Prevalence and risk factors associated with cat parasites in Italy: a multicenter study

Parasites that infect cats include protozoa, helminths and arthropods, many of which are transmissible to humans. Effective control relies on a good knowledge of parasite distribution and the risk factors for infection. The present study was aimed at evaluating the prevalence of major feline parasites in Italy and the risk factors associated with their occurrence. Over a 12-month study period, feces, hair and ectoparasites from naturally infected cats from feral colonies, shelters and private households were analyzed at 13 study centers across Italy. Samples from these cats (n = 987) were analyzed at all centers using the same diagnostic methods. Prevalence values and risk factors were evaluated statistically for the identification of predictors of risk. The overall prevalence of gastro-intestinal and broncho-pulmonary (BP) nematodes was 35.9% (354/987). Toxocara cati was the most prevalent species (253/987; 25.6%), followed by Ancylostomatidae (98/987; 9.9%). Among BP nematodes, Aelurostrongylus abstrusus was the most common (76/987; 7.7%). Approximately 35.7% (352/987) of the study population was infested by ectoparasites, of which the most common were fleas (29.4%, 290/987), followed by ear mites Otodectes cynotis (9.8%, 97/987). Predictors of risk for parasite infection included age, a predominantly or exclusively outdoor lifestyle, geographic area and lack of antiparasitic treatment. Both ecto- and endoparasites are still common in cats throughout Italy, many of them being of zoonotic concern and vectors of pathogens to humans. Given the presence of parasites throughout the entire study period, year-round treatment should be considered. Furthermore, data confirm the need to protect the human–animal bond using proper endo- and ectoparasiticides to reduce the risk of human infection, in application of the One-Health concept.

other animals and humans. Recent multicenter studies in both Europe and Italy have been carried out to define the current status of endo-and ectoparasite infections of cats [1][2][3][4]. The results of these studies indicate that infections are widespread and depend on various risk factors, including lifestyle, geographical area and frequency of antiparasitic treatment. Most studies on prevalence, distribution and risk factors for feline parasites in Italy have been carried out in the central and southern areas of the country [5,6].
Multicenter studies provide useful information on the distribution and risk of parasite infection. However, few apply the same, standard diagnostic protocols at each center, thus compromising comparability of the results. Indeed, it has been reported that different copromicroscopic techniques have differing sensitivity, specificity and accuracy for the diagnosis of GI and broncho-pulmonary (BP) nematodes.
Seasonality of parasite infection in cats has been evaluated mostly in retrospective, longitudinal studies [12,13]. It is possible that sampling and analysis over a fixed period of time may provide more useful information on the current effects of season on parasite prevalence.
The aims of the present multicenter study were therefore to: (i) determine the current prevalence of feline endo-and ectoparasites throughout Italy through the recruitment of cats from all regions; (ii) evaluate seasonal trends by recruiting a set number of cats each month consecutively over a 12-month period; (iii) use standardized diagnostic methods in order to eliminate variables associated with differences in test sensitivities/specificities/accuracy; (iv) identify those factors that significantly increase the risk of infection.

Animals and study period
The study period was from July 2019 to September 2020. Thirteen university study centers participated, and each center had a recruitment target of seven cats per month for 12 months, for a total of 84 cats per center. Each cat could only be included in the study once, with no more than two cats from the same home/shelter/colony sampled. Randomization was based on the target number of seven cats: once the target number was reached, enrollment ceased even if there were other cats that met the inclusion criteria.

Enrollment and sample collection
Inclusion criteria included: outdoor access; no antiparasitic treatment (endo and/or ecto) in the 3 months prior to enrollment; and signed informed consent. Exclusion criteria included: no outdoor access; and antiparasitic treatment (endo and/or ecto) in the 3 months prior to enrollment.
Each study center was supplied with tubes (15 ml) prefilled with 80% ethanol; flea combs and zip-lock bags; one Mini-FLOTAC kit Fill-FLOTAC [14] containing four Mini-FLOTAC and four Fill-FLOTAC devices (200 tests); and instructions for material collection, conservation and analyses.
At enrollment, general information, inclusion criteria, clinical observations, frequency of antiparasitic treatments in the previous 12 months and eventual signs of ectoparasitic infestation were recorded, and an online data collection sheet was filled in (Additional file 1: Text S1). Any observed ticks, lice and nits were collected (mites were collected by scrapings or ear wax collection while nits, lice and ticks were simply removed with tweezers) and stored in the provided 15-ml tubes containing 80% ethanol. Each cat was combed with a flea comb for 5 min, and collected material was stored in the zip-lock plastic bag at 4 °C. Owners were asked to submit at least 7 g of fresh feces, which were collected, examined for the eventual presence of proglottids and stored at 4 °C.

Laboratory analyses
All collected material was analyzed at the university laboratory of each participating center.
Material collected with flea combs was examined under a stereomicroscope and the presence of flea/flea debris recorded. Feces were examined for the presence of proglottids and identified according to Soulsby et al. [15].
Mini-FLOTAC copromicroscopic examination was carried out on 2 g of feces in 18 ml of NaCl floating solution (specific gravity: 1.200) according to the protocol described in Cringoli et al. [7]. Minimum/maximum number of eggs/oocysts/cysts per gram of feces (EPG/ OPG/CPG) were calculated. The Baermann test was carried out on 5 g of feces and the feces examined approximately 12 h later, according to Bowman et al. [16]. Larvae were identified according to Varcasia et al. [17] and Brianti et al. [18].
Study center reference personnel were asked to register with the Castor ® EDC ® database [19], which was managed by the study monitor. Each reference personnel had her/his own login credentials. Results were transcribed into the database, preferably within several days of receipt and analyses.
Relationships between infection and the variables ("predictors") were analyzed by multivariable regression analysis applying a stepwise backward elimination method (IBM SPSS Statistics for Macintosh, version 27.0; IBM Corp., Armonk, NY, USA.). Statistical significance was set at α = 0.05.

Study population
Severe restrictions on movement/activity were put into place to contain the SARS-CoV-2 pandemic (March 2020 "lockdown"), which resulted in a decrease in/lack of cat enrollment at many participating centers from March 2020 to May 2020, leading to an extension of the study for a further 2 months. A total of 987 cats were enrolled.
Cats were evenly distributed in terms of sex. Approximately half the study population (47.3%) was within the age range of 1-5 years, with the remaining cats evenly distributed between < 1 year (28.1%) and > 5 years of age (24.6%). Over 40% of enrolled cats were from southern Italy and 69.2% were privately owned cats. Over 75.8% of the study population lived predominantly outdoor/exclusively outdoor (Table 1).
Most cats, including privately owned animals, had not received antiparasitic treatment in the year before enrollment. Overall, 539 of the 987 (54.6%) cats in the study were infected with one or more parasites. Of these 987 infected animals, 13.7% (n = 135) had at least two endoparasites and 7.4% (73) had at least two arthropod infestations.
Monthly prevalence throughout the study was variable (Fig. 2), but there was no month in which parasites were not observed.
Minimum/maximum EPG/OPG/CPG results for helminths and protozoa were as follows: T. cati (from 5 up  Table 2).
Monthly prevalence throughout the study was variable (Fig. 4). Only 23.8% of the cats had received at least one treatment against endoparasites in the past year, while only 33.6% of the cats had received treatment against ectoparasites in the past year.

Risk factors for endo-ectoparasites infestation
Significant risk factors as determined from univariate analysis were entered in the multivariable logistic regression model in order to address possible confounding factors and to compute adjusted odds ratios (OR). Tables 3 and 4 report the results of the multivariate analysis, which considered overall prevalence of endoparasite infection, overall prevalence of ectoparasite infestation and prevalence of the most common endoparasites (T. cati, Ancylostomatidae and A. abstrusus) and ectoparasites (fleas and O. cynotis) as dependent variables and sex, age, provenance, lifestyle, geographical area and anti-parasitic treatment as predictors.

Discussion
The study provides a overview of the endo-and ectoparasites affecting Italian cats during a 15-month study period. Despite monthly recruitment being brusquely interrupted due to the SARS-CoV-2 pandemic, preventing several centers from reaching the established number of cats per month, nearly 1000 cats were analyzed. Importantly, laboratory analyses were carried out according to standardized protocols which were followed by all centers, thus reducing the risk of variability associated with different test sensitivities/specificities. It should be noted, however, that while using one method does improve comparability, having different people use the method can influence results.
The present study also applied univariate and multivariate analyses to evaluate overall risk for endoparasites or ectoparasites and for the most common helminths and arthropods observed.
Our findings show that feline endo-and ectoparasites are widespread in Italy, with varying prevalence across the different regions. Approximately 55.0% (539/987) of cats enrolled in our study were infected with at least one internal or external parasite. This compares well with the results of a study at the European level [1] which reported that more than half (50.7%) of the cats studied were infected with one or more endo-and/or ectoparasite.
Among the GI helminths, T. cati was the most frequently found (25.6% of enrolled cats). This prevalence is higher than that reported by a previous Italian multicenter study [4] (21.6%), and is also higher than the mean European prevalence value reported in 2017 by Giannelli et al. [2] (14.5%). The prevalence is, however, in line with that of other surveys conducted in Italy [5,6]. In the present study, risk for T. cati infection included age (< 5 years of age), an exclusively outdoor lifestyle and living in southern Italy. Pre-and perinatal transmission and age immunity are well known for Toxocara spp., while outdoor access favors exposure to both highly resistant eggs contaminating the environment and to paratenic hosts [20]. The warmer climatic conditions in southern Italy and Italian islands likely increase the presence and persistence of infective stages of the parasite [21].
Ancylostomatidae represented the second most frequent group of nematodes diagnosed in the study population (9.9%). In a study performed at the European level [2], the mean prevalence of hookworms was reported to be 4.5%, while in some selected areas of Italy the prevalence was reported to be 4.9% [4]. In the present study, Ancylostomatidae infection was associated with gender (females were at increased risk), predominantly outdoor access, living in southern Italy and lack of anthelmintic treatment. Age was not a significant risk predictor. Indeed, it is assumed that there is no transmammary transmission of Ancylostomatidae from the queen to her kittens [22].
As mentioned above, warmer climatic conditions in southern Italy may increase the presence and persistence of infective stages of Ancylostomatidae. For transmission to occur, Ancylostomatidae need to develop into infective larvae in the soil from eggs passed in the host's stool, and higher temperature and humidity (tropical and subtropical climates) provide an adequate environment for this growth stage [23].
The feline lungworm A. abstrusus was present in 7.7% of the cats analyzed in our study; in comparison, Giannelli et al. [2] reported a mean prevalence in Europe of 8.2%, while recent data from Italy [4] reported a prevalence of 10.3%. Multivariate analysis indicated that an exclusively outdoor lifestyle, living in southern Italy and lack of anthelmintic treatment were significant risk factors for A. abstrusus. Outdoor access has been reported previously as an important risk factor for A. abstrusus [24]. While the geographical distribution of feline lungworms tends to be patchy but stable in endemic hotspots [2], interpretation of geographical location as a risk factor should be done with caution, and any geographic location reporting autochthonous circulation of the parasite should be considered to be potentially endemic. Traversa et al. [4] reported a 20.0% prevalence of A. abstrusus in Piedmont, while in the present study only 1.2% of cats were infected. Giannelli et al. [2] reported that 16.7% of cats from the province of Bari were infected with A. abstrusus, compared to a prevalence of 2.4% in our study.
Values from the provinces of Sassari (SS) and Messina (ME), on the other hand, were higher in the present study (20.9 and 22.4%, respectively) ( Table 2), compared to data reported by Giannelli et al. [2] (11.6 and 15.3%, respectively), but lower than the prevalence of 25.2% reported by Tamponi et al. [25] in a previous study in Sardinia.
Interestingly, univariate analysis showed that cats aged > 5 years had a significantly higher prevalence for A. abstrusus, but age was not confirmed as a risk factor following multivariate evaluation. As expected, larvae of T. brevior were found mostly in cats from southern regions. Nevertheless, it is worth mentioning that, in the present study, this potentially fatal metastrongyloid was also found in two cats in northern Italy, indicating an apparent northward geographical expansion.
The Mini-FLOTAC technique [7] has been recently demonstrated to be a highly sensitive method for diagnosing parasitic infections of human and veterinary importance where larvae or ova of parasites are shed in the feces [8][9][10][11]. Moreover, the mini-FLOTAC technique in combination with Fill-FLOTAC has been shown to be user-friendly and safe, with a wide diagnostic range. These features are particularly useful for monitoring and control programs in which large numbers of fecal samples need to be processed rapidly and safely. In the present study, the harmonized use of the Mini-FLOTAC technique allowed the qualitative and quantitative analysis of parasite load without the need for specialized equipment.
Fleas were the most common ectoparasite found in the present study (29.4%). Cooper et al. [26] reported similar prevalence values in a recent nationwide study in the UK. Beugnet et al. [1] reported prevalence values ranging from 3.6% in Bari to 31.4% in Naples. Multivariate analysis showed that infestation was associated with young age (< 1 year), living in a colony, predominantly/exclusively outdoor lifestyle, living in central and southern Italy and lack of ectoparasitic treatment. Cooper et al. [27] reported geographical differences (prevalence declining from south to north in UK) and no treatment as significant predictors of risk for flea infestation. Beugnet et al. [1] identified outdoor access as the only risk factor in a multivariate analysis.
The ear mite O. cynotis was present in 9.8% of enrolled cats. Beugnet et al. [1] reported prevalence values of 40.3% in cats from Bari and 21.8% in cats from the area of Naples. In Tuscany (central Italy), O. cynotis was identified in 66.1% cats with otitis externa [27]. Age < 1 year, living in a colony, having a predominantly outdoor lifestyle and coming from central Italy were all factors identified as increasing the risk of ear mite infestation in cats. Otodectes cynotis is transmitted by direct contact and is highly contagious. Young cats are more playful and likely have more direct contact with other cats.
The main limitation of the present study is the potential effect of bias, based on inclusion criteria. While on the one hand cats that had received antiparasitic treatment in the 3 months preceding enrollment were excluded, the evaluation of the effect of treatment versus no treatment was carried out considering the frequency of treatment for the 12 months prior to enrollment (see Additional file 1: Text S1). Beugnet et al. had a similar study design [1], including cats that had received no treatment in the 2 months prior to enrollment and analyzing the frequency of antiparasitic treatment as a risk factor for infection. Outdoor access was another enrollment bias. Indeed, this has already been identified as a risk factor for parasite infection in cats [1][2][3]. In the present study, however, access was categorized as infrequent, frequent or exclusive. The analysis of risk was based on the frequency of access, not on outdoor versus indoor.

Conclusions
The results of this study highlight the fact that both ectoand endoparasites are still common in cats throughout Italy. Interestingly, of the 239 cats with a predominantly indoor lifestyle, 31.8% were affected by endo-and ectoparasites, suggesting that parasiticide treatment is more important than lifestyle. Therefore, taking the zoonotic implications and the clinical importance into account, it is strongly advisable to promote effective and regular parasite control in cats, with adequate frequencies of treatment for both internal and external parasites.
It is interesting to note that there was no month in which endo-and ectoparasites could not be found, suggesting that cats can be infected throughout the year. This would imply that parasite infection should not be considered seasonal, but that control should be year-round. However, only 23.8% of the cats had received at least one treatment against endoparasites in the past year, while only 33.6% of the cats had received treatment against ectoparasites in the past year. The European Council for the Control of Companion Animal Parasites (ESC-CAP) recommends "year-round, life-long" parasite control. The aim of the present study was not to associate climate and environmental conditions to risk of parasite infection, but rather to support these recommendations. Furthermore, many privately owned cats spend a significant amount of time outdoors where they are exposed to parasites. Practitioners need to inform their clients of the risks and recommend routine antiparasitic treatment. Interestingly, southern Italy continues to show higher prevalence of parasite infection in cats. This may be due to climatic, social or economic factors, and practitioners working in these areas should be particularly attentive. Finally, zoonotic parasites and vectors of human disease are still widespread in cats, confirming the need to protect the human-animal bond and the application of the One-Health concept.