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