WEST NILE VIRUS SURVEILLANCE PROGRAM IN SERBIA

Serological and virological examination of the presence of human and animal infection caused by West Nile Virus (WNV) as well as the presence of the virus in vectors, which has been conducted during the past few years, confi rmed an active virus circulation in the territory of the Republic of Serbia. Based on the obtained results and anticipated intense circulation of WNV, which poses substantial risks for both public and animal health in Serbia, and having in mind its crucial role in the protection of public health, Veterinary Directorate of the Ministry of Agriculture and Environmental Protection infront the Veterinary Service launched and funded the national WNV monitoring program starting from April 2014. Th e Program encompassed the entire territory of the Republic of Serbia and was conducted by scientifi c and specialized veterinary institutes and fi eld veterinary service in close collaboration with qualifi ed entomologists and ornithologists. Th e principal objective of the monitoring – surveillance program is early detection of WNV in monitored regions, timely reporting of the virus presence and activation of human health service institutions and local authorities aimed at establishing the control measures eradication of mosquitoes, informing the local community and taking all relevant preventive measures for human health protection. Th e surveillance program of the WNV occurrence and spread is based on direct and indirect surveillance of WNV in natural environment. Indirect surveillance encompasses serological testing of seronegative sentinel horses and poultry for the presence of WNV infection, and it is performing continuously and periodically during the most 1* Corresponding author: Tamaš Petrović, tomy@niv.ns.ac.rs Arhiv veterinarske medicine, Vol. 7, No. 2, 29 45, 2014 Petrović T. et al.: West Nile virus ...


INTRODUCTION
West Nile virus (WNV) is a neurovirulent mosquito-borne Flavivirus with zoonotic potential, which is maintained in nature in an enzootic transmission cycle between avian hosts and ornithophilic mosquito vectors.Th e virus occasionally infects other vertebrates, including humans and horses, in which it may cause sporadic disease outbreaks that may result fatal.West Nile virus (WNV) was fi rst isolated from a febrile woman in the West Nile district of Uganda in 1937 (Smithburn et al., 1940) and today is considered as the most widespread fl avivirus in the world, endemic in Africa, Asia, Europe, Middle East, Australia and Americas (Trevejo and Eidson, 2008;Calistri et al., 2010;Weissenböck et al., 2010;Papa et al., 2011).
WNV infections have been described in a wide variety of vertebrates (Komar et al., 2003).Th e virus is maintained in an enzootic cycle between ornithophilic mosquitoes, mainly of the Culex genus (Hayes et al., 2005;Ziegler et al., 2012), but also Aedes and Ochlerotatus genus, and certain wild bird species (Savini et al., 2012;Ziegler et al., 2012).WNV was found in more than 150 species of wild and domestic birds (van der Meulen et al., 2005).Wild birds are important to public health because birds migrating across national and intercontinental borders and becoming a long-range virus vectors (Linke et al., 2007).Following infection, many bird species produce levels of viraemia that are suffi cient for transmitting the virus to mosquitoes (Komar et al., 2003).Human and mammals, especially horses, are occasional, dead end hosts and play limited roles in the natural cycle because viraemia is generally too low to infect mosquitoes (Dauphin et al., 2004;Valiakos et al., 2011), however severe neuroinvasive disease and occasionally with fatal outcomes can occur.
In Europe, until the 1990´s WNV had caused sporadic outbreaks with rare reports of encephalitis but its epidemiological behaviour changed when it reemerged in Romania, Russia and the Mediterranean basin causing dozens of humans and horses deaths (Castillo-Olivares and Wood, 2004;Blitvich, 2008;Calistri et al., 2010).Also, only recently the strains of WNV lineage 2 were identifi ed in Europe: in 2004  In Serbia, WNV situation was mostly unknown until 2009.Serological testing of horses sampled during 2009-2010 by ELISA based on WNV recombinant envelope E (rE) protein and PRNT showed for the fi rst time in Serbia that 12% of 349 horses from northern part of country presented specifi c neutralizing WNV antibodies.Positive horses were found in 14 of the 28 municipalities studied, which are up to 200 km distant (Lupulović et al., 2011).In another study, presence of WNV specifi c antibodies was found in 28.6% (72) of 252 examined horse sera samples collected from 7 diff erent stables and locations in Vojvodina province and Belgrade area, during 2007-2011.WNV seroprevalence ranged per stable from 13.3% up to 40% seropositive animals (Medić et al., 2014).In addition, just one year later, to asses WNV presence in the environment immediately aft er the human WNV outbreak in 2012, during November and December of 2012, presence of anti-WNV IgG antibodies were examined by commercial ELISA test in blood sera samples of 130 horses from 6 stables and 1 settlement in Vojvodina province, northern Serbia.Positive results were obtained in 49.23% (64/130) samples.Per stable, percent of seropositive animals was from 35% to 64% (Petrović et al., 2014).Th is prevalence (49.23%) obtained in horses during 2012 was much higher than that found in horses during 2009 and 2010 (12%), including the confi rmed seroconversion in at least 8 horses tested negative in 2010, thus confi rming an intensive WNV circulation in 2012 on the territory of Serbia (Petrovic et al., 2014).Similarly, 96 horses from 5 tested stables during 2012 were tested again during 2013 with the same methodology.High prevalence of 46.88% (ranged between stables from 23.53-75.0%)with new cases of seroconversion were detected also indicating an intensive WNV circulation in 2013 (unpublished data).
WNV circulation in Serbia was also confi rmed in wild birds as virus natural host.In total, 92 blood sera and 81 pooled tissue samples were collected from 133 dead and live captured wild resident and migratory birds (45 species within 27 families) from January until September 2012 in Vojvodina Province -northern part of Serbia.WNV antibodies were detected by ELISA and PRNT in 7.6% (7/92) blood sera and virus presence was confi rmed in tissue of 8 out of 81 (9.87%) and blood of one bird.Most of the antibody or virus positive birds were strictly resident, suggesting endemic presence of WNV in Serbia (Petrovic et al., 2013).By phylogenetic analysis of genomic sequences, all WNV isolates were classifi ed as a lineage 2 strains that clusters with the viruses responsible for the most recent human and animal outbreaks reported in neighbouring countries (Petrovic et al., 2013).
Th e fi rst studies on the presence of WNV in mosquitoes as virus vectors date back to the period 2005-2010.A total of 56757 mosquitoes (841 pools of 50 individual insects) originating from 66 localities in 29 settlements in Vojvodina were examined.Th e presence of WNV genome was established in only three pooled-samples of mosquitoes collected during 2010 in the territory of Detelinara (part of the city of Novi Sad).Th e isolate was typed as lineage 2 WNV (Petric et al., 2012).Th is study was furher done during 2012 and 2013, when signifi cantly increased prevalence of WNV in mosquitoes was established.Even more than 9% of the mosquito pools examined during 2012-2013, mainly of species Culex pipiens was tested positive for WNV presence (unpublished data).
Th e history of WNV infection among human population in Serbia is mostly unknown, and only scarce historical data exists.First serological investigation of WNV infection presence in human population in Serbia was conducted in 1972 and antibodies against WNV were found in 2.6% -4.7% of human sera (Bordjoški et al., 1972).In another study, antibodies against WNV were detected, depending on location, in 1 to 8% of tested human sera in Serbia (Vesenjak-Hirjan et al., 1991).Aft er a gap of many years, more recent serological examinations show presence of anti-WNV IgG antibodies in 18 out of 451 (3.99%) human collected from 2005 to 2010 in Vojvodina province with yearly rates varying between 1.97% and 6.04% (Petrić et al., 2012).Except this data, as to our knowledge, no clinical manifestation of disease was ever reported in Serbia until 2012.In August 2012, an outbreak of WNV infection in humans was reported for the fi rst time ever in Serbia (EpiSouth Weekly Epi Bulletin -N°232 and N°240, 2012; ECDC, 2012), being the fi rst time that WNV infections in the country have been associated with clinical symptoms.As of November 30, 2012, a total of 71 West Nile fever cases were reported, among which 42 were clinically and laboratory confi rmed, and in 9 cases resulted fatal (lethality of 12.7%).All the cases were detected in central and northern part of the country, 72% of them in the Beograd district (ECDC, 2012;Obrenovic et al., 2013;Popovic et al., 2013).Th is epidemic continued, and became even more severe during 2013.As of November 2nd, 2013, a total of 303 West Nile fever cases were reported, among which 202 were clinically and laboratory confi rmed, and 103 were classifi ed as probable cases.Infection in 35 cases resulted fatal (lethality of 11.6%).Almost all of the cases were also detected in central and northern part of the country (Institute of Public Health of Serbia, 2014).
Th e aforementioned serological and virological examinations confi rmed active circulation and endemic presence of WNV in the territory of the Republic of Serbia.Based on the obtained results and anticipated intense circulation of WNV that poses substantial risks for both public and animal health in Serbia, and having in mind its crucial role in the protection of public health, Veterinary Directorate of the Ministry of Agriculture and Environmental Protection infront the Veterinary Service launched and funded the national WNV surveillance program starting from April 2014.Th e methodology of implementation and management of this surveillance program is presented in this article.

METHODOLOGY OF WNV SURVEILLANCE PROGRAM IN SERBIA
Th e surveillance program encompassed sentinel species (poultry and horses), mosquitoes (particularly species Culex pipiens, which were confi rmed as most prevalent WNV vectors in our region) and wild bird species, which are natural virus reservoirs and populate the natural habitats in Serbia, either temporarily or permanently.Th e surveillance program was conducted throughout the year according to the provided guidelines.Active surveillance was performed by serological examination of sentinel poultry and horses and by testing of virus presence in samples of mosquito vectors (sampled by dry-ice baited traps in the period of most prominent vector activity using special traps), as well as in the samples of all collected dead wild birds belonging to the species susceptible to WNV (tested throughout the year).Passive surveillance encompassed serological (testing of paired serum samples) and virological examination of clinically ill horses manifesting signs of CNS dysfunction.
Th e active and passive surveillance encompassed all municipalities in the Republic of Serbia.Th e selection and distribution of sampling localities in each county-region is defi ned by epizootiological services of scientifi c and specialized veterinary institutes according to the assessment of the risk of exposure to WNV.By assessing the exposure risk, the following is taken into consideration: 1. already available results of serological examination of horses; 2. existence of areas suitable for mosquitos such as standing waters, rivers, water fl ows, canals etc.; 3. settlements with recorded human infections (according to the data obtained from the Institute of Public Health of Serbia -"Batut" and regional Institutes of Public Health in the relevant territory) Based on the available data on the presence and circulation of WNV in the Republic of Serbia, the districts, i.e.Counties, were categorized according to risk of WNV infection outbreak (Table 1).

ACTIVE SURVEILLANCE
1. Serological surveillance Serological surveillance implicated sampling and examination of blood sera of sentinel horses and poultry for the presence of WNV specifi c antibodies (sentinel animals are individuals that have not been in contact WNV, that is, do not possess specifi c antibodies against WNV in the blood).Serological examination is performed using ELISA technique.Th e testing is performed in authorized scientifi c and specialized veterinary institutes.

Serological surveillance of sentinel poultry
Serological testing of sentinel poultry encompassed blood samples of poultry kept in extensive breeding system (backyard poultry), where only poultry hatched during the year of testing shall be tested on anti-WNV antibody presence.Th e tested population should be mainly located in the suburban areas (predominantly rural settlements).In high-risk regions (10 Counties -Table 1), the sampling is performing in ten settlements with highest risk, i.e. fi ve poultry blood samples were collected per one settlement, from at least one husbandry with extensive poultry keeping system (backyard poultry).Th e sampling and examination thereof is performing during the period May-September, i.e., in 6 sampling ocassions: one in May (by the end of the month), one in June, two in July, one in August (middle of the month) and one in September (until 15 th September), meaning ones or twice monthly, depending on the risk of infection in the relevant period of the year.In regions/Counties with lower risk of WNV infection outbreak (15 Counties -Table 1), the sampling is performing in 6 high-risk settlements by collecting up to 5 blood sera per one settlement from at least one husbandry with extensive (backyard) keeping system.Th e sampling and examination thereof is performing during the period June-September, i.e., in 4 sampling ocassions: one in June, one in July, one in August (middle of the month) and one in September (until 15 th September).Th roughout entire surveillance period (from May/June until September), the obtaining of blood samples had to be done at the same locations for all sampling ocassions.Th e term "location" in this Program considered the area of selected settlements.Sampling plan is presented in Table 2.

Serological surveillance of sentinel horses
During the preparatory period of WNV surveillance in horses and with an aim of selecting appropriate sentinel animals, mandatory serological surveillance of horses was conducted from March to May 2014 to identify WNV-seronegative animals, which are to be used as sentinel animals in the WNV surveillance program.
Serological testing of sentinel horses' blood sera implicated collecting of up to 50 samples from as many as possible locations (minimum 3) in each high-risk County (10 Counties -Tables 1 and 2) and up to 30 samples from as many as possible locations (minimum 3) in each lower-risk region (15 Counties -Tables 1 and 2).Th e collected samples are testing for the presence of anti-WNV antibodies by applying ELISA test.Th e sampling should be performed successively from the same sentinel animals, three times (fi rst sampling -during June; second sampling -during July; third sampling -during August 2014).

Virological surveillance
Virological surveillance encompassed sampling and examination of organs and tissues of dead birds or throat swabs of captured live wild birds (susceptible species), as well as examination of pooled samples of vector mosquitoes (species Culex pipiens) for the presence of West Nile Virus -WNV.Th e virus presence was also tested in samples of brain and cerebrospinal fl uids from dead horses with clinically manifested neurological dysfunction (passive surveillance).Virological testing is performing by molecular methods (real-time RT-PCR or RT-PCR) in the National reference laboratory for WNV in Specialized Veterinary Institute "Kraljevo" (VSI Kraljevo), as well as in virology laboratories of Scientifi c Veterinary Institute "Novi Sad" (NIV-NS) and Scientifi c Veterinary Institute of Serbia (NIVS).

Virological surveillance of wild birds
Dead wild birds found in the natural environment, particularly the resident species most susceptible to infection, e.g.Corvidae (magpie, crow, raven, rook.etc.), raptors (northern goshawk, falcon and eagle) and singing birds as well as birds died in rehabilitation centres, zoos or bird breeding farms (mostly raptors such as falcons, eagles, hawks…) were submitted to the aforementioned laboratories for testing for the presence of WNV.If dead birds were unavailable for laboratory examination, the sampling in high-risk regions (May -October) could be performed from captured live WNV-susceptible birds species by obtaining throat swabs or by hunting of certain bird population (Corvidae) for examination purposes (in cooperation with the hunting associations).Storage and transportation of samples to the authorized laboratory strongly requires maintenance of cold-chain conditions (refrigeration or freezing (swabs)).Samples obtained from dead birds (brain and parenchymatous organs) and throat swabs are testing for the WNV presence using molecular methods (real-time RT-PCR or RT-PCR).Collection of dead-bird samples and testing od virus presence is performing throughout the year in high-risk regions (10 Counties -Tables 1 and 2) and in the period May-October in lowerrisk regions (15 Counties -Tables 1 and 2)

Virological surveillance of mosquitoes -WNV vectors
Vector mosquitoes (Culex pipiens) are testing for the WNV presence by molecular methods (real-time RT-PCR or RT-PCR).Th e mosquitoes were examined as pooled sample (50-100 individual insects per pool) per one sampling point.Mosquitoes are collecting by dry-ice baited traps in the period of their most intensive activity (May-September) at the semi-urban and urban localities suitable for their survival and reproduction (standing waters, rivers, water fl ows, canals etc.) in the vicinity of susceptible animals (i.e., close to horse stables and poultry farms).Th e collection of mosquitoes should be performed at two-week intervals from 10 localities throughout the high-risk Counties (7 samplings, starting by end May, and then by mid and end of following months until mid September).In lower-risk Counties, the sampling should be performed monthly at 5 localities throughout the County (5 samplings, once a month, starting by end May, and then in the second half of the following months until mid September) (Tables 1 and 2).Native mosquito samples (without liquid) collected by dry-ice baited or other traps require rapid cooling (freezing) and immediate transportation to the laboratory (VSI Kraljevo, NIV-NS, or NIVS) for examination while still frozen.To the purpose of sampling and entomological examination, establishing of close cooperation with entomologists is highly recomended.

PASSIVE SURVEILLANCE
All horses with clinically manifested neurological dysfunction had to be subjected to testing for WNV infection in the framework of passive surveillance.Th e testing encompassed serological examination of paired samples of blood sera collected at 3-4 week intervals.Th e presence of WNV-specifi c antibodies is done in the corespondent scientifi c or specialized veterinary institute.In cases of lethal outcome in horses, samples of brain tissue and cerebrospinal fl uid need to be submitted for laboratory examination for the presence of WNV (laboratories of VSI Kraljevo, NIVS or NIV-NS).

SAMPLING PROCEDURE, SAMPLE DISTRIBUTION AND REPORTING
According to the provisions of the Surveillance program, sampling of calculated amount of blood samples from sentinel horses and poultry from settlements, households and stables, as well as obtaining of basic epizootiological and anamnestic data is done by authorized veterinary service and epizootiolo-gists in scientifi c or specialized veterinary institutes responsible for serological testing of blood samples of sentinel horses and poultry for the presence of WNV-specifi c antibodies.
Responsible epizootiologists of scientifi c and specialized veterinary institutes collected basic epizootiological and anamnestic data, as well as the samples of wild birds and mosquitoes in their regions.Th e samples were submitted to laboratories for testing for the presence of WNV, that is, national reference laboratory fro WNV in the VSI Kraljevo or Virology laboratories of NIV-NS and NIVS.Th e carcasses of susceptible species of wild birds and throat swabs of captured susceptible live wild birds, as well as mosquito samples (pooled samples consisting of 50-100 mosquitoes per one sampling ocassion and sampling locality) collected in the territories of North Bačka, West Bačka, South Bačka, Srem, North Banat, Middle Banat and South Banat Counties were submitted to the virology laboratory of the NIV-NS.Th e carcasses of susceptible species of wild birds and throat swabs of live susceptible wild birds, as well as mosquito samples (pooled samples consisting of 50-100 mosquitoes per one sampling ocassion and sampling locality) collected in the epizootic regions controlled by Specialized Veterinary Institutes VSI Niš, VSI Kraljevo, VSI Zaječar and VSI Jagodina were submitted to the national reference laboratory for aviary infl uenza, Newcastle disease and WNV in VSI Kraljevo.Th e samples collected in the epizootical region controlled by NIVS, that is, epizootical regions of NIVS, VSI Požarevac and VSI Šabac were submitted to the virology laboratory of the NIVS.
Th e Institutes communicated the regular monthly testing reports to the Veterinary Directorate by the 10 th of the following month.In cases of positive seroconversion fi nding (positive serological fi nding in previously seronegative sentinel animals) during testing or establishment of virus presence in wild birds, vectors (mosquitoes) or horses (during active or passive surveillance), SUSPECTED CASE on presence of infectious disease is set up and reporting immediately, without delay, to the responsible veterinary inspector, Veterinary Directorate and to the National reference laboratory of the VSI Kraljevo.Positive and suspect samples were immediately submitted to the National reference laboratory of the VSI Kraljevo for fi nal confi rmation and diagnosis.In case of positive fi nding, the National reference laboratory sends the confi rmatory offi cial report to the sender and to the Veterinary Directorate.Based on positive seroconversion fi nding, or positive fi nding for virus presence, the Veterinary Directorate declares the presence of WNV, i.e., POSITIVE CASE of WNV infection in the relevant region.Veterinary Directorate communicates the information on Suspected and Positive Cases of WNV infection (confi rmation of suspect infection -positive case) to the Ministry of Health.
Blood serum samples obtained from horses and poultry confi rmed positive for the presence of WNV-specifi c antibodies, as well as the virus-positive samples of wild birds and mosquitoes must be stored in frozen state to the purpose of further examination (including potentially virus-positive samples of diseased/dead horses tested during passive surveillance process).
To provide the unremitting and timely WNV surveillance, particularly its stages that require technically complex procedures (surveillance of wild birds and vector mosquitoes), timely planning and implementation of required resources (personnel, equipment, reagents) by all participating parties is of vital importance.Surveillance of wild birds and mosquitoes, as a technically complex segment of the procedure, requires the following preceding actions and prerequisites: -offi cial approval for capturing and sampling of wild birds; -participation of qualifi ed ornithologists or at least certifi ed ringers (binders) for performing fi eld activities and accurate identifi cation of wild bird species used for obtaining samples -cooperation and working together with hunting societies and organizations; -legally registered, i.e., reported nets for capturing wild birds; -participation of qualifi ed entomologists to perform accurate identifi cation of trapped mosquitoes (at species level) to be examined for the presence of WNV and for fi eld activities aimed at trapping and collecting of mosquitoes; -suffi cient number of adequate traps for collecting the mosquitoes (Culex pipiens); -dry ice for storing and transportation of samples to the laboratory Th e WNV surveillance program has been conducted during 2014 in the territory of Serbia.Th e program is still ongoing, and its evaluation will be performed by the beginning of 2015.Based on the data obtained in this evaluation, the eff ects of the Program will be assessed.We believe that the obtained data will enable potential corrections and amendments to the Program, thus making it highly eff ective instrument for further surveillance of this vectorborne zoonotic viral infection in the territory of Serbia.

AKNOWLEDGMENTS
Th is work is conducted within the projects TR31084 and III43007 funded by the Serbian Ministry of Education, Science and Technological development and 2005 in goshawks and birds of prey in Hungary, in 2007 in Volgograd, Russia, and in 2008 and 2009 in goshawks and a falcon in Austria (Bakonyi et al., 2006; Erdélyi et al., 2007; Platonov et al., 2008; Wodak et al., 2011).Since 2008, WNV has been heavily spreading throughout central and southeastern Europe, constituting a serious veterinary and public health problem for Europe (Barbic et al., 2012; Ziegler et al., 2012).

Table 1 .
Categorization of districts -Counties in the Republic of Serbia according to risk of WNV outbreak based on available results of laboratory examination of horses and vector mosquitoes, as well as of human cases