Molecular Characterization of Hemoparasites and Hemoplasmas Infecting Domestic Cats of Southern India

In the present study, 111 blood samples were collected from apparently healthy cats belonging to four districts of Kerala, southern India, and they were investigated for the presence of hemoparasites and hemoplasmas by light microscopic examination and polymerase chain reaction (PCR). The microscopic examination of the Giemsa-stained blood smears did not reveal any parasites/organisms. However, PCR followed by nucleotide sequencing could detect 10 (9.01%) out of 111 samples infected with Hepatozoon felis, 3 (2.70%) with Cytauxzoon spp., and 10 (9.01%) with Mycoplasma spp. None of the samples revealed amplicons specific for the Babesia spp. and Trypanosoma evansi. The phylogenetic analysis of 18S ribosomal RNA (rRNA) gene sequences of H. felis revealed the existence of two different populations of H. felis circulating in the blood of infected cats. The phylogenetic tree was constructed based on 18S rRNA gene sequences of Cytauxzoon spp. and revealed that these isolates formed a distinct clade and do not cluster with any of the isolates from other countries. Among the 10 samples positive for Mycoplasma spp. infections, 7 were detected positive for Candidatus Mycoplasma haemominutum, two for Mycoplasma haemofelis, and one for Candidatus Mycoplasma turicensis. Phylogenetic analysis of 16S rRNA gene sequences of Mycoplasma spp. showed no distinct geographical grouping of the sequences. The sequences of M. haemofelis, Candidatus M. haemominutum, and Candidatus M. turicensis identified in the study clustered along with their respective isolates from around the world. To the best of our knowledge, this study forms the first report of molecular detection of Cytauxzoon spp. and Candidatus M. turicensis in cats from India.

In the present study, 111 blood samples were collected from apparently healthy cats belonging to four districts of Kerala, southern India, and they were investigated for the presence of hemoparasites and hemoplasmas by light microscopic examination and polymerase chain reaction (PCR). The microscopic examination of the Giemsa-stained blood smears did not reveal any parasites/organisms. However, PCR followed by nucleotide sequencing could detect 10 (9.01%) out of 111 samples infected with Hepatozoon felis, 3 (2.70%) with Cytauxzoon spp., and 10 (9.01%) with Mycoplasma spp. None of the samples revealed amplicons specific for the Babesia spp. and Trypanosoma evansi. The phylogenetic analysis of 18S ribosomal RNA (rRNA) gene sequences of H. felis revealed the existence of two different populations of H. felis circulating in the blood of infected cats. The phylogenetic tree was constructed based on 18S rRNA gene sequences of Cytauxzoon spp. and revealed that these isolates formed a distinct clade and do not cluster with any of the isolates from other countries. Among the 10 samples positive for Mycoplasma spp. infections, 7 were detected positive for Candidatus Mycoplasma haemominutum, two for Mycoplasma haemofelis, and one for Candidatus Mycoplasma turicensis. Phylogenetic analysis of 16S rRNA gene sequences of Mycoplasma spp. showed no distinct geographical grouping of the sequences. The sequences of M. haemofelis, Candidatus M. haemominutum, and Candidatus M. turicensis identified in the study clustered along with their respective isolates from around the world. To the best of our knowledge, this study forms the first report of molecular detection of Cytauxzoon spp. and Candidatus M. turicensis in cats from India.

INTRODUCTION
Diseases caused by hemoprotozoan and hemoplasmal organisms are emerging problems in cats in many parts of the world (1,2). These diseases are mostly fatal vector-borne diseases and are quickly disseminated (1,3). The increased occurrence of feline vector-borne diseases in Europe was speculated for the reasons like climatic change, increased vector population, drug resistance in vector/pathogen population, and increased international transport of man and animals (3,4).
Cats having access to the outdoors are more vulnerable to these infections as a result of exposure to a variety of ectoparasites that may transmit these diseases (1,9,10). Ticks play an important role in the transmission of babesiosis, hepatozoonosis, and cytauxzoonosis (11)(12)(13). Bloodsucking insects such as Tabanus, Stomoxys, Atylotus, and Lyperosia act as vectors for trypanosomosis (14). Feline hemoplasmas are transmitted by Ctenocephalides felis, blood transfusion, and also by fighting, scratching, and biting with other cats. Other routes of infection include transuterine and transmammary transmission (1).
The population of pet cats in India was estimated to be around 2 million in 2018 (https://www.statista.com/statistics/1061172/ india-population-of-pet-cats). The majority of them are feral or semidomesticated, which wander in the area around the house from where they may get food and shelter. Rearing domestic cats with a good pedigree has increased recently.
There were only a few documented reports on the molecular detection of these infectious agents in cats from India (15,16). Hence, the present communication focuses on the generation of baseline information regarding the presence and distribution of hemoparasites and hemoplasmas in cats of southern India.

Study Area and Samples
For this study, 111 blood samples were collected from cats belonging to four districts of Kerala, India, viz., Wayanad, Kozhikode, Ernakulam, and Thiruvananthapuram (Figure 1) in the period between March 2018 and June 2019. Whole blood samples were collected in ethylenediaminetetraacetic acid (EDTA) vials from femoral or medial saphenous veins. Thin blood smears were prepared using a drop of this blood.

Staining
Thin peripheral blood smears were fixed in methanol, then stained with diluted (1:10) Giemsa's stain (Merck Life Science, Mumbai) for 45 min. The blood smears were washed with water and air dried. The stained blood smears were examined under the oil immersion objective (100×) of the light microscope (Leica DM1000 LED, Germany) for the presence of parasites. A minimum of 150 fields were examined thoroughly before declaring a sample as negative.

Genomic DNA Extraction and Quantification
Genomic DNA was isolated from blood samples collected in EDTA vials using DNeasy R blood and tissue kit (Qiagen, Germany), according to the manufacturer's protocol. Extracted DNA was eluted in 100 µL of DNA elution buffer. The DNA concentration was determined using a NanoDrop R 2000C spectrophotometer (Thermo Scientific, USA) and stored at −20 • C for further analysis.

Polymerase Chain Reaction
The genomic DNAs isolated from these samples were used for PCR, in an automated thermal cycler with a heated lid (Eppendorf, Germany). All PCRs were carried out in a final reaction volume of 25 µL containing 0.2 mM deoxyribonucleotide triphosphates (dNTPs) (Thermoscientific, Lithuania), 1 U DyNAzyme II DNA polymerase (Thermo Scientific, USA), 10×PCR buffer (containing MgCl 2 at a final concentration of 1.5 mM), 20 ng of template DNA, and 10 pmol each of forward and reverse primers. The details of the primers used, the amplification conditions, and the amplicon size are shown in Table 1.

Positive Controls
The DNA isolated from the blood samples (Qiagen DNeasy R blood and tissue kit, Germany) of infected dogs (diagnosed based on microscopical examination of Giemsa's stained blood smears) presented to the Teaching Veterinary Clinical Complex (TVCC), College of Veterinary and Animal Sciences, Pookode, Wayanad were used as positive controls for genus-specific PCRs for Hepatozoon spp., Babesia spp., and species-specific PCR for T. evansi. Polymerase chain reactions specific for Mycoplasma spp. and C. felis were standardized without any positive controls, as they were rarely reported previously from the state.

Sequencing and Sequence Analysis
Products of polymerase chain reactions (18S rRNA and 16S rRNA) were purified using NucleoSpin R Gel and PCR Clean-Up Kit (Macherey-Nagel, Germany) as per the manufacturer's protocol. They were sent to the AgriGenome Labs Private Ltd., Cochin, Kerala, for automated nucleotide sequencing by Sanger dideoxy method with both the forward and reverse primers. The resulting sequences were examined for the overlapping peaks suggestive of coinfection using Bioedit software (23) before the comparison of the new sequence of each isolate to other published sequences available in the GenBank using NCBI-BLAST (http://www.ncbi.nlm. nih.gov/BLAST). Unique sequences were deposited in the GenBank database.

Phylogenetic Analysis
For the phylogenetic analysis, the nucleotide sequences were aligned using ClustalW (24) with the previously published

Microscopical Examination
The light microscopy examination of Giemsa's stained peripheral blood smears under oil immersion (100×) could not detect any hemoparasites and hemoplasmas in the blood smears of 111 cats examined.

PCR and Sequence Analysis
None of the samples revealed amplicons specific for the Babesia spp. and T. evansi. A 358-bp fragment of the 18S rRNA gene of Hepatozoon/Cytauxzoon species was amplified by PCR using the piroplasm-specific primers from the blood of 13 cats out of the 111 samples examined. Sequencing followed by NCBI-BLAST analysis revealed an identity of 99.4-100% to H. felis (JN584475, MK724001) for the 10 sequences and identity of 92.3-92.6% to C. felis (GU903911) for the three sequences. These 13 samples were used for confirmation using primers specific for the amplification of the 18S rRNA of Hepatozoon spp. and C. felis. Ten samples identified as H. felis with piroplasmid primers were further confirmed for monoinfection using primers specific for Hepatozoon spp. Three samples detected positive, as Cytauxzoon spp. did not amplify the desired amplicon when using primers specific for Hepatozoon spp., revealing monoinfection in these samples, too. None of the samples produced amplicons specific for the 18S rRNA of C. felis. Amplicons (595, 618, and ∼595 bp) specific for the 16S rRNA gene of Mycoplasma spp. were amplified by the PCR from the blood samples of 10 cats out of the 111 samples examined. Sequencing revealed that two sequences showed an identity of 98.5-100% to M. haemofelis (MK632346, KU645929), seven sequences with an identity of 98.8-99.8% to Candidatus M. haemominutum (KU645934, KR905451, MK632386, MK632392) and one sequence with an identity of 99.8% to Candidatus M. turicensis (KR905459).
Mixed infection due to the presence of both H. felis and Candidatus M. haemominutum was identified in 4 sample (3.6%) out of 111 DNA samples by PCR. These cats were from Wayanad (one), Ernakulam (one), and Thiruvananthapuram (two) districts of Kerala. Mixed infection due to different protozoans was not detected in any samples tested in the present study. The occurrence of infection due to hemoparasites and hemotropic mycoplasmas ( Table 2) was slightly higher in male compared to female cats. The non-descript cats harbored more infectious organisms than Persian cats. Moreover, cats belonging to the age group of 1-2 years showed a higher prevalence.

DISCUSSION
No hemoparasites and hemotropic mycoplasmas could be detected based on microscopy of blood smears stained with Giemsa's stain. H. felis gamonts were difficult to be observed under a microscope, as they were less conspicuous, smaller, or low in number (11,26,27). The intraerythrocytic piroplasms of Cytauxzoon spp.
were not detected in healthy cats that were PCR positive (6,28). It is also believed that the examination of stained smears was not a sensitive diagnostic tool and cannot identify the three different hemoplasma species (29,30 (17) were reported. Based on 18S rRNA sequences, all the H. felis field isolates detected in the present study were grouped into two different clades, clades 2 and 5. The present study also disproved the concept of genetic relatedness of Indian isolates of Hyderabad with isolates from Spain and Israel (15). Thus, there might be two different populations of H. felis in Kerala that could infect domestic cats.
The detection of Cytauxzoon spp. in three blood samples of cats in the present study forms the first report from India. C. felis was endemic solely to North America for many years, where bobcats (Lynx rufus) are believed to serve as the main hosts even though reports are available from the USA, Brazil, Spain, France, Italy, and Iraq (32,33) for its presence in domestic cats. The "parasite" has also been described in felids originating from several Asian countries, including India. However, there are no comprehensive molecular data available, which could confirm the specific identity of these Asian isolates. The nucleotide sequences generated in the current study showed only 92% identity to the closest match in the GenBank database. Furthermore, the Indian sequences formed a separate clade (herein designated as clade 3) that is very distant from that of pathogenic C. felis isolates from the USA, Netherlands, and Brazil (6). Further, the primer sets targeting 18 S rRNA gene specific for C. felis did not reveal any amplification. The Cytauxzoon spp. detected in Kerala were from apparently healthy cats. However, Cytauxzoon felis infections (34) are highly fatal except for a few reports from asymptomatic cats (28, 33,34). In other words, the 18S rDNA sequences confirmed in the cats tested in this study most likely belong to another Cytauxzoon species, not C. felis.
Hemoplasmosis was previously reported from different parts of the world (7,29,30,(35)(36)(37)(38)(39)(40). In the present study, Mycoplasma spp. was detected in 10 blood samples of cats collected from all four districts of Kerala. Seven cats (6.3%) were infected with Candidatus M. haemominutum, two (1.2%) with M. haemofelis, and one (0.9%) with Candidatus M. turicensis. A previous study conducted in Thrissur, Kerala (16) detected a prevalence of 23% of Candidatus M. haemominutum and 1% of M. haemofelis by PCR. Candidatus M. haematoparvumlike organisms (0.7%) reported previously from the USA (41) were not detected in the present study. In addition, the present study reports for the first time the presence of "Candidatus M. turicensis" among the cat population in India.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Ethics Committee, College of Veterinary and Animal Sciences, Pookode. Written informed consent for participation was not obtained from the owners because the samples were collected from cats brought to the veterinary clinics are managed by registered veterinary practitioners. The blood samples were collected by the veterinarians after getting the oral consent from the pet owners for the detection of pathogenic organisms in their pets.

AUTHOR CONTRIBUTIONS
LM, AN, CB, BA, PK, RP, and MN collected the samples, conducted the experiments, participated in the data acquisition, and drafted the manuscript. KA conceived the study and supervised the protocols. AV, CD, LJ, and RR helped in the collection of samples, data acquisition, supervision of the experiments, and review of the manuscript. All authors read and approved the manuscript.

FUNDING
The research work was supported financially by Kerala Veterinary and Animal Sciences University, Pookode.