Cytauxzoon sp. and Hepatozoon spp. in Domestic Cats: A Preliminary Study in North-Eastern Italy

Knowledge on the presence of Cytauxzoon sp. and Hepatozoon spp. in Italy is scant and mostly limited to a few areas of Northern and Southern regions, respectively. The present study updated the current epidemiological scenario by investigating the occurrence of these protozoa in domestic cats from three broad regions of North-Eastern Italy. Blood samples from cats at risk of vector-borne diseases were processed by PCR to detect Cytauxzoon and Hepatozoon DNA. Blood smears were observed for haemoparasite inclusions. The influence of cat individual data (e.g., provenance, management, indoor/outdoor lifestyle) on the prevalence of haemoprotozoan infections was statistically evaluated. Among 158 cats, Cytauxzoon and Hepatozoon DNA were detected in 6 (3.8%) and 26 (16.5%) animals, respectively. No Hepatozoon gamonts were detected in blood smears, whereas all Cytauxzoon PCR-positive samples were microscopically positive, though with low levels of parasitaemia. Two species of Hepatozoon were identified, Hepatozoon felis (n = 10) and Hepatozoon silvestris (n = 16). Hepatozoon silvestris prevalence values were significantly (p < 0.05) higher in the region Friuli Venezia Giulia and in stray cats. Cytauxzoon sp. was detected in 6/39 (15.4%) stray cats from Friuli Venezia Giulia (Trieste province). These data add new information on the occurrence of these neglected protozoa in domestic cats’ populations.


Introduction
Cytauxzoon sp. and Hepatozoon spp. are two apicomplexan protozoa belonging to Orders Piroplasmida and Eucoccidiorida, respectively [1]. The genus Cytauxzoon was reported for the first time in a domestic cat (Felis silvestris catus) in 1976 in the US, and the species was named Cytauxzoon felis [2]. Then, reports of Cytauxzoon in cats were described only in some US regions [3,4], until the 2000s, when cases were also reported in Europe. More recently, cats positive for Cytauxzoon have been recorded in Spain [5,6], France [7,8], Portugal [9], Switzerland [10], and Germany [11]. In Italy, cases were limited to an area in the North-Eastern region of Friuli Venezia Giulia, where an endemic focus was described in the city of Trieste with a prevalence rate of 23% among owned and stray cats [12]. Subsequently, clinical cases were then recorded in other Italian regions, i.e., Veneto, Tuscany, and Latium [13]. Molecular analyses showed that isolates of Cytauxzoon in Europe are different from C. felis affecting felid populations in the USA. Indeed, Cytauxzoon is a monophyletic group, characterised by different isolates grouped in separate species (i.e., C. felis, Cytauxzoon manul) [14]. In addition, among the isolates from European wild felids, three genotypes of Cytauxzoon (i.e., major-EU1, minor-EU2, rare-EU3), defined as three new species, were recently detected [15]. Hepatozoon spp. was reported in domestic cats in India at the beginning of the 1900s [16], then only a few reports were published until 1973, when schizonts of Hepatozoon-like protozoa were described in the myocardium of a domestic cat in Israel [17]. Since then, Hepatozoon has been described worldwide, including in Africa [18][19][20], the US and South America [21,22], and Europe [6,7,[23][24][25][26][27][28]. In Italy, hepatozoonosis was described in the Emilia Romagna region [29] and in Southern regions, i.e., Apulia and Basilicata [30] and the Aeolian Islands [31]. Three species of Hepatozoon infect cats (i.e., Hepatozoon felis, Hepatozoon silvestris, and Hepatozoon canis) [27,30].
Due to the merit in improving knowledge on the occurrence of cat cytauxzoonosis and hepatozoonosis in populations of domestic cats, the aim of this work was to investigate the presence and distribution of Cytauxzoon sp. and Hepatozoon spp. in domestic cats in North-Eastern Italy, aiming towards an update of the current epidemiological scenario.
All Cytauxzoon blood smear samples were also positive using the molecular assay. All sequences of Cytauxzoon sp. (from MZ227613 to MZ227618), H. felis (from MZ227585 to MZ227594), and H. silvestris (from MZ227596 to MZ22611) were deposited in GenBank.
Regarding geographical distribution, Cytauxzoon sp. was found only in Site 2, in particular in one province (Trieste). Contrariwise, Hepatozoon spp. was distributed in all investigated regions (Figure 1).  Individual data of cats positive for Cytauxzoon sp. and Hepatozoon spp. are reported in Table 2.

Statistical Evaluation
Differences in the infection rate among sub-groups of animals were found by the Pearson Chi-Square test for Cytauxzoon sp. and H. silvestris for two factors: a significantly higher prevalence (p < 0.05) was found in stray cats compared to owned animals, and in the cats living in Trieste province (Site 2) compared to the other two sites. Moreover, cats infected with immunosuppressive viruses seem to be at higher risk of positivity of Cytauxzoon sp. (p = 0.051), and cats with ectoparasites had a higher prevalence of H. silvestris (p = 0.080). However, in both cases, the Fisher exact test showed a p-value slightly higher than the 0.05 threshold. No significant differences were observed for H. felis, nor for the other factors in general.

Discussion
To date, cytauxzoonosis and hepatozoonosis are neglected diseases in feline populations. Data on the Cytauxzoon species circulating among European cats are still limited [6,46,47] and information on Hepatozoon spp. in felids is also poor [48,49].
The present study confirmed that Trieste (Site 2) is an endemic site for the presence of Cytauxzoon sp. in domestic cats. As in a previous study, these results are supported both by blood smear examinations and molecular analysis, with a prevalence value similar to that reported (23%) almost ten years ago in 2012 [12].
Site 2 is the only region in which different wild felids acting as reservoirs for cytauxzoonosis are endemic, i.e., the Eurasian lynx [50] and the European wildcat [39,51]. Moreover, Site 2 is a border region, and wildlife movements from the nearby Slovenia are extensively described [52].
The significant difference in the prevalence between the type of management (owned vs. stray cats) highlights how stray cats that live mostly outdoors are more exposed to cytauxzoonosis than owned cats (10.9% vs. 0.0%). This is probably due to the sharing of the same environment with wild felids and the presence of infected vectors. Indeed, the continuous reduction of wildlife habitat due to anthropization favours the sympatric occurrence of wild and domestic cats in many areas [53], and this has the implication of sharing parasites with high pathogenic potential, as recently investigated for nematodes affecting the cardio-respiratory system [32][33][34][35]54].
Two species of Hepatozoon spp. have been found in North-Eastern Italy (i.e., H. felis and H. silvestris). The finding of H. silvestris in Northern Italy is especially noteworthy. Indeed, this species has been reported mainly in wild felids in Europe, and rarely in domestic cats. Nevertheless, it was recently described in a domestic cat in Southern Italy during an epidemiological survey [30] and in another one in Switzerland associated with a fatal myocarditis [26].
In agreement with Baneth et al. [49], who described an extremely low level of parasitaemia in felids, no sample showed Hepatozoon gamonts in blood smear examinations.
Positive cats were mostly sub-clinically infected, in apparently good physical condition, and only in one case were diarrhoea and rhinitis present (Table 2), thus evoking the infections as well-tolerated in most cases. No correlation between Hepatozoon positivity and potentially immunosuppressive infectious diseases (i.e., FIV and FeLV) was statistically found, as already reported by Baneth et al. [45]. Instead, cats positive for immunosuppressive viruses showed a higher prevalence of Cytauxzoon sp., indicating a tendency of being more at risk to becoming infected with haemoprotozoa, as previously suggested [8,12].
This result underlines the importance of investigating subclinical infections, and in parallel highlights the diagnostic limitations posed by stand-alone cytology. Differently, all Cytauxzoon-positive cats presented mild parasitaemia. Although few parasitised erythrocytes per monolayer were observed, the positivity suggests a potential epidemiological role of clinically healthy animals as carriers and sources of infection for potential vectors.
No significant differences between individual variables (i.e., provenance, management, and lifestyle) and H. felis prevalence were found. However, the high prevalence value obtained in indoor and owned cats, that are commonly less exposed to vectors' activity due to their lifestyle, suggests that alternative ways of transmission are possible, as already predicted (e.g., vertical transmission, predation of infected preys) [45]. Indeed, H. canis may also be spread through intra-uterine transmission from the mother to the offspring, and Hepatozoon americanum may be transmitted by ingestion of infected preys [49].
On the contrary, H. silvestris showed a significant difference in its distribution between regions, especially in Site 2, achieving a prevalence rate of 23.1%, most likely for a habitat/vector sharing between domestic and wild felids, as previously mentioned for Cytauxzoon sp. The presence of H. silvestris in Site 1, where the Eurasian lynx and the European wildcats are absent, indicates that the role of wildlife as reservoirs could be unnecessary. This supports new considerations, as the possibility that H. silvestris might have another route of transmission related to the predatory instinct of cats and the carnivorism of potential paratenic hosts such as small rodents could be supported, as already described for H. americanum in the US [49]. As H. silvestris was found mainly in stray cats, this hypothesis is even more appropriate due to their predatory and hunting activities.
In conclusion, this study demonstrated that Cytauxzoon sp. and Hepatozoon spp. circulate in the feline population of North-Eastern Italy involving both owned and stray cats, focusing on the risk of exposure that some individual attitude or lifestyle factors might encourage.
Information about these haemoprotozoa is still lacking, and further studies are needed to obtain important data about their lifecycles with the evaluation of their pathogenicity and their impact on cat health as well as the potential ways of transmission, including wildlife as possible reservoirs and the involved arthropod vector, to carry out adequate control measures.

Blood Collection, Blood Analysis, and DNA Extraction
K3EDTA blood and blood smears were collected in collaboration with veterinary practitioners working in the investigated regions of North-Eastern Italy, during routine clinical examinations, from cats of all ages, exposed to at least one season at risk of arthropod vectors' activity, preferably without any regular treatments against ectoparasites.
For each sampled cat region of provenance (i.e., Veneto-Site 1, Friuli Venezia Giulia-Site 2, and Trentino Alto Adige-Site 3, Figure 1), sex, age classes (<12 months, 12-35 months, ≥36 months), management (owned, stray cats), lifestyle (indoor, outdoor), clinical signs, and ectoparasite infestations were reported. Moreover, all the involved owners or veterinary health authorities for colony/stray cats signed an informed consent form for participating in the study. Recruited animals were submitted to routine veterinary procedures not depending on this research project.
Blood smears were stained using Hemacolor ® (Merck KGaA, Darmstadt, Germany) and then observed by microscope at 100× magnification with immersion oil to evaluate the presence of Hepatozoon gamonts and Cytauxzoon merozoites according to an existing key [12,40,48]. The parasitaemia level for Cytauxzoon sp. was graded as reported by Carli et al. [12], observing how many erythrocytes presented parasitic inclusions per the entire monolayer and defining based on the following scale: mild (n ≤ 5 parasitised red blood cells), moderate (n ≤ 20), marked (n ≤ 50), and very marked (n > 50). Serum obtained from each blood sample was also analysed by the SNAP ® Combo Plus FeLV Ag/FIV Ab test (IDEXX Laboratories Inc., Westbrook, ME, USA) following the manufacturer's instructions.
DNA extraction was performed starting from 200 µL of k 3 EDTA blood by the NucleoSpin ® Tissue kit (Macherey-Nagel, Düren, Germany), in accordance with the manufacturer's protocol.

Molecular Analysis and Sequencing
DNA was processed by conventional PCR targeting the 18S-rRNA gene using the Piroplasmid primers pair 5 -CCAGCAGCCGCGGTAATTC-3 and 5 -CTTTCGCAGTAGTTYGT CTTTAACAAATCT-3 , as already described by Tabar et al. [55]. Positive (i.e., DNA of sequenced field sample) and negative (no DNA added) controls were included in each PCR reaction. Amplicons were sequenced following Sanger technology (Macrogen Spain, Madrid, Spain) and the obtained nucleotide sequences were compared to those deposited in GenBank ® using BLAST software (https://blast.ncbi.nlm.nih.gov/Blast) (accessed date: 2 August 2021)..

Data Analysis
In order to evaluate the presence of differences in infection rates among subgroups of the investigated cat population, a statistical evaluation was performed by means of the Pearson Chi-Square test or the Fisher exact test, if appropriate, using SPSS for Windows, version 27.0. The factors taken into consideration were: sex (i.e., males, females), age classes (i.e., <12 months, 12-35 months, ≥36 months), region of provenance (i.e., Site 1, Site 2, Site 3), lifestyle (i.e., indoor, outdoor), management (i.e., owned, stray cat), infection with immunosuppressive virus (i.e., positive for FIV and/or FeLV, or negative), presence of clinical signs (i.e., gastro-intestinal and respiratory signs), and ectoparasite infestation.  Institutional Review Board Statement: Ethical review and approval were waived for this study, due to the involved animals being submitted to routine veterinary procedures not depending on this research project.

Informed Consent Statement:
Informed consent for participating to the study was obtained from all the involved owners or veterinary health authorities for colony cats.

Data Availability Statement:
The authors declare that data are available upon request to the corresponding author, by email.