, pathogen surveillance, and molecular epidemiological approaches, including multiple-locus variable-number tandem repeat analysis (MLVA), which has become a routine epidemiological tool for distinguishing isolates from specific geographic origins and conducting trace-back analyses (Kiliç et al., 2011; De Massis et al., 2015).

In this study, we performed a preliminary analysis of the epidemiological and clinical characteristics of human brucellosis in Ningxia. We observed three phases of human brucellosis prevalence: the epidemic phase, control phase, and re-emergence phase. The incidence of human brucellosis was high during 1955–1978, then decreased dramatically during 1979–1994, after which brucellosis reemerged in 1995. The observed re-emergence phase is consistent with that reported in Shanxi, in which the incidence increased dramatically from 7.0/100,000 in 2005 to 23.5/100,000 in 2014, with an average annual increase of 14.5% (Chen et al., 2016). However, some regions have not experienced a re-emergence phase. Notably, no positive cases have been reported after 15 years of continuous serological surveillance in Zhongwei county. Consequently, the brucellosis surveillance point in Zhongwei county was discontinued in 2005, resulting in a lack of prevention and control measures in this district. The re-emergence of human brucellosis is associated with many factors. Key contributing factors include the long-term persistence of infection in livestock (sheep and goats) and their movement. Additionally, with the continuous expansion of animal husbandry and the frequent introduction of livestock, resources for quarantine, immunization, and compensation are inadequate, resulting in the long-term persistence of animal brucellosis epidemics. Previous efforts to address this contributing factor have included newborn immunization of female lambs for three consecutive years between 2015 and 2017 to curb the rapid spread (Duan et al., 2023). This resulted in the formation of an effective immunization barrier, and consequently, the incidence of human brucellosis decreased rapidly during this period. Therefore, the implementation of ongoing animal immunization is recommended in Ningxia.

Our results revealed that the incidence of brucellosis was concentrated in Yanchi county, Hongsibao district, and Tongxin county. These data raise questions about the effectiveness of public health measures. This is consistent with a previous study that indicated that high-incidence areas were mostly concentrated in central Ningxia and displayed a certain degree of correlation with the number of sheep in stock (Zhao et al., 2020). These counties are renowned for sheep breeding; for instance, Yanchi County was designated as the National Animal and Poultry Genetic Resources Reserve in 2000. Furthermore, this county also successfully registered the Yanchi Tan Sheep breed as a National Brand within the national germplasm resources core protection area in 2021.

Moreover, Ningxia is home to the largest concentration of Hui people in China, whose diet and lifestyle practices align closely with Islamic traditions. The primary sources of income and animal protein for the local population are sheep and goat farming. While this industry forms the backbone of the county’s poverty reduction efforts, the processing, sale, and consumption of mutton may pose infection risks to humans. This offers a plausible explanation for why the strains from Yanchi county led to multiple-point outbreaks of human brucellosis. A similar effect was observed in Shaanxi, where the increased brucellosis incidence was closely related to the development of large-scale small ruminant (goat and sheep) farms in Guanzhong and some southern Shaanxi regions (An et al., 2021). Notably, Yanchi County is located in the easternmost region of Ningxia, adjacent to the Inner Mongolia Autonomous Region, Gansu (Wei et al., 2019), and Shaanxi (An et al., 2022), which are cluster areas of human brucellosis (Yang et al., 2020). Previous studies showed that the highest seropositivity was observed in Yulin City, Shaanxi, which borders regions that have a high incidence of human brucellosis, including Inner Mongolia, Ningxia, and Gansu (An et al., 2022). The high disease burden in this region raises questions about the effectiveness of public health measures. Therefore, further genome molecular association surveys of strains from the border regions are necessary to elucidate the transmission chain.

In this study, we observed high species/biovars and host diversity in Ningxia, constituting a relatively complete niche for the spread of Brucella strains. The potential for cross-host transmission of Brucella strains may create an optimal environment for their persistent circulation in this region. The results from MLVA genotyping suggested that the dominant circulating genotypes persistently expanded over an extended time span, contributing to both intra-county and cross-county human brucellosis epidemics. Moreover, the 39 singletons each represented one strain, indicating sporadic cases, including four strains from the border regions of Inner Mongolia and Shaanxi Province. This is consistent with the results of MLVA-16 genotyping of 70 B. melitensis isolates from Egypt, which revealed that 51 genotypes were represented by single isolates, while the remaining 19 genotypes were shared among 67 isolates, suggesting a combination of sporadic and epidemiologically related characteristics in B. melitensis infection (Sayour et al., 2020). Further genomic epidemiological studies are necessary to support the present results and illustrate the phylogenetic relationships of B. melitensis strains. B. melitensis has been the dominant circulating population for nearly eight decades, responsible for the majority of human brucellosis cases. A similar high prevalence of B. melitensis has been observed in small ruminants in Turkey, as evidenced by isolations from sheep, goat, and cattle abortion samples from various farms across seven geographical regions (Akar and Erganis, 2022). Furthermore, Brucella strains isolated from all regions of Kyrgyzstan were identified as B. melitensis (Kydyshov et al., 2022). Notably, Guangxi province has witnessed considerable shifts in the species/biovar composition of Brucella strains, with previously predominant populations, B. suis and B. canis, being replaced by recently emerging B. melitensis strains (Liu et al., 2019). Furthermore, the predominant circulating genotypes in this region were similar to strains from the Northwest, including Inner Mongolia, Xinjiang, and Qinghai. These data indicate that brucellosis has become a significant regional issue, with a high risk of spillover to southern areas due to animal trade and their products. Additionally, strains from this study showed complete consistency in MLVA-16 genotypes with strains from other Asian countries, such as Kazakhstan and Mongolia. The breeding and trade of small ruminants have contributed to the strain’s spread due to similar lifestyle and domestic farming practices in these agricultural and pastoral areas in China, Kazakhstan, and Mongolia. WGS-SNP analysis showed that strains from these countries lack clear territorial differentiation, demonstrating that strains continuously expand and spread in countries along the Silk Road (Liu et al., 2019). The active exchange and trade of sheep and goats among these countries are one of the main driving factors. Further genomic epidemiology investigation of strains from multiple hosts will provide a new genetic map to better depict the transmission route and evolution profile of strains.

Based on the interpretations of our results, it is recommended to strengthen publicity and education and mobilize farmers and herders to consciously participate in brucellosis control work. First, enhancing communication and education collaboration with agricultural and animal husbandry departments to raise awareness should be a focus, particularly among high-risk groups. This should include promoting healthy lifestyles to reduce the epidemic of brucellosis (Dadar et al., 2020; Gong et al., 2023). Second, high-risk populations should be educated about the risks associated with privately slaughtering, trading, transferring, or consuming animals infected with brucellosis, those that died of disease, or those that died of unknown causes, especially during the spring calving and lambing season. They should be encouraged to implement rigorous personal protection and field disinfection measures (Abu Shaqra, 2000). Additionally, the migration of sheep from high-to low-infection areas should be strictly limited to prevent further spread (Zhang et al., 2014). Moreover, targeted prevention and control measures should be implemented, including collaborative publicity campaigns with local Islamic church organizations, to ensure that local people can participate in monitoring and control while respecting different cultural differences.

Although there were some new insights in this study, there may be potential limitations to be considered. Firstly, whole genome sequencing of some representative strains is needed to better illustrate the epidemiological transmission pattern and evolution characteristics of strains. Secondly, the limited number of strains from animals suggests that future studies should include larger sample sizes to verify the results of this study.

Our analysis indicates an ongoing increase in the incidence rate of human brucellosis in Ningxia, from 0.441 (per 100,000) in 2005 to 86.83 (per 100,000) in 2022. Geographically, the affected area has continuously expanded, progressing from one affected county in 2004 to all 22 counties by 2022. B. melitensis emerged as the dominant circulating strain, responsible for the majority of cases. Molecular epidemiological investigations conducted using MLVA-16 genotyping have revealed the persistence of dominant genotypes in both intra-county and cross-county epidemics of human brucellosis. Brucellosis has become a serious regional issue, and there is a high risk of spillover to southern areas due to the trade in animals and their products.

The results of this study have provided a foundation for recommendations on the prevention and control of brucellosis epidemics. Further genomic epidemiology studies of circulating Brucella strains will provide new insights into the evolving epidemiological patterns of human brucellosis in Ningxia.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Ethics Committee of the Ningxia Fourth People’s Hospital (no. NXFPH-0624). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

GL: Methodology, Funding acquisition, Investigation, Writing – review & editing. XM: Investigation, Methodology, Writing – review & editing, Resources. RZ: Writing – review & editing, Formal analysis, Project administration. JL: Supervision, Writing – review & editing. PZ: Writing – review & editing, Data curation, Methodology. BL: Project administration, Writing – review & editing, Resources, Supervision. TL: Data curation, Writing – review & editing, Software. HR: Supervision, Writing – review & editing, Data curation, Formal analysis. ZgL: Data curation, Software, Conceptualization, Methodology, Writing – original draft. ZjL: Supervision, Writing – review & editing, Formal analysis, Project administration. XJ: Project administration, Supervision, Writing – review & editing.