<div>Transboundary and Emerging Diseases</div><div>Canine distemper outbreak by natural infection in a group of vaccinated
maned wolves in captivity</div><div>Vicente Vergara-Wilson<sup>1</sup>, Carlos R.
Sanchez<sup>2</sup>, María J. Abarca<sup>3</sup>, Carlos
Navarro<sup>3</sup>, Sebastian Celis-Diez<sup>4</sup>,
Pilar Soto-Guerrero<sup>4</sup>, Nataly
Diaz-Ayala<sup>1</sup>, Martin Zordan<sup>1,5</sup>,
Federico Cifuentes-Ramos<sup>3</sup>, Javier
Cabello-Stom<sup>6</sup>, Ezequiel
Hidalgo-Hermoso<sup>1,4</sup></div><div><sup>1</sup> Conservation and Research Department, Parque
Zoologico Buin Zoo, Panamericana Sur Km 32, Buin, Chile</div><div><sup>2</sup> Veterinary Medical Center, Living Collection Unit,
Oregon Zoo, Portland, Oregon, US</div><div><sup>3</sup> Faculty of Livestock and Veterinary Sciences,
Universidad de Chile (FAVET), Av. Santa</div><div>Rosa, 11735 Santiago, Chile</div><div><sup>4</sup> Departamento de Veterinaria, Parque Zoológico Buin
Zoo, Panamericana Sur Km 32, Buin, Chile</div><div><sup>5</sup> World Association of Zoos and Aquariums (WAZA),
Carrer de Roger de Llúria, 2, 2-208010 Barcelona,Spain</div><div><sup>6</sup> School of Veterinary Medicine, Universidad San
Sebastian, Patagonia Campus, Puerto Montt, Chile.</div><div>*Corresponding author: Ezequiel Hidalgo-Hermoso,
ezequielhidalgovet@yahoo.com</div><div>ABSTRACT</div><div>Canine morbillivirus, also known as canine distemper virus (CDV) is one
of the most important infectious diseases threat to the health and
conservation of free ranging and captive wild carnivores. CDV
vaccination using recombinant vaccines has been recommended for maned
wolf (<i>Chrisosyon brachyurus</i> ) after the failure of modified live
vaccines that induced disease in vaccinated animals. However, there has
been a lack of systematic evaluation about the response of this
preventive protocol in zoo carnivores due to ethical reasons that do not
approve vaccination trials with challenge in that species. Here we
report a CDV outbreak in a captive population of maned wolf with an
index case that was previously vaccinated with a recombinant vaccine.
Five juveniles and one adult from a group of seven maned wolves housed
in an outdoor exhibit died in April-May 2013 in a zoo in the
Metropolitan Region, Chile. Clinical signs ranged from lethargy to
digestive and respiratory signs. Diagnosis of CDV was confirmed by
histopathology, antibody assays and viral molecular detection and
characterization. The phylogenetic analyses of the nucleotide sequence
of H gene of the CDV genome identified in the two positive samples
suggest a close relation with the lineage Europe 1, commonly found South
America and Chile. CDV infections in maned wolf have not been previously
characterized. To the authors best knowledge is the first report of the
clinical presentation of CDV in a canine species previously inmmunized
with a recombinant vaccine. Further research will be necessary to
understand the impact of CDV in wild maned wolf populations and new
protocols (with boosters) that could improve the effectiveness of the
recombinant vaccine against CDV in wild carnivores.</div><div>KEYWORDS: Canine distemper virus, outbreak, <i>Chrysocyon
brachyurus</i> , zoo, vaccination.</div><div>INTRODUCTION</div><div>Canine morbillivirus, also known as canine distemper virus (CDV) is the
etiological agent of canine distemper (CD). It is a highly contagious
disease in which elevated mortality and morbidity in dogs and is, along
with rabies, the most important infectious diseases threat to the health
and conservation of free living and captive wild carnivores. This virus
is present in most countries and several <i>Carnivora</i> families are
susceptible, including the <i>Canidae, Procyonidae, Mustelidae,
Hyaenidae, Ursidae, Viverridae, Felidae, Ailuridae, Phocidae, and
Otariidae.</i> It can also affect other mammal orders like<i>Cetartiodactyla</i> , <i>Primates</i> , <i>Rodentia</i> and<i>Pilosa</i> . The maned wolf (<i>Chrysocyon brachyurus</i> ) is the
largest member of the <i>Canidae</i> family endemic/native of South
America. According to the International Union for Conservation of
Nature’s (IUCN) Red List of Threatened Species, the maned wolf is
considered to be near threatened. The four main threats affecting wild
maned wolf populations throughout their distribution range are: habitat
loss and alteration; human persecution due to livestock losses and
cultural beliefs; increasing vehicular traffic in highways resulting in
road kills; and pathogens contracted from domestic animals due to
increased contact in disturbed environments.</div><div>Canine distemper virus vaccination using recombinant dog commercial
vaccines has been recommended as a preventive protocol for captive maned
wolf and other wild carnivore species. When modified live vaccines (MLV)
were used in wild animals some of them induced disease . However, there
has been a lack of systematic evaluation about the response of this
protocol in zoo carnivores by ethical reasons that do not approve
vaccination trials with challenge in these species. Although CDV has
been historically recognized as a threat for the maned wolf
conservation, little is known of the pathological effects in the species
. To the knowledge of the authors, there are no reports documenting the
clinical disease by natural CDV infection in maned wolves. Furthermore,
this is the first report of recombinant CDV vaccine failure to protect a
wild canid species against the clinical infection produced by this virus
and the first CDV outbreak documented in maned wolves (<i>Chrysocyon
brachyurus</i> ).</div><div>Case Presentation</div><div>A 9-year-old female maned wolf part of the collection of a zoological
institution in Chile, was presented with unspecific clinical signs in
April 2013; these signs included lethargy, sialorrhea and diarrhea. This
female was kept in the same enclosure with a 7-year-old male maned wolf
and their five 1-year-old pups. Unfortunately, after 6 days of
supportive treatment that included intravenous fluid therapy,
antibiotics, and Vitamin K, the female died. Seven days later four of
the pups started showing progressive clinicals signs, except from one
female that died suddenly, without any clinical sign, 9 days after the
death of the 9-year-old female (figure 1). The remaining 4 cubs clinical
signs range from lethargy to digestive and respiratory signs and died in
a lapse of 16 days despite supportive treatment (figure 1). The
7-year-old male was observed clinically healthy during the outbreak.</div><div>In addition to the family group where the outbreak took place, there
were two additional 2-year-old maned wolves housed at a different area,
approximately 1 Km away. These animals were clinically healthy during
the outbreak, but were tested as a control population.</div><div>Bloodwork (Complete blood count and chemistry panel) were performed in
all animals (but the pup that suddenly died), along with a coagulation
panel, haemoparasites, fecal parasitology, distemper serology and
Distemper PCR. A seroneutralization tests was performed in 6 animals
after the outbreak with stored samples.</div><div>All five pups had been vaccinated with a single dosage, 9 months prior
to the outbreak (July 2012), and both adults had their annual booster 5
months prior to the outbreak (November 2012). The vaccine used in all
cases were single doses of 1 ml of Recombitek C6 vaccine, (Merial LLC,
imported and distributed by Sanofi Pasteur S.A. Andres Bello 2711, Las
Condes, Santiago, 832000, Chile) subcutaneously (SC) in the
interscapular space. All the animals were in apparent good health status
and had showed adequate body weight and normal appetite prior the
outbreak. It is important to mention that the vaccine used is the only
CDV recombinant vaccine available in South America.</div><div>A complete necropsy was performed soon after the death of 5 of the 6
animals. In all cases the most relevant macroscopic findings were
moderate pulmonary edema and mild, multifocal intestinal erosions. Due
to financial constraints tissue samples from only 2 pups/individuals
were collected for histologic evaluation. A full set of tissues from 2
pups were submitted in 10% buffered formalin for histologic evaluation.</div><div>*figure1*</div><div>Immunofluorescence assay (IFA)</div><div>Serum from 6 animals was tested for CDV IgG and IgM using MegaScreen®. A
fluorescent intensity ≥1:40 was determined to be positive (Megacor,
2006).</div><div>Seroneutralization assay</div><div>Serum from 6 animals was tested for CDV Ab. Serum was diluted and
challenged to a fix concentration of a native viral strain of CDV,
previously isolated at the Virology Laboratory of the Agricultural and
Livestock Service (SAG) of Chile. The cellular substrate for the test
was base in MDCK cells, American Type Collection, from renal cortex.
After 72 hrs. of incubation on an atmosphere of 5% CO2, 37°C and 95%
humidity, the samples were titled.
Titers of ≥1:16 of neutralizing
antibodies were consider positive .</div><div>Nucleic acid extraction and PCR assays (qPCR and RT-PCR)</div><div>qPCR: Viral RNA was extracted using the TRIzol reagent according to the
manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Reverse
transcription was performed using the Lig™ RNAse Inhibitor Solution and
Bio™ Transcriptase Solution, part of VetqPCR-realtime™ CDV Real Time
Kit, according to manufacturer’s instructions (Bioingentech Ltd. Chile).</div><div>RT-PCR: Viral RNA extraction was performed using TRIzol reagent
according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA,
USA). Reverse transcription was performed using the SuperScript III
One-Step RT-PCR System with Platinum Taq DNA Polymerase kit plus
oligonucleotide primers CDV1 (forward) and CDV2 (reverse), according to
manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).</div><div>Every fragment RT-PCR positive were then sent to a local sequencing
center (Genytec Ltda.) for sequencing in triplicate. The resulting
nucleotide sequence were aligned using the ClustalW program, and then
compared with all sequences deposited in GenBank using the BLAST
program. Pairwise and multiple sequence alignments at the nucleotide and
amino acid levels and sequence similarities were calculated using the
MEGA v6 software.</div><div>RESULTS (Table 1)</div><div>Histopathology</div><div>The histological evaluation in both cases showed severe diffuse
(bronchointerstitial) lymphocytic and histiocytic pneumonia, with the
presence of intracytoplasmic eosinophilic inclusion bodies. Similar
inclusion bodies were found in kidney, digestive epithelium, trachea,
liver, lymph nodes and urinary bladder. Other minor findings include
diffuse congestion of the liver, atrophy and moderate to severe diffuse
lymphoid depopulation on lymph nodes and spleen. In one of the 2 animals
a moderate focal ulcerative gastritis was found. The remaining samples
did not show pathological changes.</div><div>Antibody detection assays</div><div>The six individuals from the outbreak that were tested with the IFA
yielded positive to the presence of IgM and IgG, and the two animals
from the control population tested negative for IgM and IgG.</div><div>Samples from those six animals that yield positive to IFA were then
tested with SN. Four out of those six animals had titers of ≥1:16, with
the remaining two animals showing titers below 1:16, our predetermined
threshold ; the second population was not challenge with the
seroneutralization test.</div><div>Virus detection and characterization</div><div>Positive results were also obtained via qPCR in the 6 samples from the
outbreak population. From the positive, four samples were run thru a
RT-PCR for the detection of the gen H of CDV. Two samples were positive
for the detection of the gen H. Thru nucleotide alignment two consensus
sequences were obtained: “Chile/lobo crin/1” (4517) and “Chile/lobo
crin/2” (7490). The BLAST program was then used to identify the origin
of these amplified DNA fragments.</div><div>*table1*</div><div>DISCUSSION</div><div>Infectious diseases are an important threat for the health and
conservation of free ranging wildlife and under human care; in the
latter scenario it can even put in risk the success and the viability of
breading programs. The canine distemper virus (CDV) has been identified
as one of the most significant diseases for wild carnivores in zoos ,
making the implementation of vaccination protocols strongly recommended
. This manuscript describes the first report of this naturally acquired
disease in maned wolves, that culminated with the death of 6 vaccinated
animals. As such this represents a new challenge in the CDV vaccination
protocols for wild canid species.</div><div>The pathological, immunohistochemical and molecular findings observed in
maned wolves are consistent with those previously described in several
species infected with CDV . The phylogenetic analyses of the nucleotide
sequence of H gene of the CDV genome identified in the two positive
samples suggest a close relation with the lineage Europe 1, commonly
found South America and Chile (unpublished).</div><div>CDV has been described as one of the most important infectious diseases
affecting the maned wolf in captivity, however this conclusion has been
solely based in serological studies or clinical sings , without having a
complete correlation of clinical, pathological and molecular evidence in
every report. Without this triad is challenging to rule in clinical
causality of a pathogen over a host .</div><div>The current recommendation by Infectious Diseases Manual of using a
recombinant canarypox-vectored is based on the evidence that
commercially available canine MLV can cause reversion of vaccine strains
to virulent virus and vaccine-induced CDV in non-domestic carnivores.
Inactivated CDV whole-virus vaccines do not produce sufficient immunity
to prevent infection after virus challenge . The six maned wolves that
ultimately died on this outbreak were vaccinated with one dosage of
Recombitek within the previous year. Despite this inmunization with the
recommended single dosage, six out of seven maned wolves died within 3
weeks since the beginning of the outbreak.</div><div>Although this is the first report of recombinant CDV vaccine failure to
protect a wild canid species against the clinical infection, it has been
previously documented in snow leopard (<i>Panthera uncia</i> ) despite
prior vaccination with the monovalent canine rCDV vaccine 3 months prior
.</div><div>A recently published paper supports our findings by showing that a
single dosage protocol, of Recombitek CDV vaccine, did not elicit a
measurable humoral immune response and produced low nonprotective titers
in maned wolves as evidenced by the fatal outcome</div><div>To the best of our knowledge, this is the first report of the clinical
presentation of CDV in a canine species previously inmmunized with a
recombinant vaccine. The effectiveness of protection against CDV with a
single dose of this type of vaccine (canarypox vectored) has been
recently challenged by different studies where the humoral and cellular
immune response has been measured in red fox (<i>Vulpes Vulpes</i> ) and
giant panda (<i>Ailuropoda melanoleuca</i> ) respectively, and in both
studies the expected effect was not observed. Facing this new body of
evidence it’s clear the need for new studies of cellular and humoral
immune response in this and other species of wild carnivores, taking in
consideration that as wild species it’s not ethically viable to perform
challenge studies.</div><div>The present study confirms the lethal nature of CDV in maned wolf and
reinforces the necessity for new protocols of preventive medicine that
includes the vaccination for this species in captivity. It is imperative
the development of new studies on wild populations to understand the
role of this pathogen in the maned wolf conservation and population
dynamics. Finally, the protection failure of the vaccine against CDV
shown in this report makes necessary new studies that could present new
protocols that improve the effectiveness of its use in this and other
species, perhaps contemplating additional periodical boost dosages.</div><div>CONFLICT OF INTEREST STATEMENT</div><div>The authors declare no competing interests.</div><div>DATA AVAILABLE STATEMENT</div><div>Derived data supporting the findings of this study are available from
the corresponding author upon request.</div>