Anti-Leishmania spp. antibody detection in domestic cats from a visceral leishmaniasis transmission area

Feline leishmanial infection is reported worldwide, but the epidemiological role of domestic cats in the leishmaniasis cycle remains unclear, and cats might act as cryptic reservoir hosts in endemic areas with no feline leishmaniosis cases. Considering that, a serological screening for anti-Leishmania spp. antibodies was performed by indirect immunofluorescence antibody test (IFAT) in 389 necropsied cats’ serum samples from a new visceral leishmaniasis transmission area with no feline leishmanial infection reported to unveil if the cats are being exposed to the parasite. The overall seroprevalence for Leishmania spp. was 11.05% (43/389). No association was found between sex, neutering status, age group, breed, coat length, feline immunodeficiency virus (FIV) infection, and Leishmania spp. antibody detection. A positive association was found with coat color (cats within the orange spectrum with white [particolor]) (OR = 2.47, CI 95% 1 – 6.13, P = 0.044) and a negative association (OR = 0.38, CI 95% 0.18 – 0.79, P = 0.01) between feline leukemia virus (FeLV) infection and IFAT positivity for Leishmania spp. Therefore, it is concluded that the seroprevalence found was greater than 10%, indicating contact of the protozoan with cats in the region served.


Introduction
Leishmaniasis is a spectrum of chronic, infectious diseases caused by flagellated kinetoplastid protozoa of the genus Leishmania that affect a wide range of animal species, including humans (Solano-Gallego and Baneth 2012).The disease is prevalent in tropical and subtropical countries around the world (Cardoso et al. 2021), particularly in the Mediterranean Basin, Middle East, Americas, and Northern Africa (Solano-Gallego and Baneth 2012).The reservoir hosts of the parasite include wild and domestic animals, with the domestic dog (Canis familiaris) being the most important host for L. infantum, especially in urban areas (Lainson and Rangel 2005).However, the role of the domestic cat (Felis catus) in the epidemiology of the disease has been investigated, as cats may act as cryptic reservoirs in endemic regions (Asfaram et al. 2019).
Domestic cats are susceptible to the same Leishmania species as dogs and humans from similar geographic areas, as the already identified species include L. infantum, L. mexicana, L. venezuelensis, L. amazonensis, L. braziliensis (Solano-Gallego and Baneth 2012;Pennisi et al. 2015;Pennisi and Persichetti 2018), L. major and L. tropica (Paşa et al. 2015).In Brazil, three species of Leishmania have already been identified: L. infantum (Savani et al. 2004), L. braziliensis (Schubach et al. 2004), and L. amazonensis (Carneiro et al. 2020).Among these, L. infantum is responsible for the majority of the cases in the country (Nascimento et al. 2022), as has been reported worldwide, where 63% of the infections are caused by this species (Asfaram et al. 2019).
As in all susceptible vertebrates, infected phlebotomine sandfly bites are the main form of infection for cats.Other transmission forms haven't been described yet for this species.Thus, in endemic areas for canine leishmaniosis, cats are potentially susceptible to L. infantum infection (Pennisi et al. 2015).In Brazil, the most epidemiologically relevant vector for L. infantum belongs to the genus Lutzomyia (Diptera, Psychodidae), with Lutzomyia longipalpis being the main vector (Lainson and Rangel 2005) and Lu.cruzi an important vector in some transmission foci (Oliveira et al. 2018).In addition to being among the species that sandflies blood-feed on (Rêgo and Soares 2021), infected cats can transmit the parasite to the vector (Mendonça et al. 2020;Vioti et al. 2021), which, in experimental conditions, can later infect dogs after feeding on the infected cat (Batista et al. 2020).
Currently, the region where this study was conducted, the city of Santa Maria (29°41′29″S, 53°48′3″W) and the central region of Rio Grande do Sul state, Southern Brazil, is considered a transmission area for visceral leishmaniasis (VL).In 2018, when the samples were first collected, nine dogs were necropsied with leishmaniosis as the cause of death or the reason for euthanasia.In comparison, in 2021, 41 cases were registered, representing an increase of over 300% in the number of fatal cases (Souza et al. 2022), demonstrating the increasing expansion of the canine disease.Additionally, the municipality also registered two cases of human visceral leishmaniasis in 2020 and 2021 (Centro Estadual de Vigilância em Saúde 2021), and one of the cases resulted in the death of a man with a history of comorbidities and a situation of social vulnerability.
Despite the area's current status, no cases of feline leishmaniosis are registered.In contrast, autochthonous cases of feline leishmaniosis have been reported for years in other endemic and non-endemic regions of Brazil, mostly in areas where canine leishmaniosis and VL are endemic for years.The disease was first diagnosed and reported in 2004, in São Paulo State, Southeastern Brazil (Savani et al. 2004).Thenceforth, 14 states of the country reported feline leishmanial infection (Mendonça et al. 2017;Nascimento et al. 2022;Oliveira et al. 2020).It is of great importance to know if cats are possible reservoirs for Leishmania spp.infection in transmission areas, since they are the second most frequent pet in the world (Ahuir-Baraja et al. 2021).Therefore, this manuscript aimed to perform a serological screening in order to determine if the cats in new VL transmission areas are being exposed to Leishmania spp., as well as a search for possible risk factors for seropositivity, such as sex, neutering status, breed, age group, retroviral infections, and coat color and length.

Study area, feline population, and sampling
All 389 samples were obtained by blood collection of all the cats necropsied between April 2018 and May 2022 in a veterinary pathology laboratory from an enzootic area for canine leishmaniosis (Santa Maria, Rio Grande do Sul State, Southern Brazil).The blood samples were centrifuged in tubes without any additives, and the sera were separated and stored in microcentrifuge tubes at − 20 °C until the serological analysis.Data concerning sex (male or female), neutering status (neutered or intact), breed (mixed-breed or purebred), coat length (short, semi-long, or long), coat pattern and color, and cause of death or reason for euthanasia were recorded for each individual.Regarding the age group, the cats were classified as kittens (up to 1 year), adults (1 to 10 years), or seniors (over 10 years old) (Quimby et al. 2021).
None of the necropsied cats had a previous clinical history of leishmaniosis or gross lesions resembling the disease.Therefore, all the cadavers underwent a thorough gross examination during necropsy to verify the presence of any of those lesions.Multiple tissue samples were collected from all the necropsied cats and were further analyzed by histopathology.These samples were fixed in 10% neutral-buffered formalin, routinely processed for histopathology, and embedded in paraffin blocks.For tissue analysis, hematoxylin-eosin (H&E) stained slides were prepared by sectioning the formalin-fixed paraffin-embedded (FFPE) blocks, and the tissue slides were evaluated by the authors using light microscopy.
To verify possible color attractiveness by sandflies, the cats were divided into nine groups based on their coat color within the visible spectrum.Group 1 (n = 58) was represented by black solid cats only.Cats within the blue spectrum (solid blue, black smoke, blue mackerel [striped] tabby, blue mackerel tabby with white [particolor], blue tortie, and blue torbie) composed Group 2 (n = 55).Group 3 (n = 45) included dark-pointed cats such as seal point, seal tortie point, seal torbie point, seal lynx, blue point, and blue lynx.Group 4 (n = 38) comprised cats within the orange spectrum (red mackerel tabby, red classic tabby, cream mackerel tabby, and solid cream), and Group 5 (n = 32) included the orange spectrum cats with wide white parts, like red mackerel tabby with white, black tortie with white (calico), red point, and cream lynx.The red spectrum cats (brown mackerel tabby, brown classic tabby, brown torbie, and black tortie) composed Group 6 (n = 45) and the red spectrum cats with wide white parts (brown mackerel tabby with white, brown classic tabby with white, and brown torbie with white), Group 7 (n = 32).Groups 8 (n = 46) and 9 (n = 25) were the bicolor cats (black and white or blue and white), and the predominantly white cats (white solid, blue and white van, black and white van, red mackerel tabby van, and brown mackerel tabby van), respectively.Information on the haircoat color and pattern of 13 cats was not available.

Detection of anti-Leishmania spp. antibodies by indirect immunofluorescence antibody test (IFAT)
All sera were tested by IFAT for Leishmania spp.antibodies.Immunofluorescence slides were sensitized with L. infantum promastigotes maintained in cell culture and fixed on the slide with acetone.After the preparation of the slides, the samples were diluted in phosphate-buffered saline (PBS) at 1:80, which is the cut-off value recommended by the current LeishVet guideline (Pennisi et al. 2015).Following dilution, the samples were added to the slides and incubated in a humidity chamber at 37ºC for 40 min.Next, the slides were rinsed in distilled water and PBS and dried.Then, the commercial fluorescein-labeled anti-Cat IgG© (Goat Anti-Cat IgG FITC®, F4262, Sigma-Aldrich, San Luis, Missouri, USA) was added to the slides as the secondary antibody, diluted in 1:100, as recommended by the manufacturer.The slides were incubated in a humidity chamber at 37ºC for 40 min, rinsed in distilled water and PBS, submerged in Evans blue for 2 min, and then set up for evaluation.The fluorescence of each sample was evaluated using a fluorescent microscope (Optiphase INV403F) at 400 × magnification.A serum sample of a cat that previously tested positive and a sample of a cat from a non-endemic area for VL were used as a positive and negative control, respectively.The samples that showed complete cytoplasmatic or membrane fluorescence were considered positive (Fig. 1).Positive sera were serially diluted in two-fold dilutions to determine the endpoint titer.

Detection of feline immunodeficiency virus (FIV) antibodies and feline leukemia virus (FeLV) antigen
Concerning the retroviral status of the studied cats, the sera of 189/389, including all the Leishmania spp.seropositive cats, were tested using a commercial rapid assay kit (SNAP® FIV Antibody/FeLV Antigen Combo Test: IDEXX Laboratories, Westbrook, ME, USA) for the detection of p27 FeLV antigen and FIV antibodies.

Statistical analyses
Several variables were tested as risk factors for seroconversion (sex, neutering status, age group, breed, retroviral infections, coat color and length), for which the odds ratio (OR) was calculated with a significance level of 0.05 and a 95% confidence interval.Also, the chi-square was performed to identify possible associations between the studied variables and the presence of antibodies for Leishmania spp., as it was done in other serosurveys (Alcover et al. 2021;Iatta et al. 2019;Priolo et al. 2022).Statistical analyses were performed by SPSS (IBM SPSS Statistics® version 28), a commercially available software program (IBM Corp 2021).
In addition, 16.6% (8/43) of them were FIV positive and 27.91% (12/43) were FeLV positive.A negative relation was found between FeLV infection and IFAT positivity for Leishmania spp., with P = 0.008 and OR = 0.38 (95% CI: 0.18 -0.79).None of the cats had gross or microscopic lesions compatible with feline leishmaniosis.A positive association between group 5 (orange spectrum cats with wide white parts) and Leishmania spp.seropositivity was found, with P = 0.044 and OR = 2.47 (95% CI: 1.00 -6.13).Even the OR CI had a value of 1, the chi-square test showed a higher frequency of positive animals in this coat color group, suggesting a relationship.Statistical analyses from all epidemiological data and the coat color groups are described in Tables 1 and 2, respectively.

Discussion
Similar to what has been described for dogs, the prevalence of Leishmania spp.infection on feline populations has been commonly estimated by specific antibody detection, more frequently by IFAT and enzyme-linked immunosorbent assay (ELISA).ELISA is described as a proper method for diagnosing clinical leishmaniosis, while IFAT has a higher sensitivity, hence being more useful in detecting the subclinical forms and populations screenings (Persichetti et al. 2017).In VL transmission areas, the feline seroprevalence demonstrates highly variable results, ranging from 0 to 68.5% (Pennisi et al. 2015).In Brazil, some serological studies had already been performed, demonstrating the same inconsistency, ranging from 0 to 54% for L. infantum (Nascimento et al. 2022).That inconsistency may be due to the different endemicity status and the use of different diagnostic methods and cut-off values, as there are no standardized protocols nor a gold-standard method for diagnosing Leishmania spp.infection in cats (Iatta et al. 2019).Our findings were similar to the overall prevalence of feline Leishmania spp.infection in other parts of the world, estimated to be 10% (Asfaram et al. 2019).
Some comparative studies have demonstrated that cats may have greater seroprevalence than dogs exposed to the same environmental conditions, although infected dogs had a higher parasite load than infected cats, emphasizing the role of the dog as the main reservoir and most efficient spreader of L. infantum (Baneth et al. 2020).However, cats more often present lower seroprevalence than dogs in the same areas (Pennisi et al. 2015).That is the case in this VL transmission area, where in 2021, anti-Leishmania spp.antibodies were detected in 23% (250/1087) of canine serum samples (Souza et al. 2022), while 11.05% (43/389) of the cats in our study were seropositive.A cross-reaction between L. infantum and other dermotropic Leishmania strains and Trypanosoma spp. is possible by serology methods, but the IFAT 1:80 cut-off point reduces those reactions (Pennisi and Persichetti 2018).
We found out that the cats of this new VL transmission area are being exposed to Leishmania spp., but further studies are necessary to differentiate if the cats are being truly infected or just exposed, as the definition of infection in leishmaniasis is based on the combination of the detection of specific antibodies and parasite DNA (Cardoso et al. 2021).
Since no cases of feline leishmaniosis have been registered in our region, cats may be acting as cryptic reservoir hosts.Although Leishmania spp.infection is usually detected in immunocompromised cats because of overt disease, asymptomatic infection may occur in immunocompetent individuals (Asfaram et al. 2019).Therefore, molecular studies are required to confirm it, considering that cats may not show signs of overt disease even when truly infected (Cardoso et al. 2021).
As far as it's known, cats probably have a natural resistance to disease development since clinical signs are usually associated with immunosuppression (Pennisi et al. 2015).In dogs, clinical progressive disease is associated with the inability to architect an effective cell-mediated immune response, characterized by the expression of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), which induces intracellular amastigote destruction by macrophages (Day 2016;Pereira and Maia 2021).In most cats, it seems that Leishmania spp.induce a cell-mediated protective immune response (Pereira and Maia 2021).Additionally, L. infantum infection and clinical disease in cats are associated with individual susceptibility, sandfly alimentary preferences, inoculum size during blood-feeding, the number of infected sandfly bites, and co-infections with immunosuppressive agents (Baneth et al. 2020) such as FIV, that impairs the cellmediated immune response, increasing the risk of disease development (Iatta et al. 2019).
Dermatologic signs, such as alopecia and dermatitis (nodular, ulcerative, crusting, exfoliative, or a combination of them), especially in the head and limbs, are the most common lesions reported in sick cats.Additionally, mucocutaneous ulcerative or nodular lesions are frequently reported, as well as ocular signs, such as blepharitis, chemosis, keratitis, conjunctivitis, and/or uveitis.Systemic lesions such as lymph node enlargement, hepatomegaly, and nephromegaly, have also been described (Fernandez-Gallego et al. 2020;Pennisi et al. 2015;Rocha et al. 2019).Unusual to rare manifestations of L. infantum-induced granulomatous rhinitis (Arenales et al. 2018;Leal et al. 2018) and granulomatous enteritis (Tabar et al. 2022), have also been reported.None of the seropositive cats in this study had similar lesions.
A positive association between FIV (Akhtardanesh et al. 2020;Alcover et al. 2021;Iatta et al. 2019;Priolo et al. 2022;Sobrinho et al. 2012), FeLV (Akhtardanesh et al. 2020;Sherry et al. 2011), and L. infantum infection * Group 1: black solid cats.Group 2: solid blue, black smoke, blue mackerel (striped) tabby, blue mackerel tabby with white (particolor), blue tortie, and blue torbie cats.Group 3: seal point, seal tortie point, seal torbie point, seal lynx, blue point, and blue lynx cats.Group 4: red mackerel tabby, red classic tabby, cream mackerel tabby, and solid cream cats.Group 5: red mackerel tabby with white, black tortie with white (calico), red point, and cream lynx cats.Group 6: brown mackerel tabby, brown classic tabby, brown torbie, and black tortie cats.Group 7: brown mackerel tabby with white, brown classic tabby with white, brown torbie with white cats.Group 8: black and white bicolor or blue and white bicolor cats.Group 9: white solid, blue and white van, black and white van, red mackerel tabby van, and brown mackerel tabby van  is described and even pointed as a major risk factor, considering that FIV-positive cats might be 2.8 (Priolo et al. 2022) to 6.8 (Akhtardanesh et al. 2020) times more likely to be infected by L. infantum than immunocompetent cats, whereas FeLV-positive cats might be 2.2 times more likely (Akhtardanesh et al. 2020).Notwithstanding, we did not find a significant association between Leishmania spp.seropositivity by IFAT and FIV infection.However, we found a negative association between Leishmania spp.seropositivity and FeLV infection and there are some possible explanations for that finding.This retroviral infection reduces immunoglobulin production and, commonly, those animals are lymphopenic (Hartmann 2012).Consequently, false negative results may occur in indirect diagnostic tests because of the impaired immunoglobulin production, and IFAT may be an unreliable diagnostic test in FeLV-positive cats.Other possible explanation is related to the studied population.The population of our study comprises necropsied cats, and most FeLV-positive cats die in their young adult years (1-6 years) from FeLV-related diseases, with 3 years as the median death age (Lorenzetti 2021).Considering that leishmanial infection mostly occurs in mature adults to elderly cats (Akhtardanesh et al. 2020;Solano-Gallego et al. 2007;Pennisi et al. 2012), maybe those FeLV-positive cats who died at a young age from FeLV-related diseases weren't exposed enough to the vector before death.
Although cats are considered a relatively resistant host to Leishmania spp.infection in experimental studies and case reports (Pennisi et al. 2015), there are some risk factors associated with infection, seropositivity, or clinical disease other than retroviral coinfections, such as overcrowding (Matos et al. 2018), cohabitation with dogs (Morelli et al. 2020), a rural environment (Cardoso et al. 2010), immunosuppressive therapy (Pennisi et al. 2015), free access to the streets (Rocha et al. 2019), male sex (Solano-Gallego et al. 2007), non-neutered status (Otranto et al. 2017), adult and elderly age (Akhtardanesh et al. 2020;Pennisi et al. 2012;Solano-Gallego et al. 2007).External factors such as altitude (Nasereddin et al. 2008) and seasonality (Pennisi et al. 2012) also seem to play a role in seropositivity.
As for the coat color groups, the positive association between group 5 (orange spectrum cats with wide white parts) and Leishmania spp.seropositivity was intriguing since groups 4 (orange spectrum cats) and 9 (predominantly white cats) did not show significant results.Previous studies that included coat colors as associated variables with L. infantum infection were conducted on dogs but have considered only the shade of the coat as bright/light, dark, or mixed (Maia et al. 2020;Mazeris et al. 2010), demonstrating that L. infantum DNA was significantly higher in light and dark color dogs (Maia et al. 2020).Although the reason for group 5 cats' positive association with Leishmania spp.seropositivity is unclear, this was the first study to correlate coat color with the presence of antibodies for a vector-borne disease in cats.It is known that very low expected values, as found in some coat groups, can interfere with the chisquare test.Also, other variables are related to vector attraction, such as carbon dioxide and water vapor emission from the skin.Therefore, further studies on phlebotomine color preferences considering the whole visible spectrum, as has already been performed with other arthropods (San Alberto et al. 2022), may be useful for understanding this finding.
The level of awareness toward canine leishmaniosis and, consequently, the use of sandfly repellents has been hypothesized as a possible cause for an increase in the feline-phlebotomine interaction, as the sandflies must look for other blood sources in the urban environment (Ahuir-Baraja et al. 2021).Perhaps the absence of feline leishmaniosis cases in new transmission areas may be due to the lack of awareness among dog owners regarding canine leishmaniosis itself, with most dogs not wearing repellent collars.Despite being considered a L. infantum transmission area, infections are relatively recent, with a significant increase in the number of cases since 2018 (Souza et al. 2022).Furthermore, in Brazil, the Ministry of Health estimates that for each human case of visceral leishmaniasis, 200 dogs have to be infected by L. infantum (Secretaria de Estado and de Saúde 2020).A similar ratio between feline and canine leishmaniosis might exist, but there are still scant data on the epidemiology of infection in cats from new transmission areas, as most cases of clinical disease are seen where canine leishmaniosis has been enzootic for decades (Nascimento et al. 2022).

Conclusion
Our data provide substantial evidence that, besides no cases of feline leishmanial infection have been detected in this new VL transmission area, cats are being exposed to Leishmania spp.Moreover, the negative FeLV association suggests that necropsied individuals are less likely to be Leishmania spp.positive by IFAT.A positive association between cats with coat color within the orange spectrum with white (particolor) and Leishmania spp.seropositivity was also found.From now on, further molecular investigations are required to determine if cats are really infected or just being exposed since the identification of potential reservoirs of zoonotic diseases is essential to control programs.Also, a better understanding of phlebotomine whole visible color spectrum attraction would be elucidating for understanding blood meal preferences according to coat colors.

Fig. 1
Fig. 1 Complete cytoplasmatic fluorescence of Leishmania infantum promastigotes in a feline positive sample.Indirect immunofluorescence antibody test

Table 2
Associations between the coat color groups and