Infection, Genetics and Evolution 46 (2016) 130–137
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Research paper
Lethal distemper in badgers (Meles meles) following epidemic in dogs and wolves Daria Di Sabatino a, Gabriella Di Francesco a, Guendalina Zaccaria a, Daniela Malatesta a, Luca Brugnola b, Maurilia Marcacci a, Ottavio Portanti a, Fabrizio De Massis a, Giovanni Savini a, Liana Teodori a, Enzo Ruggieri a, Iolanda Mangone a, Pietro Badagliacca a, Alessio Lorusso a,⁎ a b
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (IZSAM), Campo Boario, 64100, Teramo, Italy Corpo Forestale dello Stato, Ufficio Territoriale per la Biodiversità, Viale Riviera 29, 65121, Pescara, Italy
a r t i c l e
i n f o
Article history: Received 4 May 2016 Received in revised form 8 October 2016 Accepted 24 October 2016 Available online 27 October 2016 Keywords: Canine distemper virus Badgers Whole genome sequencing Fox Wildlife Phylogeny
a b s t r a c t Canine distemper virus (CDV) represents an important conservation threat to many wild carnivores. A large distemper epidemic sustained by an Arctic-lineage strain occurred in Italy in 2013, mainly in the Abruzzi region, causing overt disease in domestic and shepherd dogs, Apennine wolves (Canis lupus) and other wild carnivores. Two badgers were collected by the end of September 2015 in a rural area of the Abruzzi region and were demonstrated to be CDV-positive by real time RT-PCR and IHC in several tissues. The genome of CDV isolates from badgers showed Y549H substitution in the mature H protein. By employing all publicly available Arctic-lineage H protein encoding gene sequences, six amino acid changes in recent Italian strains with respect to Italian strains of dogs from 2000 to 2008, were observed. A CDV strain belonging to the European-wildlife lineage was also identified in a fox found dead in the same region in 2016, proving co-circulation of an additional CDV lineage. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Canine distemper virus (CDV) belongs to the genus Morbillivirus of the family Paramyxoviridae and it is responsible for a highly contagious and severe disease, known as distemper (Barrett, 1999), that affects wild and domestic carnivores, non-human primates and marine mammals (Kennedy, 1998; Deem et al., 2000; Decaro et al., 2004; Di Guardo et al., 2005; Beineke et al., 2015; Martella et al., 2010; Monne et al., 2011). CDV has a non-segmented single-stranded negative sense RNA genome encoding for six structural proteins. Haemagglutinin (H) is part of the virus envelope and is crucial to cellular infection via attachment to signalling lymphocyte activation molecule (SLAM) (Tatsuo et al., 2001). The H protein binds the receptor resulting in cellular attachment and activation of the F protein by tissue-specific proteases leading to cellular infection. Therefore the H protein has a key role in determining host range and tropism. Recently, also nectin4 has been demonstrated to act as CDV receptor (Noyce et al., 2013). Although nectin4 expression in neural cells of the brain is still questionable (Pratakpiriya et al., 2012), it is clearly present in the epithelia of various organs explaining the pathological consequences such as immunosuppression, respiratory and gastrointestinal lesions of CDV infected animals. To better represent the evolution of currently circulating strains, ⁎ Corresponding author. E-mail address:
[email protected] (A. Lorusso).
http://dx.doi.org/10.1016/j.meegid.2016.10.020 1567-1348/© 2016 Elsevier B.V. All rights reserved.
CDV has been classified into several lineages based upon the genetic relatedness of the H protein encoding genes. Italy has experienced so far the circulation of at least three separate CDV lineages including Europe-1, Europe Wildlife and Arctic lineages. Martella and colleagues in 2006 described the first case of Arctic CDV infection in dogs in central and southern Italy (Martella et al., 2006) and, between 2000 and 2008 the same lineage was also reported retrospectively in the northern Italy (Monne et al., 2011). A large distemper epidemic occurred in Italy during 2013 and involved primarily the Abruzzi region, an area hosting several natural parks with significant animal biodiversity. In this outbreak, distemper caused overt disease in unvaccinated domestic and shepherd dogs, Apennine wolves (Canis lupus) and other wild carnivores living in natural areas of the Abruzzi and neighboring regions including foxes (Vulpes vulpes), badgers (Meles meles), beech martens (Martes foina) and European polecats (Mustela putorius) (Di Sabatino et al., 2014; Lorusso and Savini, 2014). Molecular investigations revealed that the outbreak was sustained by an Arctic-lineage strain (Marcacci et al., 2014) that had been likely circulating in the domestic dog population before the spillover in wildlife (Lorusso and Savini, 2014). This scenario was supported by the evidence of a tyrosine (Y) residue at position 549 (Y549) of the mature hemagglutinin protein (H) of viral strains isolated from dogs and wolves at the beginning of the outbreak (Di Sabatino et al., 2014; Marcacci et al., 2014). Residue 549 falls into the receptor-binding domains of the H protein (McCarthy et al., 2007), therefore it may have implications for host
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range and tropism. On the other hand, a histidine (H) residue occurs predominantly in CDV isolates from non-dog host species (McCarthy et al., 2007; Origgi et al., 2012; Monne et al., 2011). Following the epidemic, a massive vaccination campaign was implemented in summer 2013 by the regional Veterinary Services of the Abruzzi region enrolling the domestic and shepherd dogs living in the areas surrounding the protected natural areas of the region with important animal biodiversity. In this manuscript we describe the clinical, pathological and molecular findings observed in two CDV-infected Eurasian badgers (Meles meles) collected in the Abruzzi region by the end of September 2015 and discuss the likely scenario of CDV persistence in the wildlife milieu of the region. 2. Materials and methods 2.1. Animals and sample collection By the end of September 2015, two badgers were found in Pianella, a countryside municipality in the province of Pescara (Abruzzi region). The first badger (11956/2015), an adult female, was found dead in a rural area. The second badger (12254/2015), an adult male showing mild neurological signs (ataxia and prostration), was found at the edge of a road and referred to the regional wildlife rescue center of the Italian State Forestry Corps where it died four days later. Badgers were necropsied at the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (IZSAM) and samples from brain, lung, liver, intestine and spleen were collected. Feces, stomach content and samples from liver, intestine, kidney, lymph nodes content were also collected for additional laboratory investigations as described below. 2.2. Histology, immunohistochemistry (IHC) and molecular diagnosis Samples from brain, lung, spleen, liver, small intestine and kidney were fixed in 10% neutral buffered formalin and routinely processed for histopathology. Sections were cut (5 μm) and stained with haematoxylin and eosin (H&E). Formalin-fixed, paraffin-embedded samples were also processed using an IHC technique with a specific antibody directed against CDV (1:800, mouse monoclonal antibody antiCDV-NP; VMRD™, Pulmann WA) and detected by indirect streptavidin-biotin method (Dako REAL™ Detection System, Peroxidase/DAB kit, California USA). Deparaffinized and rehydrated sections were incubated in 3% hydrogen peroxide in absolute methanol for 45 min to inhibit endogenous peroxidase activity then rinsed in 0.05 M Tris-buffered saline (TBS), pH 7.6, for 5 min. Antigen retrieval was performed by heat treatment in citrate buffer 0.01 M at 121 °C for 8 min. CDV positive lung tissue sections of dog and badger available in house were used as positive controls, whereas the specificity of the immunolabeling was verified with an irrelevant Ab directed against an unrelated antigen. Samples from tissues (lung, brain and spleen) were homogenized with phosphate-buffered saline buffer (PBS) with antibiotics and 100 μl of the homogenates were used for total nucleic acid purification by using BioSprint 96 One-For-All Vet Kit (Qiagen), following manufacturer's instructions. Purified nucleic acids were screened by rtPCRCDV with specific primers and probe for the N protein-encoding gene of CDV, as described by Elia et al. (2006). Only nucleic acids purified from the lung of badger 12254/2015 were tested by rtPCRCDV. 2.3. Isolation on cell culture and sequencing Vero cells stably expressing canine SLAM (Vero. DogSLAMtag; Seki et al., 2003) were inoculated with lung homogenates and incubated until cytopathic effect (CPE) was observed. As tissue homogenates can be toxic to cultured cells, producing a CPE-like effect, a second passage was performed. Whole genome sequences of both CDV isolates were obtained by combination of sequence independent single primer amplification method (SISPA) and next generation sequencing (NGS) as
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recently described by our group (Marcacci et al., 2016) starting from total RNA purified from the supernatants of passage 2 on Vero. DogSLAMtag. Deep sequencing was performed on the NextSeq 500 (Illumina Inc.) using the NextSeq 500/550 Mid Output Reagent Cartridge v2, 300 cycles and standard 150 bp paired-end reads. Furthermore, in order to exclude that the observed amino acid (aa) changes within the H protein coding gene sequences (CDS) obtained by NGS were unwanted mutations due to cell-adaptation, RNAs purified straight from lung tissues homogenates were used for RT-PCR and Sanger sequencing of the H protein CDS. Amplicons were obtained using RH3 and RH-4 primers as described by Mochizuki et al. (1999) and sequenced using classical dideoxy Sanger method, performed onto 3130xl Genetic Analyzer (Applied Biosystem). Sequence reads were assembled with DNAStar software package (DNAStar Inc., Madison WI; USA). Whole genome sequences obtained in this study were compared to the homologous obtained from a CDV-infected dog (CDV2784/2013, KF914669) and to the full-length-H protein CDS of CDV detected in two Apennine wolves (Wa-CDV2013, KC966928; Wb-CDV2013, KC966929), originating from the CDV outbreak of 2013 (Di Sabatino et al., 2014). Moreover the almost complete genome sequence of a CDV strain (CDV599/2016, KX545421) isolated from a fox found dead in February 2016 in the area surrounding the municipality of Avezzano (L'Aquila province, Abruzzi region) and collected during the surveillance activities on wildlife routinely performed at IZSAM, was analyzed in comparison to the extant genome sequences of this study. Full-length H protein CDS of publicly available CDV Arctic-lineage strains were also retrieved for sequence comparison including sequences available from Martella and colleagues from CDV-infected dogs of 2013. H protein CDS of strains W10/1382 (JF810117) and W10/1376 (JF810116) from Switzerland and Pr78O-Lu (AB329581) from Japan, all originating from CDV isolates of badgers, were used for sequence comparison with homologous sequences obtained from badgers in this study. 2.4. Phylogenetic analysis Representative H protein CDS of geographically distinct CDV lineages were retrieved from GenBank (Supplemental file S1). These representative sequences and the H protein CDS consensus sequences obtained in this study (CDV11956/2015 and CDV12254/2015 from badgers and CDV599/2016 from fox) were aligned using MAFFT (Multiple Alignment with Fas Fourier Transform) with default settings (Katoh et al., 2009). A phylogenetic tree was inferred using MEGA 6, through the neighbor-joining (NJ) distance method (Kimura's two-parameter distance mode) and statistical support was provided by bootstrapping over 1000 replicates (Tamura et al., 2013). Other tree-building methods, including maximum parsimony and maximum likelihood, were used in order to verify the topology of the neighbor-joining tree. 2.5. Other laboratory investigations After homogenization, samples from brain, lung, spleen, liver and kidney were submitted for microbiological investigation and bacterial identification, which was carried out using an automated system (Vitek®, BioMérieux Italia). Purified nucleic acids from liver and kidney were tested by PCR for Leptospira spp. (Stoddard et al., 2009) whereas those purified from small intestine, liver and spleen were tested for canine parvovirus type 2 (Streck et al., 2013), canine infectious hepatitis virus (Hu et al., 2001) and carnivore circoviruses (Zaccaria et al., 2016), respectively. Feces were investigated for gastrointestinal parasites by flotation and for cardiopulmonary nematodes by Baermann technique. Samples of liver and stomach content were screened for organophosphate/organochlorine pesticides and for rodenticides using Gas Chromatography-Low Resolution Mass Spectrometry or High Performance Liquid Chromatography-Mass Spectrometry techniques.
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2.6. CDV positive animals 2012–2016 in the Abruzzi and Molise regions The number of dogs and wildlife diagnosed with CDV infection was retrieved from our internal database. We considered only individuals whose biological samples (blood, urine, swabs or carcasses) resulted positive for CDV by molecular techniques. Dogs and wildlife originated from Abruzzi and Molise, regions of jurisdiction of IZSAM. Samples from wildlife are regularly screened for CDV as recommended by surveillance activities whereas biological samples of domestic dogs are tested for CDV based on clinical suspect or necropsy recommendations. The period of observation is comprised between 2012 and June 2016. 3. Results 3.1. Gross and microscopic findings Necropsy examination of the first carcass (11956/2015) of about 10 kg of weight, revealed multifocal consolidation areas located in the apical and diaphragmatic lobes of both lungs. No evident signs of putrefactive phenomena were observed. Splenic congestion and hyperemia of superficial brain vessels were also detected. Histologic examination of the lung tissues revealed severe bronchopneumonia in which alveoli are filled with neutrophils and tissue debris (Fig. 1). Eosinophilic intracytoplasmic inclusions were evidenced in the bronchial and bronchiolar epithelium. Spleen lymphoid depletion and bacterial co-infection in all tissues examined were detected. In the central nervous system, only cerebellar spongiosis was observed. The second badger (12254/2015), a carcass of about 13 kg, displayed gross pathological lesions in lungs and spleen similar to those detected in the first badger. The histological examination of the lung tissue revealed diffuse hyperemia with massive alveolar exudation of macrophages. Moderate splenomegaly due to congestion with lymphoid depletion was also observed. In addition, the gastrointestinal tract was filled with blood content, associated with mucosal necrosis. Renal medullary hyperemia and hepatic centrilobular stasis were revealed, in association with microscopic evidence of septic emboli. Intracytoplasmic inclusion bodies were observed in the renal epithelial cells and in the intestinal columnar epithelial cells. 3.2. IHC and rtPCRCDV
Fig. 2. A Badger 12254/2015, Lung. Intense and diffuse CDV immunoreactivity in epithelial cells and macrophages. IHC; 20×. B Badger 11956/2015, Brain. CDV immunoreactivity in the neuro-epithelium of choroid plexus. IHC; 20×.
CDV antigen was observed by IHC in all analyzed tissues from both animals. Lung tissue samples showed strong immunoreactivity in epithelial cells and macrophages (Fig. 2A). Moreover, strong immunostaining was observed in lymphoid cells of the spleen, especially in badger 11596/2015. Viral antigen was also observed in the intestinal and biliary epithelium, as well as in the neuro-epithelium of choroid plexus (Fig. 2B), with immunoreactivity distributed in neurons and neuronal cytoplasmic processes. CDV RNA was detected by rtPCRCDV in both lung tissues processed. Lower CDV threshold cycles (CT) value was evidenced for badger 12254/2015 (Ct 19) than badger 11956/2015 (Ct 23). Moreover, CDV RNA was detected in all tested tissues of badger 11956/ 2015 (Table 1).
Table 1 Tissue samples from both badgers tested by rtPCRCDV and immunohistochemistry (IHC). CT, threshold cycle.
Fig. 1. Badger 11956/2015, Lung. Broncho-interstitial pneumonia with airways filled with inflammatory cells. HE; 40×.
Badger/Tissue
rtPCRCDV, CT
IHC
11956/2015 Brain 11956/2015 Lung 11956/2015 Spleen 12254/2015 Lung
27 23 24 19
+ + + +
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Table 2 Amino-acid comparison for selected positions in the mature H protein within available Italian CDV Arctic lineage strains from 2000 to 2015. Green, strains from badgers from 2015; light blue, strains from dogs and wolves from 2012 to 2013;dark red, strain from an imported dog from Hungary to Switzerland; dark blue, strain from Lazio region from 2011; pink, strain from northern Italy of 2008; dark green, representative of Hungarian strains from 2005 to 2006; yellow, strains from 2000 to 2005; na, not available; NI, Northern Italy; SI, Southern Italy; CI, Central Italy.
Italian CDV arctic strains
49
66
195
273
310
417
435
445
549
555
559
Badger CDV11956/2015–Italy–CI
V
N
I
T
D
T
N
M
H
T
S
Badger CDV12254/2015–Italy–CI
V
N
I
T
D
T
N
M
H
T
S
Dog CDV2784/2013–Italy–SI
I
S
I
I
D
T
N
M
Y
T
S
Wolf Wa–CDV2013–Italy–CI
I
S
I
I
D
T
N
M
Y
T
S
Wolf Wb–CDV2013–Italy–CI
I
S
I
I
D
T
N
M
Y
T
S
Dog BA376/13/ITA–Italy–SI
I
S
I
I
D
T
N
M
H
A
S
Dog BA466/12/ITA–Italy–SI
I
S
I
I
D
T
N
M
Y
A
S
Dog BA201/13/ITA–Italy–SI
I
S
I
I
D
T
N
M
Y
A
S
Dog 278/13/ITA–Italy–SI
I
S
I
I
D
T
N
M
Y
A
S
Dog BA440/13/ITA–Italy–SI
I
S
I
I
D
T
N
M
Y
A
S
Dog Bari–370–2013–Italy–SI
na
na
na
na
D
T
N
M
Y
na
na
Dog Bari–181–2013–Italy–SI
na
na
na
na
D
T
N
M
Y
na
na
Dog Bari–237–2013–Italy–SI
na
na
na
na
D
T
N
M
Y
na
na
Dog Bari–12–2013–Italy–SI
na
na
na
na
D
T
N
M
Y
na
na
Dog CDV 319.2012 Italy–CI
na
na
na
na
na
T
N
M
Y
T
S
Dog CDV 352.2012 Italy–CI
na
na
na
na
na
T
N
M
Y
T
S
Dog CDV Dog10 Switzerland 2013
I
S
V
I
D
T
N
L
Y
T
S
Dog CDV 99.2011 Italy–NI
na
na
na
na
na
T
N
M
Y
T
S
Dog CDV 08RS2382 RO 11 08–Italy–NI
I
S
V
I
D
T
N
L
Y
T
P
Dog CDV H05Bp3S–Hungary 2005/06
I
S
V
T
G
I
D
L
Y
T
P
Dog CDV 48/05 Italy–SI
I
S
V
I
G
I
D
L
Y
T
P
Dog CDV 179/04 Italy–SI
I
S
V
I
G
I
D
L
Y
T
P
Dog CDV 64.2004 Italy–NI
na
na
na
na
na
I
D
L
Y
T
P
Dog CDV 456.2003 Italy–NI
na
na
na
na
na
I
D
L
Y
T
P
Dog CDV 444.2002 Italy–NI
na
na
na
na
na
I
D
L
Y
T
P
Dog CDV 2894 BL 00–Italy–NI
na
S
V
I
G
I
D
L
H
T
P
3.3. Isolation and genome characterization CDV was isolated from lung homogenates of 11956/2015 and 12254/2015; isolates were named CDV11956/2015 and CDV12254/ 2015, respectively. CDV growth on cell culture was confirmed by
rtPCRCDV. Whole genome sequence was retrieved from both isolates and nucleotide (nt) sequences were deposited on GenBank (CDV11956/2015, KX024708; CDV12254/2015, KX024709). Full-length H protein CDS were also obtained from RNA purified straight from infected lungs of both carcasses by RT-PCR and Sanger sequencing;
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consensus sequences were identical to those obtained by Illumina from viral isolates, confirming that mutations in the H protein CDS did not occur during viral isolation. Whole genome consensus sequences of CDV11956/2015 and CDV12254/2015 showed 99.9% nt identity within each other and both showed the 99.5% of nt identity with strain CDV2784/2013. CDV11956/2015 and CDV12254/2015 sequences showed two synonymous nt substitutions at the N protein and L protein genes level, respectively and a nt change in the intergenic region separating the N and P CDS. Amino acid (aa) differences within CDVs from badgers and CDV2784/2013 are depicted in Fig. 3 whereas aa changes in the H protein of CDV from badgers with respect Italian Arctic-lineage sequences from 2000 to 2015 are listed in Table 2. In particular, whether CDV from wolves and dogs from 2013 have Y549, CDV strains isolated from badgers have H549. Interestingly H549 is present only in a CDV field strain isolated from a dog in northern Italy in 2000 (CDV 2894 BL 00, 97.3% aa identity, HM443713) and in CDV BA376/13/ITA strain (99.1% aa identity, KM115535) of a dog from Apulia region from 2013. Remaining publicly available CDV sequences (KM115534–KM115536) of 2013 from Apulia had Y549. Two unique aa changes were identified at position 49 and 66, showing V and N residues respectively (Table 2). These two aa were not present in other H protein sequences publicly available from badgers. The number and position of potential glycosylation sites in the H protein of CDV from badgers do not differ from those estimated in CDV strains from wolves and dogs from the 2013 epidemic (data not shown). By sequence comparison, 6 aa residues that may be related, as previously suggested (Balboni et al., 2014), to the temporal origin of recent CDV Arctic strains, were observed (Table 2). Excluding from the analysis single-strain unique aa residues, aa at position 195, 310, 417, 435, 445 and 559 differ in strains isolated in Italy from 2011 onward with respect strains isolated since 2000. Based on the sequences available so far, the combination of these residues, which has been observed in strain CDV2784/2013, was detected for the first time, although partially -as for the lack of sequence
for residues 195 and 310-in strain CDV 99.2011 (KF184989, Balboni et al., 2014) isolated from a dog in Lazio region (Central-Italy) in 2011 (Table 2). In an intermediate position we encountered strain Dog10 isolated from Switzerland and CDV strain 08RS2382 RO 11 08 isolated in northeast Italy in 2008 (Monne et al., 2011). Strain CDV H05Bp3S, representative of Hungarian CDV strains from 2005 to 2006 (DQ889179DQ889186, Demeter et al., 2007), is more closely related to the “old” Arctic-lineage strains isolated in Italy from 2000 including strains 48/ 05 (DQ226088), 179/04 (DQ226087) 64.2004 (KF184987), 456.2003 (KF184986), 444.2002 (KF184985) and CDV 2894 BL 00 (HM443713), this latter representative of the Arctic CDV strains isolated in northern Italy between 2000 and 2005 (Monne et al., 2011). 3.4. Phylogenetic analysis Phylogenetic analysis was inferred by NJ method employing the fulllength H protein CDS of the two isolates from badgers, a CDV strain from a fox (CDV599/2016) and sequences representative of the main CDV geographical lineages retrieved from the GenBank database (S1). CDV11956/2015 and CDV12254/2015 from badgers grouped, as expected, into the Arctic-lineage cluster whereas CDV599/2016 from a fox grouped into the European Wildlife lineage (Fig. 4). 3.5. Other laboratory investigations The two badgers tested negative for canine parvovirus type 2, canine infectious hepatitis virus, dog and fox circoviruses and Leptospira spp. No evidence of toxics including organophosphate, organochlorine pesticides, strychnine or anticoagulant rodenticides was revealed. A polymicrobial flora composed by Gram positive and negative bacteria (Aeromonas hydrophila, Streptococcus pharyngis and Escherichia coli) was detected in the liver and lungs of badger 11956/2015, as well as in the spleen and lungs of badger 12254/2015 (Clostridium perfringens,
CDV2784/2013
C P
V
M
H
F
L
N 1
15690
CDV11956/2015
C P
V
M
H
F
L
N 1
15690
CDV12254/2015
C P
V
M
H
F
L
N 1
15690
CDV2784/2013
Wa-CDV2013
CDV11956/2015, CDV12254/2015
I 49
S 66
N 244
I
S
D
49
66
244
V
N
D
49
66
244
I
H 273 I
H 273 T
H 273
N 487
Y 549
S
Y
487
549
S
H
487
549
Fig. 3. Graphic representation of CDV genome. Red indicates amino acid substitutions with respect CDV2784/2013 genome. Graph is not in scale. In the bottom, the H proteins of CDV from badgers compared to the H protein of CDV2784/2013 and Wa-CDV2013 from a dog and wolf of 2013, respectively (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
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Micrococcus luteus and Escherichia coli). Eggs of Ancylostomatidae and Trichuridae were detected in the feces of the two animals. In the feces of badger 12254/2015 larvae of Angiostrongylus spp. were also detected. 3.6. CDV positive animals in the Abruzzi and Molise regions, 2012–2016. The number of CDV positive dog and wildlife individuals is showed in Fig. 5. 4. Discussion CDV, a paradigmatic multi-host pathogen, represents a conservation threat to several animal species (Daoust et al., 2009; Beineke et al., 2015) and it is considered as the most serious infectious disease of mustelids (Williams et al., 1988; Deem et al., 2000; Hammer et al., 2004). In this manuscript we first described the pathological and virological findings of CDV infection in two Eurasian badgers collected in the Abruzzi region, central Italy. These two badgers were collected two years after the CDV epidemic, sustained by an Arctic-lineage strain, recorded in the same area. The peak of the epidemic was indeed evidenced in winter/spring 2013 and largely involved, according to our diagnostic records, the domestic dog population (78.2% of total cases, Fig. 5). The number of new CDV-positive dogs dropped in 2014, 2015 and 2016 likely due to the vaccination campaign promoted in domestic dogs during summer 2013. The vaccination campaign in dogs was primarily established to protect the threatened population of Marsican brown bear (Ursus arctos marsicanus) living in the area. The recently emerged Italian Arctic-lineage strains possess a novel amino acid constellation at position 195, 310, 417, 435, 445 and 559 of
Fig. 5. Graph showing the number of positive animals by rtPCRCDV or other similar molecular methods within the years of observation. Green, dog; red, wildlife. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the mature H protein with respect to strains that have been circulating in Italy since 2000 (Table 2). The first Arctic-lineage CDV strain presenting this constellation was revealed, although partially, in an infected dog in Lazio region (Central-Italy) in 2011 (strain 99.2011, Balboni et al., 2014). This constellation was observed thereafter in all Arctic-lineage CDV isolates including strain CDV2784/2013 from a dog (Marcacci et al., 2014) and CDV strains from wolves (Di Sabatino et al., 2014). Overall, they are genetically related to CDV strains isolated from infected dogs in Eastern Europe suggesting, as previously speculated (Martella et al., 2006), the spread of this virus to Italy by the illegal trade of infected puppies from Eastern Europe. A similar scenario has been also
Fig. 4. Neighbor-joining tree inferred from multiple nucleotide sequence alignment of the H protein CDS of selected CDV strains retrieved from GenBank. Sequences obtained in this study are marked with a circle.
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suggested to explain the re-emergence of infectious canine hepatitis in Italy (Decaro et al., 2007). In 2013, we possibly observed the spread of this virus from dogs to wildlife (Di Sabatino et al., 2014). Therefore, the detection of Arctic-lineage CDV in 2015 in badgers might suggest that this lineage is becoming endemic in the wildlife population of Abruzzi region. Alternatively, as the Eurasian badger lives in a wide variety of environments including also urban and suburban areas, this Arctic-lineage CDV infection may represent an isolate case of direct transmission from an infected dog. CDVs from badgers, unlike homologous viruses from wolves and dogs of 2013, show H549 in the mature H protein. H549 occurs predominantly in non-dog host species and it has been suggested to have correlation with host tropism. The same substitution was indeed evidenced in previous studies (Origgi et al., 2012; Martella et al., 2010; Monne et al., 2011; Sekulin et al., 2011) from naturally occurring CDV strains isolated from wildlife and, importantly, it was also demonstrated to occur in ferrets experimentally infected with a dog-adapted CDV field isolate (von Messling et al., 2003). Unfortunately we do not possess any genetic information regarding CDV strains detected in 2012 from wildlife as well as from other non-dog host species in the following years. Nevertheless, H549 was also observed in a CDV strain isolated from an infected dog of Apulia region (Dog BA376/13/ITA-Italy) in 2013. A Europeanwildlife lineage CDV strain was also detected in a fox collected in 2016, proving also co-circulation of at least two different geographic lineages in the wildlife of this area. The scenario of CDV introduction and maintenance varies according to the involved geographical areas and animal species. Indeed, once established in the wildlife milieu, CDV circulation may involve a relevant number of wild species, including badgers, as it is the case for Abruzzi and Molise regions. The presence of wildlife nearby villages, urban roads and farms is a common finding in the Abruzzi region; in addition transhumance with shepherd dogs is commonly practiced in proximity of places where wildlife lives, scenario that enhances the risk of direct contact between dogs and wildlife with potential spillover of CDV in both directions (Kapil and Yeary, 2011). Thus virus maintenance in wildlife must be taken into account and vaccination in domestic dogs has to be particularly implemented in areas with intense animal biodiversity and with presence of threatened wild animals (Di Sabatino et al., 2015). As for wildlife, the number of CDV positive animals changed within the years of observation. It ranged from 21.8% of total cases observed in 2013 to 26% observed in 2015. In 2014 the total number of wildlife positive for CDV was 6 (10% of total cases). Up to June 2016, two cases of CDV infection in wildlife have been recorded. Although the number of CDV-positive wildlife is reasonably biased by the fact that only a part of succumbed animals is found, collected and conferred to the IZSAM, it seems that novel CDV outbreaks are occurring in wildlife, phenomenon which has apparently scarce or null effects to the domestic dog population as for the immunity acquired following vaccination. In conclusion, our data confirm distemper as a serious disease for badgers and the potential establishment of Arctic-lineage CDV circulation in wildlife. CDV infection has to be suspected in badgers showing neurological and respiratory symptoms and it needs to be promptly diagnosed and adequately managed. Further investigations are reasonably requested in order to assess the role of badgers for the epidemiology and evolution of distemper in Italy. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.10.020. Conflict of interest The authors disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work.
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