Prevalence and phylogenetic analysis of Babesia parasites in reservoir host species in Fujian province, Southeast China

Babesiosis is a tick‐borne disease that mainly affects small mammals and has been reported in at least five provinces in China. However, the host range and geographical distribution of the parasite in Fujian province are unclear. Therefore, we investigated the prevalence and genetic characteristics of Babesia in Fujian province, Southeast China, between 2015 and 2020. Rodent blood samples were collected from 26 different surveillance sites across Fujian province. Genomic DNA was extracted to screen for Babesia infection using polymerase chain reaction based on 18S rRNA. DNA samples from 316 domestic goats, 85 water buffalo, 56 domestic dogs and 18 domestic pigs were examined. The prevalence of Babesia was statistically analysed using the Chi‐square test or Fisher's exact test. Babesia infections were detected in 3.96% (43/1,087; 95%CI: 2.80%, 5.12%) of rodents and 1.26% (6/475; 95%CI: 0.26%, 2.26%) of other mammals. Multivariate logistic regression analysis revealed that irrigated cropland, shrubs and forests were risk factors for Babesia microti infections. The infection rates among domestic pigs, dogs and goats were 5.56%, 1.79% and 1.27%, respectively, with no infection found in water buffalo. The 18S rRNA gene sequencing revealed that rodents were infected with Babesia (sensu lato), whereas other mammals were infected with Babesia (sensu stricto). The geographical distribution and phylogenetic relationship of Babesia was determined in Southeast China. Mammals, particularly wild rodents, maybe the main natural hosts of Babesia in Fujian. Our findings provide a foundation for public health officials to develop prevention and control measures for Babesia.

The rst human case was reported in 1957 in Zagreb, Croatia, and subsequently found on all continents, except for Antarctica. Infections are primarily found in tropical and subtropical areas [7,8]. More than 60 cases of human babesiosis have recently been reported in China including 48 patients infected with Babesia venatorum in Heilongjiang [9], 12 infected with Babesia divergens in Shandong [10] and Gansu [7], eight infected with Babesia microti in Yunnan [11], and one case infected with Babesia sp. XXB/Hangzhou in Zhejiang [12]. During the past few decades, an increasing number of B. microti species have been reported in the upper midwestern and northeastern regions of the USA [2,13]. In Europe, B. divergens is responsible for most cases of babesiosis and the parasites are transmitted by Ixodes ricinus ticks and infected cows [14,15]. Endemic infections of B. microti in rodents and ticks have been recently detected in many European countries, including Slovakia [16], Finland [17], Belgium [18], Switzerland [19], Poland [20,21], and France [22]. Cases have also been recorded in South Africa [23], India [24], and Australia [25]. Therefore, the recent emergence of babesiosis has become a worldwide public health concern.
Babesia parasites have a wide range of vertebrate hosts including rodents, horses, goats, cattle, dogs, cats, and humans [5]. More than 100 different Babesia species have been discovered; however, only a few are capable of infecting humans, including Babesia microti [26], B. divergens [7], B. venatorum [27], and Babesia duncani [28]. As the main etiological agent of human babesiosis, the rodent parasite B. microti is maintained through an enzootic cycle in nature, which involves ixodid ticks and small mammals [14,29]. The clinical characterization of babesiosis ranges from asymptomatic infection to severe morbidity (fever, chills, headache, fatigue, anemia, jaundice, thrombocytopenia, hemolysis, hemoglobinuria, and even multiple organ dysfunction syndromes [MODS]) and may result in death [2]. Susceptibility to Babesia infection is usually related to the age and immune status of the host. Neonates, people of advanced age, those undergoing immunosuppressive therapy, and individuals with acquired immune de ciency syndrome (AIDS), or cancer are more susceptible to infection [15,30]. Babesiosis is frequently overlooked in China, due to a lack of medical awareness, effective diagnostic techniques, and the low incidence of the disease [31,32]. To date, B. microti-like organisms have been reported in humans from Taiwan [33] and Yunnan [34], and B. microti-like parasites have been found in small mammals and hard ticks in Yunnan [35], Beijing [3], Taiwan [29], Heilongjiang [36], and Henan [37].
Fujian Province is located on the southeast coast of China, has a subtropical climate, and encompasses 124,000 square kilometers of land and 136,000 square kilometers of ocean. The natural and geographical environments in Fujian provide an ideal habitat for Babesia and favorable conditions for the spread of tick-borne diseases, with an average annual rainfall of 1400-2000 mm, abundant sunshine, and 65.95% forest coverage. This study aimed to investigate the infection prevalence and phylogenetic relationship of Babesia in mammals across eight cities of Fujian Province, where the host species are abundant.

Sample collection
A total of 1,087 rodents were captured from eight cities in Fujian Province between 2015 and 2020 with live animal traps. The sampling sites included four different habitats: residential areas, irrigated cropland, shrubland, and forests. Live traps were placed every night at every surveillance point for three consecutive nights at locations where rodent activities were detected and were retrieved the following morning.
Chinese monographs were used to identify the species of trapped rodents according to their morphology [38,39]. The sex, age class, and ecological habitat of the mammals were recorded. Any rare rodent species captured were identi ed using DNA barcoding technology [40]. All animal experiments were performed following the Guidelines for the Care and Use of Laboratory Animals [41]. For anesthesia, the rodents were placed in a biological safety cabinet, in a transparent plastic box together with cotton wool soaked in ether. After the rodents were anesthetized and disinfected, 1 mL of blood from each rodent was collected through cardiac puncture and stored at -80°C for further tests. Blood samples (2 mL) from 316 domestic goats, 85 water buffalo, 56 domestic dogs, and 18 domestic pigs in Fujian Province were collected, and animals were returned to their farms. Both groups of animals used in this study were part of larger surveillance projects investigating hemorrhagic fever with renal syndrome (rodents) and severe fever with thrombocytopenic syndrome (domestic animals).

DNA extraction
The Blood Genomic DNA Kit (Tagene Biotechnology, Xiamen, China) was used to extract genomic DNA from the animal blood samples according to the manufacturer's instructions. The genomic DNA was dissolved in 100 µL elution buffer and the 260/280 absorbance ratio was measured to con rm that the DNA purity was between 1.7 and 1.9 using a spectrophotometer (Denovix, DS-11, USA). Samples were stored at -20 ℃ until further use.

Detection of Babesia infection using polymerase chain reaction
Polymerase chain reaction (PCR) ampli cation of the speci c fragment of the Babesia 18S rRNA gene region was performed using the following primers: PIRO-A, 5′-AATACCCAATCCTGACACAGGG-3′, PIRO-B, 5′-TTAAATACGAATGCCCCCAAC-3′ which were used in previous studies [42,43]. Target DNA ampli cation was carried out under the following conditions: 94°C for 5 min; 40 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s; followed by a nal extension step at 72°C for 5 min. A concentration of 20-50 ng/µL of genomic DNA (5 µL) was used as a template in a 25 µL reaction, which contained 12.5 µL Premix Taq (TaKaRa Taq Version 2.0 plus dye, Beijing, China) and 1.0 µL of each primer ( nal concentration 0.4 µM). To eliminate the possibility of contamination, negative (nuclease-free water) and positive controls (a con rmed Babesia sample from the rst human babesiosis case in Fujian Province) were also included. Ampli ed products were subjected to electrophoresis on a 1.5% agarose gel stained with SYBR gold (Invitrogen, Shanghai, China) and visualized under ultraviolet light. Positive PCR products were puri ed and sequenced using the primers listed above, by Sangon Biotechnology Company (Shanghai, China) using Sanger sequencing.

Phylogenetic analysis
The sequences were assembled using the SeqMan program software 7.0.1 (DNASTAR, Inc.; Madison, WI, USA). All newly generated sequences were subjected to the Basic Local Alignment Tool (BLAST) analysis against the GenBank database. Phylogenetic trees were constructed using the neighbor-joining method, with 1,000 replications for bootstrap tests. All other parameters were set at default.

Statistical analysis
A spatial map of the prevalence of Babesia in rodents was drawn using ArcGIS 10.3.1. The geographic data used the vector map of the administrative divisions of the county boundaries of Fujian Province (1:1,000,000), and the latitude and longitude were retrieved from Google Maps. The association between rodent species, sex, age class, habitat environments, sampling locations, and Babesia infection were analyzed using the univariate analysis based on the Chi-square (χ 2 ) test or Fisher's exact test.
Multivariate logistic regression was used to analyze the risk factors for B. microti infection. All analyses were conducted using the SPSS software (version 20.0, SPSS Inc. Chicago, IL, USA). The signi cance level for all results was set at P < 0.05. Sequences used for phylogenetic analysis in the study To clarify the phylogenetic relationship of Babesia species detected in reservoir host species collected in Fujian Province, sequences of the 18S rRNA gene fragments were used for alignment and comparison to the sequences from the GenBank database and are summarized in the additional le 1: Table S1, with additional sequence details if available (species, strains, hosts, years, countries, regions, and GenBank accession numbers).

Prevalence of B. microti in rodents captured from different cities in Fujian Province
A total of 1,087 rodents were captured from 26 surveillance points in eight cities in Fujian Province (Fig. 1, Table 1, Additional le 2: Table S2). The captured rodents belonged to the order Rodentia, and they were part of two families, seven genera, and 12 species (Table 2). Sequencing analysis using BLASTn revealed that 3.96% (43/1,087) of the rodents were infected by B. microti (Table 1).  Infected rodents were captured in four cities: Sanming, Ningde, Nanping, and Fuzhou (Table 1). Rodents collected from Sanming had the highest B. microti infection rate of 9.94% (17/171). Babesia microti infection rates in the rodents from Sanming and Ningde were both signi cantly higher than in the rodents captured in Fuzhou (odds ratios: 5.96, 4.18, respectively; P < 0.05) ( Table 1).

Prevalence of Babesia in domestic animals in Fujian
The positive infection rates of Babesia in domestic pigs, domestic dogs, and domestic goats were 5.56%, 1.79%, and 1.27%, respectively. No water buffaloes were infected with Babesia (Table 2). There was no signi cant difference in the prevalence of Babesia between male and female domestic goats (P = 0.129) (data not shown).

Risk factors associated with B. microti infection
Risk factors related to B. microti infection in rodents were analyzed with respect to sex, age, and ecological habitat (Table 3). There was no signi cant difference in the prevalence of B. microti between male and female rodents (χ 2 = 0.466, P = 0.495). However, the prevalence of B. microti in adult rodents (4.53%) was signi cantly higher (χ 2 = 4.645, P = 0.031) than in pubertal rodents (1.10%). It is worth noting that the prevalence of B. microti in mammals from irrigated cropland, shrubs, and forests, was 4.70%, 11.18%, and 4.55%, respectively, and were all signi cantly higher than those in rodents from residential areas (P < 0.05, Tables 3 and 4). Furthermore, the multivariate logistic regression analysis suggested that irrigated cropland, shrubs, and forests were risk factors for B. microti infection (Table 4).   (Fig. 3).

Discussion
Our research systematically illustrated the wide prevalence and phylogenetic relationship of Babesia in reservoir host species in Fujian Province, Southeast China. Infections of B. microti parasites were observed in four cities and eight sampling sites in Fujian Province (Fig. 1, Table 1). Babesia microti has been reported in small mammals in Beijing [3], Henan [37], Yunnan [35], and Taiwan [29]. Babesia microti infections was also previously reported in the Wuyi Mountain area, Fujian [44]; however, the epidemiological features of Babesia remain unclear in the other cities in Fujian. In this study, the prevalence of B. microti in rodents (3.96%) followed the low prevalence described in Yunnan (4.31% [31] and 2.40% [35]), and the Dapan Mountains of Zhejiang (1.30%) [45]. However, the positive infection rates of B. microti in R. tanezumi in Yunnan (2.70%) and Dapan Mountains (5.56%) were higher than in our research (0.61%). Our study showed that the high prevalence of B. microti in N. confucianus in Fujian (17.02%) was similar to that found in the Dapan Mountains of Zhejiang (20.0%). This suggested that N. confucianus may be superior reservoir hosts in Southeast China [35]. The high prevalence of B. microti infection in rodents in Ningde and Sanming in this survey strongly supports the hypothesis that these surveillance points are major natural foci for human babesiosis. Furthermore, the results call for close monitoring of B. microti transmissions in Ningde and Sanming, while the epidemic of B. microti in other cities should not be ignored. It should be noted that the B. microti infection rates were zero in Putian, Quanzhou, Zhangzhou, and Longyan, which may be attributed to a lack of samples and rodent habitats ( Table 4). The prevalence of B. microti varied from district to district. Although both Fuzhou and Quanzhou are adjacent to Sanming, the B. microti infection rates were lower than 5.00% for both, while Sanming had the highest infection rate. This may be attributed to the distribution and density of the reservoir host species.
Interestingly, the infection rate of Babesia in Xiapu District, Ningde City, was 15.79%, which may provide a novel link to the rst human case of babesiosis in Fujian [46]. The patient, who was diagnosed with a B. microti infection, lived and worked in a village in Xiapu, Ningde, which was surrounded by abundant shrubs and forests. Our study revealed that the prevalence of B. microti in rodents from shrubs (11.18%), irrigated cropland (4.70%), and forests (4.55%) was signi cantly higher than in the residential areas (0.99%), suggesting that ecological habitat types played an important role in the spread of B. microti. Furthermore, B. microti would be able to live and reproduce in the wild, which is likely related to the habitat and density of the tick vector [35]. Similar results have been reported in Yunnan [35] and Beijing [3]. The prevalence of B. microti in small mammals in Yunnan from the forest (3.37%) and agricultural areas (1.79%) was signi cantly higher than in residential areas (0.93%). In Beijing, the positive rate of B. microti from different habitats was shrubs (27.4%), broad-leaved forests (23%), cropland (16%), mixed forests (8.4%), and residential areas (7.2%). It has been reported that forests are an essential risk factor for Babesia infection in Thailand, Cambodia, Lao PDR, and China (Yunnan and Heilongjiang) [32,35,36].
Considering that forest areas are burdened with tick-transmitting pathogens, people who work in or travel to forests should take appropriate protective measures. Both Babesia and Plasmodium are intraerythrocytic protozoans and elicit similar in ammatory responses with similar clinical symptoms, which allows them to be easily misdiagnosed [47]. In summary, doctors should pay attention to human babesiosis, while public health agencies should urgently formulate prevention and control measures. Our study found that the prevalence of B. microti in adult rodents (4.53%) was signi cantly higher than in pubertal rodents (1.10%), which was similar to other studies in Yunnan (2.69% (adult) and 0.37% (pubertal)) [35] and Beijing (13.3% (adult) and 6.2% (pubertal)) [3].
Our study revealed that all the collected Babesia parasites were B. microti from rodents in Fujian Province. This conclusion can be drawn from the abundance of samples detected, which was similar to previous ndings in Yunnan [31,35], Taiwan [29], and Beijing [3]. Phylogenetic analysis suggested that B. microti in this survey shared high homology with those in Zhejiang Province, where a con rmed human babesiosis case was reported in Hangzhou in 2002 following kidney transplantation [44]. Surprisingly, no water buffaloes were infected with Babesia bovis or Babesia orientalis. This varied in comparison to other studies [48,49], which might be due to insu cient samples and a single sampling location. In addition, both domestic goats and domestic pigs were infected with Babesia spp. We detected B. canis vogeli from the blood of domestic dogs for the rst time and the sequence shares high homology with B. canis vogeli from Côte d'Ivoire (GenBank MK495837.1). The prevalence of B. canis in dogs has been previously documented in Henan province [50].
Ovine babesiosis is a tick-borne disease in goats, sheep, and cattle, posing a huge threat to the livestock industry [51][52][53]. Although the infection rate of ovine babesiosis is extremely low in this study, relevant institutions should pay more attention and strengthen quarantine measures for early detection and treatment. Babesia infection can also be transmitted through blood transfusion when the infected individual is asymptomatic or in the latent period of the infection [30]. Therefore, it is necessary to test Babesia infection in donors when evaluating the risk of blood transfusion. In the future, we will investigate the prevalence of Babesia in different ticks and blood donors to provide scienti c evidence for preventing and controlling babesiosis epidemics.
There are limitations to this study. A nested PCR approach was used to investigate the rates of Babesia parasites in mammals in some reports [31,32]. A large number of samples (n = 1,562) with a high risk of contamination were part of this study, therefore the nested PCR method was not used. Our study showed that the prevalence of B. microti was higher in northern Fujian (Sanming, Nanping, Ningde, and Fuzhou) than in southern Fujian (Putian, Quanzhou, Zhangzhou, and Longyan), which could be due to altitude, as northern Fujian (495 meter) has a higher average altitude than southern Fujian (411 meter). A study in southern Norway has shown that ticks exist at an altitude much higher than previously, with an increased risk of infection of mammals with tick-borne diseases [54]. It was reported that altitude is a risk factor associated with Babesia infections [3,35]; however, altitude was not taken into account in this study. With the development of the economy and the improvement of living standards in China, the number of domesticated dogs and cats has increased, resulting in B. vogeli cases in dogs and cats [4,50,55]. Therefore, it is necessary to monitor Babesia infections in domestic dogs and cats.

Conclusions
Our study suggests a wide distribution and phylogenetic relationship of Babesia in mammals in Fujian Province, Southeast China. This research provided basic data to help the public health authorities develop prevention and control measures. Due to insu cient samples from single sampling surveillance, hosts such as livestock should be investigated. Because human babesiosis is a tick-borne disease that is transmitted through blood transfusions, it is necessary to survey the prevalence of Babesia in various tick and donor populations.