Priolo et al. Parasites & Vectors (2022) 15:107 https://doi.org/10.1186/s13071-022-05230-w Parasites & Vectors RESEARCH Open Access Association between feline immunodeficiency virus and Leishmania infantum infections in cats: a retrospective matched case‑control study Vito Priolo1, Marisa Masucci1, Giulia Donato1, Laia Solano‑Gallego2, Pamela Martínez‑Orellana2, Maria Flaminia Persichetti1, Ana Raya‑Bermúdez3, Fabrizio Vitale4 and Maria Grazia Pennisi1* Abstract Background: Feline leishmaniosis caused by Leishmania infantum is often associated with feline immunodeficiency virus (FIV) infection; however, the role and clinical significance of this coinfection remain unknown. This study aimed to assess whether FIV is associated with L. infantum infection in cats from canine leishmaniosis endemic areas and to report the clinical signs and hematological alterations associated with coinfection. Methods: A retrospective matched case-control study (ratio 1:2) was conducted. Data of clinical examination and complete blood count (CBC) were selected from a cohort of 705 cats examined for epidemiological studies on feline leishmaniosis conducted between 2012 and 2019. Ninety-one FIV seropositive cases and 182 FIV seronegative control cats were selected. Matching was done according to age, sex, lifestyle and geographic provenience of case cats. Rapid ELISA devices were mainly used to detect anti-FIV antibodies. Anti-Leishmania IgG antibodies were detected by indirect-immunofluorescence test (IFAT). Leishmania DNA was searched in blood, oral and conjunctival swabs by quantitative real-time PCR. Results: Feline immunodeficiency virus seropositive cats had no hematological abnormalities suggestive of an advanced stage of FIV infection and were statistically more frequently IFAT positive, and their risk of being L. infantum antibody positive was 2.8 greater than in the FIV seronegatives. The association of FIV seropositivity with L. infantum antibody positivity was confirmed in the univariable model of logistic regression. A multivariate model found FIV infection and L. infantum PCR positivity as predictors of a positive L. infantum IFAT result. Male outdoor cats from rural or suburban areas were at risk for FIV and L. infantum antibody positivity. Clinical signs more frequently associated with the coinfection were oral lesions, pale mucous membranes and low body condition score (BCS). Conclusions: This study documents that FIV seropositive cats with no hematological abnormalities suggestive of an advanced stage of FIV infection are more prone to be L. infantum seroreactive by IFAT in endemic areas. Therefore, FIV seropositive cats should be tested for L. infantum antibodies and treated for preventing sand fly bites. Pale mucous membranes, low BCS and oral lesions but no CBC abnormalities were significantly associated with the coinfection. Keywords: Feline immunodeficiency virus, Leishmaniosis, Feline retrovirus, Coinfection, Risk factors, Logistic models, Polymerase chain reaction, Indirect fluorescent antibody technique *Correspondence: mariagrazia.pennisi@unime.it 1 Dipartimento di Scienze Veterinarie, Università di Messina, Messina, Italy Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Priolo et al. Parasites & Vectors (2022) 15:107 Page 2 of 10 Background and hematological abnormalities associated with this Leishmaniosis is a vector-borne disease caused by pro- coinfection. tozoa of the Leishmania genus transmitted by sand fly bites. Leishmania infantum is the most widespread spe- Methods cies and is of zoonotic concern, with dogs considered Study design, cat characteristics and selection the main domestic reservoir in endemic areas. How- A retrospective matched case–control study was carried ever, other domestic and wild animals are reported to be out. Feline immunodeficiency virus seropositive (cases) infectious to sand flies [1]. In recent years, an increasing and FIV seronegative (controls) cats were selected from number of case reports of feline leishmaniosis (FeL) and our research database if they were evaluated by physical subclinical infections caused by L. infantum were docu- examination and tested for FIV as well as for L. infan- mented in endemic areas of the Mediterranean basin [2], tum by IFAT and PCR in blood, conjunctival and oral and FeL is considered an emerging feline disease [3]. swabs with the same methodology. A population of 705 Many studies have confirmed that feline L. infantum cats studied between 2012 and 2019 was examined. The infection is not negligible in areas where canine leish- assumptions were: alpha < 5%, power ≥ 80%, two con- maniosis is endemic [2]. However, different levels of trols per case, L. infantum positivity 17.3% (prevalence of endemicity and type of population under study or differ- the population where cases and controls were extracted ences in diagnostic methodologies may be responsible from) and expected odds ratio (OR) of 2.5. The target for the high variability in antibody or molecular preva- sample size was 92 cases and 184 controls. Cases and lences reported in published studies [2]. Investigations controls were matched according to age (> 6 months and have been performed on L. infantum prevalence in cats exposed to sand flies for at least one transmission sea- in Southern Italy for a long time, reporting an antibody son), sex (male/female), lifestyle (indoor/outdoor) and prevalence between 6.9 and 59% [4–10] and a molecu- geographical provenience (Sicily, Calabria, Catalonia, lar prevalence between 1.3 and 61% [5, 7–9, 11, 12]. In Andalusia) (Table 1). Cats were enrolled after their own- Spain, the antibody prevalence reported ranges between ers provided informed consent and in compliance with 3.2 and 4.8% in the Madrid area [13–16], between 2.2 the requirements of ethics committees from the authors’ and 16% in the northeast [17–19] and 28.3% in the south academic institutions. The “Strengthening the Reporting of the country [20]. The molecular prevalence reported of Observational Studies in Epidemiology” (STROBE) ranges between 0 and 0.43% in the Madrid area [13–16], recommendations were followed to describe the study between 3 and 26% in the northeast [6, 17–19, 21] and methods [33]. 25.7% in the south of the country [20]. Feline immunodeficiency virus (FIV) is a retrovirus dis- Diagnosis of feline retroviral infections tributed in feline populations worldwide and associated Different diagnostic tests were used in the period of time with adult, male and free-roaming cats because the main considered for the retrospective selection of matched transmission path is via biting [22]. Prevalence rates of cases and controls (2012–2019). In particular, anti-FIV FIV positivity are therefore influenced by the character- antibodies were investigated with commercial kits (SNAP istics of populations under study, and surveys reporting Combo Plus FeLV antigen and FIV antibody test, Idexx FIV prevalence in the same area of South Italy investi- Laboratories, Westbrook, ME, USA; Pet Check FIV anti- gated in the present study found a wide range of positiv- body test kit, IDEXX Laboratories, Westbrook, ME, USA; ity, between 7.6 and 37% [6, 8, 23]. FIV prevalence studies INgezim FIV, Ingenasa, Madrid, Spain). The FeLV posi- available from various regions of Spain reported ranges tivity was assessed by a rapid ELISA test detecting p27 between 5.1 and 20.9% [13–15, 17, 19, 24, 25]. antigenemia (SNAP Combo Plus FeLV antigen and FIV or by blood real-time PCR (U3 region LTR-genesig® Among risk factors for feline L. infantum infection, co- antibody test, Idexx Laboratories, Westbrook, ME, USA) infection with feline immunodeficiency virus is the most investigated. Many studies found a significant associa- Advanced kit, Rownhams, UK). All tests were performed tion between FIV and L. infantum positivity in cats [5, 9, according to the manufacturer’s protocol. 10, 12, 19, 26–28]. However, other studies did not docu- ment this association [8, 14, 17, 20, 29–32]. Therefore, Leishmania infantum the association between FIV and L. infantum infection in IFAT cats remains unclear. The aims of the present study were Antigen slides were produced by C.Re.Na.L (Centro to assess whether FIV and L. infantum infections are di Referenza Nazionale per la Leishmaniosi, Palermo, associated in cats living in canine leishmaniosis endemic Italy) using L. infantum strain MHOM/IT/80/IPT1. A areas of South Italy (Calabria and Sicily) and Spain (Cat- fluoresceinated anti-cat immunoglobulin G (IgG) anti- alonia and Andalusia) and to investigate clinical signs body [working anti-feline IgG (H + L)-FITC, Fuller Priolo et al. Parasites & Vectors (2022) 15:107 Page 3 of 10 Table 1 Signalment and history data of 91 feline immunodeficiency virus (FIV)-positive cases and 182 FIV-negative controls, with description of number (N) and percentage (%) of cats recorded for each variable Variable Cases Controls Total N (%) N (%) N (%) Sex Male 61 (67) 122 (67) 183 (67) Female 30 (33) 60 (33) 90 (33) Reproductive status Neutered 44 (48.3) 79 (43.4) 123 (45.1) Not neutered 47 (51.7) 103 (56.6) 150 (54.9) Hair Short 59 (64.8) 126 (69.3) 185 (67.8) Medium 18 (19.8) 21 (11.5) 39 (14.3) Long 3 (3.3) 13 (7.1) 16 (5.8) Missing 11 (12.1) 22 (12.1) 33 (12.1) Age ≤ 24 months 26 (28.6) 52 (28.6) 78 (28.6) > 24 months 65 (71.4) 130 (71.4) 195 (71.4) Geographic area Italy 80 (87.9) 160 (87.9) 240 (87.9)   Calabria 36 (39.6) 70 (38.5) 106 (38.8)   Sicily 44 (48.3) 90 (49.4) 134 (49.1) Spain 11 (12.1) 22 (12.1) 33 (12.1)   Catalonia 7 (7.7) 14 (7.7) 21 (7.7)   Andalusia 4 (4.4) 8 (4.4) 12 (4.4) Lifestyle and household Indoor 19 (20.9) 41 (22.5) 60 (21.9) Outdor 72 (79.1) 141 (77.5) 213 (78.1) Owned 19 (20.9) 45 (24.7) 64 (23.4) Stray 32 (35.2) 57 (31.3) 89 (32.6) Missing 40 (43.9) 80 (44) 120 (44) Single-cat 59 (64.8) 108 (59.3) 167 (61.2) Multi-cat 18 (19.8) 60 (33) 78 (28.6) Missing 14 (15.4) 14 (7.7) 28 (10.2) Environment Rural and suburban 32 (35.2) 67 (36.8) 99 (36.3) Urban 53 (58.2) 98 (53.8) 151 (55.3) Missing 6 (6.6) 17 (9.3) 23 (8.4) Laboratories, Fullerton, CA, USA] was used according healthy non-infected cat was always used as a negative to Persichetti et al. (2016), and the endpoint titer of posi- control. The quantitative real-time polymerase chain tive samples was determined preparing serial two-fold reaction (RT-PCR) was carried out in a CFX96 Real-time dilutions of serum starting from 1:20. The cutoff value System (Bio-Rad Laboratories s.r.l., Hercules, CA, USA) for positivity was set at 1:80 [34, 35]. Fluorescence micro- using TaqMan Master Mix (Applied Biosystems by Ther- scope readings were done by a unique operator (MM). moFisher, Waltham, MA, USA) and performed as previ- ously described [36]. PCR The EDTA blood and swab DNA was extracted using Complete blood count the PureLink Genomic DNA kit (Invitrogen, Life Tech- Complete blood count (CBC) was performed using nologies Waltham, MA, USA). Blood from a clinically a laser hematology analyzer (IDEXX ProCyte Dx Priolo et al. Parasites & Vectors (2022) 15:107 Page 4 of 10 Hematology Analyzer, IDEXX laboratories, Westbrook, according to the other variables studied. We used a uni- ME, USA). Reference intervals of statistically analyzed variate logistic regression model adjusted for all variables CBC parameters are listed in Additional file 1: Table S1. studied, and only the statistically significant ones were Blood smears were stained by May Grünwald-Giemsa reported (P ≤ 0.05). Multivariable models were computed staining and examined for morphological abnormalities using statistically significant univariate models (P ≤ 0.05) and platelet concentration estimate. considering two different categories: (1) risk factors; (2) clinical signs and CBC changes. Overall, 13 sets of mul- Variables tivariable logistic regression were constructed. Statistical Data extracted from the database and included in the analyses of L. infantum tests were carried out separately statistical analysis were: (1) FIV and L. infantum diagnos- for IFAT, PCR and positivity to both tests. P-value was tic test results: anti-FIV antibodies (positive/negative); considered significant for values ≤ 0.05. IFAT anti-L. infantum antibodies (titer ≥ 1:80/≤ 1:40); L. infantum PCR assay (positive/negative PCR test from blood and/or swabs); L. infantum IFAT and PCR tests Results (positive/negative at both tests); (2) potential risk fac- Description and analysis of clinical characteristics of cases tors for FIV and L. infantum positivity from signalment and controls and history data set: sex (male/female), age (months), A total of 273 cats were selected with a 1:2 case/control age group [two different cutoff values for age group set- ratio. The 273 sera were tested for anti-FIV antibodies, ting were analyzed as they influence the exposure of and 264 of them were also tested for FeLV and found cats to a different number of transmission seasons: negative. In particular, 144 sera were tested with SNAP 24 months (≤ 24 months/> 24 months) and 36 months Combo Plus FeLV antigen and FIV antibody rapid test (≤ 36 months/> 36 months)], hair (shorthaired, medium- (Idexx Laboratories, Westbrook, ME, USA). One hun- longhaired, longhaired), lifestyle and origin (indoor/ dred twenty cats were tested for anti-FIV antibodies by outdoor; single-cat/multi-cat household; stray/owned), Pet Check FIV antibody test kit (IDEXX Laboratories. region LTR-genesig® Advanced kit, Rownhams, UK). provenience (Sicily, Calabria, Catalonia, Andalusia), envi- Westbrook, ME, USA) and by blood PCR for FeLV (U3 ronment (rural and suburban/ urban); (3) clinical abnor- malities more frequently detected in the 273 cats (8–37% Nine sera were tested for anti-FIV antibodies by ELISA of them): poor body condition score (BCS) (< 3.5/5: (INgezim FIV, Ingenasa, Madrid, Spain). underweight, ≥ 3.5/5 normal and overweight), cutaneous The 182 controls were matched to 91 cases, and the (presence/absence of at least one of the following: ulcers, differences between the two groups according to the papules, nodules, crusts, scales and alopecia), oral (pres- matched variables were not statistically significant (Fish- ence/absence of at least one of the following: gingivitis, er’s exact test). Signalment and history data of both case stomatitis) and ocular (presence/absence of at least one and control cats are reported in Table 1. The age of cats of the following: blepharitis, conjunctivitis, keratitis, uve- was the unique normally distributed variable (range itis and panophthalmitis) lesions, enlarged lymph nodes 7–204 months; mean 66.1 months ± 48), and the differ- (presence/absence), pale mucous membranes (pale/ nor- ence of mean ages (cases: 67.2 months ± 47.4; controls: mal); (4) CBC abnormalities: anemia (and anemia sever- 64.9 months ± 48.1) of the two groups was not statisti- ity: low, moderate or severe), neutrophilia, neutropenia, cally significant (t-test). The two different cutoff values monocytosis, lymphocytosis, lymphopenia, eosinophilia, used for age group settings did not yield different results, eosinopenia, basophilia, thrombocytopenia. and we reported results obtained with age group cut- off set at 24 months. No statistically significant differ- Statistical analysis ences were found in the other clinical characteristics of Statistical analyses were performed using Stata 16 (Stata- the two groups of cats examined in addition to the ones Corp LP, College Station, TX). used to match cases and controls (Fisher’s exact test and Data set was evaluated for normal distribution by chi-square test). The clinical features considered in the skewness and kurtosis test. T-test was used in case of selected cats are listed in Table 2. The proportion of cats normal distribution and Mann-Whitney U test when with enlarged lymph nodes and skin lesions was higher data were not normally distributed to make compari- among FIV-seropositive cats than the FIV seronega- sons. According to FIV and Leishmania status, differ- tives [Fisher’s exact test: P = 0.025 (OR = 1.765; 95% CI ences between groups were evaluated, and the IFAT titers 1.05–2.944) and P = 0.023 (OR = 1.978; 95% CI 1.081– between cases and controls were also compared. Odds 3.562), respectively] (Table 2). Additionally, the ratios ratios (OR) and 95% confidence intervals (CI) of Leish- of neutrophilia and monocytosis were higher among mania and FIV positivity were calculated and stratified cases than controls [Fisher’s exact test, respectively: Priolo et al. Parasites & Vectors (2022) 15:107 Page 5 of 10 Table 2 Clinical findings considered in the 91 feline immunodeficiency virus (FIV)-positive cases and 182 FIV-negative controls, with description of number (N) and percentage (%) of cats recorded for each variable Variable Cases Controls Total N (%) N (%) N (%) Body condition score Normal or overweight 60 (65.9) 113 (62.1) 173 (79) Underweight 20 (22) 26 (14.3) 46 (21) Total 80 – 139 – 219 – Missing 11 (12.1) 43 (23.6) 54 (19.7) Mucous membranes Normal 80 (87.9) 166 (86.5) 246 (91.8) Pale 9 (9.9) 13 (7.1) 22 (8.2) Total 89 – 179 – 268 – Missing 2 (2.2) 3 (1.6) 5 1.8 Lymph ­nodesa Enlarged 37 (40.7) 52 (28.6) 89 (33) Normal 52 (57.1) 129 (70.9) 181 (67) Total 89 – 181 – 270 – Missing 2 (2.2) 1 (0.5) 3 (1.09) Ocular lesions Presence 13 (14.3) 24 (13.1) 37 (13.6) Absence 78 (85.7) 157 (86.3) 235 (86.4) Total 91 – 171 – 272 – Missing 0 – 1 (0.5) 1 (0.5) Skin ­lesionsa Presence 27 (29.7) 32 (17.6) 59 (21.6) Absence 64 (70.3) 150 (82.4) 214 (78.4) Total 91 – 182 – 273 – Oral lesions Presence 40 (44) 46 (25.3) 86 (36.7) Absence 50 (55) 98 (53.8) 148 (63.2) Total 90 – 144 – 234 – Missing 1 (1) 38 (20.1) 39 (14.3) a Statistically significant Fisher’s exact test P = 0.002 (OR = 2.513; 95% CI 1.337–4.513) and P < 0.001 Medians of IFAT titers of positive cases and controls (OR = 3.661; 95% CI 1.851–7.108), respectively] (Table 3). were not statistically different (Mann-Whitney U-test). The results of univariable and multivariable analyses of significant risk factors and clinical abnormalities are Leishmania infantum and FIV positivity association listed in Table 6. In the univariable models, L. infantum Leishmania infantum IFAT and PCR results are dis- antibody positivity was significantly associated with FIV played in Table 4. Cases were statistically more frequently antibody positivity, but this association was not found IFAT positive than controls [Fisher’s exact test: P = 0.001 with PCR positivity (logistic regression: P = 0.002). A (OR = 2.765; 95% CI 1.482–5.249)], but the difference was unique multivariable model yielding significant associa- not significant when considering the PCR test results or tions was obtained, and it was with L. infantum antibody both tests (Fisher’s exact test). Feline immunodeficiency positivity, anti-FIV antibody positivity and L. infantum virus infection was associated only with an increased risk PCR (logistic regression: P < 0.0001). Logistic regression of L. infantum antibody positivity detected by IFAT, and models did not find significant associations of risk factors the ORs of strata for selected covariates in FIV and IFAT and clinical abnormalities considered for L. infantum and L. infantum-positive cats are summarized in Table 5. FIV-positive cats. Priolo et al. Parasites & Vectors (2022) 15:107 Page 6 of 10 Table 3 Statistically significant hematological changes in 91 feline immunodeficiency virus (FIV)-positive cases compared to 182 FIV- negative controls, with description of number (N) and percentage (%) of cats recorded for each variable Variable Cases Controls Total N (%) N (%) N (%) Neutrophiliaa Presence 28 (30.8) 32 (17.6) 60 (22) Absence 47 (51.6) 135 (74.2) 182 (66.7) Missing 16 (17.6) 15 (8.2) 31 (11.3) Monocytosisa Presence 23 (25.3) 18 (9.9) 41 (15) Absence 52 (57.1) 149 (81.9) 201 (73.6) Missing 16 (17.6) 15 (8.2) 31 (11.4) a Statistically significant Fisher’s exact test Table 4 Positivity to L. infantum tests of 91 feline Table 5 Odds ratios (OR), 95% confidence intervals (CI) and immunodeficiency virus (FIV)-positive cases and 182 FIV-negative P-values for feline immunodeficiency virus (FIV) and IFAT L. controls, with description of number (N) and percentage (%) of infantum positivity (IFAT/FIV) according to the selected covariates cats recorded for each variable OR CI P Variable Cases Controls IFAT\FIV 2.765 1.399–5.459 0.001 N % N % Covariables: risk factors IFATa Sex Positive 26 (28.6) 23 (12.7)   Female 1.978 0.536–7.052 0.001 Negative 65 (71.4) 159 (87.3)   Male 3.226 1.398–7.471 0.001 PCR Lifestyle and environment Positive 7 (7.7) 10 (5.5)   Indoor 2.466 0.397–14.899 0.001 Negative 84 (92.3) 172 (94.5)   Outdoor 2.825 1.325–6.020 0.001 IFAT and PCR   Urban area 2.580 1.026–6.488 0.001 Positive 6 (6.6) 3 (1.7)   Suburban/rural area 3.352 1.026–11.04 0.001 Negative 85 (93.4) 179 (98.3) Covariables: clinical signs a Oral lesions Statistically significant Fisher’s exact test   Absent 1.736 0.583–5.005 0.014   Present 3.039 1.056–9.026 0.014 Discussion Body condition score Clinical characteristics of cases and controls   Underweight 6.273 1.193–41.415 0.019 Feline immunodeficiency virus is a RNA virus belong-   Normal or overweight 1.606 0.674–3.755 0.019 ing to the family Retroviridae, subfamily Lentiviridae, Mucous membranes a group of viruses known to cause life-long infections   Normal 2.175 1.012–4.684 0.004 with protracted incubation periods [22]. Immuno-   Pale 15 1.030–766.4 0.004 suppression is determined in FIV-positive cats by a progressive decline in CD4+ T cells number, reduc- tion in the CD4+/CD8+ ratio, generalized lymphoid showed enlarged lymph nodes and skin lesions. Periph- depletion, reduced ability to respond to antigenic eral lymphadenomegaly is reported in both early and stimulation and dysregulation of cytokine produc- more advanced stages of FIV infection and is directly tion [22]. Immunosuppression contributes to second- caused by the virus as well as by secondary infec- ary and opportunistic infections but FIV-positive cats tions, immune-mediated and neoplastic conditions remain clinically healthy for years, depending on the observed in FIV-positive individuals [22]. Miscellane- infecting isolate [22]. The FIV seropositive case cats ous skin diseases are described in FIV-positive cats, of this study differed from controls regarding clini- and they are mainly caused by secondary and oppor- cal abnormalities observed in the 273 studied cats. tunistic infections that may have a more severe and In particular, FIV seropositive cats more frequently prolonged course compared to FIV negative cats [37]. Priolo et al. Parasites & Vectors (2022) 15:107 Page 7 of 10 Table 6 Univariate and multivariate analyses of significant risk factors and clinical abnormalities according to L. infantum positivity Variable N LR ­chi2 Coef SE P CI Model P L.infantum IFAT Univariate models FIV positivity 273 9.96 1.017 0.322 0.002 0.386–1.648 0.002 L. infantum PCR positivity 273 14.19 2.101 0.554 0.000 1.014–3.187 0.000 Age group 273 4.77 0.481 0.223 0.031 0.0436–0.919 0.029 Oral lesions 234 9.32 1.020 0.336 0.002 0.361–1.679 0.002 Anemia 241 4.72 0.780 0.352 0.027 0.0909–1.469 0.030 Monocytosis 242 4.32 0.824 0.385 0.032 0.0703–1.578 0.038 L.infantum IFAT Multivariate model FIV positivity 273 23.01 0.993 0.334 0.003 0.338–1.646 0.000 L. infantum PCR positivity 273 2.064 0.560 0.000 0.046–3.181 L. infantum PCR Univariate models L.infantum IFAT positivity 273 14.19 2.101 0.554 0.000 1.014–3.187 0.000 Age group 273 6.05 0.947 0.405 0.020 0.152–1.742 0.014 BCS 219 7.92 -1.295 0.481 0.007 -2.239- (-0. 351) 0.005 Monocytosis 242 7.74 1.607 0.550 0.004 0.525–2.681 0.005 Eosinophilia 242 4.74 1.801 0.730 0.014 0.371–3.231 0.029 2 LR ­chi , likelihood ratio chi-square test; Coef, logistic regression coefficient; SE, standard error; P, P-value; CI, 95% confidence interval; Model P, P-value model Additionally, cutaneous neoplasms (particularly car- Leishmania infantum and FIV positivity association cinomas) are reported with high rates in FIV-positive We found that FIV antibody positivity was associated cats [37]. In the present study, FIV seropositive cats had with IFAT antibody positivity to L. infantum in cats from hematological abnormalities related only to increased areas endemic for both infections. In particular, FIV sero- neutrophil and monocyte concentration. In agreement positive cats had a 2.8 times higher risk to be L. infantum with the present results, neutrophilia [38] and mono- antibody positive. Similarly to a previous study, we did cytosis [38, 39] were the most common hematological not find differences in the antibody titer among the two abnormalities seen at the time of the first diagnosis in groups [6]. Additionally, this association was confirmed cats with naturally occurring FIV infection in previ- by the logistic regression analysis performed in the uni- ous studies [39, 40]. During the asymptomatic phase of variable model and in the significant multivariable model FIV infection, the number of granulocyte/macrophage constructed. Previous studies evaluating risk factors for and erythroid progenitors is unchanged [40], and cats L. infantum positivity, such as FIV seropositivity, were have a normal hematological response to concurrent prevalence studies that analyzed cross-sectional data [5, diseases [38]. Conversely, in advanced stages of FIV 9, 10, 12, 19, 26–28, 30, 42]. Large cross-sectional studies infection cytopenia (anemia, leukopenia, neutropenia, are often based on routinely collected samples and his- and lymphopenia) is more common [40]. In this study, tory, and clinical findings data can be incomplete and do the stage of FIV infection was not assessed with immu- not provide information on some confounding factors nological markers, and this is a limitation shared with [5]. We designed a specific case-control study to test the all field studies that have so far investigated the asso- hypothesis that FIV seropositivity was associated with L. ciation between FIV and L. infantum infections. Based infantum positivity in endemic areas, and we were able to on hematological findings, we assume that many of match cases and controls for confounding factors such as these cats were not in terminal stages of the disease. age, sex, lifestyle and geographic area. The study was ret- FIV-induced immunosuppression could have facilitated rospective, but data were obtained from a selected popu- a secondary or opportunistic infection, to which an lation of 705 cats homogeneous for systematic recording appropriate inflammatory response (neutrophilic and of clinical data and L. infantum and CBC assays. Unfor- monocytic) was made [38]. However, it is important tunately, methods used for testing retroviral infections to highlight that neutrophilia is also compatible with were inhomogeneous, and this is a limitation of the study. stress leukogram, immune-mediated disorders, neopla- However, all the different tests we used have high sensi- sia and tissue necrosis and that monocytosis may also tivity and specificity and are widely used in relevant stud- occur in many of these conditions [41]. ies about feline pathogens [43–45]. Priolo et al. Parasites & Vectors (2022) 15:107 Page 8 of 10 As well as cross-sectional studies, case-control studies antibody titers in dogs are associated with the progres- are not able to prove which of the associated variables sion of infection to disease, but a longitudinal clinical, has a causative role. This question is answered by longitu- serological and parasitological evaluation of cats is dinal field investigations which unfortunately are not eas- needed to correctly analyze these data [46]. However, ily performed in veterinary medicine. age can also influence host susceptibility to diseases in We did not find associations of FIV seropositivity with case of kittens or senior age [49, 50], but we did not set L. infantum PCR positivity. We performed PCR assays age groups with this aim. Iatta et al. [5] compared cats on three different tissues preferring non-invasive sam- aged between 19 and 72 months (adults) with younger ples (swabs) and residual EDTA blood, and the number and older cats. Curiously, these adult cats had the high- of PCR-positive cats was low when the sample size was est risk for L. infantum positivity compared to the other calculated according to IFAT positivity. Consequently, age groups [5]. results concerning PCR should be interpreted with cau- Akhtardanesh and others (2020) evidenced by a multi- tion, and a larger sample size should be assessed. How- variate logistic regression analysis that L. infantum infec- ever, in the multivariate model, PCR positivity was also tion was more frequent in adult (particularly cats > 3 years a FIV seropositivity predictor for L. infantum antibody old) and FIV seropositive cats [28]. Differently, when we positivity. Since 1998, a significant association between stratified results of IFAT L. infantum and FIV seropositive FIV infection and anti-L. infantum antibodies was cats according to selected covariates (Table 5), we found reported [10], and subsequently many other studies have higher odds in males compared to females, in outdoor confirmed this association with L. infantum antibody compared to indoor cats and in cats from rural and sub- [12, 19, 26, 27], PCR [28, 42], antibody and/or PCR [5, 9] urban areas compared to those from urban areas. Interest- positivity. However, sample size and FIV prevalence were ingly, males were always the sex category more frequently variable in these studies as well as diagnostic techniques found positive in other studies detecting a significant sex used to evaluate both FIV and L. infantum positivity, and difference in L. infantum positivity of cats [13, 26, 51]. this is a limitation to compare results from different stud- Concerning lifestyle and housing, other studies found a ies or make a meta-analysis. The role of FIV coinfection significantly higher L. infantum positivity in cats from in L. infantum-positive cats could therefore be better multi-cat compared to single-cat households [48], from investigated by evaluating markers useful to assay the cat rural compared to urban areas [51] and from colonies immunocompetence. In L. infantum-infected dogs, sus- compared to catteries [47]. ceptibility to the development of clinical disease is due Concerning clinical findings (Table 5), the higher odds to reduced cellular immune response and high antibody were for pale mucous membranes, low BCS and oral lesions. level [46]. Adaptive humoral and cell-mediated immune These latter findings are reported in feline leishmaniosis, response is elicited by L. infantum feline infection, but no and oral lesions are among the most frequent clinical signs difference was found in FIV seropositive cats in a study observed in clinical cases [3]. Unfortunately, the clinico- evaluating the ex vivo blood production of L. infantum- pathological data available from all 273 selected cats included specific IFN-γ [6]. only CBC, and no other clinicopathological abnormalities The selected cohort of 273 cats included only cats could be analyzed in the matched cats. This is a limitation of > 6 months in order to examine cats exposed to sand the study, and we are able to provide information only about flies for at least one transmission season, and we ana- CBC abnormalities that were not significantly associated lyzed two different cutoff values for setting age groups with positivity to both pathogens. to compare cats exposed to two or three transmission seasons with cats exposed to four or more sand fly sea- sons. In fact, age was often found to be a risk factor Conclusions for cat L. infantum positivity in previous studies. In This case-control study documents that FIV seropositive particular, cats > 12 [13, 47], 24 [7] or 36 months [9, cats with no hematological abnormalities suggestive of an 28, 48] were more frequently found infected. However, advanced stage of FIV infection are more prone to be L. we did not find significant differences in L. infantum infantum seroreactive by IFAT in endemic areas. There- positivity among age groups of case and control cats, fore, FIV seropositive cats should be tested for L. infan- similarly to other studies comparing L. infantum posi- tum antibodies and treated to prevent sand fly bites. Pale tivity in cats of various age groups [26, 27] and where mucous membranes, low BCS and oral lesions but no age analysis did not include age grouping [30]. Higher CBC abnormalities were significantly associated with the coinfection. Priolo et al. Parasites & Vectors (2022) 15:107 Page 9 of 10 Supplementary Information References 1. Cardoso L, Schallig H, Persichetti MF, Pennisi MG. New epidemiological The online version contains supplementary material available at https://​doi.​ aspects of animal leishmaniosis in Europe: the role of vertebrate hosts org/​10.​1186/​s13071-​022-​05230-w. other than dogs. Pathogens. 2021;10:307. 2. Pennisi MG. Leishmaniosis of companion animals in Europe: an update. Additional file 1: Table S1. Reference values of complete blood count Vet Parasitol. 2015;208:35–47. (CBC) parameters statistically evaluated. RV: reference values. 3. Pennisi MG, Persichetti MF. Feline leishmaniosis: is the cat a small dog? Vet Parasitol. 2018;251:131–7. 4. Iatta R, Mendoza-Roldan JA, Latrofa MS, Cascio A, Brianti E, Pombi M, Acknowledgements et al. Leishmania tarentolae and Leishmania infantum in humans, dogs The authors are grateful to Sara Montserrat‐Sangrà, (Departament de and cats in the Pelagie archipelago, southern Italy. Plos Neglect Trop D. Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma 2021;15:e0009817. de Barcelona, Bellaterra, Spain), Angela Burrascano, (Dipartimento di Scienze 5. Iatta R, Furlanello T, Colella V, Tarallo VD, Latrofa MS, Brianti E, et al. A Veterinarie, Università degli Studi di Messina), Istituto Maimònides de Inves‑ nationwide survey of Leishmania infantum infection in cats and associ‑ tigaciòn Biomèdica de Cordoba (Cordoba, Spain), and Centro de Sanidad y ated risk factors in Italy. Plos Neglect Trop D. 2019;13:e0007594. Bienestar Animal (SBA) de Sadeco (Cordoba, Spain) for technical collaboration. 6. Priolo V, Martínez-Orellana P, Pennisi MG, Masucci M, Prandi D, Ippolito D, et al. Leishmania infantum-specific IFN-γ production in stimulated blood the 16th ­CVBD® World Forum Symposium. The paper has been sponsored by Elanco Animal Health in the framework of from cats living in areas where canine leishmaniosis is endemic. Parasit Vector. 2019;12:133. Authors’ contributions 7. Otranto D, Napoli E, Latrofa MS, Annoscia G, Tarallo VD, Greco G, et al. VP conceived the research study. VP, MFP and GD collected samples and Feline and canine leishmaniosis and other vector-borne diseases in the carried out laboratory work with the supervision and/or collaboration of MM, Aeolian Islands: pathogen and vector circulation in a confined environ‑ MGP, LSG, PMO, ARB and FV. VP, MFP, MGP and GD selected data. VP and MM ment. Vet Parasitol. 2017;236:144–51. performed the statistical analysis. VP, MGP and MM interpreted the results. VP, 8. Persichetti MF, Pennisi MG, Vullo A, Masucci M, Migliazzo A, Solano-Gal‑ MGP and GD wrote the first draft of the manuscript. MGP, MM, GD and LSG lego L. Clinical evaluation of outdoor cats exposed to ectoparasites and revised the manuscript. All authors agreed to be personally accountable for associated risk for vector-borne infections in southern Italy. Parasit Vector. the author’s own contributions and to ensure that questions related to the 2018;11:136. accuracy or integrity of any part of the work, even ones in which the author 9. Pennisi MG, Lupo T, Malara D, Masucci M, Migliazzo A, Lombardo G. was not personally involved, are appropriately investigated, resolved and the Serological and molecular prevalence of Leishmania infantum infection in resolution documented in the literature. All authors read and approved the cats from Southern Italy. J Feline Med Surg. 2012;14:656–7. final manuscript. 10. Pennisi MG, Masucci M, Catarsini O. Presenza di anticorpi anti-Leishmania in gatti FIV+ che vivono in zona endemica. Atti LII Convegno Società Funding Italiana Scienze Veterinarie. 1998;52:265–6. Not applicable. 11. Latrofa MS, Iatta R, Toniolo F, Furlanello T, Ravagnan S, Capelli G, et al. A molecular survey of vector-borne pathogens and haemoplasmas in Availability of data and materials owned cats across Italy. Parasit Vectors. 2020;13:116. Data supporting the conclusions of this article are included in the report. Raw 12. Pennisi MG. A high prevalence of feline leishmaniasis in southern Italy. data are available from the corresponding author upon reasonable request. Proceedings of the second international canine leishmaniasis forum, Sevilla, Spain. 2002. p. 39–48. 13. Montoya A, García M, Gálvez R, Checa R, Marino V, Sarquis J, et al. Implica‑ Declarations tions of zoonotic and vector-borne parasites to free-roaming cats in central Spain. Vet Parasitol. 2018;251:125–30. Ethics approval and consent to participate 14. Miró G, Rupérez C, Checa R, Gálvez R, Hernández L, García M, et al. Cats were sampled during the annual health check, elective surgery or trap‐ Current status of L. infantum infection in stray cats in the Madrid region neuter‐release programs and always after the signature of informed consent (Spain): implications for the recent outbreak of human leishmaniosis? by the owner. Residual blood samples were mainly used for cats from Catalo‑ Parasit Vector. 2014;7:112. nia. Colony stray cats from Cordoba were sampled after approval by Centro de 15. Ayllón T, Diniz PPVP, Breitschwerdt EB, Villaescusa A, Rodríguez-Franco Sanidad y Bienestar Animal (SBA) de SADECO. F, Sainz A. Vector-borne diseases in client-owned and stray cats from Madrid, Spain. Vector Borne Zoonot. 2012;12:143–50. Consent for publication 16. Ayllon T, Tesouro MA, Amusategui I, Villaescusa A, Rodriguez-Franco F, Not applicable. Sainz A. Serologic and molecular evaluation of Leishmania infantum in cats from Central Spain. Ann N Y Acad Sci. 2008;1149:361–4. Competing interests 17. Sherry K, Miró G, Trotta M, Miranda C, Montoya A, Espinosa C, et al. A The authors declare that they have no competing interests. serological and molecular study of Leishmania infantum infection in cats from the Island of Ibiza (Spain). Vector Borne Zoonot. 2011;11:239–45. Author details 18. Millán J, Zanet S, Gomis M, Trisciuoglio A, Negre N, Ferroglio E. An investi‑ 1 Dipartimento di Scienze Veterinarie, Università di Messina, Messina, Italy. gation into alternative reservoirs of canine leishmaniasis on the endemic 2 Departament de Medicina i Cirurgia Animals Facultat de Veterinaria, Uni‑ Island of Mallorca (Spain): reservoirs of Leishmania infantum in Mallorca versitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain. 3 Facultad de island. Transbound Emerg Dis. 2011;58:352–7. Medicina Veterinaria, Universidad de Córdoba, Campus Rabanales, Córdoba, 19. Alcover MM, Basurco A, Fernandez A, Riera C, Fisa R, Gonzalez A, et al. Spain. 4 Centro di Referenza Nazionale per la Leishmaniosi (CReNaL), Istituto A cross-sectional study of Leishmania infantum infection in stray cats Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Palermo, Italy. in the city of Zaragoza (Spain) using serology and PCR. Parasit Vector. 2021;14:178. Received: 30 January 2022 Accepted: 2 March 2022 20. 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