Cytauxzoon spp. and Hepatozoon spp. are the etiologic agents of cytauxzoonosis in felids and hepatozoonosis in a wide range of animals worldwide. The first description of the genus Cytauxzoon was recorded in a domestic cat in the USA and was named Cytauxzoon felis (1). A different species of Cytauxzoon was molecularly recorded in Europe [Spain (2, 3), France (4–6), Portugal (7), Switzerland (6, 8), Germany (9, 10), Luxembourg (10), Romania (10), Czechia (10), Russia (11), and Italy (12–16)] in wild and domestic felids and named Cytauxzoon sp. until 2020.

In fact, three species affecting wild and domestic European felids have recently been described and defined as Cytauxzoon europaeus, Cytauxzoon banethi, and Cytauxzoon otrantorum (10).

Different clinical presentations have been associated with the American and European Cytauxzoon species. Although data are still lacking, Cytauxzoon in Europe seems to be less virulent than the C. felis described in domestic cats in the USA. In fact, no clinical findings are commonly detectable in cats infected with the European Cytauxzoon species, which usually present with subclinical infections and are rarely associated with mild anemia (17). However, erythroparasitemia may persist in supposedly healthy cats, which may serve as a reservoir for European Cytauxzoon species (12). On the other hand, C. felis infections in domestic cats present with generalized rather than specific clinical manifestations, including fever, lethargy, anorexia, dehydration, icterus, pallor of the mucous membrane, dyspnea, and progressively worsening anemia; most infected cats die within 1 week of the onset of clinical signs (18, 19).

Hepatozoon spp. has been described in several host species, such as mammals, reptiles, birds, and amphibians (20). In particular, three Hepatozoon species have been described and reported in wild and domestic felids in Europe: H. felis, H. silvestris, and H. canis. Hepatozoon infections are widespread and have been reported in Spain (3), France (4, 21), Portugal (22), Cyprus (23), Germany (24), Austria (25), Greece (26), and Italy (15, 27, 28).

Hepatozoon felis infection is often subclinical and detected accidentally by observation of stained blood smears. When present, clinical manifestations are not specific and include anemia, elevated creatine kinase, and lactate dehydrogenase values (29). Clinical manifestations of H. silvestris in case reports have been under-reported in the scientific literature. Kegler et al. (24) reported a cat in Switzerland infected with H. silvestris with fatal lymphoplasmacytic and histiocytic myocarditis, weakness, and anorexia.

Finally, H. canis has also been described in dogs and cats, although clinical signs in cats are occasionally reported (3, 27, 30).

The observation of specific parasitic inclusions (i.e., merozoites) in red blood cells suggests piroplasmic infection and cytauxzoonosis. Unfortunately, the stained blood smear is not sufficient to make a diagnosis, and molecular analysis is strongly recommended. Moreover, since cytauxzoonosis is characterized by a low burden of merozoites circulating in the bloodstream in both acute and chronic phases of the infection, molecular analysis represents the gold standard for the diagnosis due to its high sensitivity and ability to identify the Cytauxzoon species involved (31).

Although the detection of Hepatozoon gamonts may be sufficient for a hepatozoonosis diagnosis, molecular detection is strongly suggested because hepatozoonosis in felids is usually asymptomatic and characterized by low parasitemia in about 1% of infected white blood cells (32).

The literature has described conventional polymerase chain reaction (cPCR) assays for Cytauxzoon and Hepatozoon detection, usually targeting 18S rRNA, a highly conserved gene for both protozoa (8, 23, 33–38), and cytochrome B and cytochrome C oxidase subunit I (COI), mitochondrial protein-coding genes, which are more specific for Cytauxzoon species identification (10). Real-time polymerase chain reaction (real-time PCR) may be a reasonable alternative for the rapid screening of large numbers of samples. This method is time-saving because the electrophoresis gel analysis is not required, and the fluorescence analysis allows the operator to collect data while real-time PCR is running. In 2007, Criado-Fornelio et al. (39) developed a real-time PCR assay for the detection of Hepatozoon spp. in canine and feline blood samples, and the procedure appeared to be more sensitive than conventional PCR in feline samples. Recently, real-time PCR assays targeting the piroplasmid 18S-rRNA gene have been used to individually detect Cytauxzoon spp. and Hepatozoon spp. in tissue samples and blood from domestic and wild felids in Europe (6, 16). However, to our knowledge, no assay has been developed to simultaneously detect Cytauxzoon spp. and Hepatozoon spp. DNA.

In light of the above considerations, this study aimed to (i) evaluate the occurrence of Hepatozoon spp. and Cytauxzoon in felids using real-time PCR for the simultaneous detection of both pathogens in north-eastern Italy, (ii) assess the distribution of Cytauxzoon and Hepatozoon species and their genetic diversity in felids, and (iii) study the involvement of felines other than domestic cats.

Vector-borne diseases have stimulated the interest of the scientific community in the last decades, and indeed, the epidemiologic data for Cytauxzoon spp. and Hepatozoon spp. in wild and domestic felids are continuously updated (6, 10, 46). In Italy, the circulation of Cytauxzoon spp. and Hepatozoon spp. has been reported in both domestic and wildcats (12, 15, 27, 47), highlighting the need to better investigate their circulating species and genotypes. A recent survey of Cytauxzoon spp. and Hepatozoon spp. infections in asymptomatic domestic cats confirmed the presence and establishment of a domestic cycle for both protozoa in northeastern Italy. Hepatozoonosis was fairly evenly distributed in all the investigated regions, whereas cytauxzoonosis was strictly limited to the province of Trieste in the Friuli-Venezia Giulia region (15). The present study (i) updates the epidemiological data on domestic cats in those same areas using a new, fast, and sensitive molecular procedure (real-time PCR) able to detect and differentiate simultaneously both protozoa, (ii) adds information on the protozoan species and their distribution in north-eastern Italy, and (iii) investigates other potential felid hosts in the same areas and their potential role in the transmission of both protozoa.

Out of 237 animals, 4.2% were infected with Cytauxzoon. In particular, the 10 positive animals were six stray cats and four wildcats coming from the same site (Site 2 – Friuli-Venezia Giulia region), where the first Italian report of Cytauxzoon was identified in 2012 (12) and recognized as endemic about 10 years later (14). The province of Trieste is close to Slovenia and is considered an ecological corridor for wildlife movements (48). In addition, the province is characterized by a large peri-urban area overlapping with a sylvatic environment inhabited by wildcats, suggesting the possible role of these wild felids as reservoirs. Moreover, the Eurasian lynx is present in the region (49, 50) and may be involved in parasite circulation, as previously reported (51).

In fact, the stray cats at Site 2, including mostly street colony cats, were statistically more infected than domestic cats, and this supports the hypothesis that animals with an exclusively outdoor lifestyle, living virtually in sympatry with sylvatic species and without regular controls and treatments, are more exposed to the risk of infection. No captive exotic felid was found to be infected with Cytauxzoon spp. Considering that all tested animals lived in zoological parks in Site 1 (Veneto region), this could be related to the absence of cytauxzoonosis in domestic or wildcats in the considered area. On the other hand, exotic felids are susceptible hosts for other Cytauxzoon species such as C. felis and C. manul (18).

In north-eastern Italy, Cytauxzoon spp. was first reported in domestic cats in 2012 (12) and in wildcats in 2016 (47); however, species identification was not achieved until recently, when a new molecular approach targeting more variable genes (i.e., cytochrome B and cytochrome C oxidase subunit I – COI) allowed phylogenetic analyses (10). In this study, all sequences had a percentage identity of 99–100% with reference sequences of C. europeus, the most common species isolated in felids in Europe (10).

The obtained Cytauxzoon phylogenetic analysis showed a scenario that partially contrasts with what was observed in Germany, where most of the sequences were identical, with few exceptions. Unfortunately, the limited sequence availability and the lack of proper knowledge about the epidemiology of this parasite prevent any definitive conclusion, and only speculative hypotheses can be advocated. Therefore, more extensive and systematic studies should be performed to formally evaluate the prevalence of infection and to investigate the spreading patterns and the involved countries.

The presence of wildcats in the Friuli-Venezia Giulia region is known, also thanks to the possible movement of these animals across the Alps from nearby Slovenia (48). Recently, the distribution of European wildcats in Italy has been updated (52), and the data collected shows that wildcats are expanding their territory toward the northern part of the Veneto region and the southern areas of the Trentino-Alto Adige region (53, 54).

Out of 237 animals, 17.3% were infected with Hepatozoon spp. Specifically, 41 positive animals were recorded (i.e., 31 domestic cats, eight wildcats, and two tigers).

A significantly higher prevalence (p < 0.05) of H. silvestris infection was observed in stray cats and animals living in Site 2, suggesting that a free-roaming lifestyle increases exposure to vectors. H. silvestris was also isolated in domestic cats in Site 1, most of them coming from the province of Verona, followed by the provinces of Vicenza, Treviso, and Belluno. These positive results are particularly interesting because they seem to follow the recent movements of wildcats in the Italian territory. Even if the infected cats of Site 1 were evenly divided between strays and domestic animals, all of them had an outdoor lifestyle. The main risk of exposure is thus related to living outdoors and coming into contact with ticks, although it must be considered that other modes of transmission could be possible. In the H. canis and H. felis life cycles, vertical transmission from mother to offspring during pregnancy has been proven (30, 32), and in the H. americanum life cycle, predation on infected prey has been demonstrated to be a mode of transmission (32). In felids, no information is available, and it can only be assumed that vertical and/or horizontal transmission by ingestion of infected prey may be possible. These considerations justify the H. felis results. First, H. felis is fairly evenly distributed in all areas investigated and affects both domestic and wild felids. In particular, H. felis was found more frequently in domestic cats and animals younger than 1 year and older than three, with statistically significant prevalence values. If outdoor habits were a significant risk factor for domestic cat infection with C. europeus and H. silvestris, they do not seem to be significant for H. felis; in fact, positive cats were evenly distributed between indoor and outdoor animals, some of which were infected in areas where wild felids are not yet reported (e.g., the southern areas of Site 1), suggesting a domestic cycle of H. felis. In addition, younger animals were more likely to be infected by the vertical route (30) along with older animals because they had more time to be exposed to the infection through the ingestion of infected arthropods and/or infected prey. The same considerations could justify the isolation of H. felis from wildcats; indeed, all the animals were adults and came from different provinces of Site 2 (i.e., the Trieste, Udine, and Pordenone provinces).

None of the 12-36-month-old cats tested positive for H. felis. Since hepatozoonosis is still under-investigated and no data are available on pathogenesis and host immune response, it could be assumed that (i) a young cat with a competent immune system is likely to be able to face and overcome the infection, (ii) no immunological memory is formed after the infection, and (iii) repeated exposure throughout life leads cats to new infections as they become older.

Among the captive wild felids, two tigers with hepatozoonosis came from the same zoological park in the province of Venezia in Site 1, where both H. felis and H. silvestris were isolated in domestic cats. To our knowledge, this is the first description of H. felis and H. silvestris in captive tigers in Italy, as three tigers from a zoological park located in southern Italy were found positive for H. canis (55).

The H. felis sequence analysis revealed two distinct clusters, whose features support the above-mentioned scenario. One contained sequences mainly from domestic cats, with only three exceptions (i.e., the tiger and two wildcats, one from Italy and one from Spain), while the other comprised only strains from wildcats, originating from Italy and Eastern Europe, with the sole exception of one strain detected from a wildcat in the present study. Therefore, despite the separation between the domestic and wild cycles, it is likely that some strain exchange occurs in both directions. Finally, the relationship between sequences obtained from captive and domestic animals supports the involvement of a domestic cycle, although the precise contact pathway remains unclear. Similar evidence emerged from H. silvestris, where a close relationship was found between Italian strains obtained from Panthera tigris and a domestic cat. Also in this case, two clusters were observed, one containing sequences mainly, but not only, from wildcats, linked to strains collected in Eastern European countries, and another group containing only strains from domestic cats (the tiger being the only exception). In the latter, the clustering with Turkish strains can hardly be explained, supporting the speculative hypothesis about the role of human and pet travel in parasite dispersal.

The observation of merozoites in red blood cells and Hepatozoon gamonts in white blood cells in stained feline blood smears is strongly suggestive of these protozoan infections. However, because cytauxzoonosis and hepatozoonosis usually present a low parasite burden in feline hosts (8, 15, 32), the blood smear is not a sensitive method, and molecular procedures may be considered the method of choice for the diagnosis. Several studies support the higher sensitivity of molecular procedures compared to stained blood smears in the diagnosis of hepatozoonosis. In 2006, a study reported that 32% of cats were positive for Hepatozoon spp. by PCR, while only 0.7% had gamonts in blood smears (56). Similarly, Pereira et al. (57) described that 12.5% of cats in Cape Verde were PCR-positive for H. felis, with no gamonts observed in blood smears. Conventional PCR is currently the most common method for the individual detection of Hepatozoon spp. and Cytauxzoon spp.; however, it has some limitations (e.g., time and cost) that can be overcome by alternative solutions, such as a real-time PCR protocol.

The real-time PCR procedure adopted in this study was designed to guarantee a high-sensitivity protocol that can (i) simultaneously detect and (ii) differentiate both Cytauxzoon spp. and Hepatozoon spp. DNA by rapid melting curve analysis, (iii) be applied to different matrices (from blood to different tissues and organs), and (iv) quickly screen a consistent number of samples. Because of the low sensitivity of blood smear observations, the lack of serological tests on the market, and the limitation of the molecular procedures described in the literature to detect only one pathogen at a time, this protocol represents a good choice for simultaneously diagnosing hepatozoonosis and cytauxzoonosis.

Conventional PCR was successively adopted to further characterize the involved species and strains in positive samples by Sanger sequencing, allowing alignment and phylogenetic analysis. In conclusion, this study updated the epidemiologic data on cytauxzoonosis and hepatozoonosis in feline hosts through a new, fast, and sensitive real-time PCR, providing new information on the species and strains involved, their genetic correlation with European isolates, susceptible feline hosts, and their distribution in north-eastern Italy.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the animal study because blood collection on felids was performed during routine veterinary procedures not depending on this research project. Written informed consent was obtained from the owners for the participation of their animals in this study.

MG, GS, and AFdR: conceptualization. MG, GD, CT, and GF: methodology. GD and CT: validation. MG, GS, EM, and PB: investigation. GF: software. GS: resources and project administration. MG: data curation and writing—original draft preparation. GS, AFdR, and DM: writing—review and editing. AFdR and GS: supervision. All authors read and agreed to the published version of the manuscript.

This research was supported by the Department of Animal Medicine, Production, and Health of the University of Padua (BIRD193835, 2019).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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