**3. Materials and methods**

#### **3.1. West Nile virus integrated surveillance system in Northwestern Italy**

Up to 2011, according to the national WNV surveillance programme, just a small area in Piemonte, defined as a 'risk area', in the Provinces of Alessandria and Asti, had to be monitored by entomological surveillance and checking for WNV seroconversion in horses residing in the area. No active surveillance activities were planned in Liguria. Even if any animal nor human cases had not been reported before, geographical and environmental features were conducive to maintaining competent vectors for MBDs.

Since 2011, supplementary surveillance activities have been put in place to control MBDs, with particular attention to WNV.

#### *3.1.1. Area under surveillance: Piemonte and Liguria regions*

Other family members and people should use general precautions for parenterally transmit-

• wash hands with soap and water, before and after assisting the patient, and, in any case,

Risk communication, training, information and health education have a decisive role in

Since there are currently no vaccines and/or therapies for the prevention and treatment of diseases as Chikungunya and Dengue (authorized only in some endemic countries) and Zika,

Currently, therefore, the key message is: 'Protect yourself from mosquito bites' which includes both the active control of the vector (use of insecticides, reduction of breeding sites) and adoption of individual protection measures (clothes, mosquito nets, repellents and also preventive

Risk communication to travellers going to or returning from endemic areas is of primary

**1.** People travelling to endemic areas or endemic countries should inform about the circulation of ongoing epidemics (consulting ECDC and WHO sites and the Safe Travel website of the Ministry of Foreign Affairs and International Cooperation) and protect themselves

**2.** People travelling from endemic areas or endemic countries who develop suspected symp-

Up to 2011, according to the national WNV surveillance programme, just a small area in Piemonte, defined as a 'risk area', in the Provinces of Alessandria and Asti, had to be monitored by entomological surveillance and checking for WNV seroconversion in horses residing in the area. No active surveillance activities were planned in Liguria. Even if any animal nor human cases had not been reported before, geographical and environmental features were

Since 2011, supplementary surveillance activities have been put in place to control MBDs,

toms within 2 weeks of returning home should seek medical attention.

**3.1. West Nile virus integrated surveillance system in Northwestern Italy**

conducive to maintaining competent vectors for MBDs.

the most effective prevention is to reduce people exposure to mosquito bites.

• use gloves, not sterile, if contact is made with the patient's blood;

• do not use sharp products used for patient care or assistance.

measures to avoid sexual transmission, e.g. for Zika virus).

ted diseases, such as:

**Risk communication**

importance:

from mosquito bites.

**3. Materials and methods**

with particular attention to WNV.

obtaining people collaboration.

after removing the gloves if used;

60 Current Topics in Tropical Emerging Diseases and Travel Medicine

The study area includes Piemonte and Liguria regions, in Northwestern Italy (**Figure 2**).

Piemonte region is the second largest Italian Region by geographical area (25.402 km<sup>2</sup> ) and comprises eight Provinces. It is surrounded on three sides by the Alps. About 41% of the Region is mountainous (prealpine and alpine) and 59% is hilly or flat. It is crossed from the West to the East by the Po river and bounded to the East by the Ticino river. Surrounding highly urbanized areas are intensive agriculture farmlands; they cover approximately 960,000 hectares where cereals and forage are predominantly cultivated; extensive rice fields dominate the landscape in the northeast part of the Region.

Liguria is one of Italian smallest Regions. It is divided into four Provinces. It is a narrow strip of land (5.416 km<sup>2</sup> ), highly urbanized, overlooking the Mediterranean Sea: the ring of Maritime Alps and Ligurian Apennines beyond the narrow strip of coast descends almost immediately to a considerable marine depth. It represents an important touristic and commercial area.

Characteristic rocky coasts and seascape attract many travellers to spend the holidays in the most famous tourist resorts along the Italian Riviera. The Port of her capital City Genova, with a trade volume of 58.6 million tonnes, is the first port of Italy. The invasive alien tiger mosquito (*Aedes albopictus*), which is an important vector of viral MBDs, was firstly detected in Italy in the city of Genova in 1990.

Piemonte and Liguria Region neighbours on areas where WNV is historically endemic: Camargue and Var in Southeastern France [36] to the West, Lombardy and Emilia Romagna Regions in Italy to the East.

**Figure 2.** Map of Italy with regional boundaries. Piedmont and Liguria in the upper left, in white colour.

In both Regions, the climate is warm temperate, and socioeconomic and geographic features described earlier provide ideal habitats for potential mosquito vector species for MBDs.

located in sampling stations considered at a higher risk level for the introduction of exotic

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Prior to 2011, no entomological surveillance activities were conducted in Liguria Region.

Since 2011, a Regional entomologic surveillance plan was carried out during seasonal mosquito activity with the goals to gather entomological and virological data and early detection

**Figure 3.** Map of Piemonte region with the grid of 20 x 20 km quadrants. Grey dots represent trapping sites. The eight

species and exotic pathogens. Each trap worked for a 24-h period.

of the introduction of invasive vectors and MDBs.

provinces are named in uppercase letters in textboxes.

*3.1.2.2. Liguria region*

#### *3.1.2. Entomological surveillance on mosquitoes*

Entomological surveillance was activated from July to the end of October through adult mosquito collection with fortnight samplings. After every daily catch, mosquitoes were collected from each trap, and then they were transported in refrigerated boxes to the laboratories. Adult females were counted and identified to the species level, using a stereomicroscope and morphological standard classification keys [37]. After identification, mosquitoes were pooled in groups of up to 100 specimens each, sorted by species, date and site of collection, frozen, and then stored at −80°C for virological investigation.

#### *3.1.2.1. Piemonte region*

Prior to 2011, Piemonte had no a widespread entomological surveillance system for public health purposes. A local mosquito-fighting programme was carried out since 1997 for nuisance issues, only in eastern areas where rice is extensively cultivated nearby human settlements [38].

Besides the active surveillance in the 'risk area' established by the WNV National Surveillance Plan, a Region-wide systematic mosquito collection system has been carried out during seasonal mosquito activity since 2011 to gather entomological and virological data to support the Regional MDBs monitoring programme.

The Region was mapped into 73 equal quadrants (20 km x 20 km) as a proxy for geographical area, where the quadrant was defined as the epidemiological unit.

Trapping sites were selected according to risk factors of mosquito spread and the epidemiological cycle for MBDs: proximity to wetland zones, the presence of hosts, commercial areas and touristic routes (ports, airports and freight terminals), different land use and habitat features. At least one trap was placed in each quadrant at an elevation below 600 m. a.s.l., because most anthropophilic mosquito species, potential vectors of MBD, generally inhabit areas below 600 m. a.s.l. in Italy [39].

All trapping sites were mapped by latitude and longitude on a geographical positioning system (**Figure 3**).

Two types of traps were employed to collect adult mosquitoes:



#### *3.1.2.2. Liguria region*

In both Regions, the climate is warm temperate, and socioeconomic and geographic features described earlier provide ideal habitats for potential mosquito vector species for MBDs.

Entomological surveillance was activated from July to the end of October through adult mosquito collection with fortnight samplings. After every daily catch, mosquitoes were collected from each trap, and then they were transported in refrigerated boxes to the laboratories. Adult females were counted and identified to the species level, using a stereomicroscope and morphological standard classification keys [37]. After identification, mosquitoes were pooled in groups of up to 100 specimens each, sorted by species, date and site of collection, frozen,

Prior to 2011, Piemonte had no a widespread entomological surveillance system for public health purposes. A local mosquito-fighting programme was carried out since 1997 for nuisance issues, only in eastern areas where rice is extensively cultivated nearby human settlements [38]. Besides the active surveillance in the 'risk area' established by the WNV National Surveillance Plan, a Region-wide systematic mosquito collection system has been carried out during seasonal mosquito activity since 2011 to gather entomological and virological data to support the

The Region was mapped into 73 equal quadrants (20 km x 20 km) as a proxy for geographical

Trapping sites were selected according to risk factors of mosquito spread and the epidemiological cycle for MBDs: proximity to wetland zones, the presence of hosts, commercial areas and touristic routes (ports, airports and freight terminals), different land use and habitat features. At least one trap was placed in each quadrant at an elevation below 600 m. a.s.l., because most anthropophilic mosquito species, potential vectors of MBD, generally inhabit

All trapping sites were mapped by latitude and longitude on a geographical positioning sys-

trap): they attract adult females searching for a blood meal and are routinely used as the most common sampling method in WNV surveillance programmes in many Regions in the world.

attractant contains a combination of substances found on human skin that strongly attract anthropophilic mosquitoes (mainly belonging to the *Aedes* genus). Nevertheless, with the

, they are adapted to the collection of *Culex* mosquito species [40]. They were

) (approximately 0.5 kg/

(approximately 0.5 kg/trap): the BG-Lure

area, where the quadrant was defined as the epidemiological unit.

Two types of traps were employed to collect adult mosquitoes:



Each trap worked for a minimum of 12-h periods from sunset to sunrise.

*3.1.2. Entomological surveillance on mosquitoes*

62 Current Topics in Tropical Emerging Diseases and Travel Medicine

Regional MDBs monitoring programme.

areas below 600 m. a.s.l. in Italy [39].

tem (**Figure 3**).

addition of CO2

*3.1.2.1. Piemonte region*

and then stored at −80°C for virological investigation.

Prior to 2011, no entomological surveillance activities were conducted in Liguria Region.

Since 2011, a Regional entomologic surveillance plan was carried out during seasonal mosquito activity with the goals to gather entomological and virological data and early detection of the introduction of invasive vectors and MDBs.

**Figure 3.** Map of Piemonte region with the grid of 20 x 20 km quadrants. Grey dots represent trapping sites. The eight provinces are named in uppercase letters in textboxes.

As the Regional territory displays characteristic feature of a narrow hilly coastal strip, trapping sites were located along the coastline. Locations were selected according to risk factors for the introduction and spread of exotic invasive mosquitoes and MBDs: habitat features, proximity to commercial areas and touristic routes (ports, airports and freight terminals) and presence of hosts.

The surveillance system has undergone further updates over time, increasing the number of trapping sites in order to best adapt to the evolving epidemiological scenario of the Region.

Particular attention was paid to the city of Genova, due to the presence of several risk factors of importing invasive mosquitoes and viral diseases: the port, the airport and the largest ethnic Latin America Ecuadorian community outside Ecuador itself. In the last decade, South America recorded changes in spatio-temporal distribution of infectious diseases, including MBDs due to globalization and climate changes [41, 42].

All trapping sites were mapped by latitude and longitude on a geographical positioning system (**Figure 4**).

> All WNV and USUV positive pools were sent to the National Reference Centre for Animal Exotic Diseases (CESME, Teramo) for confirmation, sequencing and determination of Lineage. In addition, *A. albopictus* pools collected in sites considered at a higher risk level for the introduction of exotic invasive species and exotic pathogens (annually revaluated) were tested using a panflavivirus end-point RT-PCR, targeting the conserved Region of the NS5 gene

> **Figure 4.** Map of Liguria region with the grid of 20 x 20 km quadrants. Grey dots represent trapping sites. The four

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The SuperScript\_ III Platinum\_ One-Step qRT-PCR Kit (Thermo Fisher Scientific) was used to detect other flaviviruses of extreme importance, such as Dengue or Zika as previously

According to WNV National Surveillance system [32], a passive clinical surveillance of all neurological signs in equine, and monitoring of wild birds found dead have been carried out in the study area. Necropsy of dead animals and virological molecular testing by real-time RT-PCR was performed on EDTA whole blood (live or dead animals) or on pooled samples of target organs (brain, heart, spleen and kidney) (dead animals). All samples were analysed by a TaqMan® One-Step RT-PCR protocol distinctive for WNV Lineage 1 and Lineage 2 with WN-LCV-F1 and WN-LCV-R1 primers [44] and a TaqMan® One-Step RT-PCR protocol for

Active surveillance activities have been updated according to the epidemiologic scenario of

In 2009, the WNV National Surveillance plan introduced serological screening of horses in a small wetland area in southeast Piemonte Region. The area named 'Garzaia di Marengo' was classified as at risk for WNV introduction due to the presence of waterfowls, including species

sequences.

described by Scaramozzino et al. [48].

provinces are named in uppercase letters in textboxes.

USUV with USU F and USU R primers [45].

the previous year in each region.

*3.1.4.1. Piemonte region*

of migratory birds.

*3.1.4. Veterinary surveillance*

Three types of traps were employed to collect adult mosquitoes:


#### *3.1.3. Virological surveillance on mosquitoes in Piemonte and Liguria regions*

Virological surveillance was performed since 2011 in Piemonte and 2013 in Liguria, focusing on mosquitoes collected from a selection of traps, updated every year, according to risk-based factors. In 2016 and 2017, mosquitoes collected at least from a trap in each quadrant (20 × 20) were analysed. Pools were homogenized in phosphate-buffered saline (600 μl if pool of <30 mosquitoes or 1200 μl if pool of >30 mosquitoes) in a 2-ml microtube with round copper beads.

Total RNA was extracted from mosquito samples according to the RNeasy Mini kit (Qiagen) manufacturer's instructions, with an *automated QIAcube protocol*.

All pool were analysed by a TaqMan® One-Step RT-PCR protocol distinctive for WNV Lineage 1 and Lineage 2 with WN-LCV-F1 and WN-LCV-R1 primers [44] and a TaqMan® One-Step RT-PCR protocol for USUV with USU F and USU R primers [45].

On positive pools, two traditional RT-PCRs for the amplification of WNV [46] and USUV [47] were carried out. Amplicons of the expected size (408 and 425 bp, respectively) were sequenced using the Big Dye Terminator kit v 3.1 (Lifetecnologies) and run on a ABI3130 Genetic Analyzer (Applied Biosystems). The related sequences were employed to perform a basic local alignment search tool (BLAST) in the GenBank library to confirm the specificity of positive reaction and to estimate the degree of identity of detected strains.

Mosquito-Borne Diseases and 'One Health': The Northwestern Italian Experience http://dx.doi.org/10.5772/intechopen.78985 65

**Figure 4.** Map of Liguria region with the grid of 20 x 20 km quadrants. Grey dots represent trapping sites. The four provinces are named in uppercase letters in textboxes.

All WNV and USUV positive pools were sent to the National Reference Centre for Animal Exotic Diseases (CESME, Teramo) for confirmation, sequencing and determination of Lineage.

In addition, *A. albopictus* pools collected in sites considered at a higher risk level for the introduction of exotic invasive species and exotic pathogens (annually revaluated) were tested using a panflavivirus end-point RT-PCR, targeting the conserved Region of the NS5 gene sequences.

The SuperScript\_ III Platinum\_ One-Step qRT-PCR Kit (Thermo Fisher Scientific) was used to detect other flaviviruses of extreme importance, such as Dengue or Zika as previously described by Scaramozzino et al. [48].

#### *3.1.4. Veterinary surveillance*

As the Regional territory displays characteristic feature of a narrow hilly coastal strip, trapping sites were located along the coastline. Locations were selected according to risk factors for the introduction and spread of exotic invasive mosquitoes and MBDs: habitat features, proximity to commercial areas and touristic routes (ports, airports and freight terminals) and

The surveillance system has undergone further updates over time, increasing the number of trapping sites in order to best adapt to the evolving epidemiological scenario of the Region. Particular attention was paid to the city of Genova, due to the presence of several risk factors of importing invasive mosquitoes and viral diseases: the port, the airport and the largest ethnic Latin America Ecuadorian community outside Ecuador itself. In the last decade, South America recorded changes in spatio-temporal distribution of infectious diseases, including

All trapping sites were mapped by latitude and longitude on a geographical positioning sys-

• gravid traps baited with hay infusion added with yeast (almost 2.5 l): these traps attract *Culex* females that have blood fed and are ready to lay their eggs [43] in sites containing water high in organic matter, increasing together the likelihood of collecting infected mos-

Virological surveillance was performed since 2011 in Piemonte and 2013 in Liguria, focusing on mosquitoes collected from a selection of traps, updated every year, according to risk-based factors. In 2016 and 2017, mosquitoes collected at least from a trap in each quadrant (20 × 20) were analysed. Pools were homogenized in phosphate-buffered saline (600 μl if pool of <30 mosquitoes or 1200 μl if pool of >30 mosquitoes) in a 2-ml microtube with round copper

Total RNA was extracted from mosquito samples according to the RNeasy Mini kit (Qiagen)

All pool were analysed by a TaqMan® One-Step RT-PCR protocol distinctive for WNV Lineage 1 and Lineage 2 with WN-LCV-F1 and WN-LCV-R1 primers [44] and a TaqMan® One-Step

On positive pools, two traditional RT-PCRs for the amplification of WNV [46] and USUV [47] were carried out. Amplicons of the expected size (408 and 425 bp, respectively) were sequenced using the Big Dye Terminator kit v 3.1 (Lifetecnologies) and run on a ABI3130 Genetic Analyzer (Applied Biosystems). The related sequences were employed to perform a basic local alignment search tool (BLAST) in the GenBank library to confirm the specificity of

and BG sentinel traps baited with BG-Lure and CO2

, with the

MBDs due to globalization and climate changes [41, 42].

64 Current Topics in Tropical Emerging Diseases and Travel Medicine

Three types of traps were employed to collect adult mosquitoes:

*3.1.3. Virological surveillance on mosquitoes in Piemonte and Liguria regions*

manufacturer's instructions, with an *automated QIAcube protocol*.

RT-PCR protocol for USUV with USU F and USU R primers [45].

positive reaction and to estimate the degree of identity of detected strains.

presence of hosts.

tem (**Figure 4**).

beads.

• CDC traps baited with CO2

same rules as in Piemonte Region;

quitoes. Each trap worked for a 24-h period.

According to WNV National Surveillance system [32], a passive clinical surveillance of all neurological signs in equine, and monitoring of wild birds found dead have been carried out in the study area. Necropsy of dead animals and virological molecular testing by real-time RT-PCR was performed on EDTA whole blood (live or dead animals) or on pooled samples of target organs (brain, heart, spleen and kidney) (dead animals). All samples were analysed by a TaqMan® One-Step RT-PCR protocol distinctive for WNV Lineage 1 and Lineage 2 with WN-LCV-F1 and WN-LCV-R1 primers [44] and a TaqMan® One-Step RT-PCR protocol for USUV with USU F and USU R primers [45].

Active surveillance activities have been updated according to the epidemiologic scenario of the previous year in each region.

#### *3.1.4.1. Piemonte region*

In 2009, the WNV National Surveillance plan introduced serological screening of horses in a small wetland area in southeast Piemonte Region. The area named 'Garzaia di Marengo' was classified as at risk for WNV introduction due to the presence of waterfowls, including species of migratory birds.

A fixed number of seronegative unvaccinated equines sentinel (28 animals: 10% of prevalence, 95% confidence level) was selected in order to uniformly cover the study area. Horses were sampled three times during the transmission season: immediately before (month of May), at seasonal peak (last week of August) and at the end of the transmission season (last week of September), with the aim to monitor the raising of WNV-specific IgM antibodies that are related to recent infection by commercial competitive ELISA. Positive samples were confirmed by seroneutralization assay (SN) and IgM ELISA at the CESME. Seroconversion was confirmed if SN titre was at least 1:10 and there was evidence of IgM antibodies [49].

Local Health Authorities must establish an active surveillance system against human MBDs during the transmission season. Furthermore, passive surveillance has to be set up during the whole year, requesting physicians to report all possible, probable and confirmed cases according to national case definition [32]. Surveillance against Zika virus infection includes investigation on neurological disorders such as Guillain-Barrè syndrome in adults and micro-

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Every case confirmed by the Regional and National Reference Laboratories has to be subjected to epidemiological investigation with the aim to establish any local viral circulation. Moreover, during the transmission season, measures for vector control have to be imple-

According to the blood directive, if veterinary or human cases of WNV are detected, immediate WNV NAT screening of all blood and haematopoietic stem cell donations of solid organ donations is introduced in affected areas (provinces). Every suspected case of Chikungunya or Zika virus has to be temporarily deferred from donations for 28 days start-

In addition to surveillance actions, another important item of the MBDs surveillance is the information activity to raise awareness on population on the links between mosquitoes and

Information campaigns focused on personal protection measures against mosquito bites, especially for pregnant women, and mosquito fighting were conducted in Piemonte and Liguria since 2014 due to WNV spread in Northern Italy and in 2016 due the Zika virus

Training courses have been organized for public health physicians and veterinarians in order to early detect any MBDs clinical suspected case. Advertising as poster and leaflets was shared

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

The most abundant species detected was *C. pipiens*, mainly collected by CDC traps followed

**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*

in hospitals, offices of health authorities and travel medicine centres.

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

by *Ochlerotatus caspius*, *Anopheles maculipennis* s.l. and *A. albopictus.*

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

pools (August 27, 2014; September 10, 2015).

cephaly in infants.

mented promptly.

ing to recovery [51].

the diseases they can transmit.

emergency in South America.

**4. Results**

analysed.

*4.1.1. Piemonte region*

Since 2014, the sentinel horses system in 'Garzaia di Marengo' was replaced by a one-shot random sampling of horses in order to detect IgM antibodies suggestive of WNV early infection. The number of animals to be tested for each Province during the transmission season was assigned based on the provincial equine census, as recorded in National Livestock Database [50].

Since 2015, after the detection of WNV circulation in mosquito pools and in horses in Piemonte Region, veterinary surveillance procedures were revised, and the Region has been considered as WNV Endemic Area by the National Surveillance programme.

Serological testing on horses was suspended while active virological surveillance on synanthropic non-migratory birds was implemented. From April to November, synanthropic birds (magpies—*Pica pica* and hooded crows—*Corvus cornix*), in the framework of the Regional control programme against pest birds for cultivated land protection, were weekly captured by Larsen traps and then killed or directly shot by trained hunters. Samples of organs (brain, heart and kidney) of each bird were pooled and tested for WNV and USUV.

All samples were analysed by a TaqMan® One-Step RT-PCR protocol distinctive for WNV Lineage 1 and Lineage 2 with WN-LCV-F1 and WN-LCV-R1 primers [44] and a TaqMan® One-Step RT-PCR protocol for USUV with USU F and USU R primers [45].

In 2017, the control programme against synanthropic birds was not activated in Torino Province. Then, as alternatively recommended by the National Surveillance Plan, a specific serological monitoring activity was enforced on rural poultry, with the aim to detect IgG antibodies by commercial ELISA test.

#### *3.1.4.2. Liguria region*

Since 2014, Liguria Region was included in WNV National Surveillance plan by serological random sampling of horses in order to detect IgM antibodies suggestive of WNV early infection. The number of animals to be tested for each Province during the transmission season was assigned based on the provincial equine census, as recorded in National Livestock Database [50].

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

Human surveillance activities against MBDs in Piemonte Region are regulated by both the National Surveillance Plan of imported and autochthonous MBDs (CHIKV, DENV and ZIKV), annually published by MoH and by National Integrated Surveillance Programme against WNV and USUV.

Local Health Authorities must establish an active surveillance system against human MBDs during the transmission season. Furthermore, passive surveillance has to be set up during the whole year, requesting physicians to report all possible, probable and confirmed cases according to national case definition [32]. Surveillance against Zika virus infection includes investigation on neurological disorders such as Guillain-Barrè syndrome in adults and microcephaly in infants.

Every case confirmed by the Regional and National Reference Laboratories has to be subjected to epidemiological investigation with the aim to establish any local viral circulation. Moreover, during the transmission season, measures for vector control have to be implemented promptly.

According to the blood directive, if veterinary or human cases of WNV are detected, immediate WNV NAT screening of all blood and haematopoietic stem cell donations of solid organ donations is introduced in affected areas (provinces). Every suspected case of Chikungunya or Zika virus has to be temporarily deferred from donations for 28 days starting to recovery [51].

In addition to surveillance actions, another important item of the MBDs surveillance is the information activity to raise awareness on population on the links between mosquitoes and the diseases they can transmit.

Information campaigns focused on personal protection measures against mosquito bites, especially for pregnant women, and mosquito fighting were conducted in Piemonte and Liguria since 2014 due to WNV spread in Northern Italy and in 2016 due the Zika virus emergency in South America.

Training courses have been organized for public health physicians and veterinarians in order to early detect any MBDs clinical suspected case. Advertising as poster and leaflets was shared in hospitals, offices of health authorities and travel medicine centres.
