Prevalence And Phylogenetic Analysis of Babesia Parasites In Host Animals In Fujian Province, Southeast China

Background: Babesiosis is a tick-borne infectious disease of socio-economic importance. The clinical manifestations of babesiosis are usually intermittent fever, headache, chills, sweats, and myalgia. Babesia infections in small mammals and ticks have been reported in at least ve provinces in China. However, the host range and geographical distribution of this parasite in Fujian Province are unclear. The aim of this study was to investigate the prevalence and genetic characteristics of Babesia in Fujian Province, Southeast China, between 2015 and 2020. Methods: Rodent blood samples were collected from 26 different surveillance sites across Fujian Province. Genomic DNA was extracted to screen for Babesia infection via PCR amplication based on 18S rRNA. The prevalence of Babesia was compared using the chi-square test or Fisher’s exact test. A phylogenetic tree was constructed using MEGA 5.0 by gene sequence alignment. DNA samples from 316 domestic goats, 85 water buffalo, 56 domestic dogs, and 18 domestic pigs were also collected in this survey. Results: Babesia infection was conrmed in 3.96% (43/1087) of rodents and in 1.26% (6/475) of other mammals. Multivariate logistic regression analysis revealed that irrigated cropland, shrub, and forest were risk factors for Babesia microti infection. The infection rates of domestic pigs, dogs, and goats were determined to be 5.56%, 1.79%, and 1.27%, respectively, and no infection was found in water buffalo. Sequencing using the 18S rRNA gene revealed that rodents were infected with Babesia (sensu lato) while other mammals were infected with Babesia (sensu stricto). Conclusions: The results indicate that there is a broad geographical distribution and phylogenetic relationship of Babesia in Southeast China. This study suggests that mammals, especially wild rodents, are the main natural hosts of Babesia in Fujian. Our research provides new insights into the exposure risk of Babesia in humans and animals, laying a solid foundation for the development of babesiosis prevention and control measures.

Cases have also been recorded in China [14], South Africa, India, and Australia.
Babesia parasites have a wide range of vertebrate hosts including rodents, horses, goats, cattle, dogs, cats, and humans [15]. More than 100 different Babesia species have been discovered; however, only a few can infect humans, including B. microti, B. divergens, B. venatorum, and B. duncani [16]. 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 [9,17]. Clinical characterization of babesiosis ranges from asymptomatic infection to severe morbidity and death, including fever, chills, headache, fatigue, anemia, jaundice, thrombocytopenia, hemolysis, hemoglobinuria, and even MODS [7]. The susceptibility to Babesia infection is usually related to the age and immunity of the hosts. Neonates, people of advanced age, those undergoing immunosuppressive therapy, and individuals with HIV/AIDS or cancer are more susceptible to infection [10,18]. Due to a lack of medical awareness, effective diagnosis technologies, and the low incidence of the disease, babesiosis is often neglected in China [16,19]. To date, B. microti-like organisms have been reported in humans from Taiwan [20] and Yunnan [21], and B. microti-like parasites have been found in small mammals and hard ticks in Yunnan [22], Beijing [1], Taiwan [17], Heilongjiang [23] and Henan [24].
Fujian Province, located on the southeast coast of China, belongs to the subtropical climate zone and covers 124,000 square kilometers of land and 136,000 square kilometers of ocean. With su cient rainfall, abundant sunshine, and the best forest coverage of 65.95% in China, the natural and geographical environments of Fujian Province provide ideal habitats for Babesia and favorable conditions for the spread of tick-borne diseases. The aim of this study was to investigate the infection prevalence and phylogenetic relationship of Babesia in mammals in eight cities of Fujian Province, where the host species are abundant.

Sample Collection
A total of 1087 rodents were captured using animal snap traps from eight cities in Fujian Province between 2015 and 2020. The sampling sites included four different habitat environments: residential area, irrigated cropland, shrub, and forest. Traps were placed for three continuous nights at locations where rodent activities were detected and then retrieved the following morning. Chinese monographs were used to identify the species of trapped rodents according to their morphology [25,26]. The sex, developmental stage, and ecological habitat of the mammals were also recorded. Some rare rodent species have been identi ed using DNA barcoding technology [27]. After ether inhalation anesthesia and disinfection, the blood of the rodents was collected through cardiac puncture and stored at -80°C for further tests. Blood samples from 316 domestic goats, 85 water buffalo, 56 domestic dogs, and 18 domestic pigs were collected in this survey.

DNA extraction
Genomic DNA was extracted from the blood of the animals described above using the Blood Genomic DNA Kit (Tagene Biotechnology, Xiamen, China), according to the manufacturer's instructions for animal blood. The genomic DNA was dissolved in 100 μl elution buffer and stored at -20℃ for further use.

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 BLAST analysis against the GenBank database.
Phylogenetic trees were constructed using the neighbor-joining (NJ) method, with 1000 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 (1:1000000), and the latitude and longitude were retrieved from Google Maps. The association between rodent species, sex, developmental stage, habitat environments, sampling locations, and Babesia infection were analyzed using univariate analysis based on the Chi-square test or Fisher's exact test. Multivariate logistic regression was used to analyze the risk factors for Babesia microti infection. All analyses were conducted using SPSS software (version 20.0, SPSS Inc. Chicago, IL). 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 host animals 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 Additional le 2: Table S2, with additional sequence details if available (species, strains, hosts, years, countries, regions, and GenBank accession numbers).

Results
The prevalence of Babesia microti in rodents captured from different cities in Fujian province A total of 1087 rodents were captured from 26 surveillance points in eight cities in Fujian Province (Figs. 1 and 2, Table 1, Additional le 1: Table S1). The captured rodents belonged to Rodentia, including two families, seven genera, and twelve species (Table 2). Sequencing analysis by blastn showed that 3.96% (43/1087) of rodents were infected by B. microti (Table 1).   10%). It is worth noting that the prevalence of B. microti in mammals from irrigated cropland, shrub, and forest, which were 4.70%, 11.18%, and 4.55%, respectively, 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, shrub, and forest were risk factors for B. microti infection (Table 4).   (Fig. 4).

Discussion
Our research systematically illustrated the wide prevalence and phylogenetic relationship of Babesia in host animals in Fujian Province, Southeast China. Infections of B. microti parasites were observed in four cities and eight sampling sites in Fujian Province (Figs. 1, 2, Table 1). Babesia microti has been reported in small mammals in Beijing [1], Henan [24], Yunnan [22] and Taiwan [17]. B. microti infection was also previously reported in the Wuyi Mountain area, Fujian [29]; however, the epidemiological features of Babesia remain unclear in other cities in Fujian. 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 the 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 in Putian, Quanzhou, Zhangzhou, and Longyan were zero, which may be attributed to the insu cient samples and the habitat of trapped rodents (Table 4). Obviously, the prevalence of B. microti was vary from district to district. Although both Fuzhou and Quanzhou are adjacent to Sanming, their B. microti infection rates were both lower than 5.00%, while Sanming had the highest infection rate. This may be attributed to the distribution and density of the host animals.
Interestingly, the infection rate of Babesia in Xiapu District, Ningde City, was 15.79%, which may provide a novel clue to the rst human case of babesiosis in Fujian [30]. The patient who was diagnosed with 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 irrigated croplands, shrubs, and forests was signi cantly higher than in the residential areas, suggesting that the ecological habitat types played an important role in the spread of B. microti (Tables 4 and 5). It has been reported that forest is an essential risk factor for Babesia infection in Thailand, Cambodia, Lao PDR, and China (Yunnan and Heilongjiang) [19,22,23]. Considering that forest areas are burdened with ticktransmitted pathogens, people who work in or travel to the forests should take appropriate protective measures. Babesia and Plasmodium are both intraerythrocytic protozoans and elicit similar in ammatory responses with similar clinical symptoms, which renders them to be easily misdiagnosed [14]. In summary, doctors should pay attention to human babesiosis, while public health agencies should formulate prevention and control measures urgently.
Our study revealed that all the collected Babesia parasites were B. microti in rodents in Fujian Province. This can be concluded based on the abundance of samples detected, similar to previous ndings in Yunnan [16,22], Taiwan [17] and Beijing [1]. 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 a kidney transplantation [29]. Surprisingly, no water buffaloes were infected with Babesia bovis or Babesia bigemina. In addition, both domestic goats and domestic pigs were infected with Babesia sp.. Meanwhile, because of insu cient samples from a single sampling location, hosts such as cattle should be investigated further. 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 Cote d' Ivoire (GenBank MK495837.1). The prevalence of Babesia canis in dogs has been previously documented in Henan province [31].
Ovine babesiosis is a tick-borne disease in goats, sheep, and cattle, posing a huge threat to the livestock industry [32,33]. Although the infection rate of ovine babesiosis is extremely low in this study, relevant institutions should pay more attention and strengthen quarantine for early detection and treatment.
Babesia infection can also be transmitted through blood transfusion when the infected is asymptomatic or in the latent period of the infection [18]. 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 some limitations in our study. Nested PCR approach was used to investigate the rates of Babesia parasites in mammals in some reports [16,19]. Because of we had a large number of samples (a total of 1562 samples in this survey), with a high risk of contaminations during nested PCR, we did not use nested PCR method. In addition, there was no signi cant difference between conventional PCR and nested PCR in sensitivity and speci city [34]. It was reported that the altitude was a risk factor association with Babesia infection [1,22]. But the altitude was neglected to record in our study. With the development of economy and the improvement of living standards, the number of pet dogs and cats have increased. There were some cases of B. vogeli found in dogs and cats in China [2,15,31]. So it is necessary to monitor the epidemics of Babesia infection in pet dogs and cats further.

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 support public health authorities in developing prevention measures and other control measures. Due to insu cient samples from a single sampling surveillance, hosts such as livestock animals should be investigated further. Human babesiosis is a tick-borne disease that is transmitted through blood transfusions. Therefore, it is necessary to survey the prevalence of Babesia in different tick and donor populations.
Declarations Table   Table 5 is not available with this version Figures Figure 1 The map showed the prevalence of Babesia microti in rodents at 26 various surveillance points in eight cities in Fujian province, Southeast China. The prevalence of Babesia microti infection in each city is indicated by different colors, and special infection rate is provided in the legend at the lower right corner.
The geographic location of each surveillance point is labeled with black dots for easy recognition on the map.

Figure 2
The prevalence of Babesia microti in rodents from eight cities in Fujian province. Legend: Error bar indicate ±1.0 standard deviation Figure 3 Neighbor-joining phylogenetic tree based on Babesia microti 18S rRNA partial sequence data from Fujian isolates with Babesia microti reference strains. Babesia divergens, Babesia sp. XXB/Hangzhou and Babesia sp. venatorum were used as the outgroup. For reference, taxon names include the corresponding GenBank accession number, Babesia species, hosts and regions of isolation. The number on each branch indicates the percent occurrence in 1,000 bootstrap replicates. The black circle stands for novel sequences identi ed in this work.