Epidemiological and phylogenetic investigation of Babesia spp. in dogs of Hainan Province/Island, China

Babesiosis is a significant tick-borne disease, which is globally prevalent. Many previous research studies have discussed the presence of Babesia gibsoni, Babesia vogeli, and Babesia canis in dogs in China. In the present study, we have used distinct molecular approaches to detect the presence of Babesia spp. in dogs of Hainan Province/Island, China. A total of 1106 dog blood samples were collected from the Island, of which 61 dog samples were found to be positive for Babesia vogeli. The highest infection rate was 56.7% (17/30) detected from Tunchang, followed by 25.0% (3/12) from Baisha and 10.4% (5/48) from Wenchang. There was only one positive case of Babesia gibsoni, and the infection rate was found to be 0.1% (1/1106). The sequencing results showed that the subjected sample sequences were identical and resembled the Babesia vogeli and Babesia gibsoni sequences available in the database. The results derived from this study will be helpful for planning effective strategies for the treatment, control, and prevention of babesiosis in dogs of Hainan Province/Island.


Introduction
Babesiosis, also known as piroplasmosis, is a disease caused by Babesia protozoan parasites that use a variety of ticks as natural vectors for the disease. The disease is mainly transmitted by tick bite when they bite their hosts (Selim et al. 2022). The transmission of Babesia spp. majorly depends on the type of specific vector in action and the distribution of respective tick vectors. Babesiosis is an important parasitic disease with global significance in regards to human and animal health and the effects it causes on both. Babesiosis cases in humans have been reported primarily in the USA, Canada, and China (Yang et al. 2021). Babesia canis was first detected and described in northern Italy in 1895. Historically, based on the size of intra-erythrocytic inclusions, they were divided into small (1.0-2.5 μm) or large (2.6-5.0 μm) forms of Babesia. All large forms of Babesia infecting dogs were identified as Babesia canis, while all small forms of Babesia infecting dogs were considered as Babesia gibsoni (Baneth 2018). Many studies have found that the large forms of Babesia that can infect dogs include B. canis, B. vogeli, and B. rossi, and the small forms comprise of B. gibsoni, Babesia conradae, and Babesia vulpes (Vishwakarma et al. 2019).
The prevalence and distribution of canine babesiosis are closely related to the distribution of its corresponding tick vectors. However, the increase of host mobility also provides favorable conditions for the spread of the disease from one toother geographical areas.
Traditionally, the diagnosis of canine babesiosis is based on peripheral blood smear or serological test results (Kubelová et al. 2013). However, due to its lower sensitivity, it is considered to be unreliable. Although Babesia spp. are easy to recognize morphologically, studies have shown that the probability of detecting them in routine blood smears is very low. Additionally, due to the different levels and stages of the parasitic disease, blood smear detection might also get affected. Serological techniques, such as immunofluorescence antibody test and dot enzyme-linked immunosorbent assay, are more sensitive than blood smear tests. Polymerase chain reaction (PCR) has been proved to be the most reliable method to detect Babesia spp. infection in dogs (Rucksaken et al. 2019). In the early stages of infection, the antibody titer is lower than the threshold of serodiagnosis, which makes the molecular-based methods as the most preferable and effective method for diagnosis of Babesia spp. (Harrus and Waner 2011).
The main purpose of this study was to investigate and uncover the infection rates of Babesia spp. in dogs from Hainan Island using molecular approaches which would give insights about the prevalence and distribution pattern of Babesia spp. in dogs from Hainan Island.

Sampling and nucleic acid extraction
This study was conducted in Hainan Province from October 2017 to May 2022. One thousand one hundred six dog blood samples were collected from 18 cities and counties of Hainan Province. Samples were selected through random sampling from dogs showing symptoms and signs of having Babesia spp. infection. Post collection, the collected blood samples were stored in vacuum blood collection vessels coated with EDTA and stored at − 80 ℃ until further use. Genomic DNA was extracted from 200 μL of blood sample using a commercial DNA extraction kit, according to the manufacturer's instructions (Ezup Column Blood Genomic DNA Purification Kit, Sangon Biotech, China). DNA samples were stored at − 20 °C for further downstream applications.

Polymerase chain reaction (PCR)
ssrRNA gene was amplified using the forward primer 5′-GTC TTG TAA TTG GAA TGA TGG-3′ and the reverse primer 5′-CCA AAG ACT TTG ATT TCT CTC-3′ (Beck et al. 2009). The fragment amplification length was 550 bp. The same set of primers were used for capillary sequencing. Briefly, PCR reactions were conducted in a total of 25 μL, including 2 X Taq PLus Master Mix II 12.5 μL (Vazyme, China), 1.0 μL of template DNA, 0.5 μL of each primer (10 −8 mol/L), and 10.5 μL of double distilled water. An initial denaturation step at 94 ℃ for 2 min was followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 60 s. Final extension was done at 72 ℃ for 7 min, followed by a hold step at 4 ℃.

Sequence analysis
The amplified positive PCR products were checked using agarose gel electrophoresis in 1.5% agarose gel stained with ethidium bromide and visualized with UV light. Finally, the amplified products were subjected to sequencing using the forward and anti-sense primers. BLASTn program (NCBI) was used to identify and analyze the relevant sequences. Clustal X 2.0.12 software was used for multiple sequence alignment, and representative sequences with different homologies were deposited in the GenBank database with the accession ID's The sequence similarity search in BLASTn showed that the homology of B. vogeli from Hainan province was 99.8-100%, which was highly similar to the B. vogeli sequence published in GenBank (99% nucleotide identity). B. gibsoni was also found to be highly similar to the B. gibsoni sequence published in GenBank (99% nucleotide identity). MEGA-XI software was used to analyze the representative Babesia spp. ssrRNA gene sequences (OP781946-OP781949) obtained from the current study were used for the construction of phylogenetic tree (Fig. 2). These sequences were found to be more than 99.82% identical to B. vogeli sequences derived from cats in Brazil (KT323935), dogs from Italy (AY072925), and the USA (AY371198). The sequence of B. gibsoni was found to be 98.92% identical to the sequences of dogs from Spain (AY278443).
This study is a comprehensive investigation of Babesia spp. infections in dogs in Hainan Province, China, and covers the important aspects related to the infection rates. The use of molecular approaches especially PCR has improved the ability to diagnose canine babesiosis and identify parasite subtypes, making it the most reliable method for detection of Babesia spp.
Hainan Island has a tropical monsoon climate, with relatively high temperature and humidity throughout the year, with an average low temperature of 22 ~ 27 ℃. The climate in 1 3 this area is favorable for the development and survival of the tropical species of tick, Rhipicephalus sanguineus. Molecular phylogeny has made a significant contribution in revealing evolutionary lineages and their relationships. In our study, all PCR positive samples were sequenced targeting the ssrRNA gene fragments. Selected representative sequences were used to construct the phylogenetic tree. Our sequencing and phylogenetic results confirm the existence of B. gibsoni and B. vogeli in the Chinese canine population in our study area. At the same time, B. gibsoni clustered in the same branch as B. gibsoni found in Spanish dogs (AY278443).
Babesia spp. found in Hainan Province was B. vogeli, which is less pathogenic, and the infection is relatively mild, representing no obvious clinical symptoms and manifestations (Uilenberg et al. 1989). The reason for this is that the main vector of transmission for B. vogeli is R. sanguineus, which is a highly common tick found in Chinese dogs and widely distributed globally. The geographical location of Hainan Island provides a relatively comfortable environment for the development of R. sanguineus, which is conducive to the spread of B. vogeli.
Regarding the infection of B. vogeli, the epidemic range varies greatly from region to region, with reported infection rates in some provinces in China ranging from 0.85 to 11.0%. A research study showed that B. gibsoni was the most common species in Babesia spp. in Chinese dog population, followed by B. vogeli (Zhang et al. 2019). It is reported that in Jiangsu (Shen et al. 1997), Yunnan (Xu et al. 2015), Henan (Wang et al. 2019), Hubei (He et al. 2017), Hunan , Jiangxi (Zheng et al. 2017), Guangdong  provinces of China, dogs have been found to be infected with B. gibsoni and B. vogeli. It was also reported previously that Babesia spp. was detected in ticks in Hainan Province, with an infection rate of 3.3% (4/121) which was in proximity to the infection rate of 5.6% (62/1106) detected in dogs from this study (Wei et al. 2012). The results obtained from this study indicate that B. vogeli is commonly found Babesia spp. and may lead to extensive subclinical infection. Fig. 2 Phylogenetic tree. The tree was generated from the ssrRNA partial fragments of the autochthonous B. vogeli and B. gibsoni isolates compared with other corresponding GenBank sequences by the neighborjoining method with bootstrap of 1000 replications and the evolutionary distance adjusted using the Kimura-2-parameter. GenBank access numbers were shown in the tree. Arrows indicate the species which were reported in the present work

Conclusions
This report describes for the first time a very comprehensive and precise molecular detection and characterization of babesiosis canis in dogs in Hainan Province. A total of 1106 dog blood samples are collected in Hainan Island, and B. vogeli and B. gibsoni were found in 61 samples and 1 sample. To our best knowledge, this is the first such attempt and kind of study which will provide a lot of information about the prevalence and distribution pattern of the disease in the province.