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Published in final edited form as: Vet Parasitol Reg Stud Reports. 2024 Jun 18;53:101068. doi: 10.1016/j.vprsr.2024.101068

Prevalence of Leishmania infection in 205 cats from a referral hospital population in Brazil (2021–2022)

Brenda Lopes Paiva a, Lucas Christian de Sousa-Paula b,c, Kamila Gaudêncio da Silva Sales b,d, Karla Maria Vieira Costa a, Aline Martins Venuto a,e, Valcledes Nascimento do Oriente a, Francisco Roger Aguiar Cavalcante a, Roberta Lomonte Lemos de Brito a, Jessica Maria Leite dos Santos a,*, Filipe Dantas-Torres b
PMCID: PMC11265201  NIHMSID: NIHMS2007736  PMID: 39025541

Abstract

Leishmaniases are a group of neglected diseases of significant public health concern, with Brazil being the primary focus of this disease in the Americas. The municipality of Sobral, in the state of Ceará, is a historical focus of visceral leishmaniasis in both humans and dogs, but data on Leishmania spp. infections in cats are limited. Between April 2021 and February 2022, 205 cats from a referral hospital population were sampled and tested for Leishmania spp. by real-time PCR. Eight cats (3.9%; 95% CI: 1.7–7.5%) tested positive. Among these, three (37.5%) displayed clinical signs compatible with feline leishmaniosis. Non-domiciled cats showed significantly higher positivity compared to domiciled ones (Fisher’s exact test, P=0.0124). Considering their potential role as reservoirs of L. infantum, it is crucial to conduct further studies to understand the Leishmania spp. circulating among cats in Sobral and to implement measures for reducing their exposure to phlebotomine sand fly vectors in this important focus of leishmaniases.

Keywords: Feline leishmaniosis, Risk factors, Molecular diagnosis, Real-time PCR

1. Introduction

Leishmaniases represent a group of neglected diseases of significant public health importance, leading to high rates of morbidity and mortality in endemic regions (Alvar et al., 2012). These wide-spectrum diseases undergo complexes transmission cycles, involving several species of Leishmania parasites, vertebrate hosts, and phlebotomine sand fly vectors (Maia et al., 2018). In the Americas, visceral leishmaniasis (VL), the most severe and potentially fatal form, is a zoonosis caused by the protozoan parasite Leishmania infantum and is primarily transmitted through the bite of the phlebotomine sand fly Lutzomyia longipalpis (Sousa-Paula et al., 2020).

Over the years, there has been an increasing number of reports documenting domestic cats infected with Leishmania parasites worldwide, particularly L. infantum (Pennisi et al., 2015). Studies have demonstrated the infectivity of cats to L. longipalpis through xenodiagnoses (Mendonça et al., 2020; Batista et al., 2020). Lutzomyia longipalpis females were shown to acquire parasites when feeding on cats naturally infected with L. infantum, subsequently transmitting them to uninfected dogs (Batista et al., 2020).

Brazil remains the primary focus of human VL in the Americas, with an annual incidence ranging between 1,406–3,695 cases over the past decade (Brasil, 2023). The northeastern region of Brazil accounts for the highest number of cases (Brasil, 2023). In particular, the city of Sobral, state of Ceará, is an area where both canine and human VL are endemic (da Silva et al., 2017; Sousa-Paula et al., 2019). The city holds historical significance in VL research, being the site of the first reported outbreak of the disease in Brazil, the identification of foxes as a wild reservoir of L. infantum, and the implementation of seminal control strategies (Deane, 1956). However, limited knowledge exists regarding Leishmania spp. infection in cats in this area. Hence, the present study aimed to assess the prevalence of Leishmania spp. infection in cats in Sobral and evaluate potential associated risk factors.

2. Material and methods

2.1. Ethics statement

This study was approved by the Animal Care and Use Committee (ACUC) of the Centro Universitário INTA (UNINTA) (#: 2021-01-01-P). Additionally, cat owners and/or caretakers signed an informed consent form before the inclusion of the animals in the study.

2.2. Study area, cat population and blood sampling

This study was conducted in Sobral (3°40’58” S, 40°21’4” W), located in the northern part of the state of Ceará. Between April 2021 and February 2022, 205 cats from a referral hospital population (Hospital Veterinário do UNINTA; HOVET-UNINTA) were sampled. All cats included had undergone consultations or hospitalizations for various clinical conditions, and/or had taken part in spaying/neutering campaigns. This study included cats above 3 months of age, with a docile temperament, and both domiciled and non-domiciled (such as those living in shelters or as community-cared strays).

We gathered information on age, sex, breed, spaying/neutering status, lifestyle, reasons for hospitalization (e.g., obstructions, traumas, and others), and environmental factors (e.g., the presence of accumulated waste or organic matter near residences) for each sampled cat. The ages were categorized following the American Animal Hospital Association (AAHA)-American Association of Feline Practitioners (AAFP) Feline Life Stage Guidelines (Quimby et al., 2021). Each cat underwent a clinical evaluation, including a physical examination, followed by the collection of venous blood samples. A 2 ml-blood sample was obtained from either cephalic or jugular vein and transferred into EDTA (purple cap) collection tubes, then transferred into 1.5 ml microtubes and stored at −20°C.

The minimal sample size (n = 201) was calculated based on a 5% margin of error, a 95% confidence level, an unknown population size, and an expected prevalence of 15.4%, as determined in a previous study (Bezerra et al., 2019).

2.3. DNA extraction and real-time PCR testing

Blood samples were used for DNA extraction using the PureLink™ Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.

Real-time PCR reactions were carried out using the primers LEISH-1 (5’-AAC TTT TCT GGT CCT CCG GGT AG-3’) and LEISH-2 (5’-ACC CCC AGT TTC CCG CC-3’) along with the TaqMan® probe FAM-5’-AAA AAT GGG TGC AGA AAT-3’-non-fluorescent quencher-MGB. These primers amplify a 120-base pair fragment from the conserved region of the kinetoplast DNA (kDNA) of Leishmania spp. (Francino et al., 2006; Dantas-Torres et al., 2017). This PCR method detects various Leishmania spp., including L. infantum, a predominant species identified in dogs and humans in Sobral (da Silva et al., 2017; Sousa-Paula et al., 2019). The reaction volumes and cycling conditions were as described in Dantas-Torres et al. (2017). A no-template control (NTC), containing master mix without DNA, and a positive control (DNA extracted from L. infantum promastigotes; strain MHOM/BR/76/M4192) were included in each real-time PCR run. The reactions were conducted using the QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and the results were analysed using the QuantStudio Design and Analysis Software v1.4 (Applied Biosystems, Foster City, CA, USA).

2.4. Data analyses

The real-time PCR positivity and cats’ data were compared using Fisher’s exact test or Chi-square test, with a significance level set at P < 0.05. Calculations and statistical analyses were performed using Epi Info software (Dean et al., 2011) and GraphPad Prism. Exact 95% confidence interval (95% CI) was calculated for the overall real-time PCR positivity.

3. Results and discussion

Among the 205 cats in this study, 129 (62.9%) were females and 76 (37.1%) were males. Cats aged ≤1 year (n = 97; 47.3%) and >1≤6 years (n = 93; 45.4%) predominated, with fewer cats aged >6<10 years (n = 9; 4.4%) and ≥10 years (n = 6; 2.9%). Most of the cats (n = 203; 99.0%) were of mixed breed (see Table 1). Additionally, most cats (n = 119; 58.0%) were participating in spaying/neutering campaigns and primarily domiciled (n = 161; 78.5%).

Table 1.

Association between sex, breed, age, and spaying/neutering status of cats and molecular positivity for Leishmania spp. in the municipality of Sobral, Ceará, Brazil

Variable Category Positive/Exposed % Negative/Exposed % Fisher’s exact test
Sex Female 6/129 4.6% 123/129 95.3% P=0.7128
Male 2/76 2.6% 74/76 97.4%
Breed Mixed breed 8/203 3.9% 195/203 96.1% P=1.0000
Pure breed 0/2 0% 2/2 100%
Age ≤ 1 year 4/97 4.1% 93/97 95.9% P=1.0000
>1 year 4/108 3.7% 104/108 96.3%
Spaying / neutering status No 8/188 4.3% 180/188 95.7% P=1.0000
Yes 0/17 0% 17/17 100%
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Among the 205 tested cats, eight (3.9%; 95% CI: 1.7–7.5%) tested positive for Leishmania spp. DNA in their blood. Our overall real-time PCR positivity (i.e., 3.9%) aligns with other studies conducted in Brazil using molecular methods to detect Leishmania spp. DNA in different types of samples (Nascimento et al., 2022). For instance, da Silva et al. (2023) reported a positivity of 3.6% (n = 166), using both conventional PCR and qPCR on blood samples from cats in Ilha Solteira, São Paulo. Similarly, Coelho et al. (2011) reported a positivity of 3.8% (n = 52) using PCR on lymph node samples from cats in Andradina, São Paulo. Other PCR-based studies, however, documented positivities above 10% (e.g., Oliveira et al., 2015 and Costa-Val et al., 2020), reaching over >50.0% in a study (Marcondes et al., 2018).

In some cases, seroprevalence for anti-Leishmania antibodies in cats can be higher than real-time PCR positivity on blood samples (Iatta et al., 2019). Indeed, considering that anti-Leishmania IgG antibodies are long lasting, it is plausible to speculate that seroprevalence in cats in Sobral could be even higher than the one reported by real-time PCR positivity in the present study. Similarly, it is acknowledged that blood is not the most reliable biological sample for detecting Leishmania DNA. Nonetheless, it is a more accessible sample, being therefore widely used for epidemiological studies (Nascimento et al., 2022). As most of the animals in the present study participated in castration/spaying campaigns, blood collection would already be necessary to carry out hematological tests.

In our univariate analyses, no statistical differences were observed concerning sex, breed, or spaying/neutering status. However, in absolute values, females (n = 6; 75.0%) showed a higher incidence of positive results compared to males, likely due to the greater number of samples collected from female cats. All positive cases were of mixed breed, aged between 6 months to 3 years and were not neutered.

Among the eight cats positive by real-time PCR, five (62.5%) displayed no clinical signs suggestive of feline leishmaniosis, such as dermatological, ocular, or respiratory changes, lethargy, and/or weight loss. The remaining three cats (37.5%) were sick, exhibiting dermatological (alopecia, hyperkeratosis), respiratory (nasal discharge), and ocular alterations (blepharitis), weight loss, gingivitis, and/or lesions on the nose. Like in dogs, clinical manifestations of feline leishmaniosis are often nonspecific and may resemble other conditions such as sporotrichosis, cryptococcosis, eosinophilic granuloma, and cutaneous neoplasms (Pennisi et al., 2015).

A considerable proportion of the tested cats had outdoor access (n = 162; 79.0%) and contact with other animals (n = 186; 90.7%) (Fisher’s exact test, P=0.0124), suggesting that street-dwelling cats were more exposed to infections than domiciled cats. While some authors do not report statistically significant differences in the lifestyle of positive cats (e.g., Sousa et al., 2014), our observations indicated that non-domiciled cats were more exposed to infection than their domiciled counterparts. This suggests a higher risk of exposure to the vector L. longipalpis, which is present throughout the year in Sobral (Macedo et al., 2008; Silvino et al., 2017).

Most of the owners or caretakers (n = 169; 82.4%) reported the presence of waste or organic matter near the animals’ residence. Among the cats that tested positive for Leishmania DNA, all had outdoor access and contact with other animals. Regarding the presence of accumulated waste near the residence of positive cats, only one owner (12.5%) reported its absence (Table 2).

Table 2.

Association between the blood collection status of cats and risk factors considered and real-time PCR positivity for Leishmania spp. in the municipality of Sobral, Ceará, Brazil

Variable Category Positive/Exposed % Negative/Exposed % Fisher’s exact test
Collection status Spaying/neutering campaigns 7/119 5.9% 112/119 94.1% P=0.1424
Hopital admission 1/86 1.2% 85/86 98.8%
Housing status Non-domiciled 5/44 11.4% 39/44 88.6% P=0.0124
Domiciled 3/161 1.9% 158/161 98.1%
Contact with other animals Yes 8/186 4.3% 178/186 95.7% P=1.000
No 0/19 0% 19/19 100%
Access to the streets Yes 8/162 4.9% 154/162 95.1% P=0.2084
No 0/43 0% 43/43 100%
Waste or organic matter near homes Presence 7/169 4.1% 162/169 95.9% P=1.000
Absence 1/36 2.8% 35/36 97.2%
Clinical signs Presence 3/43 7.0% 40/43 93.0% P=0.3684
Absence 5/162 3.1% 157/162 96.9%
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Other studies focusing on feline leishmaniosis in hospital populations have been conducted in Brazil. Bezerra et al. (2019) analysed 91 cats referred to a veterinary hospital in Mossoró, Rio Grande do Norte state, reporting a seropositivity rate of 15.4% (n = 14/91). Matos et al. (2018) sought to detect anti-Leishmania antibodies in cats participating in spaying/neutering campaigns in Londrina and Telêmaco Borba, in Paraná state, and observed a positivity rate of 8.5% (n = 58/679). The authors noted that animals referred for spaying/neutering by non-governmental animal protection organizations were more likely to test positive compared to animals referred by their owners.

In conclusion, cats in Sobral, especially non-domiciled ones, are at risk of Leishmania infection. Local veterinary practitioners and public health authorities should be informed about the best-practice recommendations for the prevention, diagnosis, and treatment of feline leishmaniosis (Pennisi et al., 2015). This is pivotal to prevent the risk of adopting unfounded control measures, such as the culling of infected cats, as it has been done in the past with dogs.

Acknowledgements

The authors would like to thank the Veterinary Hospital – Small Animal Department, UNINTA for providing infrastructure and support.

Funding

BLP was recipient of a master’s degree fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; 88882-508006/2020-01).

Footnotes

Declaration of Competing Interest

None.

CRediT authorship contribution statement

Brenda Lopes Paiva: Conceptualization, Investigation, Writing – original draft.

Lucas Christian de Sousa-Paula: Investigation, Writing – original draft.

Kamila Gaudêncio da Silva Sales: Investigation, Writing – review & editing.

Karla Maria Vieira Costa: Investigation, Writing – review & editing.

Aline Martins Venuto: Investigation, Writing – review & editing.

Valcledes Nascimento do Oriente: Investigation, Writing – review & editing.

Francisco Roger Aguiar Cavalcante: Investigation, Writing – review & editing.

Roberta Lomonte Lemos de Brito: Investigation, Writing – review & editing.

Jessica Maria Leite dos Santos: Conceptualization, Resources, Writing – review & editing, Supervision.

Filipe Dantas-Torres: Conceptualization, Resources, Writing – review & editing.

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