**4.1. Entomological and virological surveillance on mosquitoes**

#### *4.1.1. Piemonte region*

Since 2011, a total of 111.676 adult mosquitoes, divided in 4620 pools, were identified and analysed.

The most abundant species detected was *C. pipiens*, mainly collected by CDC traps followed by *Ochlerotatus caspius*, *Anopheles maculipennis* s.l. and *A. albopictus.*

Details of collected species for years are shown in **Table 1**.

**USUV** was reported since 2011 [52]. After the first detection, it was found every year in fieldcollected mosquitoes (*Cx. pipiens*), confirming the establishment of the virus in the Region.

**WNV Lin2** was detected for the first time in 2014, in Alessandria Province, in two *C. pipiens* pools (August 27, 2014; September 10, 2015).


analysed with bio-molecular assays to confirm the species identity. Since 2017, considering the most abundant number of adults trapped, *A. koreicus* specimens were also screened for

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In 2014, WNV and USUV were detected for the first time in the area. A WNV-positive pool was collected by gravid traps at Genova airport on September 29. Actually, this is the only

USUV was detected in a *C. pipiens* pool in La Spezia Province by gravid traps on September 23. Unlike WNV, in the following years, USUV continued to circulate in the Region, and it was annually reported in mosquitoes. No other flavivirus of medical interest was detected [5].

**Table 2.** Total amount of mosquitoes collected and tested in Liguria region since 2013, sorted by species and number.

**N Pool**

flavivirus.

report of WNV circulation in the Region.

**Mosquito species Total**

*Ae. albopictus* 9054 493 *Ae. koreicus* 26 15 *Aedes spp* 92 11 *An. claviger* 4 4 *An. maculipennis s.l.* 3 3 *An. plumbeus* 10 8 *Anopheles. spp* 1 1 *Cq. richiardii* 19 3 *Cx. hortensis* 5 5 *Cx. impudicus* 21 4 *Cx. mimeticus* 1 1 *Cx. pipiens* 13,191 536 *Culex spp* 184 34 *Cx. territans* 2 2 *Cx. theileri* 3 3 *Cs. annulata* 9 9 *Cs. longiareolata* 381 99 *Culiseta sp.* 2 2 *Oc. caspius* 35 15 *Oc. geniculatus* 1 1 *Oc. mariae* 2 2 *Ochlerotatus spp* 4 4 Total 23,050 1255

**Table 1.** Total amount of mosquitoes collected and tested in Piemonte region since 2011, sorted by species and number.

In 2015, WNV Lin 2 was detected in six pools of *Cx. pipiens*. They were collected with CDC dry ice-baited traps from July in four provinces: Novara (July 29, 2015, and August 26, 2015), Alessandria (August 6, 2015), Vercelli (August 21/26, 2015) and Torino (September 23, 2015).

In 2016 and 2017 in Alessandria and Novara provinces, respectively, positive *Cx. pipiens* pools were found. No other *flavivirus* of medical interest was detected [53, 54].

#### *4.1.2. Liguria region*

Since 2011, a total of 33,244 adult mosquitoes were collected.

Virological investigations were performed since 2013 on 23,050 adult mosquitoes, split in 1255 pools (**Table 2**).

The most abundant species detected was *C. pipiens*, mainly collected by gravid traps followed by *A. albopictus* collected in Bg-Sentinel. Since 2015, in the city of Genova, adult female *A. koreicus* specimens were detected [55]. Pool collected in 2015 and 2016 was analysed with bio-molecular assays to confirm the species identity. Since 2017, considering the most abundant number of adults trapped, *A. koreicus* specimens were also screened for flavivirus.

In 2014, WNV and USUV were detected for the first time in the area. A WNV-positive pool was collected by gravid traps at Genova airport on September 29. Actually, this is the only report of WNV circulation in the Region.

USUV was detected in a *C. pipiens* pool in La Spezia Province by gravid traps on September 23.

Unlike WNV, in the following years, USUV continued to circulate in the Region, and it was annually reported in mosquitoes. No other flavivirus of medical interest was detected [5].


In 2015, WNV Lin 2 was detected in six pools of *Cx. pipiens*. They were collected with CDC dry ice-baited traps from July in four provinces: Novara (July 29, 2015, and August 26, 2015), Alessandria (August 6, 2015), Vercelli (August 21/26, 2015) and Torino (September 23, 2015). In 2016 and 2017 in Alessandria and Novara provinces, respectively, positive *Cx. pipiens* pools

**Table 1.** Total amount of mosquitoes collected and tested in Piemonte region since 2011, sorted by species and number.

**N Pool**

Virological investigations were performed since 2013 on 23,050 adult mosquitoes, split in 1255

The most abundant species detected was *C. pipiens*, mainly collected by gravid traps followed by *A. albopictus* collected in Bg-Sentinel. Since 2015, in the city of Genova, adult female *A. koreicus* specimens were detected [55]. Pool collected in 2015 and 2016 was

were found. No other *flavivirus* of medical interest was detected [53, 54].

Since 2011, a total of 33,244 adult mosquitoes were collected.

**Mosquito species Total**

68 Current Topics in Tropical Emerging Diseases and Travel Medicine

*Ae. albopictus* 8692 875 *An. maculipennis s.l.* 12,126 502 *An. plumbeus* 74 38 *Ae. vexans* 4074 288 *Ae cinereus* 1 1 *Cx. hortensis* 4 2 *Cx. pipiens* 50,660 1672 *Cx. territans* 16 2 *Cx. theileri* 34 10 *Cs.annulata* 6 3 *Cx.modestus* 1621 100 *Cs.subochrea* 14 6 *Cs. longiareolata* 5 4 *Culiseta sp* 2 2 *Oc.caspius* 34,085 1053 *Oc.geniculatus* 198 51 *Oc.cantans* 56 8 *nd* 8 3 Total 111,676 4620

*4.1.2. Liguria region*

pools (**Table 2**).

**Table 2.** Total amount of mosquitoes collected and tested in Liguria region since 2013, sorted by species and number.

## **4.2. Veterinary surveillance**

### *4.2.1. Piemonte region*

Since 2009, in the framework of passive surveillance, 35 suspected equine clinical cases were notified: three in 2014, 24 in 2015, six in 2016 and two in 2017.

the Amedeo di Savoia Hospital, Torino. From 2015 to date, four WNND autochthonous cases were detected: one fatal case in 2015 in Torino Province, one fatal case in 2016 in Novara

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For Liguria Region, the Regional Reference Centre for MBDs diagnosis is located at the

Notified human cases of CHIKV, DENV and ZIKV since 2011 in Piemonte and Liguria Regions

As WNV surveillance involved both Veterinary and Human Public Health Regional Authorities, specific flow charts were prepared and shared among working group with the aim to coordinate the notification of viral circulation triggers and the implementation of con-

> DEN 1 0 ZIK 0 0

> DEN 5 0 ZIK 0 0

> DEN 12 0 ZIK 0 0

> DEN 5 1 ZIK 0 0

> DEN 12 0 ZIK 0 0

> DEN 14 0 ZIK 14 1

> DEN 18 \* ZIK 2 \*

**Table 3.** Notification of Chikungunya (CHIK), dengue (DEN) and ZIKA (ZIK) human cases in Piemonte and Liguria

**Arbovirosis human surveillance Piemonte Liguria** 2011 CHIK 0 0

2012 CHIK 0 0

2013 CHIK 0 0

2014 CHIK 1 0

2015 CHIK 1 0

2016 CHIK 2 0

2017 CHIK 1 \*

\*data not available.

regions from 2011 to 2017 (all imported cases).

Province and two fully recovered cases in 2017 in Asti province [56].

Institute of Hygiene of the Policlinico San Martino Hospital, Genova.

are displayed in **Table 3** (all imported cases).

trol measures. Hierarchy of actions is shown in **Figure 5**.

Nine of the suspected cases were confirmed: six in 2015 (Alessandria Province), one in 2016 (Vercelli province) and two in 2017 (Asti and Cuneo Provinces).

Within the passive surveillance on wild birds that were dead, from 2009 no WNV infection cases were detected.

The active surveillance from 2009 to 2013 on 28 sentinel horses selected in the risk area sited in 'Garzaia di Marengo', annually tested three times during the transmission season, did not detect any seroconversion. In 2014 and 2015, a total of 1819 equines sentinel were screened by ELISA IgM serological test. The only seropositive horse detected (2014) lived in Alessandria Province, within 4 km of radius from the first WNV-positive mosquito pool.

Since 2016, the Surveillance Plan was selectively targeted on the active surveillance of wild, synanthropic and rural birds, and then sentinel horses system was dropped out.

A total of 2451 wild birds were tested by RT-PCR for WNV, mainly corvids species: 14 corvids were confirmed WNV infected, in 3 out of 8 Piemonte Provinces (Alessandria, Torino and Vercelli).

In 2017, the serological monitoring activity on rural poultry in Torino Province allowed to detect 17 WNV positive hens; 4 animals out 17 tested SN positive both for WNV and for USUV by the CESME, providing demonstration of co-circulation of both viruses in the same areas.

#### *4.2.2. Liguria region*

Since 2009, in the framework of passive surveillance, seven suspected clinical cases were notified: none was confirmed by laboratory investigation.

A total of 97 wild birds found dead were virologically tested by RT-PCR for WNV. In 2013, the necropsy of an Eurasian hobby (*Falco subbuteo*), a long-range migratory species, found dead at the end of spring in Imperia Province, accidentally allowed to identify WNV Lin2. This report has not been considered a significant proof of virus circulation, because the infection was probably contracted in Africa. This is to date the only WNV virological positivity in birds in Liguria region.

By active surveillance, since 2014, 611 horses were screened by ELISA IgM serological test: no positivity was detected.

## **4.3. Human surveillance of West Nile virus and others MBDs in Piemonte and Liguria region**

Every WNV or MBDs suspect case was promptly reported to the Regional Public Health Authority. For Piemonte Region, biological samples were transmitted to the Laboratory of Microbiology and Virology, within the Regional Reference Centre for Infectious Diseases of the Amedeo di Savoia Hospital, Torino. From 2015 to date, four WNND autochthonous cases were detected: one fatal case in 2015 in Torino Province, one fatal case in 2016 in Novara Province and two fully recovered cases in 2017 in Asti province [56].

**4.2. Veterinary surveillance**

70 Current Topics in Tropical Emerging Diseases and Travel Medicine

Since 2009, in the framework of passive surveillance, 35 suspected equine clinical cases were

Nine of the suspected cases were confirmed: six in 2015 (Alessandria Province), one in 2016

Within the passive surveillance on wild birds that were dead, from 2009 no WNV infection

The active surveillance from 2009 to 2013 on 28 sentinel horses selected in the risk area sited in 'Garzaia di Marengo', annually tested three times during the transmission season, did not detect any seroconversion. In 2014 and 2015, a total of 1819 equines sentinel were screened by ELISA IgM serological test. The only seropositive horse detected (2014) lived in Alessandria

Since 2016, the Surveillance Plan was selectively targeted on the active surveillance of wild,

A total of 2451 wild birds were tested by RT-PCR for WNV, mainly corvids species: 14 corvids were confirmed WNV infected, in 3 out of 8 Piemonte Provinces (Alessandria, Torino and

In 2017, the serological monitoring activity on rural poultry in Torino Province allowed to detect 17 WNV positive hens; 4 animals out 17 tested SN positive both for WNV and for USUV by the CESME, providing demonstration of co-circulation of both viruses in the same areas.

Since 2009, in the framework of passive surveillance, seven suspected clinical cases were noti-

A total of 97 wild birds found dead were virologically tested by RT-PCR for WNV. In 2013, the necropsy of an Eurasian hobby (*Falco subbuteo*), a long-range migratory species, found dead at the end of spring in Imperia Province, accidentally allowed to identify WNV Lin2. This report has not been considered a significant proof of virus circulation, because the infection was probably contracted in Africa. This is to date the only WNV virological positivity in birds

By active surveillance, since 2014, 611 horses were screened by ELISA IgM serological test: no

Every WNV or MBDs suspect case was promptly reported to the Regional Public Health Authority. For Piemonte Region, biological samples were transmitted to the Laboratory of Microbiology and Virology, within the Regional Reference Centre for Infectious Diseases of

**4.3. Human surveillance of West Nile virus and others MBDs in Piemonte and** 

Province, within 4 km of radius from the first WNV-positive mosquito pool.

synanthropic and rural birds, and then sentinel horses system was dropped out.

notified: three in 2014, 24 in 2015, six in 2016 and two in 2017.

(Vercelli province) and two in 2017 (Asti and Cuneo Provinces).

fied: none was confirmed by laboratory investigation.

*4.2.1. Piemonte region*

cases were detected.

Vercelli).

*4.2.2. Liguria region*

in Liguria region.

**Liguria region**

positivity was detected.

For Liguria Region, the Regional Reference Centre for MBDs diagnosis is located at the Institute of Hygiene of the Policlinico San Martino Hospital, Genova.

Notified human cases of CHIKV, DENV and ZIKV since 2011 in Piemonte and Liguria Regions are displayed in **Table 3** (all imported cases).

As WNV surveillance involved both Veterinary and Human Public Health Regional Authorities, specific flow charts were prepared and shared among working group with the aim to coordinate the notification of viral circulation triggers and the implementation of control measures. Hierarchy of actions is shown in **Figure 5**.


**Table 3.** Notification of Chikungunya (CHIK), dengue (DEN) and ZIKA (ZIK) human cases in Piemonte and Liguria regions from 2011 to 2017 (all imported cases).

Two National Surveillance Plans regulate MDBs management in Italy: the first regarding WNV and USUV, which integrates human and veterinary surveillance and the second regarding MBDs specifically transmitted by *Aedes* mosquitoes, mainly CHIKV, DENV and ZIKV,

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In Piemonte and Liguria Regions (Northwestern Italy), a WNV multisectoral task force, including representatives of Public and Animal Health and Vector Control, was established in 2011 and then strengthened annually according to epidemiological findings. Since 2013, the working group cooperates with experts of four neighbouring Regions, sharing integrated WNV and USUV surveillance guidelines (human, entomologic and veterinary) throughout the whole Po river valley area. The main goal of this standardization has been the reduction of the transmission risk through blood transfusions, quickly implementing local preventive measures when the virus (in animals, vectors or humans) is detected in a specific Province. This allows to definitely decrease the risk of human transmission and consequently results in a significant reduction of health-care costs [58]. The active virological surveillance on mosquitoes and birds is considered an important tool to early detect virus circulation in a specific area, since it has been shown that the virus in mosquitoes and birds appears much earlier than the occurrence of clinical cases in dead-end

Passive surveillance of clinical cases in horses also can be considered a useful tool for the detection of WNV activity, but it will be less sensitive, and a positivity regarding the proof that the cycle of the virus is ended must be considered. Being dead-end hosts, the probability of infection is similar for horses and humans; then, an early detection tool especially in an endemic area cannot be considered. Furthermore, considering that in affected countries, vaccinations of horses are progressively adopted, it is estimated that surveillance in equidae will

The entomological surveillance can be a valuable additional tool also for the surveillance of MBDs caused by non-endemic viruses (CHIKV, DENV and ZIKV) in which humans are the reservoirs. Even if the syndromic surveillance on human cases is considered the most adequate approach to detect the introduction of these viruses, the presence of risk factors, as competent vectors and suitable climatic conditions, can significantly increase the potential risk for the

Indeed, the selection of the most suitable mosquito-trapping method and the identification of areas with major risks of introduction of exotic mosquitoes and pathogens are crucial, and surveillance should be planned in response to a recognized risk and carried out to support subsequent actions. In Piemonte and Liguria, the entomological surveillance in such sites (ports, international airports, international connection points and hospitals) revealed the presence of *A. albopictus*, competent vector of CHIKV, DENV and ZIKV, but no positive mosquito was found. In Liguria region, Genova city, this risk-based surveillance allowed to detect in 2015 the introduction of *A. koreicus*, an Asiatic mosquito which has become invasive in Europe in the recent years, proven to be an effective transmitter of Japanese encephalitis virus, *Dirofilaria immitis* and CHIKV [60]. While the origin of these specimens remains unknown, the presence of *A. koreicus* in such an important commercial and tourism hub is worrisome, as it might strongly accelerate the species' spread in Italy and in the rest of Europe, as already

local transmission in vectors, as happened in Emilia Romagna (2007) and Lazio (2017).

based on the surveillance of both imported and autochthonous human cases.

hosts (humans and equines).

gradually become irrelevant [59].

happened with *A. albopictus*.

**Figure 5.** Flow chart for the management of WNV viral circulation trigger event in Piemonte and Liguria regions, since 2015.

During transmission season, following the detection of WNV viral trigger by the national Integrated Surveillance Plan, the NAT test was progressively introduced throughout affected Provinces of Piemonte Region. According to National legislation, it was stopped on November 30.

In 2014 and 2015, only Alessandria province was involved in blood screening.

In 2016, four (Alessandria, Novara, Torino and Vercelli) of the eight Provinces in the Piemonte Region were involved in NAT testing of blood donors.

In 2017, blood screening was enforced in Novara, Vercelli, Cuneo and Asti Provinces.

Since 2014, a total of 99,882 blood bags were screened by NAT (5172 in 2014; 38,623 in 2015; 35,812 in 2016 and 20,275 in 2017). No infected blood bags were detected.
