Molecular Detection of Babesia gibsoni in Cats in China

Simple Summary Tick-borne diseases in companion animals have been increasing globally. Domestic dogs and cats as potential reservoir hosts of tick-borne pathogens might transfer zoonotic diseases to humans. There are currently few reports of feline babesiosis in China. To investigate the incidence of Babesia spp. infection in cats, blood samples were collected from Chongqing, Fujian, Hubei and Shandong, and Babesia gibsoni was detected. These findings will be useful for understanding the epidemic situation of babesiosis in China and provide a theoretical basis for undertaking effective disease control measures in the interests of public health. Abstract As there are few studies of Babesia spp. infection in cats in China, or anywhere in the world, the aim of this study was to explore the epidemic features of babesiosis in pet cats in China. In total, 429 blood samples were randomly collected in four different geographical regions. The 18S rRNA gene fragment of Babesia spp. was amplified by nest polymerase chain reaction (PCR), and haplotype and phylogenetic analysis of Babesia were performed to analyze the relationship of this protozoa. The total positive rate of infection was 2.8%. BLAST analysis indicated that Babesia gibsoni was detected in 12 cats. Among these, 4.3%, 3.1%, 0.8% and 2.0% were from Chongqing, Fujian, Hubei and Shandong, respectively. Haplotype and phylogenetic analysis showed that there were nine haplotypes and no obvious genetic variation among B. gibsoni populations. These findings will be helpful for understanding the epidemiology of Babesia spp. in China, and provide a foundation for developing effective preventative strategies.


Introduction
Tick-borne diseases are recognized as important infectious diseases, posing a potential threat to the health of humans and animals. Tick-borne infections in companion animals have been increasing worldwide, which could be due to the distribution of vectors influenced by climate change, environmental and artificial factors [1] and increased detection capacity with the popularization of molecular biology. In China, there were 74 million dogs and 67 million cats as companion pets at the end of 2018 [2]. As potential reservoir hosts of tick-borne causative agents, domestic dogs and cats can transfer zoonotic diseases to humans [3,4], so the risk of feline and canine tick-borne pathogens has drawn attention from veterinary and public health research organizations.
Babesiosis, caused by the intracellular hemoprotozoa of the genus Babesia, is transmitted by ixodid ticks and has been widely described [5,6]. The severity of this disease ranges from mild infections to severe illness characterized by anorexia, fever, icterus, pallor, splenomegaly, hemolytic anemia and hemoglobinuria [7][8][9]. The species of Babesia are divided into the large types, including Babesia canis, Babesia rossi and Babesia vogeli and small types, including Babesia conradae, Babesia gibsoni and Babesia vulpes [10][11][12][13]. In China, B. canis, B. gibsoni and B. vogeli have been described in dogs and are endemic in the central,

PCR Amplification and Sequencing
To identify the Babesia infection in cats, a nested PCR was employed to amplify a region of the 18S rRNA gene as previously described [16,18,25]. Primary amplification was conducted using Piro1-S: 5 -CTTGACGGTAGGGTATTGGC-3 , Piro3-AS: 5 -CCTTCCTTTAAGTGATAAGGTTCAC-3 to amplify a gene fragment of 1379 bp, and sec-ondary amplification was performed using Piro-A: 5 -ATTACCCAATMCBGACACVGKG-3 and Piro-B: 5 -TTAAATACGAATGCCCCCAAC-3 to amplify a gene fragment of 405 bp. The PCR reaction conditions were described previously [25]. All PCR products were analyzed on 1.5% agarose gels stained with GoldView II dyes (Solarbio, Beijing, China) and visualized under ultraviolet light. A sick dog was clinically diagnosed as babesiosispositive by microscopic analysis and a positive PCR test for B. gibsoni. Genomic DNA extracted from the blood sample of the sick dog was used as a positive control and distilled water was used as negative control.
The positive amplicons were purified using a Hipure Gel Pure DNA Mini Kit (Magen, Guangzhou, China). The purified products were cloned into the pMD19-T vector (TaKaRa, Dalian, China), and then transformed into Escherichia coli Trans5α competent cells (Trans-Gen, Beijing, China). Three positive clones were sequenced using universal M13 forward and reverse primers (PRISM3730XL, ABI).

Sequences Analysis
The obtained 18S rRNA sequences were analyzed using the NCBI BLASTN program (https://blast.ncbi.nlm.nih.gov, accessed on 27 January 2022 and 22 June 2022) and the sequences were deposited in GenBank under the accession numbers OM403679-OM403682 and ON810481-ON810488.
Multiple sequences were aligned using Clustal W within MEGA 11 software [26]. Nucleotide sequence analysis was performed by Genedoc program [27]. A haplotype network was drawn using the TCS network within PopArt software [28,29]. To evaluate the phylogenetic relationships, the sequences were compared with the registered sequences from different countries in the GenBank database. A phylogenetic tree was constructed using the neighbor-joining method based on the Tamura 3-parameter substitution model with gamma distributed (G) rates in MEGA 11, with bootstrap values of 1000 replicates [26]. A 50% cut-off value was performed for the consensus tree.

Detection and Identification of Babesia spp.
The results showed that the total prevalence of Babesia spp. infection was 2.8% (12/429) of sample cats, shown in Table 1. Among these 12 positive samples, 4.3%, 3.1%, 4.0% and 2.0% were from Chongqing, Quanzhou, Wuhan and Linyi cities, respectively, but there were no positive samples found in Xiangyang city. All the positive pet cats were under one year of age. Blast analysis showed that the obtained sequences were 98.8% to 99.8% identical to that of B. gibsoni from dogs in China (KP666166), India (MN134517), Japan (AB478328) and the USA (DQ184507). Compared with the sequences from China, India, Japan and the USA, these results showed 11 substitutions at nucleotide sites 11,13,19,83,109,163,198,250,285, 289 and 360, as seen in Figure 2. All obtained 18S rRNA sequences showed 98.5% to 100% nucleotide identity with each other. Haplotype analysis indicated that nine haplotypes existed in B. gibsoni isolates in this study, as shown in Figure 3.

Phylogenetic Analysis of B. gibsoni Using 18S rRNA Sequences
A neighbor-joining (NJ) tree was constructed using the 18S rRNA sequences gibsoni isolates derived from cats, together with the data deposited in GenBank (se 4). The results showed that no obvious sub-clusters were observed among B. gib lates from this study and those from other geographic regions including China, I pan and the USA. The NJ tree also indicated that there was no apparent genetic v

Phylogenetic Analysis of B. gibsoni Using 18S rRNA Sequences
A neighbor-joining (NJ) tree was constructed using the 18S rRNA sequences of 12 B. gibsoni isolates derived from cats, together with the data deposited in GenBank (see Figure 4). The results showed that no obvious sub-clusters were observed among B. gibsoni isolates from this study and those from other geographic regions including China, India, Japan and the USA. The NJ tree also indicated that there was no apparent genetic variation among the B. gibsoni isolates between dogs and cats. The other Babesia species such as B. felis, B. hongkongensis, B. leo, B. microti and B. vogeli formed separate clades with high bootstrap support (Figure 4). Original references order: 33-39. 50 Revised references order: 34-40. 51 With this correction, the order of some references has been adjusted accordingly.

Discussion
For feline Babesia species, B. vogeli was found in three of 203 cats in China [22], and has been reported in Thailand, Portugal and Brazil [30][31][32]. In other studies, one cat was diagnosed with B. hongkongensis in Hong Kong and two cats were positive for B. hongkongensis in Hunan [23,24]. For the presence of B. gibsoni in cats, there were related reports in China, Singapore and St Kitts [21,33]. B. gibsoni was considered to be a species responsible for canine babesiosis in China, including Shanghai, Jiangsu, Shandong, Anhui, Zhejiang, Jiangxi, Fujian, Hubei and Shaanxi with positivity 0.72% to 64.2% [15,34,35].
In the present study, a small-scale survey was conducted in pet animals to investigate the epidemiology of babesiosis. BLAST analysis showed that the obtained 18S rRNA sequences shared high identity with the 18S rRNA of B. gibsoni, indicating that only B. gibsoni was found in cats and the total prevalence of B. gibsoni infection was 2.8%, indicating that the prevalence of B. gibsoni infection in cats was lower than in dogs in China. According to clinical records, the pet cats spent most of their time indoors and had limited chance to roam around outside, so were exposed to a low-risk environment with regard to active Haemaphysalis longicornis and Rhipicephalus sanguineus. All 12 positive cats were under one year of age, which was consistent with a previous study indicating young animals were susceptible to B. gibsoni infection [22].
Nucleotide sequence analysis suggested that nucleotide variations were found within 18S rRNA sequences of B. gibsoni ( Figure 2). Haplotype analysis demonstrated that genetic differences were observed among B. gibsoni sequences (Figure 3). Phylogenetic analysis displayed that all B. gibsoni 18S rRNA genes from cats belonged to a clade consisting of those from dogs in other parts of China, India, Japan and the USA (Figure 4). These data were in agreement with previous studies, showing a limited genetic relationship of B. gibsoni populations in Asia and the USA [36]. B. gibsoni has been detected in dogs from Fujian, Shandong and Hubei in the previous studies [15,34]. In this study, B. gibsoni was also found in cats from the same three regions. H. longicornis occurred in Hubei and Shandong and R. sanguineus was endemic in Fujian and Shandong [37,38]. Therefore, it is hypothesized that B. gibsoni might be circulating in dogs and cats in these three sampling sites. These results provided evidence of the occurrence of cross-species transmission in the different hosts, which could be related to the movement of humans carrying pets, the mobility of hosts with ticks and shared habitats between different hosts. No ticks were found on the bodies of these pet cats, and what causes Babesia infection in pets will be examined in further research. Possibilities include that the ticks had already dropped off the cats before they were taken to the clinic, and it can be inferred that cats could become positive from direct transmission such as fighting and blood transfusion according to the transmission routes of B. gibsoni [39,40].

Conclusions
B. gibsoni was found in a low proportion of asymptomatic cats in China, as nine haplotypes found among 12 isolates. Phylogenetic analysis indicated that there was no obvious genetic variation among B. gibsoni populations based on 18S rRNA sequences. These findings can provide greater insight into the distribution of Babesia and its genetic relationship in these four regions of China, and will be also useful for making effective control approaches to improve the health and welfare of companion animals.