New insights on severe clinical manifestations and deaths from visceral leishmaniasis in free-living crab-eating foxes (Cerdocyon thous) in Brazil

Visceral leishmaniasis (VL) is a zoonotic and severe neglected tropical disease, with worldwide distribution, that still cause many deaths among dogs and humans. Brazil is the country responsible for about 97% of the cases of leishmaniasis in the Americas and the disease is still considered a serious public health concern. Among wild mammalians, studies demonstrate the involvement of Cerdocyon thous in the biological cycle of Leishmania. Nevertheless, several authors consider the clinical manifestation of the disease to be rare or mild in free-living animals. Herein, we demonstrate the occurrence of severe clinical sings and deaths caused by VL in free-living crabResearch, Society and Development, v. 10, n. 16, e108101622869, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.22869 2 eating foxes. Three specimens of foxes collected from periurban areas were diagnosed with VL. The animals presented cutaneous, ophthalmological, gastrointestinal, locomotor and hematological alterations, and died after clinical progression. We identified the presence of anti-Leishmania antibodies by immunochromatographic test in all specimens. We also observed intra and extracellular amastigotes in skin cytology and lymph node aspirate. Furthermore, Leishmania infantum DNA was identified in all samples by the polymer chain reaction technique. Additionally, we performed blood count and stool parasitological tests and observed hematological disorders common to VL, such as anemia and lymphopenia. Taken together, our data demonstrate that VL can induce clinical complications and even cause death in C. thous and corroborate that this crab-eating fox is an adequate host for L. infantum.


Introduction
Leishmaniasis is a group of severe and neglected tropical parasitic diseases, caused by protozoa of the genus Leishmania, and transmitted by the bite of an infected female phlebotomine sandfly (WHO 2020). Clinically, leishmaniasis can present in three different forms: cutaneous leishmaniasis (CL) and mucocutaneous (MCL), which cause lesions especially in the skin and mucous membranes; and visceral leishmaniasis (VL), which affects mostly the liver, spleen, lymph nodes, and hematopoietic tissue. VL is the most severe form and can progress to death in 100% of cases within two years if left untreated (Dantas-Torres 2007; WHO 2020). Otherwise, animals affected by leishmaniasis can develop different clinical signs, not well delimited by clinical form as in humans, such as asymptomatic to severe and lethal forms of the disease (Jorge et al. 2010;Souza et al. 2014).
Notably, several infectious and parasitic diseases can affect wild and domestic animals and have increasing interest of the scientific community in the last decades, especially the zoonoses, in virtue of their potential for transmission to humans and impact on public health. Among the zoonoses, visceral leishmaniasis (VL) deserves special attention due to its high rate of morbidity and mortality among dogs and humans. More importantly, is estimated that one billion people are living in VLendemic regions and about 20,000 deaths annually due to the disease. In addition, Brazil is among the ten countries with the highest incidence of VL worldwide and, in 2018, registered about 97% of all cases in the Americas (Santos 2019;WHO 2020;. Although domestic dogs are the main reservoir of Leishmania, the parasitosis has already been described in different species of wild mammalians, especially in foxes of the species Cerdocyon thous (crab-eating). C. thous is widely distributed in Brazil and easily adaptable to several environments, including periurban areas (Lainson et al., 1990;Facure & Monteiro-Filho 1996;Travi et al. 1998;Dantas-Torres & Brandão-Filho 2006;Soares et al. 2017). Furthermore, Leishmania infection has been described in C. thous since 1954 and is considered a natural reservoir of the parasite by many authors. Despite this, there are still many lacks regarding the real importance of this specie into the epidemiological chain of the disease and the risk of transmission to humans and other animals (Deane & Deane 1954;Courtenay et al. 1996;Courtenay et al. 2002). Additionally, there are few reports in the scientific literature regarding to clinical complications of VL in specimens of crab-eating foxes in Brazil.
Importantly, regardless of being preventable and treatable, leishmaniasis still causes serious concerns in many regions, mainly in developing countries and areas of high social vulnerability . Considering the high significance of VL for public health, as well as its high incidence in a synanthropic species widely distributed in the Brazilian territory, we aimed herein to describe and present new insights about clinical aspects and complications of VL in free-living crab-eating foxes from Brazil.

Study area, animal sampling, and design
This study was carried out with samples collected from three specimens of crab-eating foxes (C. thous), two females and one male, all adults, and already presenting clinical signs of leishmaniasis. The foxes (named as CF1, CF2, and CF3) were patients at the Wild Animal Screening Center (CTAS) and had been rescued in the metropolitan region of the capital of the state of Sergipe, city of Aracaju (21° 12' 41'' S and 50° 25' 34'' W), Northeastern Brazil.
The animals were clinically evaluated, following four steps: i) anamnesis and history; ii) general clinical examination; iii) assessment of the skin and cutaneous adnexa; and iv) collection of biological samples (blood and tissues) for the diagnosis of inflammatory processes, lesions, and confirmation of VL infection. In addition, complementary tests such as blood count (hemogram) and fecal parasitology (by Hoffman's spontaneous sedimentation method, 1934) were performed in all specimens.
For the diagnosis of VL, we perform three types of tests: i) serological diagnosis; ii) parasitological examination; and iii) molecular diagnosis by polymerase chain reaction (PCR). The clinical sings were photographed, measured, and characterized (type, number, and body distribution of lesions) in individual files.

Serological diagnosis
For the diagnosis of leishmaniasis infection, we first perform a serological evaluation of the blood samples using the DPP immunochromatographic test (Dual Plate Platform -DPP ® Biomanguinhos/Fiocruz). This serological test aims to identify anti-Leishmania antibodies.

Parasitological examination
Initially, we conducted clinical evaluation to identify palpable lymph nodes in the popliteal region of the foxes. The lymph nodes that presented increased volume underwent fine needle aspiration puncture (FNAB). The material collected was deposited on microscope slides (in duplicate) and stained with fast panoptic. Subsequently, the slides were analyzed under an optical microscope to search amastigote forms of the parasite (at 400x and 1000x magnification). Similarly, the specimens that presented skin lesions were also submitted to amastigotes research in cells, through cytology analysis of the lesion edge.
Likewise, the material collected from the lesions by scraping was deposited on slides (in duplicate), stained with fast panoptic, and evaluated under an optical microscope at 400x and 1000x magnification.

Molecular diagnosis by polymerase chain reaction
The polymerase chain reaction (PCR) was carried out with genomic DNA extracted from all samples and using a commercial kit (Kit Go Taq® Green Master Mix, Promega, Madison, WI, USA). We followed all the manufacturer's instructions. First, the fragment of 447 bp of Leishmania infantum was amplified using the primers (MC1: 5'-GTTAGCCGATGGTGGTCTTG-3' and MC2: 5'CACCCATTTCGATTTTG-3') (Cortes et al. 2004). Next, the amplified fragments were visualized in 1% agarose gel electrophoresis through the UV transilluminator. Afterwards, the amplified fragments were purified using ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems) and sequenced in both directions using the Sanger method in automatic sequencer ABI 3130 (Applied Biosystems). The chromatograms were analyzed using BioEdit v.7.2.5 software (Hall 1999) and consensus sequences were submitted to BLASTn search (Altschul et al. 1990) to determine the sequence identity, based on comparisons with orthologous sequences available in the GenBank database.

Data analysis
Data were tabulated and stored in Microsoft Excel version 2017 spreadsheets. We calculated the absolute and relative frequencies of the results of the diagnostic tests and clinical signs observed in the foxes.

Search for manuscripts about leishmaniasis in foxes in Brazil
The literature review was carried out by searching for scientific manuscripts on cases of leishmaniasis in foxes in Brazil, in the main scientific databases (Academic Google -Scholar, The Scientific Electronic Library Online (SciELO), ResearchGate e PubMed. The data search descriptors were: "crab-eating fox" and "leishmaniasis" and "Brazil". The studies were categorized by study area in Brazil, number of foxes assessed, and leishmaniasis diagnosis method.
However, the presence of amastigotes (of Leishmania) was detected in only one (33.3%; 1/3) of them. In this sample, an average of 26 amastigotes/cell was observed (Figure 1). Furthermore, all samples submitted to the PCR were positive for L.
Additionally, the CF1 specimen showed an increase in the count of band and segmented neutrophils (1,080 and 13,140 nº/uL, respectively), while the CF3 showed low values in the lymphocytes count (685/uL). Importantly, all specimens were negative for intestinal parasites and died after progression of clinical signs.  Development, v. 10, n. 16, e108101622869, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.22869 9 This study describes new insights on clinical manifestations of VL on C. thous naturally infected by L. infantum and rescued in a fragment of Atlantic Forest close to an urban area of Northeastern Brazil.
Leishmaniasis are a group of severe parasitic diseases that still represents a serious public health concern, mainly in low-income countries from Africa, Asia, and Latin America. Importantly, in 2018, about 83 countries or territories were considered endemic for, or had previously reported cases of VL. Currently, it is estimated about 30,000 new cases of VL and more than 1 million new cases of CL annually (WHO 2020). Remarkable, domestic dogs are important hosts of Leishmania species and can maintain the disease transmission cycle (Almeida et al. 2018). Despite several studies investigating the occurrence and clinical manifestations of leishmaniasis in dogs, there are few studies that describe the clinical evolution of the disease in other canids in Brazil (Oliveira 2012;Nicolato et al. 2013). Herein, we present new insights into clinical aspects and mortality caused by leishmaniasis in free-living crab-eating foxes.
Molecular diagnosis by PCR identified L. infantum DNA in the animals assessed in our study and therefore confirms the occurrence of VL in foxes (Gontijo and Melo 2004). Likewise, some studies have already demonstrated the existence of a wild VL cycle, and the species C. thous as a potential reservoir for the parasite (Kruse et al. 2004;Gomes et al. 2007;Luppi et al. 2008;Oliveira et al. 2012;Ferreira et al. 2013;Almeida et al. 2018). Several factors can be associated with the occurrence of Leishmania among foxes. These canids have a generalist feeding profile and the ability to walk long distances in search of prey. Furthermore, they can easily adapt to different environments. This behavior increases the risk of infection by the parasite, the transmission of the disease to other hosts, and may corroborate the relevance of this species in the epidemiology chain of the disease (Juarez & Marinho-filho 2002;Figueiredo et al. 2008;Santos 2013).
Interestingly, previous studies suggest that free-living crab-eating foxes infected with Leishmania rarely develop clinical symptoms and that, when they do, they have a milder clinical manifestation and, most of them, spontaneously heal (Fornazari & Langoni 2014;Santos 2015;Almeida et al. 2018;Lima et al. 2018). On the other hand, our findings demonstrate that the specimens evaluated herein developed VL severe form and evolved with death after progression of clinical signs.
Considering this, we suggest that new studies should be carried out, assessing a larger sample, for a precise investigation about the impact of VL or wild canids.
Regarding the hematological findings, we observed a significant decrease in the components of the red series on the hemogram (RBC and hemoglobin) from all specimens assessed in the study. This data suggests the occurrence of anemia in foxes, that can be caused by leishmaniasis. According to Nicolato and colleagues (2013) anemia is a common clinical finding among symptomatic and VL-affected dogs. Similarly, some animals showed changes in white blood cell count, such as an increase in segmented and band neutrophils, and a decrease in the number of eosinophils and lymphocytes. These data may be associated with inflammatory or immunosuppressive conditions in foxes affected by VL.
Notably, domestic canids are important hosts in the VL transmission cycle. This is mainly justified due to the high parasite load that the dogs can have on the skin. Considering that superficial blood vessels are the main feeding site for sandfliessuch as Lutzomyia longipalpis, the main vector of VL in Brazilthis consequently enables the infection of phlebotomine with amastigotes and the transmission of the parasite among mammalian hosts (Tafuri et al. 2001;Porozzi et al. 2006). Likewise, and corroborating data from prior studies, we also identified amastigotes in the cytology of a skin lesion of one of the C. thous. Conversely, data based on the xenodiagnoses suggest that the transmission rate of Leishmania from C. thous to L. longipalpis is rare and, therefore, the species would not be a relevant reservoir for the parasite (Courtenay et al. 1996;Courtenay et al. 2002). Despite this, our results suggest that foxes may also be reservoirs at risk for Leishmania transmission to sandflies (Tenório et al. 2011;Roque & Jansen 2014).
According to Mana and colleagues (2009), the parasite load is an important factor associated with the severity of clinical presentation in canine VL. Moreover, we must highlight that due the increasing human exploratory activity, such as Development, v. 10, n. 16, e108101622869, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.22869 10 urban expansion and destruction of forest areas, there is an invasion of the natural habitat of C. thous and hence a synanthropic behavior of the species. Considering that the foxes assessed herein were captured in periurban areas, and that in recent years there has been a process of urbanization of leishmaniasis, this species may be exposed to a greater parasite load in areas with many infected domestic dogs and sandflies (Cerqueira et al. 2000;Rodrigues & Daemon 2004;Gomes 2006;Santos 2015).
Hereupon, high exposure to sandflies can result in an infection with a high parasite load and greater severity of the clinical manifestations of the disease in those animals, unlike the asymptomatic conditions observed in exclusively wild specimens.

Discussion
Researches carried out in several countries, including Brazil, have been carried out to determine the occurrence of parasitism in wild animals in zoos by means of coproparasitological analysis. In general, Cystoisospora sp., Toxocara cati, Strongyloides stercoralis, Toxascaris leonina, and hookworms are commonly reported for mammalian carnivores. In artiodactyls, protozoans such as Eimeria spp. and Cryptosporidium spp., as well as nematodes such as Trichuris sp., Toxocara vitulorum, Strongyloides sp., and parasites of the Paramphistomidae family have also been observed. Eggs of the Ancylostomatidae family, Trichuris sp., Strongyloides sp., Prosthernorchis elegans, and Bertiella spp. have been found in primates. In Brazil, Capillaria spp., Ascaridia sp., Heterakis sp., Libyostrongylus spp., Raillietina spp., Eimeria spp., and Isospora spp. have already been reported in birds (Fagiolini et al., 2018, Marques et al., 2019. Among the findings of this study, the first record of gastrointestinal endoparasites from Tapirus terrestris in Northeast Brazil stands out. This study reports the occurrence of non-sporulated oocysts of coccids, and cysts and trophozoites of Balantidium, both being reported for the first time in Brazil. Generally, infections by endoparasites in free-living tapirids are asymptomatic, but signs of parasitic disease have been observed in captive animals. We also highlight the first record of Bertiella spp. in Alouatta caraya in the Northeast, Brazil. Clinical signs of this parasitic disease have not been demonstrated for this primate species, but the importance of this zoonosis has been reported, which may occur mainly in patients with direct or indirect contact with these animals, resulting in abdominal discomfort, diarrhea, gastroenteritis, and anorexia (Oliveira et al., 2011;Fernandes-Santos et al., 2020).
Further, this is the first occurrence of Hymenolepis spp. and Aspiculuris spp. as spurious parasites from reptiles in Brazil, exhibiting pseudoparasitism in these animals from the moment they ingest rodents, which are the definitive hosts.
Although these pseudoparasites do not infect them, reptiles can be regarded as dispersers of viable parasitic forms that may cause infection in their respective hosts, such as rodents, non-human primates, and humans. In addition, this study also highlights the parasitism by Entamoeba coli and Eimeria spp. in Iguana iguana. Although infection by the latter is usually asymptomatic, generally when Entamoeba coli affects the gastrointestinal system, it may cause necrotic enteritis, hepatitis and liver abscesses, lethargy, diarrhea, regurgitation, convulsions, depression, hematochezia, and death in Squamata (Rinaldi et al., 2012;Lima et al., 2017;. Despite the great relevance of Zoological Parks and the struggle of these institutions to maintain the health of their animals, implementing prevention, diagnosis, and treatment programs, it is well known that many captive animals are housed close to each other, making parasitic infections inevitable. In addition, captive animals are often under considerable stress, which decreases their immunity and makes them more susceptible to infections. These parasites can pose a serious threat to the captive animals, occasionally causing fatalities. Many of these diseases, besides interfering with the welfare of the host species and affecting considerably their birth and mortality rates, may be zoonotic, posing a risk to animal handlers and animal care workers. In the present case, there was still the aggravating factor of the enclosures being within a forest reserve, which favors Research, Society and Development, v. 10, n. 16, e108101622869, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.22869 11 a greater contact of the animals with the droppings of other individuals, or even with intermediate hosts (Fagiolini et al., 2010;Oliveira et al., 2011;Snak et al., 2014;Schieber, Štrkolcová, 2019;Dashe & Behanu, 2020;Patra et al., 2020).
The parasitological diagnosis of wild and exotic animals kept in captivity is essential to assist decisions related to their treatment, since they provide important information about the health of the herd and the immune resistance of the hosts. In addition, this information collaborates with the scientific community and contributes to provide ecological data for each of the species herein studied, favoring their conservation and preservation (Barros et al., 2017).
Although there are previous works that have carried out parasitological surveys in animals from zoos in Brazil, these studies, besides being very scarce, are much spaced. Typically, these investigations did not evaluate most of the animals kept at those zoobotanic parks. The present research was the pioneer, in Brazil, to perform the parasitological diagnosis of most of the vertebrate animals of the same zoobotanic park, with species from the Mammalia, Aves, Archelosauria, and Lepidosauria taxa.

Conclusions
Taken together, our data demonstrate the occurrence of severe clinical signs of VL in free-living crab-eating foxes, which died after the clinical course of the disease. Considering this, we concluded that leishmaniasis can also severely affect wild mammalians of the species C. thous. Additionally, the presence of amastigotes in the skin lesions of them indicates a risk of transmission to sandflies and maintenance of the VL cycle. Importantly, the fox C. thous is one of the wild species susceptible to Leishmania in Brazil and that, despite the rarity of clinical manifestations in free-living specimens, our data demonstrate that in animals with synanthropic behavior, there is a higher risk of severe clinical forms and death. Regardless of our findings, the real role of these animals in the VL transmission cycle remains uncertain, and more studies are extremely required. Furthermore, understanding the biological, epidemiological, and social aspects involved in maintaining the transmission chain of leishmaniasis are necessary for the effective control of this still neglected zoonosis.