"Endangered large carnivores and scavenging raptors in
Europe“
Faculty of Veterinary Medicine, Teramo – 13-15 October2016
CANINE DISTEMPER VIRUS
INFECTION IN WOLF IN
ABRUZZO
Cristina E. Di Francesco, Francesca Profeta, Leonardo Gentile, Vincenza
Di Pirro, Simone Angelucci, Marco Innocenti, Fulvio Marsilio
THE DISTEMPER OUTBREAK IN 2013 IN
CENTRAL ITALY
• Wolf (Canis lupis) population
of Parco Nazionale d’Abruzzo,
Lazio e Molise (PNALM)
• N. 24 animals 6 months -5
years old
• Positives for Canine distemper
virus (CDV) by means RT-PCR,
IFI, and IHC test from mucosal
swabs and tissues sample (Di
Sabatino et al., 2014)
THE DISTEMPER OUTBREAK IN 2013 IN
CENTRAL ITALY
THE DISTEMPER OUTBREAK IN 2013 IN
CENTRAL ITALY
• F irst report of CDV Arctic lineage epidemics
in the wild population in Europe
“Wildlife Europe 2006-2009”
(WE/06-09)
Monne et al., 2011
Di Francesco et al., 2012
1. RETROSPECTIVE SURVEY IN DOGS
• GENETIC CHARACTERIZATION OF CDV STRAINS FROM
DOMESTIC DOGS
Conjunctival swabs
Nasal swabs
Rectal swabs
n. 40 samples from
CDV POSITIVE dogs
from 2010 to April
2013
Amplification of H gene using 4 couples of
primers
GENE
PRIMER*
SEQUENCE 5’-3’
(GenBank F378705)
72f
TACTCTGGTCACACGTCTTA-3
74-93
798r
TAGCTCCACTGCATCTGTAT-3
574-555
472f
CCGTACATCACCAAGTCATA-3
248-267
1172r
TAGAACACCACCTTGTGAAC-3
948-929
1113f
GTAGATGAGAGCACCGTATT-3
889-908
1771r
TGTGTAGGCAACACCACT AA-3
1547-1530
1629f
GGAGACCAGTTCACTGTGTAAT-3
1405-1424
2221r
GATGGACCTCAGGATATAGA-3
2223-2204
TARGET
LENGHT
FRAGMENT
H
500 bp
H
700 bp
H
658 bp
H
818 bp
*Sekulin et al., 2011
Sequencing
Phylogenetic Analysis
GENETIC CHARACTERIZATION OF CDV STRAINS
IN DOMESTIC DOGS IN ABRUZZO REGION
Cluster in Arctic lineage,
with sequence of the
CDV wolf strain
•
•
Identity of 100 %
CDV wolf strain
Among CDV strains of dogs
during 2012 - 2013
Identity of 97,11%
strains of 2010
2. MONITORING ACTIVITIES OF WILDLIFE
POPULATION – FOXES IN MOLISE REGION
• October 2012 and January
2013
• 120 fecal samples of foxes
– 104 environmental 16 rectal
swabs
• Eminested RT-PCR for N gene
(Di Francesco et al., 2012)
• 5 pool of fecal samples and n.
2 rectal swabs positives for
CDV
GENETIC CHARACTERIZATION OF CDV FOX
STRAINS IN MOLISE REGION
High identity(99%) with the
CDV wolf strain belonging to
Artict lineage during
2012/2013 in PNALM area
3. MONITORING ACTIVITIES IN WILDLIFE –
FOXES AND MUSTELIDS IN PNALM AREA
• Between May and June
2014
• n. 224 environmental
fecal samples of foxes
(n.100) and mustelids
(n.124)
• Emi-nested RT-PCR for
CDV
3. MONITORAGGIO SANITARIO NELLA FAUNA
SELVATICA - VOLPI E MUSTELIDI NEL PNALM
• Tra la fine di Maggio e gli
inizi di Giugno 2014
• n. 224 campioni
ambientali di feci di Volpe
(n.100) e Mustelidi
(n.124)
• Emi-nested RT-PCR per
CDV
4. MONITORING ACTIVITIES IN WOLVES OF
THE PARCO NAZIONALE DELLA MAJELLA
WOLVES
• September 2010 –
December 2013
• n° 23 fecal samples
collected from 3 rendezvous sites (July-Oct 2013)
• n° 22 fecal samples
collected from other sites of
PNM (Sept 2010-Dec 2013)
DOGS
• Mucosal swabs
– 3 conjunctival
– 2 rectal
– 1 nasal
SEQUENCE DETECTION OF N AND H GENES OF CDV
WOLVES: 6 positive fecal samples
• Emi-nested RT-PCR for CDV
- 5 collected from rendez-vous sites (July-Oct 2013)
- 1 from young wolf (Aug 2012)
CANI: 1 positive rectal swab
Sequencing and analysis of a
180 bp fragment of N gene
for viral identification
Sequencing and analysis of a
323 bp fragment
of H gene (Sekulin et al., 2011)
for characterization
of viral strain
AF478548 Dog Denmark
Z77671.1 Dog Germany Z77672.1 Dog Germany Z77672 Dog Germany
Z77673 Dog Germany
AF178038.1 Giant panda China
AB016776.1 Raccoon dog Japan
EU545142 Fox HLJ1 06 China
Asia 1
AB212964 Dog Japan
AB212963 Dog Japan
AF164967.1 Dog USA
AB252718 Dog Japan
AY297453.1 Dog Japan
AB040768.1 Dog Japan
Asia 2
EU743935 Fox HLJ2 China
AF172411.1 Dog China
GU270850.1 Ferret Germany GU270848.1 Ferret Germany
DQ228166 RedFox Italy
Wildlife
Z47760.1 Greenlandic dog Denmark
X84998.1 Phoca sibirica Siberia
Artico
DQ226087 Dog Italy
Wolf PNM F184989.1 Dog Italy KC966928.1 Wolf Italy
DQ226088 Dog Italy
AY438597.1 Raccoon USA
AF259552.1 Snyder Hill Germany.
Z35493.1 Convac vaccine strain
America 1
AF378705.1 Onderstepoort virus strain
Specific for Arctic lineage
Identity of 100% with similar sequences obtained from CDV infected wolves
of PNALM
DISTEMPER IN PNM AREA
CDV spreading in all sampled wolf packs
• Viral detection in fecal samples collected from rendez-vous sites
• Probable exposition of wolf puppies
• No evidence of clinical signs or mortality episodes in monitored packs
Transmission of CDV infection from dogs to wolves
• Arctic lineage in all strains investigated
• Identity of 100% with strains detected in wolves of PNALM and from 2 dogs
• Simultaneous spreading of virus in dog population of the same area
IN CONCLUSION
• Domestic dog is the
reservoir of CDV infection
from wildlife
• The wolf is the spill-over
of the viral spreading and
new viral strains
• The monitoring of more
abundant species (fox and
mustelids) is an additional
source of information
– Possible adaptation of
new variants in wildlife
• Absence of Y549H
aminoacidic mutation
(Tyrosine
Hystidine) of
H protein observed in
strains adaptated to the
wild host (Monne et al., 2011)
IN CONCLUSION
• New lineages of CDV can be a threat for
endangered or critically endangered species
• The vaccination of domestic dogs is essential
to prevent the loss of the endangered wild
species
CONTRAST TO
STRAY DOGS
VACCINATION
GRAZIE PER L’ATTENZIONE
