l'infezione da canine distemper virus nel lupo appenninico
Transcript of l'infezione da canine distemper virus nel lupo appenninico
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
"Endangered large carnivores and scavenging raptors in Europe“
Faculty of Veterinary Medicine, Teramo – 13-15 October2016
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
• 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
THE DISTEMPER OUTBREAK IN 2013 IN CENTRAL ITALY
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
PRIMER* SEQUENCE 5’-3’ (GenBank F378705)
GENE
TARGET
LENGHT
FRAGMENT
72f
798r
TACTCTGGTCACACGTCTTA-3
TAGCTCCACTGCATCTGTAT-3
74-93
574-555 H 500 bp
472f
1172r
CCGTACATCACCAAGTCATA-3
TAGAACACCACCTTGTGAAC-3
248-267
948-929 H 700 bp
1113f
1771r
GTAGATGAGAGCACCGTATT-3
TGTGTAGGCAACACCACT AA-3
889-908
1547-1530 H 658 bp
1629f
2221r
GGAGACCAGTTCACTGTGTAAT-3
GATGGACCTCAGGATATAGA-3
1405-1424
2223-2204 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
High identity(99%) with the CDV wolf strain belonging to
Artict lineage during 2012/2013 in PNALM area
GENETIC CHARACTERIZATION OF CDV FOX STRAINS IN MOLISE REGION
• 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. MONITORING ACTIVITIES IN WILDLIFE – FOXES AND MUSTELIDS IN PNALM AREA
• 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
3. MONITORAGGIO SANITARIO NELLA FAUNA SELVATICA - VOLPI E MUSTELIDI NEL PNALM
4. MONITORING ACTIVITIES IN WOLVES OF THE PARCO NAZIONALE DELLA MAJELLA
WOLVES
• September 2010 –
December 2013
• n° 23 fecal samples
collected from 3 rendez-
vous 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
• Emi-nested RT-PCR for CDV
WOLVES: 6 positive fecal samples
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
CANI: 1 positive rectal swab
- 5 collected from rendez-vous sites (July-Oct 2013)
- 1 from young wolf (Aug 2012)
GU270845.1 Fox Germany FJ416339.1 Fox Germany FJ416338.1 Badger Germany FJ416336.1 Fox Germany
HM443704.1 Budger TN Italy
JQ966309.1 Marmota Switzer JF810111.1 dog Switzerland W10/339 Fox Switzerland JF810111.1 dog Switzerland
HM443708.1 Stone Martin Italy
GU270844.1 Fox Germany HM443705.1 Fox Italy JF810110.1 Martes Switzer JF810108.1 Fox Switzer JF810106.1 Fox Switzer HM120874.1 Fox 09 Italy FJ416337.1 Fox Germany VIR3514 Fox BZ Italy VIR3498 Badger BZ Italy HM443707.1 Fox Italy HM443709.1 Fox TN Italy
JN153025.1 Fox Germany JN153024.1 Fox Germany JN153025.1 Fox Germany
DQ889177.1 Dog Hungary DQ889177 H04Bp1F Hungary
W10/2733 Lynx Switzerland
Z47765.1 Raccon USA
AY498692.1 Raccoon USA
AY649446.1 Raccoon USA
AF478543 Dog Denmark
HM443723.1 Dog Italy DQ494318 Dog Italy DQ494319 Dog Italy
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
AB212964 Dog Japan
AB212963 Dog Japan
AF164967.1 Dog USA
AB252718 Dog Japan
AY297453.1 Dog Japan
AB040768.1 Dog Japan
EU743935 Fox HLJ2 China
AF172411.1 Dog China
GU270850.1 Ferret Germany GU270848.1 Ferret Germany
DQ228166 RedFox Italy
Z47760.1 Greenlandic dog Denmark
X84998.1 Phoca sibirica Siberia
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
AF378705.1 Onderstepoort virus strain
Asia 1
Asia 2
Artico
Wildlife
America 1
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