Brucellosis (Malta fever) is a globally distributed zoonotic disease. It has severe adverse effects on public health and the agriculture field, including population, livestock, and wildlife health. Since David Bruce first isolated the organism (Micrococcus melitensis) in 1887 (Godfroid et al., 2005), 12 species have been identified in the genus Brucella, that is, six classical and six novel species (Occhialini et al., 2022). Brucella melitensis is the most common pathogenic species in humans and animals, followed by Brucella abortus and Brucella suis (Xavier et al., 2010). Brucellosis can be transmitted to humans through the food chain or by direct/indirect contact with infected animals, such as consumption of animal raw milk and meat products or aerosol transmission (Minas et al., 2007). For centuries, brucellosis has been critically endangering human health and has led to substantial economic losses (Wareth, 2019). In some industrial countries, brucellosis has been effectively controlled, but it remains a serious public health risk to the majority of the population in developing areas. For example, in China, brucellosis is widely endemic in all 32 provinces (Rossetti et al., 2017).

Qinghai (QH) Province is located in northwestern China, and animal farming is the main source of income for the local population. Because of low development levels, poverty, and poor hygiene conditions, animal and human brucellosis is highly prevalent in this region (Ma et al., 2016). Although a comprehensive control plan for brucellosis was carried out during 2005–2010, which utilized the Brucella S2 vaccine for the immunization of ruminants combined with a serology test, and brucellosis-positive animals were eliminated, persistent funding is a great obstacle to control programs. Subsequently, the prevalence of human brucellosis increased annually in QH from 2005 to 2019 (Ma et al., 2020). Since 2010, the epidemic situation of human brucellosis has gradually become severe: the number of cases increased from 3 in 2010 to 756 in 2021, the incidence rate increased from 0.054/100,000 to 12.76/100,000 accordingly, and the affected geographic territory is currently expanding. Based on the national brucellosis surveillance sites, data indicate that the prevalence rate of human brucellosis was 3.35% (54/1,612) in 2019 and 4.77% (80/1,677) in 2020. However, the ability to tailor a cost-based brucellosis control program requires accurate and robust molecular typing tools to investigate the relationships between strains involved in common outbreaks and determine the source of infection and transmission routes (Pelerito et al., 2021). Generally, classical microbiological techniques allow researchers to obtain and bio-type the strains and facilitate molecular epidemiological investigations of the disease. The multilocus variable-number tandem repeat analysis (MLVA) has been used as the gold standard for genotyping Brucella strains, with the results combined with epidemiological data to investigate the relationships between the Brucella strains (Kiliç et al., 2011; Liu et al., 2017). Furthermore, single-nucleotide polymorphisms (SNPs) based on whole-genome sequencing have excellent power to discriminate strains and allow for the characterization of the phylogenetic relationships of strains from different scales (Janowicz et al., 2018; Abdel-Glil et al., 2022). Importantly, molecular typing tools can not only limit control costs and test time but also, improve the surveillance and evaluation of control measure effects. Therefore, classical bio-typing assay, MLVA, and whole-genome sequencing–SNP (WGS–SNP) were used to illustrate the species/biovars’ genetic diversity and the phylogeography pattern of B. melitensis from humans in QH to better assess the epidemiology profile and enhance brucellosis surveillance and control.

In the present study, both classical bio-typing procedures and two molecular tools (MLVA and WGS–SNP) were used to characterize the B. melitensis strains from QH, China, to explore the molecular epidemiological relationship. The results of this work provide the crucial evidence necessary to formulate a targeted surveillance and control program. In the present study, the B. melitensis species was a predominant pathogen that was isolated from three different host species, and it was widely distributed. In particular, B. melitensis was isolated from samples from many hosts, including yaks, sheep, blue sheep, and Tibetan gazelle (Ma et al., 2016; Cao et al., 2018). These data revealed that there was a high diversity of natural reservoir hosts that allowed B. melitensis to continue circulating in this province. Indeed, animals are a natural reservoir host for zoonotic organisms and the majority of human brucellosis infections originate from animal hosts (Recht et al., 2020). Sheep and goats are optimal hosts for B. melitensis, and they can infect many different hosts such as cattle, swine, and deer (Liu et al., 2020). Thus, brucellosis control in regions with multiple existing hosts is a significant challenge. A targeted and comprehensive control program for infected sheep and goats should be implemented as a priority strategy to curb the spread of this disease to humans. Several further measures should be implemented, such as prohibiting the blind expansion of the breeding industry, strengthening the inspection and quarantine of animals for importation and exportation, widening the wildlife surveillance zone, improving the awareness of disease prevention among practitioners, and banning the circulation of sick animals.

In this present study, four MLVA-8 genotypes and six MLVA-11 genotypes were identified: 42 (MLVA-8) and 116 (MLVA-11) are predominant genotypes, accounting for 90.7% (49/54) and 81.5% (44/54), respectively. These data indicated that all strains were of Eastern Mediterranean lineage. With the use of MLVA-8, the majority of strains (84/105) were genotyped, and 42 were clustered into the East Mediterranean lineages (Jiang et al., 2011). Furthermore, MLVA-11 genotype 116 is a predominant circulating genotype in China, accounting for 69% (951/1382) (Zhu et al., 2020). Importantly, most B. melitensis strains belong to the East Mediterranean group, which comprises strains from Europe, the Middle East, and Asia (Vergnaud et al., 2018). MLVA genotype 116 is predominant in Asian countries (e.g., 86.8% in Kazakhstan) (Shevtsova et al., 2019). Furthermore, MLVA analysis data suggested that epidemiologically related characteristics of B. melitensis infection and cross-regional transmission events are caused by common sources of infection, suggesting that the predominant circulating genotype is endemic in different regions; this indicates a lack of control over infected animals’ movement and exchange between regions. In the Middle East, uncontrolled animal transportation through “open” borders is a main risk factor for brucellosis spread between some regions (Gwida et al., 2010). We hypothesize that the nomadic lifestyle (which often involves the consumption of raw milk) and production methods (e.g., homemade dairy products) in highland pastoral areas may have caused these infection events, and a detailed field survey is needed to verify the conclusion of this molecular investigation. A nomadic lifestyle may favor the spread of brucellosis among different animals and populations (Liu et al., 2022) because animals and people live in close contact. A 2018 report found that the consumption of raw milk from smuggled sick goats caused human brucellosis epidemics (25 cases) in Douz, Tunisia (Charaa et al., 2022). The purchase and consumption of cheese and milk from non-regulated sources are very common in specific communities among Israeli Arabs, with nearly 41 and 16.1% of respondents consuming cheese and milk, respectively, from non-regulated sources (Baron-Epel et al., 2018). Therefore, ruminant vaccination, control of cross-border animal movements, and control of non-regulated goat milk sales must be strengthened to prevent the spread of brucellosis.

WGS–SNP analysis has proved to be a robust molecular tool for illustrating the phylogenetic patterns of Brucella strains (Tan et al., 2015; Georgi et al., 2017). It demonstrated that multiple B. melitensis lineages were circulating in QH. Similarly, WGS–SNP phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages (Sun et al., 2017). In addition, strains from humans and marmots were clustered in the same sub-clades, implying the possible existence of direct and/or indirect contact between sheep (and goats) and wildlife (marmots). Indeed, wildlife has a crucial role in the epidemiology of brucellosis in animals and humans (Galarce et al., 2021). Therefore, strengthening prevention, surveillance, and control of wildlife is recommended.

Global phylogenetic analysis indicated that 54 B. melitensis strains were clustered into genotype II (Pisarenko et al., 2018) and further divided into six sub-lineages, revealing the existence of multiple circulating lineages in QH. Additionally, many sub-lineages were shared by strains from this study and strains from Inner Mongolia, Hainan Province, Shandong Province, and Hebei Province, indicating that these strains have a potential phylogenetic relationship (Liu et al., 2020). The cross-border movement and transfer of animals between Côte d’Ivoire and Mali for grazing and/or trade have exacerbated the spread of brucellosis across the region (Oyetola et al., 2021). Therefore, genome sequencing in more strains and building a local genome database are necessary to improve the surveillance capacity and control the spread of brucellosis (Liu et al., 2023).

Although we obtained important insight in our investigation, some limitations are worth acknowledging. First, most strains in genome sequencing were obtained over a 2-year period, and more strains involved during a longer period could facilitate a more profound description of the brucellosis epidemiological profile of this region. Second, obtaining field epidemiological data in many cases is challenging. However, these data provide crucial auxiliary support for genome epidemiology.

Brucella melitensis is a predominant species, and its distribution has been widespread in all nine regions in QH. B. melitensis strains belonged to the East Mediterranean lineages, and the human brucellosis epidemic in recent years was potentially caused by many native circulating lineages. Strengthening the genome sequencing of strains from a variety of host sources will facilitate the identification of transmission routes and determine potential ongoing outbreaks, which is vital for formulating targeted surveillance and countermeasures.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The research protocol was reviewed and approved by the Ethics Committees of the Institute for Endemic Disease Control and Prevention of Qinghai (No. 2022006). Informed consent was obtained from all patients. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. The animal study was approved by the Ethics Committees of the Institute for Endemic Disease Control and Prevention of Qinghai (No. 2022006). Informed consent was obtained from all patients. The study was conducted in accordance with the local legislation and institutional requirements.

HX performed the strain isolation, collection, and epidemiology data process. ZhiL and ZheL participated in the design of the study, analyzed the sequencing data, and drafted the manuscript. ZZ, JW, LM, JL, XY, and LR participated in the field epidemiology survey. ZhiL, ZheL, and LX critically reviewed the manuscript and managed the project. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of Qinghai, China (No. 81860588). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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