TYPE Original Research PUBLISHED 10 November 2022 DOI 10.3389/fvets.2022.976173 Clinicopathological findings and OPEN ACCESS risk factors associated with Cytauxzoon spp. infection in EDITED BY Münir Aktaş, Firat University, Turkey REVIEWED BY Kaori Sakamoto, cats: A case-control study University of Georgia, United States Leah A. Cohn, University of Missouri, United States (2008–2021) *CORRESPONDENCE Erika Carli Erika Carli1*, Laia Solano-Gallego2 , Stefano De Arcangeli1 , erika.carli@sanmarcovet.com SPECIALTY SECTION Laura Ventura3 , Elisa Ligorio1 and Tommaso Furlanello1 This article was submitted to 1 San Marco Veterinary Clinic and Laboratory, Padua, Italy, 2 Departament de Medicina i Cirurgia Parasitology, Animal, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Department a section of the journal of Statistical Sciences, University of Padova, Padua, Italy Frontiers in Veterinary Science RECEIVED 23 June 2022 ACCEPTED 22 September 2022 In Europe, Cytauxzoon spp. infection was documented in domestic and PUBLISHED 10 November 2022 wild felids. Cats often develop a subclinical infection, while fatal disease CITATION Carli E, Solano-Gallego L, De is rare. Currently, information on the epidemiology, risk factors and Arcangeli S, Ventura L, Ligorio E and clinicopathological findings of Cytauxzoon spp. infection remains limited Furlanello T (2022) Clinicopathological findings and risk factors associated and obtained by a single subject or small groups of cats. The objective of with Cytauxzoon spp. infection in cats: this case-control study was to evaluate clinicopathological findings and to A case-control study (2008–2021). describe risk factors associated with Cytauxzoon spp. infection in domestic Front. Vet. Sci. 9:976173. doi: 10.3389/fvets.2022.976173 cats. Infected cats (n = 39) and non-infected (n = 190) cats were selected COPYRIGHT from the database of the referral San Marco Veterinary Laboratory between © 2022 Carli, Solano-Gallego, De 2008 and 2021. Demographic information, a preset questionnaire considering Arcangeli, Ventura, Ligorio and lifestyle, environment, and clinical status, and a CBC performed contextually Furlanello. This is an open-access article distributed under the terms of with the PCR analysis were recorded for all cats. Data on the biochemical the Creative Commons Attribution profile and serum protein electrophoresis were also evaluated when available. License (CC BY). The use, distribution Compared to the control group, infection was more likely to occur in stray or reproduction in other forums is permitted, provided the original cats (24/39, 61.5%, P < 0.001), living totally/partially outdoors (36/39, 92.3%, author(s) and the copyright owner(s) P < 0.001), in an urban context (37/39, 94.9%, P = 0.002), taken or recently are credited and that the original publication in this journal is cited, in adopted from colonies (34/35, 97.1, P < 0.001), with irregular or absent parasite accordance with accepted academic preventive treatments (39/39, 100%, p = 0.005), without fleas (28/35, 80%, practice. No use, distribution or P = 0.047) and without clinical signs (22/39, 56.4%, p = 0.026) at the time reproduction is permitted which does not comply with these terms. of medical evaluation. Anemia was not associated with infection, but in cats without clinical signs, the percentage of anemic-infected cats (7/22, 31.8%, P = 0.009) was higher compared to non-infected cats (5/65, 7.7%). Furthermore, a decrease in total iron serum concentration approximating the lowest reference interval [median values (IQR): 79 µg/dL (52.25) vs. 50.5 µg/dL (34), P = 0.007] was likely in infected cats. No other laboratory findings were associated with infection. Interestingly, a partial/total outdoor lifestyle was a risk factor for infection (OR: 8.58, 95% CI: 2.90–37.0, P < 0.001). In conclusion, the present Frontiers in Veterinary Science 01 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 study revealed that Cytauxzoon spp. infection manifests itself prevalently as a subclinical infection, based on physical examination and laboratory findings, in domestic European cats. However, subclinical infected cats were more likely to be anemic compared to non-infected. KEYWORDS Cytauxzoonosis, feline, clinical status, laboratory findings, Europe Introduction database of PCR analysis performed at the San Marco Veterinary Laboratory (SMVL) referred from May 2008 to July 2021. Cytauxzoonosis is a protozoan tick-transmitted disease The source database included the records of blood molecular that affects wild and domestic felids, traditionally caused by analysis of Cytauxzoon spp. obtained from (1) cats published Cytauxzoon felis. It is particularly reported in central, south- in previous works (14, 15) (n = 120) and (2) cats collected by eastern, and south-central USA (1, 2), although it was described clinicians distributed throughout Italy during routine veterinary in other parts of the world such as Brazil (3), Iran (4), and procedures or illnesses, with or without suspected Cytauxzoon China (5). Domestic cats generally develop a severe disease spp. infection (n = 526). The SMVL provides services to that rapidly progresses to death (6) while wild felids are mostly veterinary clinics spread throughout Italy. Approximately 1,400 affected by a subclinical infection that rarely has a fatal evolution molecular analyzes per year are performed in feline samples (7). However, sick cats that survive C. felis infection (8, 9) and at SMVL. subclinical infected (10–13) have been increasingly documented. A focus of Cytauxzoon spp. (14) was previously described In Europe, a piroplasm named Cytauxzoon spp. has been in Trieste (45◦ 39′ 01′′ N, 13◦ 46′ 13′′ E), a seaport city in reported in both domestic felids (14–16) and wild felids (17–20). northeastern Italy. It is located in the northern part of the high The protozoan was molecularly similar, but not superimposable, Adriatic Sea near the border with Slovenia. It is characterized to C. felis (14). The first focus of infection was found in cats by a long coastline bordered by grasslands, forests, and karstic living in Trieste in northeastern Italy with a prevalence of 23% areas (https://en.wikipedia.org/wiki/Trieste). Ticks (29) and (14). Later, Cytauxzoon spp. infection has been reported in wild felids such as Eurasian Lynx (Lynx lynx) and the wild cat cats from Portugal (21), France (22), Spain (16), Switzerland (Felis silvestris) (20) are reported in the region named Friuli (23, 24), Germany (25), and Russia (26). Although sometimes Venezia Giulia, where Trieste is located. lethal (14, 21, 25), subclinical infection appeared to be more likely and frequent in domestic felines (14, 22, 23). Recently, three Cytauxzoon spp. genotypes indicated as Cytauxzoon Patient selection, inclusion criteria, europeus, Cytauxzoon otrantorum, and Cytauxzoon banethi were sampling, questionnaire, and variable described in European wild cat (Felis silvestris) based on 18S characteristics rRNA, mitochondrial cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) genes (27). In addition, domestic The selection of cats included in the present study is shown cats infected with C. europeus were documented in Switzerland in Figure 1. The inclusion criteria were the following: (1) a blood (24). The available information on the epidemiology and piroplasm PCR analysis performed on the 18S rRNA gene, (2) a clinicopathological findings of Cytauxzoon spp. infection in preset questionnaire, and (3) a CBC performed contextually to Europe is currently limited and often based on the evaluation the PCR analysis. of a single subject or of small groups (21–23, 28). Therefore, according to the inclusion criteria described Given this context, the case-control study reported here above, the studied data set included random healthy/unhealthy, was aimed at (1) evaluating clinicopathological findings and (2) stray/owned (1) cats living in the Trieste focus (n = 90) (14, 28), describing risk factors associated with Cytauxzoon spp. infection and (2) cats (n = 139) living in the north (n = 112), in the center in domestic cats in Italy. (n = 20), and in the south (n = 7) of Italy. Based on the results of the PCR analysis, eligible cats were Materials and methods assigned to Cytauxzoon spp. infected (case) or not infected (control) groups. For the purposes of the case definition, Study design and setting 18S rRNA gene sequencing was performed randomly for the identification of piroplasm species in cats living in Trieste This case-control study compared cats with and without (14, 28), while it was considered mandatory for the others. Cytauxzoon spp. infection. Data were extracted from the The unmatched control group involved cats enrolled between Frontiers in Veterinary Science 02 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 FIGURE 1 Diagram of the selection of the cats included in the study. 2008 and 2011, while cases were obtained throughout the retrieved through a telephone call from one of the authors study period. (EC). Only questionnaires based on clinical records collected by Blood samples were taken from stray cats that lived in clinicians at the time of sampling were considered suitable. urban Trieste colonies as part of spay/neuter programs and from When available, the biochemical profile and serum protein owned cats that underwent routine blood screening for various electrophoresis performed at the same time as the other assays medical reasons. were also included. The clinicians completed a multiple choice questionnaire at All samples were collected solely for the cat’s health benefit. the same time the blood sample was collected. The questionnaire When the age of the cats was not available, the clinicians concerned patient characteristics (age, sex, breed), type of cat provided an estimated age value. For this reason, this variable (owned or stray), lifestyle (indoor or partially/total outdoor), was not considered reliable for statistical analysis. environment (rural/city), type of housing (taken/recently Cats were also stratified according to their geographical adopted from colony or cattery/household), parasite preventive origin (north, central, or southern Italy) and the focus of Trieste. treatments (regular or irregular/absent), ticks and/or flea The dichotomic variable “anemia” was created using the red presence and detection of clinical signs at the time of medical blood cell (RBC) concentration value of 6.35 × 106 /µL (lower examination. The results of the questionnaire were already limit of the reference interval of SVML) rather than using available on the cats observed between 2008 and 2011. The hematocrit (Hct) as discriminator, to avoid possible incorrect questionnaire of infected cats diagnosed subsequently was classification due to storage artifacts (30). Frontiers in Veterinary Science 03 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 The analytic methods 2.5 µL of DNA template, 0.4 µM of each primer, 1.8 mM of MgCl2, 200 µM of each dNTP, 2.5 U of FastStart High Fidelity All clinicopathological tests were performed at the SMVL Enzyme, and 1X of FastStart High Fidelity Reaction Buffer within 24–36 h after collection. The CBC was performed (Roche Diagnostics GmbH, Mannheim, Germany). The thermal on K3EDTA whole blood samples using automatic cell cycling conditions consisted of an initial denaturation at 95◦ C counters (ADVIA R 120, ADVIA R 2120, and ADVIA R 2120i for 2 min, 35 cycles of denaturation at 95◦ C for 30 s, annealing Hematology System, Siemens Healthineers GmbH, Erlangen, at 58◦ C for 30 s and extension at 72◦ C for 30 s, followed by a Germany) and was always accompanied by a microscopic final extension step at 72◦ C for 5 min. Positive controls and evaluation of the blood smear. The biochemical profile was negative controls (water) of the PCR were included in each run. assessed on serum samples using an automated analyzer All reactions were performed using an automated thermal cycler (Olympus AU2700, Olympus Diagnostic, Hamburg, Germany (Applied Biosystems 2720, Carlsbad, CA, USA). The amplicons and Atellica CH930, Siemens Healthineers, GmbH, Erlangen, were visualized by UV transillumination after electrophoresis Germany). Serum protein electrophoresis was performed using of 5 µL of the reaction solution in a 2.2% agarose FlashGel Capillarys 1 and Capillarys 2 (Sebia, Florence, Italy). DNA cassette (Lonza, Rockland, ME, USA). All PCR products Laboratory parameters evaluated included the following: containing expected size amplicons were submitted to an RBC, hemoglobin, Hct, mean cell volume, mean corpuscular external laboratory for direct sequencing on both strands (BMR hemoglobin concentration, white blood cell concentration, Genomics srl, Padua, Italy). The nucleotide sequences obtained neutrophils, lymphocytes, monocytes, eosinophils, platelet were compared with those available in the GenBank database count (PLT), mean platelet volume, creatine kinase, using the BLAST program (32) for species identification. aspartate aminotransferase, alanine aminotransferase, alkaline To perform phylogenetic analysis, Cytauxzoon spp. positive phosphatase, γ −glutamyltransferase, total bilirubin, total DNA extracts from −20◦ C stored still available samples were protein, albumin, globulin, cholesterol, triglycerides, blood urea subjected to amplification and sequencing of the nearly complete nitrogen, creatinine, glucose, calcium, magnesium, phosphorus, 18S rRNA gene as previously described (33). The contigs were sodium, potassium, total iron (Fe), serum amyloid A, albumin assembled and edited using BioEdit 7.2.5 software, and the (by serum protein electrophoresis), α-globulin, α1 -globulin, sequences obtained were aligned with a set of 34 relevant α2 -globulin, β-globulin, and γ -globulin. corresponding sequences of Cytauxzoon spp. longer than 1,000 bp retrieved from GenBank. Alignment was carried out using the MUSCLE algorithm (34) implemented in MEGA version 11 (35) and a maximum likelihood (ML) tree was inferred with the same Molecular analysis, sequencing, and software. Gaps or missing data in the alignment were deleted phylogenetic analysis and the substitution model with the lowest Bayesian information criterion (BIC), calculated using the specific function of MEGA The molecular investigation was performed by (1) 11, was selected. The reliability of the tree was evaluated by panpiroplasm end-point PCR, followed by sequencing of performing 1,000 bootstrap replicates. amplicons for species identification, carried out throughout the The nucleotide sequences obtained in this study were study period and used for the detection of Cytauxzoon spp. with submitted to the GenBank database under accession a diagnostic purpose, (2) a phylogenetic analysis evaluated on numbers OM004051-OM004057. the sequences of the nearly complete 18S rRNA of the stored samples still available at the time of the conceptualization of the study. DNA was obtained from 200 µL of blood from each Statistical analysis cat using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany). Statistical analysis was performed using R for MacOS Blood was incubated with 40 µL of proteinase K and (version 4.1.0). After rejecting the normality assumption for 200 µL of binding buffer at 72◦ C for 1 h. Subsequent quantitative variables with the Shapiro-Wilk test, quantitative steps were performed according to the manufacturer’s data were expressed as medians and interquartile ranges instructions. DNA was eluted in 50 µL of elution buffer. (IQR). Categorical variables were expressed as counts and Molecular detection of piroplasms was carried out using percentages in each category. The differences between the a conventional PCR assay using the sense primer PIROA infected Cytauxzoon spp. and the control groups were analyzed (5′ -AATACCCAATCCTGACACAGGG-3′ ) and the antisense in bivariate analysis with the Mann-Whitney rank sum primer PIROB (5′ -TTAAATACGAATGCCCCCAAC-3′ ) that for quantitative variables and with Pearson’s chi-square test amplify a fragment of ∼412 bp of the 18S rRNA gene (31). PCR with Yates continuity correction or Fisher’s exact test with reactions were carried out in a final volume of 25 µL containing categorical variables. Frontiers in Veterinary Science 04 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 Two sub datasets that included subclinical and sick cats, respectively, were created to better assess the role of the anemic status during infection. Categoric variable anemia was analyzed referring to the presence/absence of Cytauxzoon spp. in each sub dataset. The percentages of dichotomous variables such as sex, breed, type of cat, lifestyle, environment, ticks, fleas, clinical signs, and anemia that occur in the infected cat group were analyzed using the one sample proportion test considering a true proportion not equal to 0.5 as an alternative hypothesis. For each estimated proportion, the 95% confidence interval (95% CI) was calculated to provide a range of values that is likely to include the proportion of the real population of each variable. To identify the risk factors for Cytauxzoon spp. infection, a multivariable logistic regression model was estimated, after FIGURE 2 backward variables selection. The presence of anemia was Map of Italy showing the cities where Cytauxzoon spp. infected cats were diagnosed. The map was obtained by Microsoft 3D included in the model due to its clinical relevance during Maps in Excel 2016 software. AP, Ascoli Piceno; CH, Chieti; GR, infection. The results of the fitted logistic regression were Grosseto; MC, Macerata; SI, Siena; TS, Trieste; VE, Venezia; VT, Viterbo. expressed as odds ratio (OR), 95% CI and p-value. The statistical significance was declared for P < 0.05. 65.7–92.8%, P < 0.001) at the time of clinical examination Results (Figure 3). Anemia was present in 14/39 (35.9%) of infected cats and Case and control cats was not associated with infection compared to the control group (41/149, 21.6%, P = 0.089). When the sub dataset of cats During the evaluation period, piroplasm PCR analysis was without clinical signs was analyzed at the time of diagnosis performed in 646 feline blood samples (Figure 1), with a (n = 87), the percentage of anemia was likely higher in cats diagnosis of Cytauxzoon spp. infection in 47 cats (7.3%). infected with Cytauxzoon spp. compared to those not infected Four hundred and seventeen cats were excluded for various (7/22, 31.8% infected vs. 5/65, 7.7% not infected, P = 0.009). reasons. Finally, 229 cats met the inclusion criteria and, In the subgroup of cats that presented clinical signs at the according to the PCR results, were divided into case (infected, time of diagnosis (n = 134), anemia was not associated with n = 39) and control (non-infected, n = 190) groups (Figure 1). infection (7/17, 41.2% infected vs. 36/117, 30.8% not infected, Cytauxzoon spp. infected cats were detected in the northeast P = 0.401). The clinical signs observed by clinicians for (n = 32, 82.1%) and in the center (n = 7, 17.9%) of Italy cats infected with Cytauxzoon spp. were a non-specific (i.e., (Figure 2). neurological signs, slimming, diarrhea, stomatitis, dermatitis, The demographic information and clinical characteristics among others). of the case and control groups are summarized in Table 1. Laboratory findings are shown in Table 2. Compared The cats infected with Cytauxzoon spp. were predominantly to the control group, cats infected with Cytauxzoon spp. female (25/39, 64.1%) and mixed breed (37/39, 94.9%) with showed a higher platelet concentration [median values a median age (IQR) of 24 months (60). Compared to the (IQR): 357 × 103 /µL (227) vs. 288 × 103 /µL (172.5), P control group, infection was more likely to occur in stray = 0.013] and a lower total iron [median values (IQR): cats, living partially/total outdoors, in an urban context, taken 50.5 µg/dL (34) vs. 79 µg/dL (52.25), P = 0.007]. No or recently adopted from colonies, with irregular or absent other statistical differences were observed with respect to parasite preventive treatments, without fleas and without laboratory findings. clinical signs at the time of the medical evaluation. By the In the multivariable analysis Cytauxzoon spp. infection one sample proportional test the Cytauxzoon spp. infected was strongly associated with a partially/total outdoor lifestyle cat population was likely represented by high percentages of (OR: 8.58, 95% CI: 2.9–37, P < 0.001) and living in mixed breed cats (94.9%, 95% CI: 81.4–99.1%, P < 0.001) urban areas (OR: 11.9, 95% CI: 3.38–76.7, P = 0.001). The that live prevalently or totally outdoors (92.3%, 95% CI: 78– categorical variable anemia included in the logistic model 98%, P < 0.001) in urban areas (94.9%, 95% CI: 81.4– has shown the following OR: 2.22, 95% IC: 0.97–5.05, P = 99.1%, P < 0.001), with no presence of flea (80%, 95% 0.056. The AUC (area under the curve) of the model was CI: 62.5–90.9%, P < 0.001) and ticks (82.9%, 95% CI: 0.78 (95% IC: 0.71–0.85). Frontiers in Veterinary Science 05 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 TABLE 1 Characteristics of patients at baseline in the total study population and in the case and control groups. Variable Overall cats Cases Controls P-value Age, months, (n = 229), median (IQR) 60 (132) 24 (60) 73.50 (126) ND Sex (n = 223) Male (%) 87 (39) 14 (35.9) 73 (39.7) 0.796 Female (%) 136 (61) 25 (64.1) 111 (60.3) Breed (n = 229) Mixed breed (%) 205 (89.5) 37 (94.9) 168 (88.4) 0.387 Purebred (%) 24 (10.5) 2 (5.1) 22 (11.6) Type of cat (n = 229) Owned (%) 157 (68.6) 15 (38.5) 142 (74.7) <0.001 Stray (%) 72 (31.4) 24 (61.5) 48 (25.3) Lifestyle (n = 226) Indoor (%) 72 (31.9) 3 (7.7) 69 (36.9) <0.001 Partially/total outdoor (%) 154 (68.1) 36 (92.3) 118 (63.1) Living area (n = 229) North (%) 202 (88.2) 32 (82.1) 170 (89.5) 0.057 Center (%) 20 (8.7) 7 (17.9) 13 (6.8) South (%) 7 (3.1) 0 7 (3.7) Living in the TS focus area (n = 229) No (%) 139 (60.7) 8 (20.5) 131 (68.9) <0.001 Yes (%) 90 (39.3) 31 (79.5) 59 (31.1) Environment (n = 220) Rural (%) 57 (25.9) 2 (5.1) 55 (30.4) 0.002 City (%) 163 (74.1) 37 (94.9) 126 (69.6) Housing type (n = 164) Colony (%) 91 (55.5) 34 (97.1) 57 (44.2) <0.001 Cattery, family (%) 73 (44.5) 1 (2.9) 72 (55.8) Preventive treatment of parasites (n = 229) Regular (%) 38 (16.6) 0 38 (20) 0.005 Irregular/absent (%) 191 (83.4) 39 (100) 152 (80) Ticks presence (n = 216) Absent (%) 185 (85.6) 29 (82.9) 156 (86.2) 0.802 Present (%) 31 (14.4) 6 (17.1) 25 (13.8) Fleas presence (n = 218) Absent (%) 139 (63.8) 28 (80) 111 (60.7) 0.047 Present (%) 79 (36.2) 7 (20) 72 (39.3) Clinical signs (n = 221) Absent (%) 87 (39.4) 22 (56.4) 65 (35.7) 0.026 Present (%) 134 (60.6) 17 (43.6) 117 (64.3) Anemia (n = 229) Absent (%) 174 (76) 25 (64.1) 149 (78.4) 0.089 Present (%) 55 (24) 14 (35.9) 41 (21.6) The infected group with Cytauxzoon spp. (case) and not infected (control) were compared using the Pearson’s chi-square test with Yate’s continuity correction or Fisher’s exact test for categorical variables. The P-value < 0.05 was considered significant. IQR, Interquartile Range; TS, Trieste; ND, Not Determined. Frontiers in Veterinary Science 06 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 FIGURE 3 Estimated proportions for each dichotomous variable obtained by the one-sample proportional test from cats infected with Cytauxzoon spp. included in the study. The light blue bars represent the estimated proportions for each dichotomous variable obtained by the one-sample proportional test, considering a true proportion not equal to 0.5 as an alternative hypothesis. The orange lines (error bars) represent the width of the 95% IC calculated for each variable and provide interval estimations (and the uncertainty of the measurement) for the real population proportion of each variable. The P-value < 0.05 was considered significant and was indicated by *. CL, clinical. Sequencing and phylogenetic analysis description of the Trieste focus (14) and a recent paper mainly focused on the epidemiology and genetics of the parasite (24), The DNA amplicon sequences obtained for diagnostic most of the papers available in domestic cats are single case purposes based on pan-piroplasm end point PCR (31, 33) of cats reports (21, 22, 25) or descriptions of a small group of infected living in the Trieste focus (n = 13) and in other parts of Italy (n = subjects (16, 23, 28, 36). 8) showed a nucleotide identity >99% with reference sequences Although in the present study a wide range of hematological from Cytauxzoon spp. available in GenBank. and biochemical parameters were analyzed, few laboratory Sequencing of the nearly complete 18S rRNA gene was findings were statistically associated with Cytauxozoon spp. successful in seven cases. infection, with values not exceeding the reference interval. The ML phylogenetic tree (Figure 4) was estimated at 979 Infection appears more likely in cats without clinical signs sites, due to the complete deletion of gaps and missing data in the and with alterations in few or absent laboratory parameters at alignment; the Tamura 3-parameter (35) nucleotide substitution the time of diagnosis. Therefore, this study supports, from a model with discrete gamma distribution showed the lowest BIC laboratory perspective, the idea that Cytauxzoon spp. infection and was selected. in Italy is often subclinical (14, 28), as is occasionally the case for Phylogenetic analysis showed that the seven 18S rRNA infection with C. felis in the USA (10, 11, 37, 38). piroplasmid gene sequences obtained in this study grouped with At a significance level of 0.05, anemia does not appear other European Cytauxzoon spp. from domestic and wild felids to be associated with Cytauxzoon spp. However, it should be and with two sequences of C. manul detected in Pallas’ cats noted that the P-values obtained for this categorical variable from Mongolia, while C. felis sequences from North and South are borderline for significance by both the Pearson chi-square America grouped together in a clearly distinct cluster. test and the multivariable logistic regression. Furthermore, while for the most part non-anemic, subclinical infected cats showed a significantly higher percentage of anemia compared Discussion to those not infected. It could be reasonable that in apparently healthy infected cats, parasitized erythrocytes are removed from To the best of the authors’ knowledge, this is the first case- the bloodstream or hemolyzed, giving rise to a variable rate control study and, until now, the most substantial work on of reduction of RBC that is not necessarily so marked that demographic and clinical data on Cytauxzoon spp. infection in it evolves in an anemic state. Furthermore, considering the domestic cats carried out in Europe. In fact, apart from the first low concentration of iron associated with this infection, the Frontiers in Veterinary Science 07 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 TABLE 2 Laboratory findings of all cats and infected and non-infected Cytauxzoon spp. groups. Laboratory parameters (reference Overall cats Cases Controls P-value interval, unit) median (IQR) median (IQR) median (IQR) CBC (n = 229) (n = 39) (n = 190) RBC (6.35–9.5 × 106 /µL) 7.54 (2.19) 6.90 (3.05) 7.65 (2.01) 0.263 HB (9.6–14.3 g/dL) 10.90 (3.1) 10.80 (3.75) 10.90 (2.95) 0.298 HCT (28.0–42.5%) 34.60 (10.4) 33.90 (13.4) 34.60 (10.38) 0.150 MCV (38.0–49.5 fL) 45.20 (6.78) 45.30 (8.05) 45.15 (6.73) 0.941 MCHC (31.0–35.0 g/dL) 31.20 (2.6) 31.70 (5.4) 31.00 (2.48) 0.260 WBC (5.0–11.0 × 103 /µL) 11.47 (7.22) 12.82 (5.74) 10.96 (7.15) 0.097 Neutr (2,500–7,000/µL) 7,027 (6,247) 8,182 (5,557) 6,950 (6,299.25) 0.151 Lymph (1,300–5,500/µL) 2,520 (2,014) 2,556 (2,593) 2,407 (1,863.75) 0.390 Mon (65–250/µL) 306 (363) 389 (483.50) 293 (359) 0.085 Eos (70–800/µL) 540 (520) 610 (504) 521.50 (539.25) 0.094 PLT (130–430 × 103 /µL) 296 (193) 357 (227) 288 (172.5) 0.013 MPV (7.9–17.5 fL) 16.60 (5.20) 18.10 (4.80) 16.30 (4.95) 0.054 Biochemical profile n = 157 n = 13 n = 144 CPK (90–320 IU/L) 140.50 (136.75) 198 (148.25) 140 (129.25) 0.181 AST (15–35 IU/L) 31.50 (23.50) 24.50 (22.25) 32 (23.50) 0.502 ALT (32–87 IU/L) 57 (52.5) 48 (28.5) 58 (51.75) 0.220 ALP (19–70 IU/L) 24 (16–39) 22.50 (21.75) 24 (15–39) 0.926 GGT (0.1–0.6 IU/L) 0.50 (0.72) 0.50 (1) 0.50 (0.72) 0.563 TB (0.14–0.26 mg/dL) 0.21 (0.10) 0.21 (0.10) 0.21 (0.10) 0.272 TP (6.3–7.8 g/dL) 7.40 (1.20) 7.20 (0.70) 7.45 (1.30) 0.473 Albumin (3.0–4.0 g/dL) 3.0 (0.7) 3.0 (0.7) 3.0 (0.63) 0.846 Globulin (3.0–4.5 g/dL) 4.4 (1.2) 4.1 (1) 4.4 (1.2) 0.536 Cholesterol (95–210 mg/dL) 145 (65.25) 148.50 (93) 145 (64.75) 0.583 Triglycerides (19–81 mg/dL) 51 (46.5) 54 (59.5) 51 (45.25) 0.561 BUN (32–64 mg/dL) 59 (30) 53 (46) 59 (29.25) 0.367 Creatinine (0.95–1.85 mg/dL) 1.51 (0.71) 1.40 (0.6) 1.52 (0.72) 0.295 Glucose (86–116 mg/dL) 118 (56.5) 130.5 (44.25) 117 (58.5) 0.487 Ca (9.3–11.2 mg/dL) 9.5 (1) 9.6 (1.57) 9.5 (1) 0.590 P (3.5–6.6 mg/dL) 4.5 (1.8) 5.0 (2.47) 4.5 (1.82) 0.216 Na (145–152 mEq/L) 151 (5) 150 (3.48) 151.5 (4.25) 0.056 K (3.5–4.7 mEq/L) 4.10 (0.8) 4.45 (0.50) 4.00 (0.73) 0.013 Mg (0.81–1.05 mmol/L) 0.90 (0.15) 0.90 (0.18) 0.90 (0.15) 0.976 Total iron (50–118 µg/dL) 77.50 (57) 50.50 (34) 79 (52.25) 0.007 SAA (0.1–0.5 µg/mL) 0.40 (29.30) 0.70 (1.45) 0.40 (33.10) 0.812 Serum protein electrophoresis n = 154 n = 12 n = 142 Albumin (39.4–54.8%) 49.80 (12.04) 48.35 (6.67) 49.85 (12.65) 0.340 Alpha globulin (17.8–27.6%) 16.85 (6.34) 16.75 (4.28) 17 (6.6) 0.893 Alpha_1 globulin (0.8–1.6%) 1.80 (1) 1.85 (1.30) 1.80 (1) 0.721 Alpha_2 globulin (17.0–26.0%) 14.85 (5.62) 14.50 (3.43) 14.90 (6) 0.726 Beta globulin (6.4–9.4%) 11.40 (3.54) 11.30 (2.38) 11.40 (3.64) 0.981 Gamma globulin (16.2–28.2%) 20.15 (10.88) 23.20 (9.18) 20 (10.95) 0.467 Quantitative variables, not normally distributed by the Shapiro-Wilk test, were reported as median and interquartile range (IQR). Cytauxzoon spp. infected and non-infected groups were compared by the Mann-Whitney rank sum test. The P-value < 0.05 was considered significant. IQR, interquartile range; CBC, Cell Blood Count; RBC, Red Blood Cell count; HB, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cell count; Neutr, neutrophils; Lymph, lymphocytes; Mon, monocytes; Eos, eosinophils; PLT, platelet count; MPV, mean platelet volume; CK, creatine kinase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, γ −glutamyltransferase; TB, total bilirubin; TP, total protein; BUN, blood urea nitrogen; Ca, calcium; Mg, magnesium; P, phosphorus; Na, sodium; K, potassium; SAA, serum amyloid A. Frontiers in Veterinary Science 08 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 FIGURE 4 Phylogenetic relationship of Cytauxzoon spp. inferred from an alignment of 979 sites of the 18S rRNA gene. The maximum likelihood nucleotide tree inferred from an alignment of 979 sites of Cytauxzoon spp. 18S rRNA gene sequences using the Tamura 3-parameter substitution model with discrete gamma distribution. The GenBank accession number, species, host, and country of origin are included for each Cytauxzoon spp. sequence. The nucleotide sequences generated in this study are in bold and marked with a triangle. Plasmodium falciparum was included in the analysis as an out group. Bootstrap values <70% are indicated in the respective branches. Frontiers in Veterinary Science 09 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 mechanism of anemia of inflammatory disease (39) could be Thus, Cytauxzoon spp. could be introduced more easily in another explanation for our findings. It is important to note a geographic area where it was not endemic, for example, that anemia needs to be further investigated, by adding more during owner relocation (12) or after the adoption of an subjects to the sample and evaluating the presence/absence of infected subject from a colony or a cattery. More studies regeneration, to confirm and understand its real role and the are needed to determine the dynamics of the spillover of mechanisms involved during Cytauxzoon spp. infection in both Cytauxzoon spp. from felines in sylvatic ecosystems to domestic subclinical and sick cats. cats in urban locations, the role of domestic infected cats as Although many biochemical parameters were evaluated in reservoirs, and if there is another potential competent vector for the present study, even if just above the minimum value, this infection. only the lower iron concentration observed in the case group Cats living in an urban area appeared to be strongly seems to be clinically relevant compared to the control group. associated with Cytauxzoon spp. compared to other settings Hypoferremia could be caused by a shift of iron to the storage (rural, hilly, home). This result disagrees with previous papers site (e.g., inflammation disease) while other factors, such as the reporting that C. felis and Cytauxzoon spp. are more likely in young age of the animals or Fe deficiency (39) appeared to outdoor cats, especially in low-density residential or rural areas be less likely. It is important to note that the biochemical and where contact with ticks and wild felids might be more frequent serum electrophoresis parameters were obtained from a lower (16, 48). It is interesting to note that, in the present study, number of cats compared to the CBC parameters. Data from cats living in the colony, at the time of evaluation or recently more subjects might be needed to find mild differences between adopted from it, were more likely to be infected by Cytauxzoon infected and uninfected cats. spp. than cats living in the cattery or family. As most of the In the present study, living partially or completely outdoors colony cats were from the Trieste urban area focus (14, 28), was strongly associated with Cytauxzoon spp. infection. a bias referring to the result of the urban environment could Additionally, infection was more likely in stray cats. These be suspected. Consequently, it is likely that our observations results agree with previous works on both Cytauxzoon spp. might mainly reflect the characteristics of a particular habitat (14, 16, 28) and C. felis (4, 40). They suggest that this lifestyle as a colony rather than the context of the urban environment. is a risk factor for Cytauxzoon spp. infection. Outdoor cats In colonies, the interaction of cats with each other or with could be exposed to the sources of infection traditionally other animals (as wild felids) or a vector appears possibly more reported for C. felis as the tick vectors (6, 41) and wild felids like a “natural” condition. This could lead to some reflections as reservoirs (42). However, to our knowledge, the highly on the pathogenesis of Cytauxzoon spp. and could remark competent vectors Amblyomma Americanum (41, 43) and on the possible key role domestic cats play over wild cats as Dermacentor variabilis (44) that experimentally transmitted C. reservoirs of infection (9, 12, 14). Furthermore, based on feline felis are not found in Europe. Furthermore, Cytauxzoon spp. behavior lifestyle in environments such as colonies (49), other has not yet been isolated from ticks collected in European means of transmission, rather than traditional ones involving countries (36, 45). However, Cytauxzoon spp. was isolated from vectors and wild felids, could be hypothesized to contribute to European wild cats (Felis silvestris) in Italy (20), Romania the spread of Cytauxzoon spp. infection in domestic cats. We (19), Bosnia and Herzegovina (46), and France (24) as well suspected that the close co-existence between cats in colonies as in the Eurasian lynx (Lynx lynx) in Romania (19) and in is characterized by friendly and agonistic behaviors and could the Iberian lynx (Lynx pardinus) in Spain (17, 18). Domestic create a favorable environment for horizontal transmission of cats and wild cats or European lynx are considered to live in the parasite by blood. In fact, infection and parasitemia, without sympatry, inbreed, and share pathogens (20, 47). Consistent clinical signs, were experimentally obtained by inoculating with previous findings, the infected cats reported in the present blood infected with C. manul from Pallas’ cats (Otocolobus work lived interestingly in the north-east and central Italy manul) to cats (50). Furthermore, since blood transfusions could in areas almost overlapping those in which Cytauxzoon spp. represent a route of transmission, as a precaution, only cats was isolated from wild cats (20). These findings reinforce the negative for PCR were recommended as donors in endemic areas possible role of the wild felid as a reservoir for this infection (23, 51, 52). Vertical transmission is another possible way for in Italy. Cytauxoon spp. to spread in a colony setting where, despite However, the frequent subclinical Cytauxzoon spp. infection spay/neuter control programs, mating continues to occur. described in the present work is consistent with previous studies However, vertical transmission has not been fully confirmed (6, 9, 12, 14, 40) and contributes to assigning to apparently even if young cats infected with Cytauxzoon spp. (23) and C. healthy infected cats the role of “long-life domestic reservoir” felis (53) were reported. Future studies are needed to better for this parasite. This role appears to be more important than understand the main ways of transmission of Cytauxzoon spp. the one played by wild felids, due to the greater possibility in cats, especially when they live in a socially complex society as of contact between infected and non-infected domestic cats. a colony. Frontiers in Veterinary Science 10 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 A limitation of the present work is that we have not Ethics statement performed a more in-depth molecular study than the routine PCR analysis and sequencing used to make the diagnosis. Ethical review and approval and written informed consent A molecular analysis of the 18S rRNA gene was completely from the owners of the animals was not required for the evaluated only in the most recent samples, and it was not study on animals in accordance with the local legislation and possible to evaluate other genes such as CytB and COI, institutional requirements. although it would be interesting to know if the C. europeus, C. otrantorum, and C. banethi subspecies (27) were present in Italy in domestic cats. Different genotypes could vary Author contributions in virulence and geographical prevalence and could cause diseases with different pathogenicity and clinical course and EC: study design, sample collection, data analysis and different responses to treatment (6, 12, 24, 54). Future interpretation, statistical analysis, and writing manuscript. LS-G: studies are necessary to deepen our understanding of the study design, data analysis and interpretation, and revised genotypes present in Italy, their biological behavior, and their manuscript. SD: molecular biology and phylogenetic analysis role in the evolution of the disease. In addition, results of and interpretation. LV: statistical analysis. EL: molecular biology biochemical and serum electrophoresis were obtained a smaller sample processing. TF: data analysis and interpretation and number of cats than CBC. Even if our observations appear revised manuscript. All authors contributed to the article and plausible and not anecdotal, data from more subjects must be approved the submitted version. considered to find mild differences between infected and not infected cats. Dedication This paper is dedicated to the memory of Dr. Marco Caldin Conclusions (1961–2022), a mentor and a great teacher to all of us. This study revealed that Cytauxzoon spp. commonly cause subclinical infections in domestic cats in Italy. In fact, no Acknowledgments difference in clinical signs or laboratory findings was found in infected cats vs. non infected cats. Interestingly, although most The authors acknowledge the staff and the molecular biology of the cats were not anemic, the subclinical infected cats showed workgroup of the San Marco Laboratory for technical support, a significantly higher percentage of anemia compared to those and special mention goes to Dr. Michele Trotta. The authors are not infected. also grateful to Dr. Stefania Uccheddu for the feline behavioral The infection was more likely to occur in stray cats living medicine input and to all clinicians who kindly responded to the partially/completely outdoors, taken or recently adopted, from questionnaire. colonies, with irregular or absent parasite preventive treatments, without fleas and without clinical signs at the time of the medical evaluation. Conflict of interest The authors declare that the research was conducted in the Data availability statement absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author. The accession number(s) can be found Publisher’s note below: https://www.ncbi.nlm.nih.gov/genbank/, OM004051; https://www.ncbi.nlm.nih.gov/genbank/, OM004052; https:// All claims expressed in this article are solely those of the www.ncbi.nlm.nih.gov/genbank/, OM004053; https://www. authors and do not necessarily represent those of their affiliated ncbi.nlm.nih.gov/genbank/, OM004054; https://www.ncbi. organizations, or those of the publisher, the editors and the nlm.nih.gov/genbank/, OM004055; https://www.ncbi.nlm. reviewers. Any product that may be evaluated in this article, or nih.gov/genbank/, OM004056; https://www.ncbi.nlm.nih.gov/ claim that may be made by its manufacturer, is not guaranteed genbank/, OM004057. or endorsed by the publisher. Frontiers in Veterinary Science 11 frontiersin.org Carli et al. 10.3389/fvets.2022.976173 References 1. Birkenheuer AJ, Le JA, Valenzisi AM, Tucker MD, Levy MG, Breitschwerdt EB. 22. 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