Background

Due to human growing population and cat ownership habits, cats are a significant part of social life in Türkiye. Although there is no precise data on the number of stray cats in this country, it is estimated that almost one-fifth of the population owns cats. A study conducted in metropolitan Ankara indicates that there are thousands of stray cats in this province, giving an idea of the problem nationwide [1, 2]. Although cats are an essential part of human life as pets, they are hosts and reservoirs for many vector arthropod-transmitted bacterial, viral, protozoal, and helminthic infectious haemopathogens, some of which are of zoonotic concern. In different regions and socio-economic contexts, these companion animals, which share the same environment with humans, play an important role in the transmission of zoonotic agents [3].

Despite the presence of a large number of life-threatening and zoonotic infectious agents in cats, studies on this topic have not kept pace with the renewed interest in feline vector-borne diseases (VBDs), and therefore, the lack of knowledge on feline VBDs in particular dramatically hinders developing and implementing effective preventive and control measures at regional and national level [4]. The primary vector-borne pathogens (VBPs) of cats include Babesia, Cytauxzoon, Hepatozoon, Anaplasma, Ehrlichia, Rickettsia, Bartonella, and Mycoplasma species, but this list can be further expanded [3,4,5,6,7,8].

Serological and molecular epidemiological studies contribute to increasing the recognition of feline VBPs worldwide [9, 10]. In Türkiye, data on the molecular epidemiology and phylogeny of feline VBHs are limited, although there have been studies of some FVBHs, including clinical cases of hepatozoonosis [11] and ehrlichiosis [12] in Türkiye [13,14,15,16,17]. Most of the investigations have been carried out on feline zoonotic Bartonella species [13, 17,18,19], but much remains to be done on other feline vector-transmitted bacterial and parasitic pathogens. The most comprehensive study on the molecular epidemiology of FVBHs was carried out in the province of Tekirdağ, Türkiye, in 2021, in which Babesia canis canis, B. microti, Hepatozoon felis, Cytauxzoon felis, Bartonella henselae, Anaplasma platys, Anaplasma phagocytophilum, Rickettsia felis, Borrelia burgdorferi, and Mycoplasma spp. were identified in symptomatic cats by species-specific conventional polymerase chain reaction (PCR) [16]. A study material representing a wider geographical area, including phylogenetic analyses, is thought to provide valuable data on FVBHs of Turkish cats. Therefore, this study aimed to molecularly investigate the vector-borne haemopathogens of owned and shelter cats from Türkiye and to perform phylogenetic characterization of the identified species.

Results

Prevalence of feline vector-borne haemopathogens

The present study molecularly screened 250 cats for FVBHs, and 40 cats (16%) were found to be positive for at least one pathogen. The study unexpectedly revealed that four cats (1.6%) tested positive for B. ovis deoxyribonucleic acid (DNA), the primary etiological agent of ovine babesiosis. The overall prevalences of Mycoplasma spp. and Bartonella spp. were identified as 11.2%, and 4.8%, respectively. Co-infections were also detected in four cats (1.6%) as CMhm + B. koehlerae (n = 2), CMhm + B. henselae (n = 1), and CMhm + Bartonella spp. (n = 1), respectively. The DNA of other piroplasma species, Anaplasma spp., Ehrlichia spp., and Rickettsia spp. was not detected in the study (Table 1).

Table 1 Prevalences of feline vector-borne pathogens
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Phylogenetic analysis

In the present study, the phylogenetic trees of B. ovis and Mycoplasma spp. were constructed based on their respective genes (18S rRNA and 16S rRNA) by comparing them with the sequences deposited in the NCBI GenBank database. Four sequences of B. ovis 18S rRNA gene obtained in the study (OR984759- OR984762) established a well-supported clade with the 18S rRNA gene sequences from Spain (sheep and goats) and Türkiye (sheep, horse) with a 97.93–99.82% nucleotide sequence identity (Fig. 1).

Fig. 1
figure 1

A maximum likelihood phylogram of Babesia ovis inferred from the 18S rRNA gene. The tree was constructed by MEGA version X software using the Kimura 2-parameter and evolutionary rates among sites (K2 + G). Bootstrap analysis with 1000 replications was employed to ascertain the confidence of the nodes and branches within the tree. The sequences of this study are shown in boldface. The 18S rRNA gene sequence of Plasmodium yoelii (XR004618869) was used as an outgroup

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For Mycoplasma species, 11.2% of cats were detected as positive in terms of Mycoplasma spp., and most of the PCR-positive products were sequenced. As a result of the sequence analysis, 17 of the 21 Mycoplasma spp. positive PCR products were CMhm, two were M. haemofelis, and one was uncultured Mycoplasma sp. On the other hand, one Mycoplasma isolate was confirmed as a bovine species, M. wenyonii. The 16S rRNA gene sequences of Mycoplasma species are clustered in four separate clades. The CMhm sequences OR979160- OR979168, OR979170- OR979173, OR979176, OR979177, OR979179, OR979180 formed a well-supported clade with CMhm 16S rRNA gene sequences obtained from cats in Hungary (EU128752), Italy (KR905451), Switzerland (DQ157144), and Thailand (MK632396) with a nucleotide sequence identity ranging from 99.49 to 100%. The M. wenyonii isolate of the study clustered together with isolates reported from buffaloes (OM747881, OM747882) and cattle (OM468183) in Türkiye with a high level of nucleotide sequence similarity (98.0-99.46%). Although the uncultured Mycoplasma sp. isolate was located close to the M. turicensis and M. haemofelis clades, it formed a separate branch from the main clades. Finally, our M. haemofelis isolates (OR979169 and OR979175) showed 99.59–100% nucleotide sequence identity with feline M. haemofelis isolates from countries such as India (MN240855), Iran (KX253966), Romania (MT926038), Spain (AY150065) and Thailand (KU645929) and 99.77–99.80% nucleotide sequence identity with some M. haemocanis isolates from countries such as Iraq (MW784616) and Japan (AY529641) (Fig. 2).

Fig. 2
figure 2

A maximum likelihood phylogram of Mycoplasma sp. inferred from the 16S rRNA gene. The tree was constructed by MEGA version X software using the Kimura 2-parameter and evolutionary rates among sites (K2 + G). Bootstrap analysis with 1000 replications was employed to ascertain the confidence of the nodes and branches within the tree. The sequences of this study are shown in boldface. The 16S rRNA gene sequence of Anaplasma bovis (KM114613) was used as an outgroup

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No phylogenetic tree was constructed for Bartonella spp. due to the short amplified fragments of 16–23S rRNA intergenic space region. Direct sequence analysis of half of the PCR amplicons of 12 Bartonella spp. positive samples identified in the study revealed B. henselae and B. koehlerae. Blast analysis of our B. henselae sequences (OR978401, OR978402, and OR978403) showed 99.42–100% nucleotide sequence identity to a rodent (Mus macedonius) isolate from Türkiye (OR198887), human isolates from Korea (JQ638927), Australia (AJ439687), France (AF312496), and a dog isolate from China (JQ009430). On the other hand, the B. koehlerae isolates from this study (OR978398, OR978399, and OR978400) have 99.33–100% nucleotide sequence similarity with Bartonella isolates from cat, lions (Panthera leo), and hard ticks reported from the USA, South Africa, and Palestine, respectively.

Statistical analysis results

The distribution of the species detected in the study in relation to cat breeds, age, sex, and status was statistically analyzed and the findings are shown in Table 2. The study findings revealed that Mycoplasma spp. had a statistically significant high prevalence in males (p = 0.031) and cats older than one year (p = 0.022), and piroplasms had a statistically significant high prevalence in shelter cats (p = 0.015). Although statistically insignificant, it is noteworthy that all Bartonella infections were detected in owned cats older than one year.

Table 2 Statistical findings on the distribution of infections according to different variables
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Discussion

Cats, especially those living outdoors, are exposed to various arthropods, including fleas, ticks, blood-sucking flies, and the pathogens they harbor. Cats may pose a risk of infection to humans or other animals that share the same habitat with vector arthropods or may act as reservoirs of VBPs, thereby posing a risk of infection for living organisms in these environments [20, 21]. Notably, studies on the VBPs’ epidemiology in pet animals have increased in recent years. However, feline VBPs have yet to be investigated as much as canine VBPs [21]. This situation is similar both on a large scale and on a Turkish scale, where there are serious gaps in the molecular epidemiology and phylogeny of feline VBPs. One of the priorities in keeping diseases under control is to analyze the situation, identify the presence of pathogens and risk factors, and try to keep this information as up-to-date as possible. Conducting large-scale studies increases the quality of the results to be obtained due to varying climatic conditions, structure, and density of vector arthropod populations [22]. Therefore, VBPs were investigated by molecular methods in cats brought to Konya, Selçuk University, Faculty of Veterinary Medicine, Animal Hospital from many provinces of Türkiye, and unique epidemiological data were obtained in this study. The study revealed the presence of Babesia spp. (1.6%), Mycoplasma spp. (11.2%), and Bartonella spp. (4.8%) in cats, respectively. Sequencing and phylogenetic analyses also revealed the presence of ovine babesiosis agent B. ovis and bovine severe anemia-associated M. wenyonii DNA in cats for the first time. Moreover, significant feline hemotropic Mycoplasma species such as CMhm, M. haemofelis, uncultured Mycoplasma sp., and Bartonella species, including B. henselae responsible for cat-scratch disease (CSD) in humans and B. koehlerae causing human endocarditis were identified by sequencing.

Babesia is one of the most common haemoprotozoan parasites in mammalian hosts and infects the erythrocytes of many vertebrate hosts, including cats [23]. Although babesiosis transmitted by ticks is one of the main problems for livestock farming, infections in pet animals can cause serious economic losses and deaths [7, 23]. Feline babesiosis cases with severe clinical symptoms associated with erythrocyte destruction are particularly prevalent in South Africa, sporadic clinical cases have also been reported in Europe and Asia [9, 24, 25]. To date, various Babesia species such as B. felis, B. cati, B. canis canis, B. gibsoni, B. canis vogeli, B. honkongensis, B. lengau, B. leo, B. microti, B. presentii, and unidentified Babesia spp. have been detected in cats, and it is noteworthy that especially canine Babesia species cause clinical infections in cats and are frequently encountered [24, 26,27,28,29]. However, the number of new species is increasing in parallel with advances in molecular technology. Genetic diversity among Babesia species also indicates that new Babesia species can emerge [26, 30]. Epidemiological data on feline babesiosis in Türkiye are limited and no molecular data on feline-specific Babesia species have been detected. However, a rodent species B. microti (2.4%) and a canine species B. canis canis (24%) have recently been reported molecularly in cats with symptoms such as weight loss, fever, haematological abnormalities, and lymphadenopathy in Tekirdağ province of Türkiye [16]. Furthermore, a study investigating the microscopic prevalence of Babesia in apparently healthy cats from Van province of Türkiye about 20 years ago revealed a prevalence of 10.8% for B. felis [31]. Unlike previous studies, the current study reports for the first time the presence of B. ovis DNA, the most pathogenic ovine babesiosis agent, in the four cats without any signs of clinical infection. The higher molecular prevalence of B. ovis DNA detected in shelter cats with statistical significance may be attributed to the different implementations applied to owned cats and shelter cats in terms of vector control. Our B. ovis isolates were also phylogenetically analyzed, and the four sequences constructed a well-supported clade with other Turkish isolates from sheep with the nucleotide sequence identity 97.93–99.82% and horse with the identity of 99.19–99.57%. There are many studies indicating that haemoprotozoa can be found in different hosts other than the original host [30, 32, 33], and some of these studies have recently reported that B. ovis has been detected in unspecific hosts such as donkeys and horses in Türkiye [34, 35]. This study provides valuable molecular epidemiological data, including cats among the unspecific hosts in which B. ovis DNA was detected, and the detection of B. ovis in cats opens new avenues for research into the epidemiology and pathogenicity of this and related species in unspecific host populations.

Feline haemotropic mycoplasmosis, which had been diagnosed cytologically before the 2000s in Türkiye [36], is now being investigated using PCR in parallel with advances in molecular diagnostic methods. It has been noted that the disease can cause mortality in domestic cats in this country [37, 38], and molecular prevalences have reached high levels in cats suspected of having the disease [39, 40]. When the studies on the subject were compiled, few studies regarding the molecular epidemiology of hemotropic mycoplasmas in domestic cats (Felis catus) have been encountered in Türkiye. In these studies, haemoplasmas were molecularly detected in Ankara (23.1%), Antalya (17.6%), Bursa (7.7%), İzmir (17.5%) [40], Kayseri (9.5%) [41], İstanbul (19.3%) [15] and Tekirdağ (11.4%) [16] provinces. In most of these studies, CMhm was found to be the main species responsible for infections [37, 39, 40, 42], and in some studies, the species has not been identified [16]. Species-specific PCR analysis revealed that the haemotropic Mycoplasma species molecularly detected in cats in Türkiye consisted of Mhf, CMhm, and Candidatus Mycoplasma turicensis (CMt). Cetinkaya et al. [15] reported molecular prevalences of 9.9%, 17.7%, and 0.8% for each species in their study conducted in İstanbul province, respectively. Although some molecular epidemiological studies have been carried out at the provincial level in Türkiye [15, 16, 40], there is a scientific research gap in the molecular phylogenetic characterization of the identified Mycoplasma species. The present study revealed that the molecular prevalence of Mycoplasma spp. in Turkish cats was 11.2% by Mycoplasma genus-specific PCR analyses targeting the 16S rRNA gene fragment and revealed remarkable findings to fulfill the scientific gap on the subject in Türkiye. The study findings also revealed that Mycoplasma spp. infections were statistically more common in males and cats older than one year. This is thought to be due to a higher chance of exposure to vector arthropods with increasing age and to aggressive interactions between male cats.

Direct sequencing of most of the PCR amplicons (n = 21) of positive Mycoplasma spp. samples confirmed that two samples belonged to Mhf from Konya and Siirt provinces, 17 samples to CMhm from Adana, Amasya, Ankara, Antalya, Burdur, Diyarbakır, İstanbul, Karaman, Konya, and Manisa provinces, one sample to uncultured Mycoplasma sp. from Konya province and one sample to M. wenyonii from Konya province, one of the bovine haemotropic Mycoplasma species. Sequence analysis could not performed on the remaining seven PCR-positive samples. The specific identification of Mhf, CMhm, uncultured Mycoplasma sp., and M. wenyonii in our study provides insight into the diversity of Mycoplasma infections affecting cats in Türkiye and calls for a deeper understanding of their clinical and epidemiological significance. The findings of the study showed that CMhm is the main species playing a role in feline haemoplasmosis, and this is consistent with previous studies conducted in Türkiye [37, 39, 40]. The fact that M. wenyonii DNA, which is one of the remarkable findings of the study, was detected for the first time in a female Tabby cat owned in Konya province points to the need for further studies on the transmission dynamics of feline haemotropic Mycoplasma species, the vector arthropods involved in the transmission, and whether this species is important in terms of clinical infection outside its original host. Detecting M. wenyonii in a domestic cat also highlights potential cross-species transmission and underscores the complex ecosystem interactions facilitating the spread of vector-borne diseases. To the best of the authors’ knowledge, this study is also the most comprehensive study on the phylogenetic analysis of feline haemotropic Mycoplasma species in Türkiye to date, showing that feline-specific Mycoplasma species cluster together with isolates reported from different countries with high nucleotide sequence similarities (99.49–100%). The M. wenyonii isolate reported for the first time in cats clustered with Turkish buffalo (OM747881, OM747882) [43] and cattle (OM468183) [43] isolates with 98.00-99.46% nucleotide sequence similarity. The diversity of Mycoplasma species, identified in this study ranging from well-documented feline pathogens like Mhf and CMhm to the less common M. wenyonii, emphasizes the complex nature of feline haemotropic mycoplasmosis. Further investigations are essential to elucidate the clinical relevance and transmission dynamics of M. wenyonii infections in cats.

Bartonella species are emerging opportunistic bacterial haemopathogens transmitted by blood-sucking arthropods, have a wide range of hosts, including cats, and can colonize endothelial cells, migrate into the bloodstream, and infect erythrocytes [8]. Cats are the main reservoirs of public health-relevant Bartonella species such as B. henselae and B. clarridgeiae causing CSD and B. koehlerae causing human endocarditis [3, 44]. While the transmission of the infection is mainly through fleas of the species Ctenocephalides felis felis, known as cat fleas [45], it has been scientifically confirmed that ticks of the species Ixodes ricinus can transmit B. henselae transtadially [46]. There are several serological and molecular studies investigating Bartonella spp. in humans and cats in Türkiye [13, 14, 16, 18, 47, 48]. Atıcı et al. [49] reported a systemic CSD characterized with hepatic mass in an immunocompromised child. Kaynar et al. [50] reported CSD in a patient with hypotension, pancytopenia, and acute kidney injury who had a history of being scratched more than once by stray cats and pointed out that this infection, for which there is limited data, should be more widely recognized among Turkish clinicians to prevent delays in diagnosis. In Türkiye, some other case studies emphasize the importance of Bartonella spp. infection and show that it is a pathogen that needs attention [51, 52]. Moreover, Sayın-Kutlu et al. [47] detected high levels of B. henselae seroprevalence among cattle breeders (19%) and veterinarians (29.6%) and therefore pointed out that bartonellosis should be considered in the differential diagnosis list of fever of unknown origin in humans in rural areas of Aydın and Denizli provinces of Türkiye, where the study was conducted. A recent study has reported that the prevalence of B. henselae is statistically high in people working in agriculture and animal husbandry and exposed to arthropods such as ticks and sandflies [53]. Although most of the studies are on human medicine, clinical infection with B. henselae characterized by pyogranulomatous myocarditis, and diaphragmatic myositis has also been reported in a flea-infested cat in Türkiye [54]. In light of the literature above, studies indicate the significance of feline-associated B. henselae infections in both human and veterinary medicine in Türkiye. The fact that most of the case reports, serologic and molecular epidemiological studies have been conducted in the last ten years reflects the emerging public awareness about bartonellosis in the country. However, further scientific studies are still needed to increase the data on this emerging vector-borne infection. This study reports a molecular prevalence of 4.8% for Bartonella spp. in a sample representing a wide geographical area of Türkiye, for which a seroprevalence of 18.6–36.2% [13, 14, 18, 48] and a molecular prevalence of 12.1–40.1% [16, 17, 19, 55] have previously been detected at provincial levels in cats. Sequence analysis of PCR amplicons from half of the positive samples (n = 12) confirmed that apparently healthy cats are reservoirs for B. henselae (n = 3) and B. koehlerae (n = 3) species. The presence of B. henselae and B. koehlerae in the feline population in Türkiye underscores the critical role of companion animals in the epidemiology of zoonotic diseases. While B. henselae is widely recognized for its zoonotic potential, particularly concerning CSD, our findings on B. koehlerae highlights an expanding horizon of concerns and interests in the veterinary and medical communities. Although less documented, the implications for human health necessitate a vigilant approach to understanding and managing B. koehlerae infections. This underscores the complexity of Bartonella as a genus, with multiple species impacting animal and potentially human health, and stresses the importance of a One Health approach to address these zoonotic threats effectively.

In addition to single infections, a total of four cats (1.6%) were co-infected with three different combinations of CMhm + B. koehlerae, CMhm + B. henselae, and CMhm + Bartonella spp. in the study. Pennisi et al. [56] stated that co-infections caused by multiple vector-borne haemopathogens can occur in various animals including cats, but there are uncertainties about their clinical outcomes and further studies are needed to clarify this situation, especially in cats. Although there are studies reporting that Bartonella spp. and haemotropic Mycoplasma spp. coinfection is common in cats, the predisposing factors are not known [57]. No clinical signs were observed in cats co-infected with Mycoplasma spp. and Bartonella spp. in this study. However, it should be noted that different types of co-infection may have a negative impact on the prognosis of VBDs in animals, especially when compared to animals infected with a single species, co-infected animals may be adversely affected under stressful conditions [58,59,60]. Qurollo et al. [61] suggested that co-infection in a cat with Anaplasma platys, B. henselae, B. koehlerae, and CMhm may be associated with immunosuppressive conditions such as myeloma-related disorders. Such an inference cannot be made about the co-infection status in this study, because the cats were apparently healthy and further clinical diagnostic laboratory analyses were not performed. However, co-infections may be attributed to infections following previous ectoparasite infestations and carrier animals status due to preimunition.

It is noteworthy that although the detection of B. ovis DNA in cats indicates a history of tick infestation, no Rickettsia spp. or Anaplasma/Ehrlichia spp. DNA was detected in this study. A study on the subject reported that this may be related to transient rickettsemia [62]. In addition, the fact that anaplasmosis, ehrlichiosis, and rickettsiosis are not common in cats supports the findings of the study [57, 63,64,65].

The highly conserved 18S rRNA gene was utilized for the identification of piroplasms, as it is widely used in molecular epidemiological studies as a marker for the primary screening [66,67,68]. However, it is important to note that the 18S rRNA gene has limited sequence variation among different species within the same genus. Consequently, this gene may not provide sufficient resolution to effectively distinguish piroplasm species [69, 70]. Previous studies showed that Babesia duncani n.sp. is phylogenetically indistinguishable from other Babesia and Theileria species when analyzed using 18S rRNA sequencing. However, it can be distinctly identified through internal transcribed spacer 2 (ITS2) sequencing [70]. Moreover, Niu et al. [71] successfully described the differentiation of B. motasi and B. sp. Xinjiang based on ITS region sequences. Mitochondrial genes evolve faster, resulting in higher sequence divergence compared to 18S rRNA [72]. Based on the cytochrome c oxidase subunit 1 (COI) gene, B. canis and B. vogeli infections can be diagnosed faster and more reliably [73]. Furthermore, Sivakumar et al. [69] successfully used the mitochondrial cytochrome b gene to distinguish between Theileria annulata and Theileria sp. yokoyama. Thus, future research should consider targeting other faster evolving genes of B. ovis for approach confirming the presence of B. ovis in unexpected hosts.

Conclusion

Our study is one of the most comprehensive epidemiological surveys conducted for molecular screening and characterization of multiple feline VBPs from a wide geographical area of Türkiye and revealed that the cat population harbors a wide range of VBPs. In the study, Babesia ovis, Mycoplasma spp. including Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, uncultured Mycoplasma spp., Mycoplasma wenyonii, and Bartonella spp., including Bartonella henselae and Bartonella koehlerae were detected as single infections and co-infections. This study also reported Babesia ovis and Mycoplasma wenyonii for the first time in cats. Considering the zoonotic concern of some of the haemopathogens detected in the current study, further epidemiological studies should be advocated in humans and pets to determine the distribution status of VBPs, identify risk factors, and implement effective vector arthropod control strategies for prevention.

Methods

Cats and blood samples

The study material consisted of 250 randomly selected cats (Owned = 203, Shelter = 47) brought to Selcuk University, Faculty of Veterinary Medicine, Animal Hospital for routine procedures from 44 different provinces of Türkiye. Selcuk University is located in the province of Konya (37° 53′ N, 32° 31′ E), which is localized in the central part of Türkiye. Konya province is close to some of the tourism resorts bordering the Mediterranean Sea and is located on the transit route of domestic tourists going to tourism regions, especially during the summer seasons. Therefore, tourists from many different provinces and their pet cats visit the animal hospital for various reasons such as chipping, shaving, vaccinations, surgical procedures or various diseases. Comprehensive details regarding the sampling sites and sample numbers are shown in Fig. 3; Table 3. The body surface of cats with no skin lesions was visually inspected for relatively large ectoparasites such as ticks, fleas, and lice. No ectoparasite infestation was recorded in cats enrolled in the study. Blood samples were collected from clinically healthy cats without symptoms of infectious diseases. Blood samples (0.5–1.5 mL) were collected into ethylenediamine tetraacetic acid (EDTA) coated vacuum vials from each cat through the cephalic or saphenous veins. All collected samples were centrifuged (3000 rpm for 15 min) to separate red blood cells and plasma and kept at -20℃ until use.

Fig. 3
figure 3

Provinces where the sampled cats are registered

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Table 3 Detailed information regarding the sampled cats
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Genomic DNA isolation

High Pure PCR Template Preparation Kit (Roche, Germany) was used for genomic DNA extraction from feline blood samples following the manufacturer’s instructions. Until the molecular analysis, the genomic DNAs were held at − 20oC. The extracted genomic DNA concentrations were quantified using a NanoDrop 2000 spectrophotometer (Colibri TITERTEK BERTHOLD).

PCR assays for detection of feline haemopathogens

Each feline genomic DNA was screened by genus- or species-specific PCR/nPCR targeting to amplify Babesia/Theileria/Hepatozoon spp. 18S rRNA, Babesia/Theileria spp. 18S rRNA (V4), Anaplasma/Ehrlichia spp. 16S rRNA, Mycoplasma spp. 16S rRNA, Rickettsia spp. and Rickettsia felis gltA gene fragments, and Bartonella spp. 16–23S rRNA intergenic region. The molecular screening was carried out following the references, correspondingly listed in Table 4. The final volume of the PCR reaction mixture was prepared as 10 µL containing 6.35 µL of double-distilled water (Invitrogen, Ultrapure™ Distilled Water, DNAse, RNAse, Free), 2 µL of 5X One Taq Standard Reaction buffer (BioLabs, New England), 0.2 µL of dNTP mix (Deoxynucleotide solution mix, BioLabs, New England), 0.05 µL One Taq DNA polymerase (BioLabs, New England), 0.2 µM of each primer, and 1 µL of DNA template. Previously sequence-confirmed genomic DNAs obtained from the National Research Center for Protozoan Diseases (Obihiro, Japan) and double-distilled water (Invitrogen, Ultrapure™ Distilled Water, DNAse, RNAse, Free) served as positive and negative controls, respectively. The PCR amplicons were run on a 1.5% agarose gel. The gels were stained with ethidium bromide after electrophoresis and visualized under a UV transilluminator.

Table 4 Primer pairs used in the standard and nPCR assays
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Cloning and sequencing procedures

After performing PCR analysis, we selected some of the PCR amplicons giving clear and strong bands on the agarose gel for direct sequencing. To ensure that the concentrations were suitable for direct sequencing, we repeated PCR analysis at higher volumes and purified the DNA from the gel. A gel extraction kit (NucleoSpin™ Gel and PCR Clean Up, Macherey-Nagel, Germany) was utilized to purify amplified PCR products from agarose gel, and the eluted DNA concentrations were quantified using a NanoDrop 2000 spectrophotometer. All PCR amplicons with sufficient DNA concentration were directly sequenced, while samples with low concentration were cloned. For this aim, the commercial protocol of pGEM®-T Easy Vector System (Promega, USA) to clone the extracts into a pGEM vector was followed. The template DNA (6 µL) was ligated into pGEM-T easy vector (2 µL) using restriction buffer and T4 DNA ligase. The mixture was then incubated at 16 °C for 2.5–3 h and kept at 4 °C overnight. Next, plasmid was transformed into Escherichia coli DH5α competent cells and incubated them in Luria-Bertani (LB) broth at 37 °C for at least 1 h. While the cells were incubating, LB agar plates were heated to 37 °C. After centrifugation (2500 rpm for 3 min) and removal of the supernatant, we spread the remaining mixture on LB agar plates using a spreader and incubated them overnight at 37 °C. Recombinant clones were then selected to be sequenced. These clones were transferred to LB broth with 50 µg/mL ampicillin (Wako, Saitama, Japan), and incubated overnight at 37 °C. By using the Nucleospin® Plasmid QuickPure Kit (Macherey-Nagel-German), the plasmid was purified from this culture, and the samples were sequenced with the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, USA).

Phylogenetic analysis

The comparison of the nucleotide sequences obtained from the present study with previously deposited GenBank sequences to determine identities and similarities was performed using the BLASTn Analysis tool. Phylogenetic trees for Babesia ovis (18S rRNA) and Mycoplasma spp. (16S rRNA) were constructed by employing the maximum likelihood (ML) method of the MEGA version 11 software [80]. To estimate the confidence in the nodes and branches of the phylogenetic trees, bootstrap analysis with 1000 replications was used. Finally, the sequences obtained in this study were registered to the National Center for Biotechnology Information’s GenBank database using BankIt.

Accession numbers for FVBHs

The sequences have been deposited to GenBank under the following accession numbers: OR984759, OR984760, OR9847561, and OR984762 for B. ovis; OR979169 and OR979175 for Mycoplasma haemofelis; OR979160, OR979161, OR979162, OR979163, OR979164, OR979165, OR979166, OR979167, OR979168, OR979170, OR979171, OR979172, OR979173, OR979176, OR979177, OR979179, and OR979180 for Candidatus Mycoplasma haemominutum; OR979174 for uncultured Mycoplasma sp.; OR979178 for Mycoplasma wenyonii; OR978398, OR978399, and OR978400 for Bartonella koehlerae; OR978401, OR978402, and OR978403 for Bartonella henselae.

Further analysis for confirmation of Babesia ovis presence in cats and endogenous control of feline DNA

All blood samples underwent initial screening using a universal primer that targets a 403 bp region of the piroplasmid 18S rRNA genes. Out of these, four samples tested positive. All PCR-positive amplicons with sufficient DNA concentration were directly sequenced, revealing the presence of Babesia ovis. A nested PCR amplifying approximately 1600 bp was performed on the positive samples to corroborate these findings. Subsequently, one sample was selected for direct sequencing, while three samples underwent cloning procedures. In addition, all four piroplasm-positive DNAs were molecularly tested using cat’s SSR primers for endogenous control following the protocol described by Abdel-Rahman et al. [81].

Statistical analysis

A cross-tabulation evaluation was conducted using categorical data, numbers, and percentages. In cases where the expected cells were less than 20%, the data were analyzed using the Monte Carlo Simulation Method. The data were analyzed using the SPSS 25 statistical package program (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.). Pearson Chi-square p-values were calculated to determine the statistical significance of the relationship among cat breed, age, gender, and status in the distribution of FVBHs. Statistical significance was considered as p < 0.05.