Morphological and molecular diagnosis of diseases of free-ranging crab-eating foxes ( Cerdocyon thous )

The aim of this study was to investigate the occurrence of diseases in free-ranging wild canids that were roadkill on highways in the State of Espírito Santo, Brazil. PCR tests were performed for the detection of Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. in the spleen. Morphological evaluation and identification of parasites was performed in the liver and lung. Twenty specimens of C. thous were necropsied at the Animal Pathology Sector of the Veterinary Hospital of the Universidade Federal do Espírito Santo. Tissue samples were processed for histopathological examination and polymerase chain reaction (PCR) analysis. There was no PCR amplification of genomic DNA sequences of Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. using DNA extracted from the spleen as template. Histologically, lesions associated with parasitism by Platynosomum sp. and Angiostrongylus sp. were observed in the liver and lung, respectively. This is the first report of Platynosomum sp. and Angiostrongylus sp. parasitism in C . thous in the state of Espírito Santo, Brazil. Therefore, this study demonstrated parasitism of crab-eating foxes by Platynosomum sp. and Angiostrongylus sp. Importantly, no evidence of infection with Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. was obtained by PCR analysis.


INTRODUCTION
Some of the diseases that affect wild animals may also affect human or domestic animal populations.Therefore, the diagnosis and study of these diseases may provide valuable knowledge on circulation of diseases among domestic and wild animals.The zoonoses with a wildlife reservoir constitute a public health issue worldwide.The importance of these diseases has been recognized, demanding attention to obtain data on natural occurrence of diseases and assessment of risk factors in certain ecosystems (Almeida et al., 2018).The crab-eating fox (Cerdocyon thous) is a wild canid that, in addition to its natural broad feeding behavior, also have carcasses of domestic animals, processed foods, and human-generated food waste as food sources.
This situation favors the occurrence of infectious diseases that may affect domestic dogs since C. thous often lives near human habitats.This species has been suffering from loss of habitat, hunting, and trampling.They are considered one of the wild mammal species that suffer the most from the impact of roads and motor vehicles in the Brazilian territory (Beisigel et al., 2013).Considering the scarcity of information on diseases affecting the crab-eating fox, the aim of this study was to investigate the occurrence of diseases in free-ranging crab-eating foxes that were road-killed on highways in the State of Espírito Santo, Brazil.Samples of the spleen, liver, and lungs (1 cm × 1 cm in duplicates) were stored at −80°C.Tissue samples were also fixed in 10% buffered formalin solution for histological processing, and 3 m sections were stained with hematoxylin and eosin (HE).Liver and lung samples containing helminth-associated lesions were photo documented using a Dino Eye capture camera, and the slides containing parasites were selected.Then, the length and width of all the eggs and fragments of adult parasites were measured using the ImageJ 64-bit Java 1.8.0_112 software and the mean value was obtained.

Twenty
Frozen tissue samples were processed at the Laboratório de Patologia Molecular at the Universidade Federal de Minas Gerais (Belo Horizonte, State of Minas Gerais, Brazil) for DNA extraction according to the guanidine extraction protocol (Pitcher et al., 1989).PCR for brucellosis was conducted as previously described by Bayliet et al. (1992), and PCR for hemoparasites (Babesia sp.Rangelia sp. and Hepatozoon sp.) was performed according to the protocol described by Harrus et al. (2011).PCR was performed using template DNA samples from 19 out of the 20 animals because samples from one animal had advanced degree of autolysis.Positive control for Brucella sp. was a previously confirmed case affecting the canine placenta, and positive control for piroplasmida was a confirmed case of Theileria equi in equine liver.
One animal had evidence of traumatic lesions, including fractures that were inflicted by an attack from a domestic dog.PCR for Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. had negative results for all spleen samples.Microscopic evaluation of the liver revealed that 75% of the animals (15/20) had hepatic congestion, 65% (13/20) periductal fibrosis, 15% (3/20) biliary ductal proliferation, 5% (1/20) periductal hyperplasia and 5% (1/20) lymphocytic inflammatory infiltrate.In 30% (6/20) of the animals, helminths with morphologic features of trematodes were observed in the lumen of bile ducts.Many trematodes with an oral sucker and eggs with a yellow to brown shell were observed within the organism (Gardiner and Poynton, 2006).Cross-sections of the parasites demonstrated an average length of 204.03 μm and average width of 166.27 μm of suckers.In addition, 17 egg fragments (measuring on average 32.64 ± 1.32 μm length and 18.73 ± 1.95 μm width) with thick brown shell were observed with miracidia in interior (Figure 1).Sections of these parasites demonstrated elongated, spiral-shaped larvae, totaling six parasites, measuring on average 176.21 ± 9.14 μm in length and 14.24 ± 1.37 μm width.The larvae were surrounded by a severe granulomatous inflammatory reaction with numerous macrophages, multinucleated giant Langhans cells, and marked diffuse hemosiderosis (Figure 2).Neither adult parasites nor egg were observed.

DISCUSSION
The occurrence and reports of hepatic and pulmonary parasites in wild canids are extremely rare, and this is the first record of Platynosomum sp. and Angiostrongylus sp. in C. thous in the state of Espírito Santo, Brazil.Infections with these parasites have been found in domestic dogs and cats, but in wild canids (especially crab-eating fox), there are just a few reports in Minas Gerais, Brazil.In this study, morphological features of the hepatic helminths allowed identification as trematodes.The most common trematodes in wild canids belong to the family Dicrocoeliidae, including Platynosomum sp. and Athesmia heterolecithodes (Duarte, 2007).According to Duarte (2007) there few and subtle differences between Platynosomum and Athesmia, such as the more elongated and larger body and the intraceacal ventral sucker of Athesmia.However, the absence of fresh adult parasites for morphometric analysis makes this differentiation difficult.Therefore, epidemiological aspects should be considered.There are no reports of A. heterolecithodes in Espírito Santo, Brazil, but Platynosomum sp. has already been report in domestic cats (Gava et al., 2015;Campos et al., 2018).In addition, there are reports of infection of canids with Platynosomum sp., as described by Duarte (2007), in Juiz de Fora, MG, Brazil.Curi (2010) eggs of Platynosomum sp. in C. thous and in Chrysocyon brachyurus (maned wolf), by fecal examination in the region of Serra do Cipó -MG, Brazil.
Hepatic lesions observed in this study, including periductal fibrosis, periductal hyperplasia, ductal proliferation, presence of eggs, and intraductal adult parasites, were compatible with biliary parasitic diseases, such as Platynosomum sp. in cats (Ramos et al., 2017) and lesions caused by Fasciola hepatica and Capillaria hepatica.However, the histological characteristics of these two last parasites are quite different (Duarte, 2007).In relation to the pulmonary lesions found in the animals of this study, the most evident changes such as congestion, edema, and hemorrhage may also be associated with traumas resulting from running over.However, one of the cases had a multifocal granulomatous pneumonia associated with parasite larvae.Ferreira Júnior et al. (2017) found that multifocal to coalescing granulomatous arteritis in C. thous was associated with the presence of parasites measuring 100-200 μm that were identified as Angiostrongylus vasorum.In this study, lavae found in the lung had sizes compatible with the findings of Ferreira Júnior et al. (2017) and therefore it was morphologically compatible with A. vasorum.
In the Brazilian territory, Angiostrongylus sp. had been described in C. thous in the state of Paraná (Duarte, 2007).In São Paulo, Angiostrongylus sp. was diagnosed by histopathology (Ferreira Júnior et al., 2017).These studies further support the interpretation that Angiostrongylus sp. was the pulmonary parasite identified in this study.Oliveira-Filho et al. (2012)

that were road killed between January and July 2018 and obtained from the Reserva Biológica de
Sooretama (Sooretama, State of Espírito Santo, Brazil), Museu de Biologia Professor Mello Leitão (Santa Tereza, State of Espírito Santo, Brazil), Universidade Federal do Norte Fluminense Darcy Ribeiro (UENF; Campos dos Goytacazes, State of Rio de Janeiro, Brazil), and the Secretaria do Meio Ambiente de Marataízes (Marataízes, State of Espírito Santo, Brazil), were included in this study.This experimental protocol has been approved by the Ethics Committee on Animal Use of Universidade Federal do Espírito Santo (UFES) under protocol number 065/2017.All carcasses were kept frozen at the place of origin and sent to the Laboratório de Patologia Animal do Hospital Veterinário da UFES, where they were subjected to necropsy.