The first report of hepatozoonosis in domestic cats was made in India in 1908. Subsequently, the infection was reported in felids in many countries, including South Africa, Asia, Southern Europe, and the USA^3,5-9. To date, three Hepatozoon species, Hepatozoon felis, H. canis, and H. silvestris, have been reported in domestic and wild cats^2,7,10-13. In addition, H. felis has been reported as the most frequently diagnosed species in feline hepatozoonosis cases in different countries of the world^14,15. The infection is mostly subclinical in cats. More pathogenic effects can be seen in the case of under stress, reduced immune systems or with secondary infections (FIV, FeLV, and feline hemoplasma etc.) in cats⁶. Hepatozoon species cause diseases characterized by clinical symptoms such as fever, diarrhoea, vomiting, weight loss, lethargy, anorexia, jaundice, lymphadenopathy, and gait disturbances^14,16. Hepatozoon spp. in cats is particularly associated with infection of muscle tissues. The agents can be found in the skeletal muscles and myocardium of the hosts⁶. Studies in Switzerland² and Italy¹⁷ have shown that H. silvestris causes severe lymphoplasmacytic and histiocytic myocarditis in domestic cats and deaths due to intestinal invagination^2,17.

It is very important to use rapid and reliable diagnostic methods to minimize the negative results caused by hepatozoonosis¹⁸. In cases of hepatozoonosis, it has been reported that the level of parasitemia in cats is generally low, with fewer than 1% of neutrophils and monocytes containing gamonts. In addition, microscopic examination cannot detect low-level infections. Serological methods may cause cross-reactions and produce erroneous results^4,14,19. The fact that microscopic examination and serological diagnostic methods do not give sufficiently accurate results has led researchers to use molecular diagnostic methods such as PCR, which have both higher sensitivity and specificity^3,20. Molecular methods are based on the principle of amplifying the DNA of the target pathogen in vitro and making it detectable. These methods allow the detection of very few amounts of DNA in the sample, the identification of pathogens causing infection in the hosts at the species level and the detection of mixed infections. In addition, by revealing the nucleotide sequences of pathogens with sequence analyses, it contributes to the investigation of the genetic diversity of pathogens circulating in hosts, the detection of new strains, genotypes or species, and the understanding of their taxonomic positions and phylogenetic relationships^20-24.

Hepatozoonosis is a disease that has been detected in different hosts, including domestic animals (such as cats, dogs) and wild animals (such as jackals, foxes) worldwide^3,25. Although Hepatozoon spp. have been detected in many different species, it is seen that most of the studies have been carried out on dogs²⁶. Türkiye has a large number of vectors due to its geographical location, diverse climate, and vegetation. Several studies have been carried out in Türkiye to investigate the prevalence, distribution, and genetic diversity of vector-borne pathogens, and many pathogen species have been detected in different hosts^21,23,27. Moreover, several Hepatozoon species, H. canis, Hepatozoon spp., and H. viperoi sp. nov., have been identifies in different hosts from several parts of Türkiye^28-34. According to the literature review, there is a limited number of studies on the presence and prevalence of feline Hepatozoon species in Türkiye. The aims of this study was i) to investigate the molecular prevalence of Hepatozoon species in cat blood samples obtained from different parts of Türkiye by using conventional PCR, ii) to perform phylogenetic analyses of positive samples identified in the study.

Study Area and Samples: Türkiye is geographically located between Europe and Asia and has a subtropical climate. The country is divided into seven different geographical regions (Eastern Anatolia, Central Anatolia, the Black Sea, Southeastern Anatolia, Marmara, the Aegean, and the Mediterranean). This geographical structure and climatic characteristics allow various vector species to be live in the country. Therefore, vector-borne diseases are quite common in the country³⁵. In this study, a total of 320 cat blood samples from five regions of Türkiye, Central Anatolia [Sivas (n: 60), Ankara (n: 70)], Eastern Anatolia [Malatya (n: 60)], Aegean [Denizli (n: 60)], and Marmara [İstanbul (n: 70)] were collected in different studies (Figure 1). Genomic DNA was isolated from blood samples in accordance with the protocol outlined by Sambrook et al.^36, with minor modifications. Data about modifications during DNA extraction process were detailed in the study conducted by Erol et al. ²⁷.

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Figur 1: The locations of sampling area were indicated in the Türkiye maps

PCR Assay for the Research of Hepatozoon spp. in Cat Blood Samples: The obtained DNA samples were researched in terms of Hepatozoon species with conventional PCR using primers Hep-F (5′-ATACATATGAGCAAAATCTCAAC-3′) and Hep-R (5′-CTTTATTATTCCATGCTGCAG-3′) primers³⁷. These primers were amplified partial parts of 18S SSU rRNA gene region of Hepatozoon species.

The PCR assay was carried out in a total volume of 25 μL, which included 10× PCR buffer (Invitrogen™, Carlsbad, USA), 2.5 μL MgCl2 (50 mM) (Invitrogen™, Carlsbad, USA), 200 μM of each dNTP (Cat.No.: R0181, Thermo Scientific™, Lithuania), 1 μL (10 pmol/μL) of each of the primers, 1.25 U of Taq DNA polymerase (5U/ μL) (Ref.No.: 100021276, Invitrogen™, Carlsbad, USA), 2.5 μL template DNA, and DNase-RNase-free sterile water. To prevent false results, positive [H. canis (GenBank Accession Number MW350128, (33)] and negative (DNase-RNase-free sterile water, Cat. No.: 129114, Qiagen®, Hilden, Germany) control groups were added to each PCR mix.

The thermal cycling protocol utilized for the PCR comprised an initial denaturation step at 94°C for a duration of 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The protocol was concluded with a final extension step at 72°C for 5 min. The resulting PCR products were loaded onto a 1% agarose gel and then electrophoresed at 90 volts for 60 min. Subsequently, the agarose gel was subjected to staining with ethidium bromide for a period of 20 min. The results were then subjected to visualization using a UV transilluminator.

Sequencing and Phylogenetic Analysis: Nucleotide sequence analysis of all positive samples was done with primers used PCR assay. Sequence analyses were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 3730XL analyzer (Applied Biosystems, Foster City, CA) according to the standard instructions recommended by the manufacturer. Prior to the nucleotide sequencing, the purification of all PCR products was achieved through the utilization of the HighPrep™ PCR Clean-Up System (Cat. No.: AC-60005, MagBio), in accordance with the manufacturer's instructions. The resulting chromatograms were then subjected to a quality assessment using the FinchTV (version 1.4.0) program, developed by Geospiza Inc. (Seattle, Washington, USA). Nucleotides exhibiting substandard chromatogram quality scores were subjected, and nucleotides had low chromatogram quality scores were trimmed. The MEGA-11 software³⁸ was utilized to generate the consensus sequence. These consensus sequences were subsequently submitted to the GenBank database, and accession numbers were obtained.

Phylogenetic trees were constructed using a maximum likelihood (ML) analysis implemented in MEGA-11 software³⁸. The resulting trees were evaluated to reveal genetic variation between the Hepatozoon species identified in this study and those in GenBank and to determine phylogenetic relationships. Pre-construction of the phylogenetic tree involved the determination of the optimal algorithm for use in the phylogenetic tree of related pathogens. Prior to the construction of the phylogenetic tree, it was determined that the most appropriate algorithm to be employed in the phylogenetic tree of related pathogens was the Tamura-Nei (TN93) + G parameter model³⁹ using the Find Best-Fit Substitution Model in MEGA-11. This algorithm was subsequently utilized in the construction of the phylogenetic tree. The bootstrap analysis (1000 replications) was conducted.

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Figure 2: PCR products of H. felis species. M. Marker, 1. Hepatozoon spp. positive control, 2. Negative control, 4-7-10. Hepatozoon spp. positive cat samples. 3-5-6-8-9-11-12. Negative cat samples

The consensus sequence belonging to three PCR positive samples were uploaded to the GenBank under accession numbers PV639385-PV639387. These three sequence had 100% nucleotide identities each other.

The BLASTn analyses of sequence identified in this work revealed that 97.72-100% nucleotide similarities were seen with our sequence and H. felis sequence present in the GenBank reported form different regions of the world. Hepatozoon felis sequences detected in this study had 100% nucleotide identities with H. felis reported in China (PP528683, Eurasian lynx), India (ON075470, Asiatic lion), and Spain (OR263294, Felis catus). Furthermore, our sequences showed high levels of nucleotide similarity to those from Israel (99.84%, KC138533, Felis catus), China (99.84%, PP528682, Hyalomma asiaticum), Spain (99.83%, OR263293, Felis catus), Spain (99.67%, AY628681, AY620232, OR263291, and OR263276, Felis catus), India (99.67%, KX017290, Asiatic lion), Spain (99.51%, OR263292, OR263286, OR263288, and OR263280, Felis catus), India (99.00%, OK036954, Panthera tigris tigris), Namibia (98.99%, MW872057, Parahyaena brunnea), Korea (98.99%, GQ377216, Korean leopard cat), Italy (98.99%, KY511259, cat), Japan (97.72%, AB771543, Prionailurus iriomotensis), and Japan (97.72%, AB771552 and AB771562 Prionailurus bengalensis euptilurus).

Phylogenetic analysis of the 18S SSU rRNA gene region was performed by aligning our H. felis sequences with different H. felis isolates present in GenBank. The sequences obtained from H. felis in this study share the same branch as H. felis from various countries (Figure 3).

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Figure 3: Phylogenetic tree of H. felis 18S SSU rRNA gene sequence. Hepatozoon felis isolates identified in the study are underlined. Numbers at the nodes represent the bootstrap values with 1,000 replicates. The evolutionary history was inferred by using the Maximum Likelihood method and TN93 + G model 39. The scale bar represents 0.050 substitutions per nucleotide position. Evolutionary analyses were conducted in MEGA-11 38.

Rapid and reliable detection of vector-borne diseases, such as Hepatozoon spp., in different hosts, is very important. In recent years, molecular identification techniques such as PCR have been used for the identification of Hepatozoon species due to their high sensitivity and specificity compared to microscopic methods²⁰. In recent years, various molecular studies have been carried out to the determination of Hepatozoon species in cats in different countries like Angola^43, Brazil^44-46, Cyprus^5, France^15, Greece^15,47, Germany^48, Hungary^49, Israel^15,50, Italy^12,13,15, Portugal^15,51, Republic of Cape Verde (Africa)^19, Spain^6,15,52, and Thailand¹⁰. In Türkiye, few studies have been conducted to determine the presence of Hepatozoon spp. in cats. In these studies, the prevalence was found as 10.8%, 2.37%, and 18.64% in Tekirdağ^42, İzmir⁹ and Samsun^20, respectively. In the present study, Hepatozoon spp. was detected in 0.93% (95% CI 0.19-2.72) of cat blood samples obtained from diverse regions throughout Türkiye using a PCR test. Our prevalence value was lower than above-mentioned studies conducted in Türkiye. Studies performed in different parts of the world revealed that the prevalence of vector-borne pathogens, like Hepatozoon species, may be changed several factors, such as distribution and abundance of vector species in sampling areas, methods used in the studies, number of animals, genders and age of animals, health status of animals, predatory activities of animals, and characteristics of cats (i.e., shelter or owned cats) included studies^1,20,47,48. It has also been reported that the low prevalence of Hepatozoon spp. infections in cats may vary depending on the seasonal time of sampling (as it may be affected by the seasonal variation in the activity of ticks and other vectors)⁵². In this study, all animals researched in terms of Hepatozoon spp. were owned cats. These animals are under the control of veterinarians. Therefore, it is thought that our prevalence value was lower than above-mentioned studies.

In recent years, DNA sequence analyses are used for several reasons, such as to verify PCR results, to perform phylogenetic analyses of pathogens, to detect novel genotypic variants, and to identify new species by researchers ^13,20,34,47. The 18S rRNA gene region has been widely used in the detection of Hepatozoon species in hosts^11-13,20,50. In this study, the sequence of the partial region of the 18S rRNA gene was performed in order to verify the genus-specific PCR results, to identify Hepatozoon species circulated in cat population, and to perform phylogenetic analyses of the positive samples. Hepatozoon felis consensus sequences obtained in this study were compared with other H. felis isolates uploaded to the GenBank, and high nucleotide similarities (97.72-100%) were seen. Despite the 18S rRNA gene region has low genetic diversity among H. felis species, this gene is mostly used to identify Hepatozoon species in hosts from various parts of the world^{2,14,20,47,50}. However, the phylogenetic analyses were based on only three Hepatozoon spp. positive samples, which limits comprehensive inferences regarding the genetic diversity and phylogenetic relationships of Hepatozoon species circulating in Türkiye. Therefore, the phylogenetic findings should be interpreted with caution and supported by further molecular studies including a larger number of positive samples from different geographic regions.

As a result, in this study, Hepatozoon spp. were researched in cats using molecular and phylogenetic analysis. Hepatozoon spp. were found in cats (0.93%, 3/320) in different provinces of Türkiye. Furthermore, phylogenetic analyses indicated that the species belongs to H. felis. Hepatozoon felis was identified for the first time in Sivas and Malatya provinces with this study. This result will contribute to the understanding of the epidemiology of H. felis in Türkiye. The main limitation of this study is the low number of Hepatozoon spp. positive samples. The detection of only three positive cases among 320 samples limits the statistical power and reduces the reliability of regional comparisons. Therefore, the prevalence and regional distribution findings should be interpreted with caution, and further studies involving larger sample sizes, broader geographic coverage, and seasonal sampling are needed to confirm these results. Considering that H. felis can cause clinical symptoms in cats, it is thought that measures should be taken to protect cats from this pathogen. H. canis and H. silvestris, other Hepatozoon species seen in cats, were not detected in this study. However, it is thought that large-scale molecular studies are needed to understand the epidemiology of Hepatozoon species in cats.

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