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The veterinary experience on West Nile and Usutu viruses in vectors
(mosquitoes) and reservoirs (wild birds) in northern Italy (2013-2014)
Mattia Calzolari1, Romeo Bellini2, Mario Chiari1, Gioia Capelli3, Cristina Casalone4, Michele Dottori1,4
1. Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “B. Ubertini”2. Centro Agricoltura Ambiente “G.Nicoli”
3. Istituto Zooprofilattico Sperimentale delle Venezie3. Istituto Zooprofilattico Sperimentale delle Piemonte Liguria Valle d’Aosta
4. Secretary of the “Società Italiana di Medicina Tropicale”
Vector-borne diseasesFriday 21 November 2014
School of Oriental and African Studies, London, UK
Very complex cycle: WNV was isolated in 51 arthropods, including ticks in the old world (Hubalek 1999), and detected in a wide range of wild birds, and also in mammals and in reptiles
2
From 2010 to 2013 ECDC reported more than 600 human cases in Europe, in 2014 (until September) 55 human cases were reported3
Surveyed areas (I)Since 2008, besides the activities supported by the Ministry of Health at national level, more comprehensive surveillance programs, including mosquito monitoring, are carried out at Regional level.In 2013-2014 this surveillance was active in Emilia-Romagna (ER),Veneto (VEN), Friuli Venezia-Giulia (FVG) Lombardy (LOM), Piedmont (PIE) regions.
4
VEN
FVG LOM
ERPIE
Surveyed areas (II)Pianura Padana
Pianura Padana (or Pianura Padano-Veneta) is the wider plan of Italy (46.000 km2)
It is an homogeneous territory with a characteristic morphology and hydrology
Pianura Padana is a densely populated area, i. e. 20 million inhabitants, 500 per km2
Productive and urbanized areas are abundant, and environment is strongly influenced by human activity
The climate of this area is described as humid subtropical climate (Köppen climate classification)
5
In Northern Italy
• WNV was detected in 1998 at Padule di Fucecchio, Toscana (no Human cases)
• In Emilia-Romagna human cases were reported in 2008-2009 (Lineage I) and in 2013-2014 (Lineage II)
• In Veneto human cases were continuously reported in 2008-2014 period (Lineage I & II)
• In Lombardy human cases were reported in 2008 then in 2013-14
• In Friuli Venezia-Giulia in 2011-2012• No human cases were detected in Piedmont
6
Usutu virusSince 2009, both systems recorded the circulation of Usutu
virus (USUV), a flavivirus closely related to WNV with a not yet defined pathogenic capacity
Vectors: mosquitoes especially Culex (Culex pipiens)
Reservoirs: wild birds
Possible mass mortality in birds (blackbird)(Austria 2001).
WNV differential diagnosis (it can be cross-reactive also in PCR)
Human disease: 2 cases of encephalitis reported in immunosuppressed individuals in Italy in 2009 (Pecorari et al. 2009, Cavrini et al. 2009)
Anti-USUV response in healthy persons were recorded in ER, 4/359 in 2009; 14/6000 in 2010-11 (Gaibani et al. 2012, Pierro et al. 2013)
7
Surveillance targets• Mosquitoes• Birds, active and passive surveillance (mass mortality not
recorded in Europe)• Horses (not applicable after large vaccination campaigns)
8
Source: United States Centers for Disease Control and Prevention, US CDC
USA Northern Italy
Wild birds, actively collected
Humans
Horses
• Collection of mosquitoes (attractive traps carbon dioxide baited) overnight, every two week
•Identified and pooled according to species, place and date of sampling (with a maximum of 200 specimens per pool)
•Pools were ground and an aliquot of the samples were submitted to species specific (USUV and WNV) or genus (flavivirus) specific PCRs•The remaining part was stored for the viral isolation 9
Mosquito surveillance
Sampling of mosquitoes
(CO2 Baited traps)
Sampling of mosquitoes
(CO2 Baited traps)
Identification and pooling (species,
date, sites)
Grinding
Identification and pooling (species,
date, sites)
Grinding
Aliquot for biomolecular
analysis
(specific and genus-PCR)
Aliquot for biomolecular
analysis
(specific and genus-PCR)
Viral isolation on cell culture
Viral isolation on cell culture
Other experimental investigation (whole
genome sequencing,
mosquito vectorial capacity,
production of serological test)
Other experimental investigation (whole
genome sequencing,
mosquito vectorial capacity,
production of serological test)
Remaining part of homogenate stored
at -80 °C
Remaining part of homogenate stored
at -80 °C
POSITIVE PCRPOSITIVE PCR
Epidemiological data
Epidemiological data
Sequencing of obtained amplicons
(GB Blast)
Sequencing of obtained amplicons
(GB Blast)
Mosquito surveillance
10
Public Health Strategy and
Actions
Public Health Strategy and
Actions
Regional surveys features
Stations Max
specimens per pool Region
Activity period 2013 2014
Sampling interval Trap model USUV_PCR
Emilia-Romagna June-September 157* 94 2 weeks CDC-CO2; GT Yes 200
Lombardy June-October 30 31 2 weeks CDC-CO2 2013 Yes
2014 No 200
Veneto May-October 54 29 1 week CDC-CO2 Yes 50
Friuli Venezia-Giulia
May-October 12 10 1 week CDC-CO2 Yes 50
Piedmont July-October 33 40 2 weeks CDC-CO2; BG Yes 50-200
286 204
* 52 sampled for 1-2 timesIn 2014 in Emilia-Romagna and Lombardy only Cx. pipiens and Cx. modestus mosquitoes were tested
11
Sites for mosquito sampling in 2013/2014
12
1 year
both years
Sampled/Tested mosquitoes 2013 2014 Total Sampled Tested Pools Sampled Tested Pools Sampled Tested PoolsAe. cinereus/geminus 111 95 6 0 111 95 6Ae. rusticus 2 2 0 0Ae.(Och.)berlandi 1 1 1 1 1 1Ae.(Och.)cantans 171 74 26 11 182 74 26Ae.(Och.)caspius 44.108 31.842 844 21.350 10.321 412 65.458 42.163 1.256Ae.(Och.)detritus 693 117 19 7 700 117 19Ae.(Och.)flavescens 1 0 1 0 0Ae.(Och.)sticticus 3 3 1 1 4 3 1Ae.(St.)albopictus 4.659 2.648 463 4.110 1.148 120 8.769 3.796 583Ae.geniculatus 214 130 9 131 119 16 345 249 25Ae.koreicus 26 6 5 0 26 6 5Ae.vexans 14.436 11.694 257 3.153 630 43 17.589 12.324 300Aedes spp. 7 7 3 7 14 7 3An.claviger/petragnani 16 10 8 0 16 10 8An.maculipennis s.l. 5.445 2.704 156 3.373 678 81 8.818 3.382 237An.plumbeus 55 35 12 56 12 10 111 47 22Cq.richiardii 335 247 39 63 398 247 39Cs. annulata 246 203 82 70 3 1 316 206 83Cs. subchorea 2 2 2 2 2 2Culiseta sp 2 2 2 2 2 2Cx.hortensis 1 1 1 1 1 1Cx.modestus 2.231 1.450 75 1.054 911 42 3.285 2.361 117Cx.pipiens 684.660 510.773 7.256 435.114 432.237 5.307 1.119.774 943.010 12.563Cx.territans 42 39 5 0 0 0 42 39 5Total 757.461 562.079 9.2680 468.506 446.063 6.036 1.225.967 1.008.142 15.30413
Mosquito results
• In 2013-2014, 1.294.808 mosquitoes belonging to 23 species were sampled and the 77,9% were tested (1.008.142 sorted in 15.304 pools)
• The species Culex pipiens represent 90,3% of the collected mosquitoes
14
Mosquito results
2013 2014 Total
Pools WNV+ Pools WNV+ Tested Pools WNV+
Ae.(Och.)caspius 844 1 412 42.163 1256 1
Cx.modestus 75 1 42 1 2361 117 2
Cx.pipiens 7256 177 5307 128 943.010 12563 305
Total 9268 179 6036 129 100.8142 15304 308
15
• In 2013 WNV was detected in 177 pools (in ER, VEN,LOM)• In 2014 WNV was detected in 129 pools in all surveyed
regions (ER, VEN, LOM, PIE, FVG)• WNV was almost exclusively detected in Cx. pipiens• Possible active involvement of other mosquito tested
positive for WNV need elucidation, particularly for Aedes caspius
16
2013
Mosquito sites sampled and WNV-positive (red) in 2013
17
2014
Mosquito sites sampled and WNV-positive (red) in 2014
Bird surveillanceActive surveillance on sinathropic species (target of control plans), particularly corvids:
• European Magpie (Pica pica)• Hooded Crow (Corvus cornix)• Eurasian Jay (Garrulus glandarius)
Passive surveillance: birds found dead in the field or dead in Wildlife Rehabilitation Centres
18Magpie Hooded Crow Jay
19
Region Active surveillance Activity period Passive
surveillance USUV_PCR
Emilia-Romagna Yes May- November Yes Yes
Lombardy Yes May- November Yes No
Veneto 2013 Yes, 2014 No July-September Yes Yes
Friuli Venezia-Giulia Yes July-September Yes Yes
Piedmont No Yes No
Birds surveillance
20
Moreover, 21 magpies and 6 jays were USUV positive (ER data)
Active birds surveillance
2013 2014 tested WNV+ tested WNV+European Magpie (Pica pica) 1725 115 1772 26Eurasian Jay (Garrulus glandarius) 232 1 440 1Hooded Crow (Corvus cornix) 1113 31 1401 24Common Raven (Corvus corax) - - 2 0
21
• In 2013-14 a total of 1127 (694 in 2013 and 433 in 2014) wild birds were tested
• Species of positive birds reported in the table
• Moreover 32 of these birds were USUV+ (15 in 2013 and 17 in 2014)
Passive birds surveillance2013 2014
Order, Common name, Species nWNV
+ nWNV
+Passeriformes
Blackbird Turdus merula 68 5 67 0European goldfinch Carduelis carduelis 3 2 10 0
Great grey shrike Lanius excubitor 3 1 -Common starling Sturnus vulgaris 44 1 27 0Barn swallow Hirundo rustica 6 1 7 0Common house martin Delichon urbicum 4 1 3 0
Garden warbler Sylvia borin 1 1 -
European serin Serinus serinus 3 1 -Strigiformes
Little owl Athena noctua 32 3 31 1Eurasian scops owl Otus scops 4 1 6 0
Accipitriformes Common kestrel Falcus tinnunculus 33 2 46 2Common buzzard Buteo buteo 6 1 1 0
ColumbiformesDove Streptopelia spp. 48 4 40 4
Other ordersCommon swift Apus apus 25 1 59 0Gull Laridae 26 1 28 0Common pheasant Phasianus colchicus 6 0 4 1
Little egret Egretta garzetta 1 1 -
22
Lineages• Sequences obtained by field samples in 2013-2014
belonged mainly to lineage II• Also lineage I sequences were detected in these period• In previous outbreaks of WNV in Northern Italy only
lineage I strains were detected gi|341869845|gb|J F719067.1|-1_|dnas_scaffold_ID_1_|dnas_scaffold_POS_1
gi|341869845|gb|J F719067.1|-1_|dnas_scaffold_ID_1_|dnas_scaffold_POS_1(4)
gi|268306332|gb|GU011992.2| West Nile virus strain Ita09 complete genome
gi|341869845|gb|J F719067.1|-1_|dnas_scaffold_ID_1_|dnas_scaffold_POS_1(2)
WN_208846/2009-1_|dnas_scaffold_ID_1_|dnas_scaffold_POS_1
gi|341869845|gb|J F719067.1|-1_|dnas_scaffold_ID_1_|dnas_scaffold_POS_1(3)
gi|110338948|gb|DQ786572.1| West Nile virus isolate France 405/04 polyprotein gene complete cds
gi|21929240|gb|AF404757.1| West Nile virus isolate WN Italy 1998-equine complete genome
gi|399764449|gb|J Q928174.1| West Nile virus strain Italy/2011/Livenza complete genome
gi|30349731|gb|AY278442.1| West Nile virus isolate LEIV-Vlg00-27924 complete genome
gi|9930137|gb|AF260969.1|AF260969 West Nile virus strain RO97-50 complete genome
gi|359390908|gb|J N858069.1| West Nile virus isolate Italy/2011/AN-1 complete genome
gi|399764451|gb|J Q928175.1| West Nile virus strain Italy/2011/Piave complete genome
gi|471272485|gb|KC407673.1| West Nile virus isolate Sad/12 complete genome
gi|550822491|gb|KF179640.1| West Nile virus strain Austria/2008_gh complete genome
gi|551681910|gb|KF588365.1| West Nile virus strain Italy/2013/Rovigo/32.1 complete genome
WN_210912_2013
WN_244558_2013
WN_178907_2013
WN_223278-3/2013
100
100
96
89
100
100
100
80
100
99
100
100
0.02
Lineage I
Lineage II2013 -14 strains
Previously detected strains
2013 2014
23
WNNDhuman
cases
WNVpositive
birds
WNVpositive
mosquitopools
Tested birdsTested m
osquitoesWNV neuroinvasive human cases, positive birds, positive mosquito pools in surveyed area
Positive samples and human casesPeriod of detection of WNV-positive pool of mosquito and WNV-positive birds respect to human cases for surveyed Regions
24
2013 2014
WNV detection in mosquitoes WNV detection in birds
25
Estimated cost for Emilia-Romagna region
• If The WNV circulate in an area, tests on the blood bags and on transplanted organs are required
• Costs of these tests were estimated in 2.560.000 euro for the 2009-2013 period in the Emilia-Romagna region (test from 1 July to 30 November in provinces with viral circulation the previous year and provinces with human cases, according to the Surveillance National Plan)
• This sum is higher than the costs of the integrated surveillance system estimated in 2.093.000 euro in Emilia-Romagna for the same period 2009-2013, obtained by entomological surveillance and bird surveillance costs (582.215 euro), plus the blood screening surveillance linked to viral circulation detection at province level (1.512.226 euro)
From: Bellini et al. Eurosurveillance 2014
26
Added values of the surveillance
• Detection of other viruses by genus PCR and sequencing (Tahyna virus, Batai virus)
• Isolation of new viruses• Definition of culicidic fauna of the surveyed area (Anopheles
maculipennis complex, different sibling species with different vectorial competence for Malaria)
• Evaluation of influence of weather factors on the circulation of the detected viruses (in 2013 more dry and hot conditions were recorded in the WNV circulation area)
• Indication of possible reservoir and amplification role of tested birds
Conclusions
• Obtained data highlighted the role of Cx. pipiens as the main vector for WNV and USUV in Northern Italy
• The systems provided epidemiological data and allowed to assess the geographical spread of the viruses providing indication on the potential risk for humans and animals
• Obtained results show a good performance of the two systems in term of sensitivity and early detection of viral circulation; confirming that, if mosquito trapping effort is intensive, these systems are useful and reliable to address the planning of public health policies
27
28
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “B. Ubertini”Lavazza Antonio, Tamba Marco, Zanoni Mariagrazia, Lelli Davide, Giulia Maioli, Francesco Defilippo, Bonilauri Paolo, Galletti Giorgio, Pinna Marco , Moreno Ana, Varisco Giorgio.
Istituto Zooprofilattico Sperimentale delle VenezieMontarsi Fabrizio, Silvia Ravagnan, Stefania Cazzin, Paolo Mulatti, Stefano Marangon, Monne Isabella, Bonfanti Lebana.
Istituto Zooprofilattico Sperimentale Piemonte Liguria Valle d’AostaPautasso Alessandra, Modesto Paola, Prearo Marino, Cristina Radaelli, Iulini Barbara Bertolini Silvia.
Centro Agricoltura Ambiente “G.Nicoli” Albieri Alessandro, Pezzin Alex, Puggioli Arianna, Perulli Simona.
Entostudio Martini Simone.
IPLAMosca Andrea.
Regione Emilia-RomagnaAngelini Paola, Natalini Silvano.
Regione LombardiaGemma Brenzoni Laura, Farioli Marco.
Regione VenetoRusso Francesca, Cester Giorgio .
Regione Friuli Venezia-GiuliaManlio Palei.
Acknowledgements