Low prevalence of Babesia hongkongensis infection in community and privately-owned cats in Hong Kong

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Pet ownership is reported in 9.4 % of households of Hong Kong, with cats comprising 42.6 % of owned pets (HKSAR, 2019).Babesia infection of dogs in Hong Kong has a higher prevalence in the stray population compared to owned dogs (Muguiro et al., 2023;Wong et al., 2011).To the best of our knowledge, molecular evidence that Babesia species are circulating in the cat population in Hong Kong has been reported in only two cases to date (Almendros et al., 2023;Wong et al., 2012).The objectives of this study were to determine the prevalence and species of Babesia detected in healthy community-owned cats and in privately-owned cats with and without anaemia in Hong Kong.

Group selection and distribution
Ethical approval for this study was granted by the Animal Ethics Committee of City University of Hong Kong (approvals A-0478 and AN-STA-150 00000015).Two groups of cats were investigated in this study.All data collected was treated confidentially and was deidentified.

Privately-owned cats
Since babesiosis in dogs is often associated with anaemia, samples were collected from anaemic and non-anaemic cats to determine if anaemia was a risk factor for Babesia infection.Residual diagnostic EDTA blood samples and blood smears were collected from anaemic and non-anaemic cats presented to CityU Veterinary Medical Centre (VMC) between July 2022 to March 2023.Anaemia was defined as a haematocrit (HCT) ≤ 25 % (Tasker, 2012).This mixed population of cats included healthy cats presented for routine healthcare (e.g.desexing) and sick cats undergoing diagnostic investigations.
Age, breed, sex, and neuter status were recorded.Cats were categorised into two age groups (≤ 3 and > 3 years).Haematocrit and platelet count (PLT) were recorded for all cats.Where available, absolute reticulocyte count was also recorded.

Community cats
Community cats are free-roaming cats living in colonies that are cared for by members of the public.Whole blood samples were collected, with consent, from community cats presented to a trap-neuterrelease program at the Society for the Prevention of Cruelty to Animals (SPCA), Hong Kong between January and May 2021.Whole blood was centrifuged and separated sera and blood clot were stored separately at -80 • C. Sera from each community cat was tested for Feline Leukaemia Virus (FeLV) antigen and Feline Immunodeficiency Virus (FIV) antibody (SNAP Combo, IDEXX, Westbrook, ME).Age, breed, sex, and neuter status were recorded.Cats were categorised into two age groups (≤ 3 and > 3 years).Cats were clinically examined by a veterinarian and found to be healthy prior to surgery.

DNA extraction
DNA was extracted from 100 µL of EDTA blood or blood clot using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.DNA was eluted in 50 µL and stored at -80 • C until analysis.Conventional PCR of feline glyceraldehyde-3phosphate dehydrogenase (GAPDH) was performed to confirm the integrity of extracted DNA (McLuckie et al., 2018).

PCR assays
Babesia spp.detection was performed using two PCRs as previously described, one targeting the mitochondrial cytochrome b gene (cytb) and the other targeting 18S rRNA (Wong et al., 2012).PCR primers and cycling conditions are presented in Table 1.Each reaction contained 1 µL of template DNA, DreamTaq™ Hot Start Green DNA Polymerase (Thermo Fisher Scientific, Graciuno, Vilnius, Lithuania), and dNTP (Thermo Fisher Scientific, Graciuno, Vilnius, Lithuania) at a final concentration of 200 µM and a final primer concentration of 0.5 µM.For all PCRs, a positive control (synthetic DNA construct, Thermo Fisher Scientific, Graciuno, Vilnius, Lithuania) and negative control (molecular grade water) were included.For samples that were Babesia positive, an additional pan-Babesia nested PCR was performed to obtain a longer 18S rRNA sequence (Li et al., 2015).
Bands of the expected target size obtained on gel-electrophoresis after PCR were sequenced by Sanger sequencing (BGI Genomics, Hong Kong).Geneious software (version 2023.1.1)was used to assemble the consensus sequence.The nucleotide homology and coverage of sequences generated were compared by Basic Local Alignment Search Tool (BLAST) against sequences in the NCBI database in GenBank.

Microscopic examination
Blood smears from samples testing Babesia positive on PCR from privately-owned cats were fixed with methanol and stained with modified Giemsa prior to examination under a light microscope to identify piroplasms or intraerythrocytic inclusions.Blood smears were not available from community cats.

. Data analysis
The proportion of the total number of cats testing positive for Babesia spp.(frequency) was calculated.Descriptive analyses of age, breed, sex, neuter status and haematological values (range and mean) were performed.Associations between FIV and FELV status and a positive Babesia result were tested by Fisher's exact test.

Results
The sample population comprised 364 cats, of which 125 were privately-owned and 239 were community cats.

Privately-owned cats
Amongst the 125 privately-owned cats, age ranged from 9 weeks to 19.5 years (mean 7.8 years, median 8 years).Sex, neuter status, breed and numbers of cats with and without anaemia are listed in Table 2.

Community cats
Of the 239 community cats, age ranged from 1 to 9 years (mean 2.2 years, median 2 years).Signalment details and results of FIV and FeLV testing are presented in Table 2. Four community cats (1.7 %) tested PCR positive for Babesia, and all were confirmed by sequencing to be B. hongkongensis (Table 3).For each positive sample, a CytB sequence and a 18 s rRNA sequence was obtained except for one, for which we were unable to obtain the 18 s rRNA by PCR (Table 3).
The B. hongkongensis positive community cats were all sexually intact DSH including three males and one female aged 1, 2, 5 and 6 years old.At the time of blood sampling, fleas were detected on one of these cats while ticks were detected on none.One cat also tested positive for FIV, compared to 40/235 Babesia negative cats that also tested positive for FIV positive.No significant statistical association was found between FIV and Babesia infection by Fisher exact test (p=0.5314).

Discussion
The key findings in this study were that Babesia infections occur in cats in Hong Kong and are caused by B. hongkongensis and B. gibsoni.The infection rate (1.4 %) amongst cats sampled in this study was low.In dogs in Hong Kong, B. gibsoni is by far the most common Babesia species detected (Almendros et al., 2020;Hussain et al., 2023;Muguiro et al., 2023).By contrast, B. hongkongensis was discovered serendipitously in cats in 2012 after piroplasms were identified in histologic stains of kidney tissue from a community cat in an unrelated study (Wong et al., 2012).Molecular surveillance of 300 community cats at that time failed to identify any other Babesia-infected cats (Wong et al., 2012).Despite molecular surveillance spanning over a decade, B. hongkongensis has never been detected in dogs (Almendros and Burchell, 2021;Almendros et al., 2020;Hussain et al., 2023;Muguiro et al., 2023), suggesting that it has a particular tropism for cats, or that cats are more likely to come into contact with its vector.In Hong Kong, ixodid ticks from four genera have been reported -Rhipicephalus, Haemophysalis, Ixodes and Hyalomma (Chan et al., 2011).However, surveillance to determine the relative prevalence of tick species parasitizing dogs in Hong Kong is lacking.Rhipicephalus sanguineus and Haemophysalis longicornis are the most common tick species identified on dogs in East and Southeast Asia (Cheng et al., 2018;Colella et al., 2020;Nguyen et al., 2020;Wang et al., 2020;Zhang et al., 2017).Whether these or other tick species could be the vector for B. hongkongensis remains to be determined.
Infestations with ticks were not identified on any of the cats in our study.In contrast to dogs, ticks are infrequently found on cats due to their fastidious self-grooming behaviours.In dogs, B. gibsoni is also transmitted horizontally through fights and bites, blood transfusions or by transplacental infection (Birkenheuer et al., 2005;Fukumoto et al., 2005;Matsuu et al., 2004;Stegeman et al., 2003).Horizontal transmission for B. hongkongensis amongst community cats may be possible too.
The infection rate of Babesia in cats in our study was low (1.4 %) compared to what has been reported in other regions like the Western Cape, Eastern Cape and KwaZulu-Natal provinces of South Africa, where feline babesiosis is a major concern with high infection rates (>50 %) (Bosman et al., 2007;Jacobson et al., 2000).Other regions that have reported high infection rates include St Kitts (31.0 %), Iraq (26.0 %) and Portugal (9.4 %) (Kelly et al., 2017;Maia et al., 2014;Suliman, 2009).The higher infection rates in these regions might be due to differences in exposure to and prevalence of tick vectors and the Babesia species they carry as well as the differences in frequency of tick prophylactic control.
The Babesia infection rate in cats in geographic locations closer to Hong Kong, such as mainland China and Thailand, are reported in several studies to be similarly low (Simking et al., 2010;Yin et al., 2022;Zhang et al., 2019).In mainland China, B. gibsoni was detected in 2.8 % of feline blood samples from four different provinces (Yin et al., 2022;Zhang et al., 2019).A previous study investigating the association between age and Babesia detection reported that cats less than 3 years old were more susceptible to infection (Schoeman et al., 2001).The low number of Babesia positive samples in our study precluded the investigation of any age association.
There were several limitations in our study including that the healthy status designation of community cats was based on clinical examination only as clinicopathological tests other than for FIV and FeLV were not performed.Also, since low volumes of residual blood were used for testing of privately-owned cats, FIV and FeLV testing could not be performed.Since B. honkongensis was only detected in community cats in our study, whether it could be a risk factor for anaemia and/or thrombocytopenia in cats could not be determined and further investigations are warranted.
Babesia gibsoni has been reported previously to cause subclinical infections in 5/119 (4 %) healthy cats in St Kitts in the Caribbean, 12/ 429 (3 %) cats in China and in one cat in Singapore (Colella et al., 2020;Kelly et al., 2017;Yin et al., 2022).Clinical signs were identified retrospectively in the single B. gibsoni positive privately-owned cat in our study, including lethargy, pallor, hyporexia, fever and weakness with associated anaemia and thrombocytopenia (Almendros et al., 2023).Further surveillance is required to determine whether B. gibsoni has a pathogenic role in cats.

Conclusion
This study confirms the molecular presence of B. gibsoni and B. hongkongensis in cats in Hong Kong, with a low infection rate overall (1.4 %) in the studied population.The higher proportion of Babesiapositive community cats compared to privately-owned cats could be due to a higher risk of vector exposure or horizontal transmission during fighting, amongst community cats.Identified B. hongkongensis infections were subclinical.Further research on the virulence of B. gibsoni and B. hongkongensis infection in cats, as well as their potential routes of transmission and determination of the potentially involved arthropod vector(s) is warranted.

Table 1
Primers and cycling conditions used to obtain the genomic sequence of Babesia spp.detected in cats in Hong Kong.

Table 2
Signalment, anaemia status and FIV status of privately-owned cats and community cats, respectively, tested for Babesia spp. in Hong Kong.

Table 3
Percentage of nucleotide identity and coverage using the highest-match on the NCBI GenBank database in positive Babesia PCR samples identified in 5 cats in Hong Kong.