First detection of canine parvovirus 2b DNA in a crab-eating fox pup (Cerdocyon thous, Linnaeus, 1766)

Abstract

The crab-eating fox (Cerdocyon thous) is a small wild mammal present in all Brazilian biomes and in some countries of South America. This study aimed to verify the involvement of viral infectious agents in the death of a wild crab-eating fox pup (Cerdocyon thous) in Brazil. The Center for Medicine and Research of Wild Animals of the Universidade Estadual Paulista received a free-living crab-eating fox aged approximately 21 days and apparently healthy. After 13 days, the animal presented anorexia, diarrhea, fever, prostration, and neurological signs progressing to death with an inconclusive diagnosis. In a retrospective study, tissue fragments stored at − 80 °C were used to identify nucleic acids from major canine viruses, such as canine parvovirus-2 (CPV-2), canine adenovirus A types 1 and 2, canid alphaherpesvirus 1, and canine distemper virus. The amplified product with the expected length for CPV-2 was obtained from the heart fragment. After performing nucleotide (nt) sequencing of the amplicon, it was possible to demonstrate that the crab-eating fox strain exhibited high (99.8%) nt identity with the CPV-2b prototype (CPV-39 strain). Additionally, deduced amino acid (aa) sequence analysis showed the GAT codon for the aa Asp (D) at position 426 of the CPV-2 viral protein VP2, which characterizes the subtype 2b. To the best of the authors’ knowledge, this report describes the first detection of CPV-2b DNA in tissue fragments from a crab-eating fox.

Introduction

The crab-eating fox (Cerdocyon thous, Linnaeus, 1766) is a small wild mammal that can be found in all biomes of Brazil and in other South American countries. This wildlife species is listed on the Red List of Threatened Species by the International Union for Conservation of Nature as “Least Concern” regarding its risk of extinction [1]. Despite being apparently tolerant of anthropic disturbances, the vulnerability of this species is associated with destruction of its natural environment, deforestation for the extension of agricultural frontiers, dissemination of urban communities in the natural environment, and fragmentation of its habitat by the construction of roads [2]. Moreover, viral diseases that commonly affect domestic dogs represent an important threat to wildlife, mainly the carnivore population, leading to a reduction of the population of these animals [3].

Canine parvovirus (CPV) belongs to the species Carnivore protoparvovirus 1, genus Protoparvovirus, subfamily Parvovirinae, family Parvoviridae. CPV is a nonenveloped icosahedral small virus with a diameter of 25 nm, and it has a linear single-stranded DNA genome of 5.2 kb in length that encodes three structural (VP1, VP2/VP3) and two nonstructural (NS1 and NS2) proteins [4]. Canine parvovirus-2 (CPV-2) has a global distribution, and in domestic dogs, this virus is commonly associated with acute hemorrhagic enteritis, panleukopenia, and lymphoid necrosis [5]. In domestic dogs, until newborn puppies are 2 weeks old, CPV-2 can infect cardiomyocytes and cause necrotizing myocarditis frequently associated with cardiac failure or sudden death [6].

CPV-2 emerged as a variant of the feline panleukopenia virus (FPV) that adapted to the canine host by wild carnivores [7] and previously was largely replaced by more virulent variant strains, such as CPV-2a, CPV-2b, and CPV-2c [8, 9]. The original CPV-2 differed from the new variants in some nucleotide (nt) changes (positions 3045, 3685, 3699, 4062, and 4449) in the gene encoding the VP2 coat protein [10]. However, the main antigenic difference observed between CPV-2a, CPV-2b, and CPV-2c is at position 426 of the amino acid (aa) sequence of viral capsid VP2 protein [5]. The CPV-2 and CPV-2a show aa Asn (N), the CPV-2b shows aa Asp (D), and the CPV-2c shows aa Glu (E) in this position [7]. The three variants have been reported to affect domestic dogs in Brazil [11–13].

The important role in determining viral hosts and tissue tropisms of the CPV is performed by highly antigenic protein capsids, such as VP2 [9]. In the order Carnivora, six families are suspected of being susceptible to CPV: Felidae, Canidae, Procyonidae, Mustelidae, Ursidae, Ailuridae, and Viverridae [5]. Serological surveys in countries around the world report the high prevalence of CPV-2 in several species of captive and free-ranging wild carnivores [14–17]. In South American countries, including Brazil, serological studies show a high prevalence of antibodies against CPV-2 in this species [17–19]. Although serological studies against CPV antibodies are common in wild carnivores, molecular studies remain scarce.

The aim of this study was to identify the presence of nucleic acid from several of the most important canine viral agents in tissue fragments from a crab-eating fox pup with undetermined cause of death.

Materials and methods

A free female crab-eating fox (Cerdocyon thous) aged approximately 21 days was referred to the Center for Medicine and Research of Wild Animals (CEMPAS) of the Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP) in Botucatu-SP. At the initial clinical examination, the animal was apparently healthy and had good general conditions. After 13 days, the animal showed initial clinical manifestations, such as anorexia, yellowish fetid diarrhea, fever, and prostration. The hemogram showed the presence of hypochromic microcytic anemia, leukopenia, and hypoproteinemia compared to the reference values for the species [20] (Table 1). Five days after the initial clinical manifestations, the animal showed neurological signs characterized by motor incoordination with pedaling movements, evolving to hypovolemic shock, convulsion, and death. At the time, the etiological diagnosis was not conclusive. Therefore, tissue fragments were stored at − 80 °C in a bank of biological samples of wild animals until further research could be performed.

Table 1.

Hematological values of the Cerdocyon thous pup

Hematological parameters	Cerdocyon thous pup	Reference valuesa
RBC (106/μL)	2.06	3.73–4.56
Hemoglobin (g/dL)	4.7	10.3–12.6
PCV (%)	17	34–40
MCV (fL)	82.5	87.47–93.67
MCH (pg)	22.8	26.71–29.11
MCHC (%)	27.6	30.29–31.50
Metarubricytes	0	0
WBC (103/μL)	5.3	10.3–16.5
Band neutrophils (103/μL)	0	0–0.33
Segmented neutrophils (103/μL)	2.6	6.49–13.53
Lymphocytes (103/μL)	1.9	1.47–2.47
Eosinophils (103/μL)	0.4	0–0.93
Basophils (103/μL)	0	0
Monocytes (103/μL)	0.4	0.21–0.66
Platelets (103/μL)	249	249–291
Total Protein (serum) (g/dL)	2.0	5.9–8.2

RBC red blood cell, PCV packed cell volume, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, WBC white blood cell

^aMattoso et al. (2012)

In a retrospective study, tissue fragments (brain, lung, kidney, liver, spleen, heart, and tonsils) stored at − 80 °C were used to attempt to identify nucleic acids from important canine viruses, such as CPV-2, canine adenovirus A types 1 and 2 (CAdV-1 and CAdV-2), canid alphaherpesvirus type 1 (CaHV-1), and canine distemper virus (CDV).

Tissue fragments (100 mg) were treated with proteinase K (Ambion, Kaufungen, DE), and nucleic acid extraction was performed using a combination of phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate techniques [25]. The primers used for molecular identification of CDV, CAdV-1 and CAdV-2, CaHV-1, and CPV-2 are shown in Table 2 and the conditions were maintained according to the references. Aliquots of sterile ultrapure water and positive samples for each of the viruses searched were included as negative and positive controls, respectively.

Table 2.

Primers used for virological analysis

Agent	Target gene	Amplicon (size bp)	References
CDV	Nucleoprotein	287	[21]
CAdV-1	E3	508	[22]
CAdV-2	E3	1030	[22]
CaHV-1	Glycoprotein B	450	[23]
CPV-2	VP2 protein	583	[8, 24]

CDV canine distemper virus, CAdV-1 canine adenovirus type 1, CAdV-2 canine adenovirus type 2, CaHV-1 canid alphaherpesvirus 1, CPV-2 canine parvovirus 2, bp base pairs

The amplicon was sequenced with BigDye Terminator v3.1 Cycle Sequencing Kit in ABI3500 Genetic Analyzer sequencer (Applied Biosystems, Foster City, CA, USA). Sequence quality analysis and contig assembly were performed using the PHRED/CAP3 tool (http://asparagin.cenargen.embrapa.br/phph/). Multiple and pairwise alignments were performed using ClustalW in MEGA7 software and the nt sequence identity matrix was performed in BioEdit version 7.2.6.1. The nt sequence obtained in this study was deposited in GenBank under the accession number MT543040.

Results and discussion

The molecular assays to detect CDV, CAdV-1, CAdV-2, and CaHV-1 were negative in all tissue fragments evaluated. However, a 583-bp amplicon for the gene encoding the VP2 protein of CPV-2 was yielded from the heart tissue fragment. The nt sequence analysis of the CPV-2 strain showed high identity (99.8%) with the prototype CPV-2b (CPV-39 strain) and 99.4 to 99.8% nt identity with other CPV-2b strains deposited in GenBank. The deduced aa sequence analysis showed the presence of the codon GAT (aa D—Asp) at position 426 of the VP2 protein, supporting that the CPV-2 strain detected in the present study belongs to CPV-2b (Table 3).

Table 3.

Alignment of the deduced amino acid (aa) sequences of the PCR-amplified VP2 gene fragment (aa 407-440). The aligned sequences correspond to a CPV-2 vaccine strain (CPV-b, M38245), a CPV-2a (CPV-15, M24003), a CPV-2b (CPV-39, M74849), and a CPV-2c (56/00, FJ222821). Sequence from this study was deposited in GenBank with MT543040 accession number. Position 426 is highlighted in bold.

Sequence	Amino acid position
GenBank accession number	407	426	440
CPV–2 (M38245)	GRYPEGDWIQNINFNLPVTNDNVLLPTDPIGGKT
CPV–2a (M24003)	.................................................N..................................
CPV–2b (M74849)	.................................................D..................................
CPV–2c (FJ222821)	.................................................E..................................
BRA–UEL01 Cerdocyon thous (MT543040)	.................................................D..................................

This report presents the first description of CPV-2b DNA in tissue fragments from a free-living crab-eating fox (Cerdocyon thous), considered to be a common Brazilian wild species that travels in packs, is not vaccinated, and usually lives near inhabited places to obtain food. CPV-2 is the etiological agent of one of the most important infectious diseases affecting young dogs [26–28] and other members of the Canidae family worldwide [29].

Serological tests were performed on several animal species, including wolves (Canis lupus), red foxes (Vulpes vulpes), pampa foxes (Pseudalopex gymnocercus), maned wolves (Chrysocyon brachyurus), crab-eating foxes (Cerdocyon thous), and other wild carnivores [19, 30–32]. Molecular studies have shown that the three variants of CPV-2 (CPV-2a, CPV-2b, and CPV-2c) coexist at different rates in wild carnivores worldwide, as occurs in domestic dog populations [32–35].

In Italy, there was a positive correlation between the variants of CPV-2 identified circulating among domestic dogs and in wolves, reinforcing the importance of the domestic dogs as a reservoir of this viral etiological agent [36]. The same correlation was observed in the USA, which demonstrated the presence of CPV-2b and CPV-2c in feces of puma (Puma concolor), gray wolf (Canis lupus), and coyote (Canis latrans), while among domestic dogs, the three variants (CPV-2a, CPV-2b, and CPV-2c) were detected [5, 34]. In dogs from Namibia, the main circulating variant is CPV-2b, and among wild carnivores from the same country, CPV-2b was detected in cheetahs (Acinonyx jubatus) and in a bat-eared fox (Otocyon megalotis), making it possible to establish a positive correlation [5, 33]. In addition, in a Siberian tiger (Panthera tigris tigris) from Germany, CPV-2a was detected, which correlates with the variant detected in dogs from this country [5, 33].

The three variants of CPV-2 were found circulating in the domestic canine population in Brazil, though in different proportions [5, 11, 12, 27, 37]. On the other hand, studies of the molecular characterization of CPV-2 strains isolated in Brazilian wild carnivores are scarce. Therefore, this report describes the first molecular study of CPV-2b in a wild carnivore in Brazil.

Severe leukopenia is common in viral infections once viral replication occurs in bone marrow, promoting the destruction of young blood cells and the lymphoid system. The studied crab-eating fox showed 5.300 total leukocytes, with the normal value for the species being 10.300 to 16.500 [20]. This condition favors the occurrence of secondary bacterial infection, and when associated with dehydration conditions, it may lead to electrolyte imbalance, endotoxic shock, and sepsis [4].

In the present study, only the heart tissue fragment was positive for CPV-2b DNA. Currently, myocarditis with heart failure in puppies is uncommon among domestic dogs. However, when observing the emergence of CPV-2 as a variant of FPV, that is, when the virus is adapting to a new host species, this condition was much more frequent. Nevertheless, unfortunately, the absence of formalin-fixed and paraffin-embedded tissue of the crab-eating fox did not allow histopathological analysis to confirm the presence of myocarditis.

Although the diagnosis was not conclusive when the crab-eating fox died, the clinical signs and some hematological parameters were suggestive of a viral infection. However, in this retrospective study, nucleic acid screening of five major viruses for domestic dogs demonstrated the presence of CPV-2b DNA in cardiac tissue. To the best of the author’s knowledge, this report presents the first description of CPV-2b in a crab-eating fox in Brazil. Thus, CPV-2 should be included as a differential diagnosis in wildlife clinics.

Funding

This study was financially supported by the following Brazilian institutes: the National Council of Scientific and Technological Development (CNPq), the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), the Financing of Studies and Projects (FINEP), and the Araucaria Foundation (FAP/PR). Alfieri, A.A. and Alfieri, A.F. are recipients of CNPq fellowships. Lorenzetti, E. is recipient of Funadesp fellowship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

Supervision of the ethical committee to conduct this study was not required because this work was conducted with organ fragments of an animal that died spontaneously and was submitted for routine diagnosis by Center for Medicine and Research of Wild Animals (CEMPAS). All applicable international and national guidelines for the care and use of animals were followed.