Feline immunodeficiency virus, feline leukemia virus and Leishmania spp. prevalence in cats from shelters in Mato Grosso do Sul, Brazil

Abstract Diseases such as those caused by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) represent health problems for cats. Feline leishmaniasis (FL) has been reported in several cities across the country. The objective was to carry out a clinical-epidemiological and laboratory study of FIV, FeLV and FL in cats from shelters in Dourados, Mato Grosso do Sul, Brazil. Blood samples and swabs from the conjunctival and nasal mucosa were obtained from 75 cats, from four animal shelters. Serology for FIV and FeLV was performed. For Leishmania, polymerase chain reaction (PCR) was performed on blood, conjunctiva and nasal mucosa. In the immunochromatographic serological test, seven cats tested positive for FIV and none for FeLV. No samples was positive in PCR for Leishmania. The study showed that despite the presence of human and canine leishmaniasis in the studied region, Leishmania spp. were absent in the cats studied. To avoid an increase in contagion in shelters, it is essential isolate cats with FIV.


Introduction
Common retroviral diseases, such as feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), pose health problems for cats.The most affected animals are adult males with access to the external environment (Gleich & Hartmann, 2009).
Diseases caused by hemoparasites and retroviruses are emerging challenges that affect cats in many parts of the world.Free-range cats are highly vulnerable to infections because of exposure to a variety of ectoparasites that can transmit pathogens (Otranto & Dantas-Torres, 2010;Tasker, 2010).Leishmaniasis is endemic in the state of Mato Grosso do Sul (MS) and should be further investigated.Feline leishmaniasis (FL) has been reported in several cities in the Brazil, including Campo Grande/MS (Coelho et al., 2011;Benassi et al., 2017;Metzdorf et al., 2017;Asfaram et al., 2019;Mendonça et al., 2020).
Therefore, owing to the endemic characteristics of leishmaniasis in the state and as the main causes of infectious diseases in cats, this study aimed to conduct a clinical-epidemiological and laboratory study of FIV, FeLV and FL in cats from shelters in Dourados, MS, Brazil.

Study population
Males and females domestic cats, different ages and mixed breed were examined in all cat shelters in the city of Dourados, Brazil: 25 cats in shelter 1 (22.22680°S,54.84430ºW) with 95 cats sheltered, 25 cats in shelter 2 (22.27064ºS, 54.75491ºW) with 68 cats, all 15 cats from shelter 3 (22.21412ºS, 54.75907ºW), and 10 cats in shelter 4 (22.20158ºS, 54.87498ºW) with 12 cats sheltered.The sample for the study was calculated based on a total population of 190 cats, considering a confidence level of 95% and a margin of error of 6%, resulting in a sample of 75 individuals.The cats belonged to shelters, according to the owners, but had free access to the street.A cat was collected that did not belong to any shelter but had free access to it and was there at the time of sample collection.The cats were selected for convenience, from less skittish animals, which showed more apparent clinical signs and were in the shelter at the time of collection.
The animals were clinically evaluated by a veterinarian through anamnesis and physical examinations, to analyze body score, mucosal color, oral and nasal cavities, and ophthalmological and dermatological alterations.
This study was approved by the Ethics Committee on the Use of Animals (CEUA) of the Universidade Federal da Grande Dourados (protocol number 29/2020).

Obtaining biological samples
Biological sample collection was conducted from April to May 2021.The animals were catalogued in individual files and identified by name, sex, and age.After performing clinical analysis, the animals were physically restrained to collect peripheral blood, and conjunctival and nasal swab samples.
Peripheral blood (3 mL) was collected by jugular venipuncture and placed in tubes with and without EDTA to obtain whole blood and serum, respectively.The samples were kept at -20°C for subsequent serological and molecular tests.Conjunctival and nasal samples were obtained by swab collection and stored at -20ºC in microtubes for molecular diagnosis.

Serology for FIV and FeLV
Serological analyses were performed in serum using the Alere FIV/FeLV TM Test Kit (BioNote, Abbott Diagnósticos Rápidos, Belo Horizonte, Brazil), which detects FIV IgG antibodies and FeLV antigens, following the manufacturer's protocol.
Seven cats tested positive for FIV and no cat tested positive for FeLV.Of the FIV-positive cats, only three had clinical signs and presented oral lesions, alopecia and ear tip lesions.None of the animals tested positive for Leishmania spp. in the analyzed samples.The internal control (PCR actinF/V) showing the presence of mammalian DNA in all samples tested.

Discussion
Cats are common domestic animals in homes, and approximately 27.1 million felines in Brazil are estimated to share the same environment as humans (Instituto Pet Brasil, 2022).It is important to understand and investigate feline pathogens that may be involved in human diseases, owing to this close contact, which can facilitate the sharing of pathogens (Brum et al., 2007).In addition to the overcrowding of animals, shelter environments are usually precarious and favorable for FL and other diseases, because of the accumulation of organic matter (environments with fruit trees and shady places), a worrying fact because leishmaniasis vectors lay eggs in these places (Brasil, 2014).
Owing to the role of dog as a primary reservoir and the severe disease that can occur, leishmaniasis in dogs has been well studied.In contrast, cats have been considered, for many years, as less susceptible or resistant animals, and their role in the epidemiology of leishmaniasis is considered insignificant.Despite the initial reluctance, cases of FL have been increasingly reported worldwide.
Most cats with FL are subclinically infected, possibly because of their natural resistance to the development of disease (Solano-Gallego et al., 2007).The most common clinical signs include lymphadenomegaly, cutaneous and mucocutaneous lesions (nodular and/or ulcerative dermatitis), with or without visceral signs (Pennisi, 2015), generalized weakness, weight loss, anorexia, and ocular and oral lesions (Pennisi & Persichetti, 2018).However, not all clinical signs in cats with FL are necessarily due to this disease; some may be caused by comorbidities, such as FIV and FeLV (Marcos et al., 2009).Retroviruses are associated with various clinical problems such as anemia, lymphoma, chronic inflammatory disease, oral inflammatory disease such as gingivostomatitis, and secondary and opportunistic infections (Little et al., 2020).In the present study, three cats showed clinical signs of FIV (oral lesions, alopecia, and ear tip lesions), and one of them did not live in the shelter but had free access to and contact with the resident cats.This wandering animal, which has free access to the shelter, becomes a source of infection for other cats, demonstrating a health risk.Although none of the cats examined tested positive for Leishmania spp., studies have demonstrated the presence of Leishmania in cats.A study carried out in Israel showed that 75% (50/67) of cats housed in a shelter were seropositive for L. infantum, and 16% (11/67) were positive in the DNA analysis using PCR (Baneth et al., 2020).Peripheral blood PCR was negative for Leishmania spp., a very low result compared to the rates observed in another region of MS (Metzdorf et al., 2017).Little information is available on the mechanisms responsible for the susceptibility or resistance of felines naturally exposed to infection by L. infantum (Persichetti et al., 2017).The PCR positivity of peripheral blood samples is generally lower than that of lymph node and bone marrow aspirates or local skin impressions (Alcover et al., 2021); however, they are less invasive.None of the shelters studied received a complaint from the city's Zoonosis Control Center regarding precarious conditions, such as accumulation of organic matter and waste.Therefore, although the shelters have agglomerations of animals, there may be sandfly vectors in these locations.
Because retroviral diseases weaken animals and are immunosuppressive diseases, they represent a challenge for the health of felines.However, most cats naturally infected with FIV do not present a serious clinical condition; with proper care, infected cats can live for many years.As observed in the current study, although some animals tested positive for FIV, they did not present characteristic symptoms of the disease.Care must be taken in places with a high density of animals as immunosuppressive diseases favor the development of opportunistic diseases.

Conclusion
Leishmania spp.were not detected in the examined cats.A negative PCR ITS1 for Leishmania, in blood and conjunctival and nasal samples, does not exclude Leishmania infection, and it is important serology, kDNA PCR and PCR of other tissues.Further investigations of FL are necessary owing to the high number of cases of human and canine leishmaniasis in Dourados, in addition to reports of the presence of sand fly vectors.Cats from shelters can have viral diseases, such as FIV and FeLV, and rapid diagnosis and early isolation of felines is important to prevent contagion to other animals.The presence of immunosuppressive diseases in cats from shelters with high animal density requires greater monitoring of the animals' health.