Introduction

Amongst vector-borne zoonoses, visceral leishmaniasis (VL) by Leishmania infantum is a major global disease potentially fatal to humans. VL is one of the most important threats among the neglected tropical diseases causing an estimated 300,000 new cases and about 20,000 deaths in humans each year [1]. Its distribution is associated with the occurrence of the phlebotomine sand fly vectors of the genus Phlebotomus spp. and Lutzomyia spp., in the Old and New World, respectively. Developing countries take the brunt of VL considering that malnutrition and low hygienic conditions represent risk factors for the spreading of the infection in human patients [2]. Dogs are the main reservoir hosts of the parasite [3] with usually more than 30% seropositive animals in endemic areas [4]. Other animal species, such as cats and some wild animals (e.g., foxes and hares) have been implicated in the epidemiology of the infection [5,6], with hares involved as reservoir hosts in the outbreak of VL in south-western Madrid, Spain [7]. Where canine leishmaniasis (CanL) is endemic, cats are often exposed to L. infantum, with seroprevalence ranging from 0.7% to 30% according to animal life style and diagnostic technique used [8]. In a study conducted in southern Italy (i.e., the Aeolian Islands, Sicily), a cumulative L. infantum serological and molecular prevalence of 25.8% in cats was reported [9]. Differences in the feline immune function, such as an effective Th1 immunity which often allows spontaneous resolution of lesions, may play a role for the reduced clinical signs in infected cats [10] resulting in subclinical forms with only few reports of overt illness, mainly characterised by skin lesions and lymphadenomegaly [8]. Though cats are exposed to sand fly bites [11], the overall prevalence of L. infantum infection is generally lower than in dogs living in the same areas [8,9,12,13]. Consequently, feline leishmaniasis (FeL) has been for long time disregarded by veterinary practitioners and parasitologists and, as a result, the current distribution of this disease may be underestimated. Examples of neglected zoonotic VL are well embodied by the recent first report of L. infantum in dogs and a cat from Bosnia and Herzegovina, where human leishmaniasis is known to occur in people visiting the country and in the local population [14]. Further, the subclinical presentation of FeL makes the diagnosis of the infection a complex task [8]. Concomitant infections with viral agents such as feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline coronavirus (FCoV) and the protozoon Toxoplasma gondii have been diagnosed with FeL [13,15,16]. Since the first description of FeL in a domestic cat (Felis silvestris catus) [17], the number of reports of clinical cases and prevalence of L. infantum infection in cats have steadily increased in endemic areas, such as in the Mediterranean basin [12,13,18–20], the Middle East [21] and Brazil [22]. However, these data have been gained using different diagnostic methodologies since there is currently no consensus about the method of choice for diagnosing FeL. Serological [i.e., immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA)], and molecular (e.g., real time-PCR, qPCR) tests are primarily employed for the diagnosis of FeL either for clinical and research purposes [9,23] though few investigations have used multiple tests in combination [9,15,24]. CanL is endemic in Italy with up to 40% of L. infantum infected dogs in the highly endemic regions of south-central Italy [25] where the infection in cats has also been recorded [9,23,26]. However, the lack of information about the distribution of L. infantum subclinical infections in cats is an hindrance to a clear understanding of the role of cats in the epidemiology of zoonotic VL in endemic areas. In this study we assessed the prevalence of L. infantum infection in a large number of cats across Italy by serological and molecular tests and identified potential risk factors for FeL.

Materials and methods

Results

The number of sampled cats (i.e., 2,659) enrolled from northern (n = 1,543), central (n = 471) and southern (n = 645) Italy exceeded the minimum sample size (i.e., 457, 385 and 246 samples from northern, central and southern Italy, respectively). Owned cats enrolled in the study (i.e., n = 1,302 males and n = 1,357 females) aged from 1 month to 21.3 years (mean 8.3 years, median 9 years). Of these, 359 (13.5%) were less than 18 months old (G1), 599 (22.5%) between 18 months and 6 years old (G2) and 1,701 (64%) elder than 6 years (G3). Most of the animals were common European breed (n = 2,329, 87.6%) and neutered (n = 2,275, 85.5%) (Table 1). The prevalence of L. infantum infection by serology and/or molecular test in association with age, sex, breed, neutering status and cat origin is reported in Table 1. Overall, 104 (3.9%) cats were positive to L. infantum by IFAT (88/2,659, 3.3%) and/or by qPCR (22/2,659, 0.8%) with statistically significant (McNemar test p<0.0001) difference and poor concordance between the techniques, K = 0.097 (95% CI: 0.0176–0.1766). Out of these 104 positive cats, 6 (5.8%) were positive to both qPCR and IFAT with IgG titres of 1:80 (n = 3), 1:640 (n = 2) and 1:5120 (n = 1). Out of the 22 PCR positive cats, 16 (72.7%) were seronegative. Of the seropositive cats, 84.1% (74/88) had an antibody titre of 1:80, whereas in the remaining the titres varied from 1:160 to 1:5120 (Table 1). The prevalence of positive cats detected by serological and/or molecular tests in the different geographic areas was 10.5% (68/645) in the South, 2.3% (11/471) in the Centre and 1.6% (25/1543) in the North of Italy.

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Table 1. Association between variables: Age, sex, breed, reproductive status and cat origin and the serological and molecular positivity for Leishmania infantum.

https://doi.org/10.1371/journal.pntd.0007594.t001

The results of the univariate and multivariate logistic regression analyses are shown in Table 2. The risk of L. infantum infection in cats, in the multivariate model (i.e., accounting for sex and age class) was significantly associated with the geographical areas (p<0.0001). In particular, cats living in the South of Italy were related to higher risk of L. infantum infection than those in the North by the univariate (OR = 7.14; 95% CI: 4.48–11.49) and multivariate analysis (OR = 2.66, 95% CI: 1.59–4.44), and those in the Centre by the univariate analysis (OR = 4.93; 95% CI: 2.58–9.43). Animals positive to L. infantum (n = 104; 3.9%) aged from 6 months to 20.1 years old (mean 7.5 years, standard deviation 5 years), while negative cats aged from 1 month to 21.3 years (mean 8.4 years, standard deviation 5.4 years) old with no statistically significant difference between the two groups (t = 1.795, p = 0.0728). For cats in G2 (i.e., 19 months to 6 years old) a higher L. infantum prevalence (5.7%, 34/599) (χ² = 6.806; p = 0.0333) was recorded. The multivariate regression analysis showed that these G2 cats were related to a higher risk of L. infantum infection than those in G1 (i.e., ≤18 months) (OR = 3.69; 95% CI: 1.65–8.27). The neutering status was a statistically significant factor associated with positivity to L. infantum. In particular, the risk of not neutered compared to neutered cats was OR = 1.76 (95% CI: 1.06–2.93) and statistically significant (p = 0.028). No statistical association was found between Leishmania infected cats and sex in neither the univariate (p = 0.016), nor the multivariate model (p = 0.3015). Equally no association was observed for the breed variable (univariate analysis: p = 0.1413; multivariate analysis: p = 0.9947). Overall, 115 and 101 cats were positive to FeLV (4.3%) or FIV (3.8%) respectively, of which 6.1% (7/115) for FeLV and 12.9% (13/101) for FIV were also positive to Leishmania. Out of the 13 cats coinfected with FeLV and FIV, three (23.1%) were also Leishmania positive. Leishmania infantum infection was significantly associated only to FIV infection resulting in the multivariable model with an OR = 2.65 (95% CI: 1.34–5.22, p = 0.0051).

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Table 2. Odds ratio and 95% confidence interval from logistic regression analysis for cats positive to Leishmania infantum by IFATand/or qPCR.

https://doi.org/10.1371/journal.pntd.0007594.t002

At the GIS analysis, the highest number of cats (i.e., >100 cats) was sampled in 8 out of 83 provinces examined in the study area (i.e., n = 4 in the North, n = 1 in the Centre and n = 3 in the South of Italy). The geographical analysis identified the presence of three areas with a significant higher proportion of L. infantum seropositive cats, located in three provinces of southern Italy (i.e., 13.5% in Bari, 11.4% in Messina and 6.4% in Lecce; Chi-square test for linear trend = 23.4224, df  = 1, p = <0.0001) (Fig 1). In two of these three provinces (i.e., Messina and Lecce) a higher proportion of qPCR positive samples (3.8% and 1.6%, respectively) was also recorded (Fig 1).

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Fig 1. Geographical localization of areas, indicated by provinces, from where cats positive to Leishmania infantum by serological and molecular tests were collected.

The location of L. infantum positive cats was geo-referenced using a geographical information system (GIS, ArcGIS version 10.3 ESRI).

https://doi.org/10.1371/journal.pntd.0007594.g001

Discussion

The present study represents the largest epidemiological survey combining serological and molecular testing to assess the occurrence of L. infantum in cats to date.

The prevalence of FeL recorded by serology (3.3%) and qPCR (0.8%) indicates that cats are exposed to, or infected by, L. infantum throughout Italy. The overall seroprevalence of 3.3% is similar to that recorded in cats from central Spain (1.3–3.2%) and Portugal (2.8%) [12,31]. Higher seroprevalence data have been reported in cats from highly endemic regions of the Mediterranean basin for CanL, such as in southern Spain (28.3%) [19], central (16.3%) [26] and southern Italy (25.8%) [9] and Turkey (15.2%) [24]. It means that in areas where CanL is endemic, cats are likely to be more exposed to L. infantum as compared to areas where the prevalence of CanL is low [9]. Nonetheless, in our study different proportions of the population of infected animals were recorded in northern (1.6%) and centre (2.3%) versus southern (10.5%) Italy. Although L. infantum has spread throughout Italy [32], the southern and insular regions are still considered more endemic areas for human VL and CanL [25, 33], as a result of the favourable geographical climate conditions that allow the presence and abundance of sand fly vectors in the whole country (e.g., Phlebotomus perniciosus, Phlebotomus perfiliewi, Phlebotomus ariasi, and Phlebotomus neglectus) [25]. Accordingly, the occurrence of L. infantum in wild animals has been reported mainly in the centre and south of Italy [34–36].

In addition, current data available in the literature on the seroprevalence of FeL have been gained using different diagnostic methodologies and cut-off values, representing a limiting factor for data comparison. For example, for the IFAT, the percentage of seropositive animals was higher at a cut-off of 1:40 (i.e., 25.8% and 28.3%) [9,19], than that at 1:64 (i.e., 0.7%) [37], 1:100 (3.2%) [31] and 1:80 (3.3%; present study). Though, an IFAT cut-off (1:80) has been recommended by LeishVet group [8], more comprehensive and standardized protocols and ideally a defined gold standard procedure would allow a more consistent diagnosis of L. infantum infection in cats and contribute to a better understanding of the role of cats in the epidemiology of zoonotic VL.

The higher seroprevalence recorded in cats from southern (9.6%) versus central (1.3%) and northern (1.3%) regions of Italy suggests that cats are more exposed to L. infantum in the southern regions, as reported by previous serologic investigations conducted in Liguria and Tuscany (i.e., 0.9%) [18] and in Sicily and Calabria (southern Italy) (i.e., 6.9%) [38]. This is further supported by the GIS analysis that indicates a significant higher proportion of L. infantum positive cats in three areas of southern Italy. Likewise, though the distribution pattern of L. infantum infection is changing throughout the country as a result of many biological and ecological factors [32], CanL mainly occurs in southern Italy [25].

Since no information on the cats’ life style (i.e., outdoor/indoor access) and on sand fly population during the sample collection are available, the role of age and neutering status as risk factors for the occurrence of infections in cats, may be explained by differences in behaviour. Indeed, not neutered and older than 18 months cats are more prone to an outdoor life style than younger and neutered cats, due to more pronounced predatory instinct [39]. Therefore, these cats are more exposed to sand fly bites, which over time, results in a higher risk of L. infantum infection. Undoubtedly, the prevalence of cats positive for FeL at the molecular tests also depends on the cat life style and therefore exposure to sand fly bites with the higher number of animals positive to DNA recorded in feral and stray cats from Spain (i.e., 8.7% and 26%) [5,15] and Portugal (30.4%) [40].

Leishmania infantum DNA has been detected in blood samples of only a few cats (0.8%) as already reported in Portugal (0.3%) [41], northern (1.1%) [42] and southern Italy (2.1%) [9] and Cyprus (2.3%) [20]. This may suggest that blood is not the ideal tissue for molecular diagnosis of L. infantum infection in cats, as for dogs. Indeed, when comparing different matrixes for the molecular diagnosis of FeL, conjunctival swab (16.7%) and lymph node aspiration (11.7%) showed to be more sensitive than blood (7.8%) [37], as also reported for the diagnosis of CanL [27,43]. Though the number of samples positive with molecular diagnosis (n = 22) was limited in this study, the molecular positivity for L. infantum parallels the occurrence of higher seroprevalence in the examined animals, as reported previously [5,15]. The high percentage of molecular positivity and thus occurrence of L. infantum DNA in seronegative animals (72.7%, 16/22) might either indicate that these cats were at an early stage of the infection or endorse that they are less susceptible compared to dogs [10]. While there is a correlation between molecular and serological positivity in dogs infected for CanL [9,13], there is none between molecular and serological tests for FeL, supporting the different immune responsiveness of dogs and cats to L. infantum.

Longitudinal studies in cats could contribute in determining the course of L. infantum infection after natural exposure over time. Further, transmission studies with competent sand fly vectors could provide more information about the role of cats as reservoir hosts of L. infantum. So far, P. perniciosus has shown to feed on cats [44] and allow the developing of the parasite after the blood ingestion [11] thus potentially enabling further transmission of L. infantum. Since P. perniciosus is the main vector of L. infantum in Italy [45] and the most abundant phlebotomine sand fly species in some areas of southern Italy [46], further observations are needed to elucidate the role of naturally infected cats in sustaining the L. infantum life cycle.

The significant association between L. infantum and FIV infections (p = 0.0051) was previously reported [16,47] and indicates that immunosuppressive agents, such as FIV, impair the cellular immune response, thus increasing the risk for FeL. This correlation is supported by literature showing a high prevalence of FIV infection (i.e., 30%) in L. infantum infected cats [8]. Therefore, FIV and L. infantum coinfections might predispose animals to visceral forms, as an effect of the viral immunosuppression, as recognized in HIV seropositive patients [48].

In conclusion, differences in immune responses between dogs and cats and scant data on the ability of vectors in the transmission of L. infantum on natural infected cats, complicate the appreciation of the role of cats in L. infantum epidemiology. As future perspectives, the standardization of procedures for a prompt diagnosis of L. infantum infection and for screening cat populations is a crucial task for a better understanding of the epidemiology of FeL, and the role of cats as reservoir hosts. In addition, prevention measures for providing protection against the infection and treatment strategies for cats infected by L. infantum need to be further addressed.

Acknowledgments

The authors would like to thank Anna Attanasi (Centro Veterinario Salentino, San Cesario di Lecce, Lecce, Italy), Chiara Cavallaro (Laboratorio Analisi Veterinarie PET Diagnostic LAB, Catania, Italy), Carmela Coccioli (Azienda Sanitaria Locale, Bari, Italy), Raffaele Maglione (Laboratorio Analisi Veterinarie LABFORVET, Napoli, Italy) and Romana Romano (Laboratorio Analisi Veterinarie Rtr Vet, Lecce, Italy) for their support with the sample collection.