Evaluation of the first oral rabies vaccination campaign of the red foxes in Greece

Vaccine 34 (2016) 41–48

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Evaluation of the first oral rabies vaccination campaign of the red foxes in Greece Laskarina-Maria Korou a,∗ , Konstantia E. Tasioudi b , Myrsini Tzani a , Athanasios Konstantinidis c , Aikaterini Plevraki d , Peristera Iliadou b , Petroula Kostoglou a , Dimitrios Kaimaras e , Spyridon Doudounakis a , Olga Mangana-Vougiouka b a

Animal Health Directorate, Ministry of Rural Development and Food, Athens, Greece Virology Laboratory, Department of Molecular Diagnostics, FMD, Virological, Rickettsial and Exotic Diseases, Athens Veterinary Center, Ministry of Rural Development and Food, Athens, Greece c Veterinary Department, Regional Unit of Larissa, Greece d Veterinary Department, Regional Unit of Thessaloniki, Greece e Directorate of Technical Studies, Structures and Topography, Ministry of Rural Development and Food, Athens, Greece b

a r t i c l e

i n f o

Article history: Received 7 July 2015 Received in revised form 31 October 2015 Accepted 12 November 2015 Available online 24 November 2015 Keywords: Oral vaccination Foxes Antibodies Tetracycline Rabies Greece

a b s t r a c t Following the late 2012 recurrence of rabies in wild foxes (Vulpes vulpes) in central and north-western Greece, the first oral fox vaccination campaign co-financed by the European Union (EU) and the Greek state budget, was implemented. Initially, it involved 24 regional units of the Greek territory during the period October–December 2013. Vaccine-baits were aerially distributed by fixed-wing aircrafts. Vaccines were scattered along parallel flight paths 500 m apart in order to optimize aerial missions and achieve homogeneous distribution. A geographical information system was used to objectively evaluate bait distribution. This system identified areas of inadequate bait density that would require additional flights. A total number of 1,504,821 baits were distributed covering an area of 54,584.29 km2 . To assess the effectiveness of oral vaccination campaign a monitoring program was introduced, which entailed examination of serum samples and canine teeth derived from red foxes collected in the field. The laboratory analysis revealed 60% seropositivity and detection of tetracycline biomarker in 70% of the foxes tested. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Following 25 years of rabies-free status in Greece a rabid red fox (found in Kozani regional unit) was first detected and laboratory confirmed in October 2012 [1]. The recurrence of rabies in Greece was not an unexpected phenomenon. During the last decades rabies had already been established mainly in wildlife animal population of central and eastern Europe and rabid animals had been detected in the neighboring countries. Red fox currently

Abbreviations: EURL, European Union Reference Laboratory for rabies; NRL, National Reference Laboratory for Rabies (NRL) in animals, which is the Virology Laboratory of Athens Veterinary Center of the Ministry of Rural Development and Food; DZ, Department of Zoonoses, Animal Health Directorate, Ministry of Rural Development and Food; FAT, direct fluorescent antibody test; FYROM, Former Yugoslav Republic of Macedonia; ORV, oral rabies vaccination; RFFIT, Rapid Fluorescent Focus Inhibition Test; Hellenic CDC, Hellenic Center for Disease Control. ∗ Corresponding author at: Department of Zoonoses, Animal Health Directorate, Ministry of Rural Development and Food, Veranzerou 46, 10438 Athens, Greece. Tel.: +30 210 212 57 25; fax: +30 210 82 52 614. E-mail address: [email protected] (L.-M. Korou). http://dx.doi.org/10.1016/j.vaccine.2015.11.031 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

remains the main virus reservoir in many countries across Europe including Greece [2]. Although rabies transmission within wildlife is considered to be density-dependent, radical reduction of reservoir species population has failed to eliminate the disease or its spread to uninfected areas [3,4]. Additionally, humane and ecological aspects should be taken into consideration before engaging such methods of rabies control in large-scale culling campaigns. Mass vaccination of the principal wildlife hosts could be a more effective control method than reducing their population [4,5]. Vaccination strategies of wildlife were applied in Europe since the late 1970s in order to control the spread of rabies virus. Switzerland was the first European country to implement oral vaccination in 1978 [6]. The aforementioned programs contributed to achievement of rabies free status after years of oral vaccination in many EU and non EU countries of Europe [7]. Oral rabies vaccination campaigns in Europe are usually conducted twice a year, in spring and autumn with bait distributed mainly by air [4]. To address the epidemic and prevent its spread, a Greek National Rabies control and eradication program was implemented based on passive surveillance of the disease, mandatory vaccination of all

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Fig. 1. Vaccination map in Greece. The twenty-four Regional Units of Central and North Greece involved in the first Oral Rabies Vaccination Campaign of red foxes in 2013 are presented in dark color.

dogs and cats, management of all rabies cases – suspected cases and control of animal movements. Furthermore, an Oral Vaccination project for the immunization of red foxes against rabies was launched in Greece and the first vaccination campaign took place in autumn 2013. Vaccine-baits were aerially distributed in 24 regional units of the country. An assessment of the efficacy of this oral vaccination program was achieved by detection of tetracycline in the canine teeth of the red foxes, which is a biomarker incorporated in the vaccine bait and detection of rabies antibodies in animal’s serum collected from areas of vaccination [8]. The aim of this study is the detailed demonstration of the application of oral vaccination in foxes for the first time in Greece and the evaluation of the outcomes of this program. 2. Materials and methods 2.1. Pre-vaccination period The red fox oral vaccination campaign of autumn 2013 was the first to be carried out in Greece and as a result there was an increased need for preparation, proper design and collaboration of different competent authorities. The Department of Zoonoses (DZ) of the Ministry of Rural Development and Food coordinated the program. Initially, it was decided to implement the vaccine campaign in 24 regional units of the country, which encompassed locations of positive animal rabies cases as well as areas in close proximity to the previous ones. The program was co-financed by the EU and the Greek state budget. The DZ addressed to different services that could support the program by kindly providing their experience. The Greek Army provided information regarding the design of flights and the permissions required for the implementation of aerial missions. The topography service of the Ministry worked in parallel with the DZ

in order to define the total area of the country to be covered by aerial distribution of vaccines. They had to take into account the maximum altitude where red foxes inhabit and the lower temperatures corresponding to each altitude, so as to avoid freezing of the liquid content of the vaccine. It was assumed that the fox density in the landscape over 1000 m above sea level was relatively low and these areas were excluded from the vaccination program. In addition, the altitudes selected were below the freezing level according to previous practices [9]. Urban and suburban areas as well as roadways and water surfaces (sea, rivers and lakes) were excluded from the target area. The cartographical background of national topography service was used in order to estimate the areas that had to be covered. Specifically, polygon-shaded maps were created. Afterwards, the areas of the polygons in the maps, whose the use was not urban, semi-urban or water surfaces, were added. Thus, the total area to be covered was initially calculated to 59,603.51 km2 (Fig. 1). The density of distributed baits per km2 was initially estimated to be over 20 (25 baits on average), according to experience gained from other EU and non EU countries [10], since accurate data on fox population in Greece are limited. The Hellenic National Meteorological Service provided detailed weather forecasts throughout the vaccination program concerning the selected areas. Additionally, they supplied average temperatures derived from different meteorological stations, located in different altitudes, in the areas included in the vaccination schedule. The vaccine selected for the first oral vaccination campaign in Greece was the SAG2 vaccine (RABIGEN® SAG2), a modified live attenuated rabies virus vaccine, derived from the SAD Bern strain in a two-step process of amino acid mutation using neutralizing monoclonal antibodies. This vaccine has been shown to be non pathogenic after experimental exposure of multiple target and non target species, including non-human primates [11]. However, given

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Fig. 2. Bait density per km2 in the Regional Unit of Thessaloniki. The density of baits distributed per square (1 km × 1 km) in the map of Thessaloniki is indicated through a colored gradient.

the fact that the vaccine contains live strain and the lack of relevant studies in humans, it becomes apparent that exposure to the vaccine requires adequate post-exposure anti-rabies prophylactic treatment [11]. Furthermore, since baits could be released in the environment and people might come in contact with them, they were labeled with a note stating, “do not touch” and provided a phone number of the Hellenic CDC center. A communication campaign (posters and leaflets, TV spot, special educational material and courses for children in schools) was implemented in collaboration with the Ministry of Education, the regional veterinary services and the students’ associations of the two Veterinary Schools. The aim was to raise public awareness with regards to rabies and present the rationale of red fox oral vaccination program, the time schedule of the vaccination strategy, the areas involved and the necessary prophylactic measures to be adopted in order to avoid potentially harmful exposures to the live attenuated vaccine distributed by air. Furthermore, two veterinarians and a technician from the NRL were trained on tetracycline detection, age determination and phylogenetic analysis of rabies virus in the EURL in Nancy, France. The vaccination campaign started immediately after receiving the analytical results of titration of the vaccines by the European Reference Laboratory for Rabies in animals.

2.2. During vaccination The first oral vaccination campaign started on 11 October 2013. Official Veterinarians from the competent central veterinary authority of FYROM as well as laboratory staff from the EURL visited Greece during this first campaign to give advice on the proper implementation of the program. The vaccines were maintained in a cold-chain [11] in Thessaloniki regional unit, until distribution and were transported daily with freezing vehicles to the selected airfields. The duration of the vaccination campaign was initially defined to a month. Baits were distributed by Cessna fixed-wing aircrafts. The aircraft flying lines were separated by 500 m, flight altitude was set at 150 m and the average speed at 100–150 km/h. The baits were manually dropped one after another following a sound signal (alarm), in order to meet the predetermined density of vaccine baits per square kilometer. GPS equipment existed in each aircraft registering the coordinates of individual bait position. Official veterinarians supervised the whole procedure. GPS data of dropped baits were analyzed on a daily basis by the topography service of the Ministry to calculate bait densities/km2 . The surveyors entered the GPS data on the map of Greece and more specifically of each regional unit using the Geomedia GIS software.

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The areas covered by the baits were estimated conceding to the assumption that each of bait covers a radius of 300 m. The circles formed by the software, were merged in every regional unit resulting in the estimation of the areas covered. The number of baits dropped in each square (1 km × 1 km) of the map was calculated by the topography service and the densities were displayed using a colored gradient (Fig. 2). In cases that the achieved distribution was not in compliance with the initial target (>20 baits per km2 ), corrective actions were applied. During the vaccination period, press releases both in central and local level informed the public regarding the schedule of vaccines distribution. Simultaneously, the vaccination program was distributed to all competent authorities (regional veterinary services, municipalities, hunting associations and forestry authorities as well as public health departments) on a daily basis. In case of human contact with the baits, the Hellenic CDC center followed specific algorithms in order to administer the appropriate post-exposure prophylactic treatment. In many cases, the communication between veterinary and health authorities, at both central and local level, was required to define the proper management of different incidents.

2.3. Post-vaccination period Following the completion of the oral vaccination campaign, the DZ in collaboration with the topography service of the Ministry further assessed the maps, depicting the baits’ densities per km in all regional units involved in the program. One month following the end of oral vaccination in each regional unit, the monitoring program (active surveillance) started. To investigate the efficacy of the oral vaccination campaign, samples from 2 red foxes/100 km2 need to be tested, according to WHO recommendations [12]. Therefore, the total number of animals to be collected was estimated at 1212 in all 24 regional units. Initially the duration for the sampling was set up to two months, however it was later extended up to the end of April 2014. In each one of the twenty four regional units involved in the red fox vaccination program, teams consisting of official veterinarians, gamekeepers and forestry officers organized missions for hunting foxes and collecting the necessary blood and head samples. The missions were mainly carried out late in the afternoon, or in the evening. These teams used different means so as to attract the foxes such as parts of carcasses derived from slaughterhouses. Ideally, official veterinarians pumped field blood samples directly from the heart of freshly killed red foxes (Fig. 3). The samples were dispatched by the official veterinarians to the NRL. The brain samples were examined in advance for the detection of rabies virus by FAT [13]. Moreover, the laboratory staff conducted serological screening, which is important in order to record seroconversion rate in fox serum, after the consumption of baits. To detect the rabies antibodies, a commercially available blocking ELISA kit (BioPro Rabies ELISA Ab kit, Czech Republic) was used, as described previously [14]. Subsequently, the detection of tetracycline in canine tooth and in part of alveolar bone tissue, obtained from the lower jaw of each fox, was performed according to the protocol of the EURL. Tetracycline binding was assessed in teeth and in bones by ultraviolet light examination by inverse microscopy. Moreover, the age of all animals collected was determined on the basis of teeth histological examination. Age estimation is typically performed along with tetracycline detection in order to evaluate vaccination effectiveness across various red foxes’ age groups [15].

Fig. 3. Sampling procedure. An official veterinarian collects the blood sample from the fox’s heart for the needs of the active surveillance program.

The NRL estimated the age class (0–1, 1–2, 2–3, >3 years) of the animals based on the following critical parameters: the date of animal death, the diameter of the pulp cavity, the presence of cementum and the number of dark lines in the cementum [16]. The first age class (0–1 year) represents the juveniles and the remaining three (1–2, 2–3, >3 years) the adults. Regarding the passive surveillance program, samples collected by dead or suspect for rabies animals were provided by the regional veterinary services in collaboration with gamekeepers and hunting associations to the NRL. 3. Results 3.1. Evaluating the effectiveness of oral vaccination campaign The vaccination period started on 11th October 2013 and was completed on 16th December 2013. The size of vaccinated area was eventually determined to be 54,584.29 km2 . The total number of vaccines-baits distributed was 1,504,821. The average number of baits distributed per km2 in all 24 regional units was 27.22. The baits’ densities as well as the total number of baits dropped and the total area covered in each regional unit are presented in Table 1. The highest bait density was noted in Larissa regional unit (132 baits/km2 in a small part of the target area) while the average density in this regional unit was 26.3 baits/km2 . Within the framework of monitoring the effectiveness of oral vaccination, 119 brain, 121 serum, and 120 teeth samples derived from a total of 123 hunted red foxes were submitted to the NRL and further examined. All the 123 foxes examined during the active surveillance program for the presence of rabies virus by FAT gave negative results. Tetracycline was detected in 84 out of 120 samples tested. On average, 70% of tested red foxes were positive for the tetracycline biomarker, with values ranging from 33.33% to 100%, in all regional units (Table 2). 60% of tested samples (73 out of 121 serum samples) showed rabies antibody titers over 0.5 EU/ml. Seropositivity values ranged from 28.57% to 100%. It is worth mentioning that 69 out of 84 cases displayed positivity for both tetracycline and antibodies titers while 13 samples were positive for tetracycline and serologically negative.

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Table 1 Aerial baits’ distribution in the Regional Units involved in the Oral Rabies Vaccination program of 2013 in Greece. Regional Unit

Number of baits distributed

Size of vaccinated area (km2 )

Bait density (per km2 )

Preveza Florina Kastoria Aitoloakarnania Pieria Imathia Arta Thesprotia Kavala Evrytania Grevena Trikala Magnisia Karditsa Pella Fthiotida Chalkidiki Kozani Drama Ioannina Thessaloniki Kilkis Serres Larissa

24,764 25,625 27,034 34,881 34,894 37,603 37,706 39,503 40,886 43,342 51,299 56,770 57,344 62,119 62,303 75,369 79,843 83,396 85,152 85,947 99,805 102,217 114,829 133,257

988.6 998.5 1034.5 1270.75 1167.0 1409.1 1580.1 1524.5 1628.4 1464.9 1955.1 2124.0 2250.0 2198.5 2027.4 2804.96 3195.2 3128.05 3377.2 3196.3 3339.0 2453.3 3757.7 5343.4

25.04 25.66 26.13 27.44 29.90 26.68 23.86 25.91 25.10 29.58 26.23 26.72 25.48 28.25 30.73 26.86 24.98 26.66 25.21 26.88 29.89 41.66 30.55 24.93

Interestingly, two samples were positive for antibodies but negative for tetracycline. Regarding age determination, it is known that it cannot be definite when foxes are collected during the period January–April, as in our study, since during this time, dark line production takes place in the cementum and therefore the distinction of two successive age classes remains inconclusive [16]. In total, age determination demonstrated clearly that 92 out of 120 samples were adults (77%) and 12 out of 120 were juveniles (0–1 years) (10%). In addition, there were 16 (13%) cases where a firm conclusion could not be drawn. They were characterized to be either juveniles (0–1 years) or adults (1–2 years). There were also 33 samples whose age could not be definitely determined. In specific, 19 cases were categorized as either 1–2 or 2–3 years old and 14 samples were evaluated as either 2–3 or >3 years old. Out of the 73 serum samples showing seroconversion, 71 samples were accompanied by their respective teeth samples that allowed the determination of the animal age. Out of these 71 samples, 7 sera belonged to juveniles, 55 sera belonged to adults and 9

sera belonged to animals aged either 0–1 or 1–2 years old. Respectively, out of the 84 samples, in which tetracycline was detected, 10 samples belonged to juveniles, 63 samples belonged to adult foxes and 11 samples belonged to animals 0–1 or 1–2 years old. Differences were noted among the different regional units. For instance in Kozani area, the number of samples collected was 29 instead of the target number which was defined to 68. Despite this difference, the number of samples collected in Kozani was significantly higher in comparison to the majority of the other regional units. Tetracycline was detected in the 93% of the teeth samples tested, and the 83.7% of serum samples were positive for antibodies. However, the distribution of the samples collected was not homogenous but concentrated mainly on the north-eastern part of the prefecture. In contrast, in Arta regional unit, the number of hunted foxes was 26 while the target was 31 samples. The distribution of the samples could be characterized better than Kozani’s. Nevertheless, the evaluation of the laboratory results revealed that both bait uptake and seroprotection were relatively low (52% and 38% respectively).

Table 2 Bait uptake and seroprotection rates of red foxes collected for the evaluation of the effectiveness of the first Rabies Oral Vaccination campaign in Greece. Regional Unit

Serological testing Number of tests

Florina Grevena Aitoloakarnania Arta Drama Evrytania Thessaloniki Ioannina Kavala Kastoria Kilkis Kozani Magnisia Serres Trikala Fthiotida Total

7 3 1 26 6 7 4 3 2 3 1 29 9 15 1 4 121

Presence of Biomarker Number of seropositive samples

% seropositive

2 2 1 10 3 3 2 1 1 3 1 24 5 12 1 2

28.57 66.67 100 38.46 50 42.86 50 33.33 50 100 100 82.76 55.56 80 100 50

Number of tests 6 3 3 25 5 7 4 3 2 3 1 29 9 15 1 4 120

Biomarker present

% marker presence

2 2 2 13 5 3 2 1 2 3 1 27 5 13 1 2

33.33 66.67 66.67 52 100 42.86 50 33.33 100 100 100 93.1 55.56 86.67 100 50

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3.2. Passive surveillance results before and following the first oral vaccination campaign The number of suspected animals investigated in the frame of passive surveillance in Greece the period between January 2012 and June 2015 were 1364. In detail, 237 animals were tested in 2012, 587 in 2013, 410 in 2014 and 130 from the beginning of 2015 until early June 2015. The number of red foxes tested was 140 in 2012, 314 in 2013, 169 in 2014 and 60 from the beginning of 2015 to early June 2015. Starting from 2012 and up to the end of the first oral vaccination campaign in Greece on December 2013 (pre-vaccination period), a total of 38 rabies cases in different animal species (32 red foxes, 3 dogs, 2 bovines and one cat) were detected. The affected areas included the regional units of Kilkis, Thessaloniki, Pella, Trikala, Kozani, Kastoria and Serres. During the post-vaccination period, that is from January 2014 to June of 2015, 10 rabies cases (2 dogs and 8 red foxes) were laboratory confirmed in the regional units of Kilkis, Thessaloniki, Pella, Trikala and Larissa. The last positive case was diagnosed in a red fox in the regional unit of Pella on May 2014. Since then, all samples, received in the frame of passive surveillance system, have given negative results. A marked decrease (3.5%) in the positivity of the red fox samples was recorded between pre-vaccination period (7%) and post-vaccination period (4.5%).

4. Discussion Undoubtedly, the whole project constituted a major challenge for the Greek veterinary authorities, mainly because of two reasons. First of all, there was lack of previous experience on the implementation of wildlife oral vaccination programs. Secondly, the extent of the area needing to be covered by aerial bait distribution in Greece was larger as compared with other countries where similar programs have been carried out in the past [9,17]. In total the technical parameters regarding this large-scale vaccination strategy were in accordance to the recommendations of the EU and the experience of other MSs and non-EU countries [17–19]. The evaluation of the distribution of the vaccines confirmed a proper dropping system that covered the areas initially included in the vaccination program, in the majority of the regional units. In addition, the average density of vaccines per km2 was higher than 20 baits/km2 in all cases. The collaboration of the companies supplying the vaccine and performing the dropping along with the veterinary authorities (on a central and local level) could be judged satisfactory. Coordination of all actions to be taken and thorough design were the cornerstones of overcoming every obstacle. In addition, the excellent collaboration among veterinary authorities, the topography service of the Ministry and the public health authorities should be highlighted. Nevertheless, a lot of difficulties could be mentioned that retarded the vaccination campaign. The process for the final selection of both the vaccines’ supplier and the aerial distribution companies lasted several months, due to legislative and bureaucratic procedures. This, combined with the reduced pre-allocation of funds from the state budget, may explain the delay of the start of the campaign, which finally took place in October instead of September 2013. In addition, the importation of vaccines batches in Greece by the supplier company in six different dispatches, the adverse weather conditions and the limited daylight hours, extended the vaccination period. Furthermore, the deficient facilities of small, local airfields that were used for the campaign and the limited financial means and basic equipment, (vehicles, fuel, mobile phones and computers

for communication with the supervising authorities) hindering the flight operations as well as the custodial work of all the involved stuff (pilots, mechanics, veterinarians and ancillary stuff). Evidently, Greece being a rabies free country for more than 25 years, it had no experience of such mass wildlife vaccination program. There was significant help though, as already mentioned, from other EU members states e.g. France or countries already applying the program, such as Italy and FYROM [20]. Despite the aforementioned difficulties, the ORV program was applied and completed successfully. Until 2012, rabies was an unknown and unfamiliar disease in the majority of Greek population [21]. For this reason, the first priority was given to raise public awareness regarding the severity of the disease and its impact on Public Health as well as the oral vaccination program. Public information was disseminated frequently on television and local and national radio broadcasts. Further reports were issued to daily and weekly press outlets. On top of this mass media campaign, a great number of talks and lectures were given to hunting and farmers’ associations, relative commissions and primary and secondary directorates of education. The lack of previous public experience led to a small number of adverse events including direct contact with vaccine and a few citizens requesting medical help. The Hellenic CDC managed all these cases with success, always in collaboration with the central or local veterinary authorities. Collection and examination of adequate number of samples during active surveillance state probably constitutes the most determinant factor for the evaluation of a mass oral vaccination program success [22]. Unfortunately, during the first year of fox vaccination in Greece, the number of samples collected and examined by the NRL did not reach the target. Specifically, in six out of the 24 regional units involved, no sample for monitoring was collected. This phenomenon can be explained by the lack of human resources of the responsible services in charge of recruiting staff for sample collection. In contrast to other EU and non EU countries where hunters were allowed to hunt foxes and collect the samples receiving a remuneration for each sample provided, this was not the case for Greece in the first campaign. Samples were exclusively collected by the regional veterinary officers along with the local forestry officers and the gamekeepers. Moreover, lack of transport means and basic equipment (vehicles, mobile phones, etc.) in combination with the unfavorable weather conditions during monitoring process (January–April 2014) could further justify the limited number of collected samples. An additional parameter that hindered the active surveillance procedure was the absence of human anti-rabies vaccines in the country, needed for the pre-exposure vaccination of all the staff involved, leading to fear and lack of compliance in some regional units. The reduced number of samples collected in the field is likely to be due, at least in part, to the geomorphological relief of Greece, although the inability to use ridged guns for distant targeting of red foxes, due to relevant Greek legislation, could be considered as an additional reason. It should also be mentioned that in some regional units involved in the program, the collecting teams reported that they could not even see any fox during their missions, even if they were trying to attract them by using miscellaneous means. The limited number of samples submitted from many regional units limits the conclusions that can be drawn for the ORV campaign. Assessing the results of the active surveillance that followed the autumn campaign of 2013, and in spite of the small number of available samples, bait uptake as well as the percentage of seropositivity in foxes could be considered satisfactory especially in comparison to the vaccination outcomes in other countries [23]. By all means

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the major indicator for evaluating the success of these campaigns remains the incidence of rabies [7]. It could be assumed that the significant reduction in the number of animal rabies cases following the first ORV and the non detection of rabies cases in Greece, since May 2014, may be related at least in part, to the protective immunity developed in wildlife. The second vaccination campaign that took place in Greece during autumn 2014 may also have contributed to this. However, according to the experience gained by other countries, rabies elimination requires the implementation of ORV programs for many consecutive years [7,19,24]. In accordance to previous international experience [25] and taking into account the results of monitoring in the Greek territory, the implementation of only one campaign is not expected to have significant impact on rabies virus circulation in animal’s population. Certainly, the decreased rabies incidence after the first vaccination is quite encouraging, especially if we bear in mind that the last rabies case recorded in FYROM, which shares the same borders with Greece, was in 2012. However, it may be justified, at least in part, by the small sample size of foxes collected in Greece in the frame of passive surveillance in 2014 as compared to 2013. The development of a number of mathematical and computational models indicates an association between rabies virus dynamics and fox population density. Following the appearance of rabies in a closed fox population, the animal population will decrease until the fox density falls below the minimum population density at which rabies can persist, known as threshold value of rabies persistence. Thus, the disease disappears. Afterwards, the number of animals increases again until it reaches the carrying capacity of the habitat and the disease reappears [24]. In reality, in a non isolated fox population, the reappearance of the disease can occur at an earlier stage [24]. This could provide an explanation for the reduction of animal rabies cases, although currently there is no available accurate data on red fox population in Greece. Evaluating the laboratory data from the monitoring program it was observed that the number of analyzed samples derived from adult animals was significantly higher in comparison to those of cubs. Moreover, antibodies were more often detected in serum of adults than younger animals. Finally, tetracycline was also more frequently detected in adults compared to cubs. Taking into account that mating in foxes takes place between mid-December and March in the northern hemisphere, and that gestation period in this animal species is 51–53 days on average, cubs are mainly born the period mid-March to mid-April [26,27]. In Greece, the collection of animal samples took place between late December 2013 and late April 2014, a period that coincided mainly with the gestation period and to a lesser extent with the lactation period of the litters. In this period, the cubs do not leave the den and are dependent on their mother. The young animals that could be collected, were only these cubs born during the spring of 2013 and collected in the beginning of the monitoring program. The analysis of serum and teeth samples collected during the monitoring program revealed lower seropositivity rates as compared to the tetracycline positivity rates in foxes of all ages and especially in young animals. This finding could be associated with either ingestion of the bait and not of the capsule containing the vaccine, or ingestion of the baits without further puncturing of the capsule [28]. Another parameter responsible for the discrepancies among serological and tetracycline detection results is the fact that in some instances, foxes may hide the baits in order to eat them later in time, resulting in inactivation of the vaccine [29]. Published studies and anecdotal data also indicate that the detection of tetracycline in teeth can occur because of consumption of other food containing this antibiotic rather than intake of vaccine-baits. Besides, there are studies reporting fluorescence detection in teeth without previous tetracycline absorption [30,31].

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Regurgitation is the way by which the mother fox feed its cubs. This process may destroy SAG2 strain due to the acid pH of the stomach and this could justify, to a certain degree, the low seropositivity in juveniles. In addition, the contact between the vaccine suspension and the oro-pharyngeal mucosa may be sometimes insufficient for triggering the serological response [31]. Finally other studies report lower sensitivity in the detection of rabies antibodies using some commercially available ELISA kits that are commonly used, as compared to the Rapid Fluorescent Focus Inhibition Test (RFFIT) [31,32]. International organizations also recommend that “all rabies virus isolated should be typed in areas where attenuated rabies virus vaccines are used, in order to distinguish between vaccine and field virus strains” [31]. Preliminary data on the phylogenetic analysis of all field isolates from Greece found positive by FAT and molecular techniques following the first vaccination campaign belong to the classical rabies virus (genotype 1) and are all closely related. This finding implies that all positive animals were wild rabies strains rather than vaccine induced. In concluding the first ORV campaign implemented in Greece resulted in satisfactory baits uptake and immune coverage in the fox population although the number of animals tested for evaluating its effectiveness did not reach the targets. The importance of wildlife monitoring and surveillance has been recognized and constitutes main tool for the epidemiological investigation of the disease. The disease outbreak prompts for effective implementation of the above control measures, with a view to limit its further diffusion, and eventually eradicate it. Conflict of interest None declared. The ORV program, the monitoring and surveillance programs were all partially funded by the European Commission. Authors’ contributions LMK participated in the coordination of the program, in the design and drafting of the manuscript, in the analysis of the data derived by the topography service, and the evaluation of the laboratory results. KET coordinated the analysis performed in the NRL, performed the laboratory testing, participated in drafting the manuscript and in the evaluation of the laboratory results, MT was involved in the coordination of the program and drafted the manuscript, AK participated in drafting the discussion of the manuscript and supervised the aerial distribution in the airfield of Larissa. AP collected samples during the monitoring process, drafted the manuscript and evaluated the vaccination outcomes. PI performed the laboratory testing, participated in drafting the manuscript and evaluated the laboratory results. PK participated in drafting the manuscript and coordinated the project, DK determined the size of the vaccinated area, processed the data derived from the flights, prepared the maps for the program and drafted part of the manuscript. SD coordinated the project and drafted the manuscript. OMV provided scientific assistance during the implementation of the program, evaluated the laboratory results and drafted the manuscript. All authors have approved the final article. Acknowledgements We wish to thank all the official veterinarians of the 24 regional units involved in the program that participated either on the supervision of the baits distribution at the airfields or on the collection of samples from foxes. We also thank the Head of the Directorate General of Sustainable Animal Production and Veterinary Services

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of the Ministry, Dr. Thomas Alexandropoulos for his assistance during the initiation and implementation of the vaccination program. We would like to thank the technician Mr Dimitrios Bakakos as well as the seasonal veterinarians of the NRL, Dimos Papatheodorou and Gerasimos Markantonatos who assisted in the laboratory analysis of the samples. In addition, we thank all the hunters, forestry officers and gamekeepers who also contributed to the collection of the samples for the monitoring process as well as the Greek Public Health Authorities for their collaboration in the management of human contact with the vaccines. We thank the vaccine supplier company and the company responsible for the aerial distribution of the baits/vaccines for their collaboration during the implementation of the project. We thank Dr. Vassiliki Roussi for her assistance in the procedures during the dispatch of the vaccines in Greece. We thank Dr. Sofia Makridima for her assistance in editing the manuscript. Finally we are really grateful for the assistance and the support of Dr. F. Cliquet and her team from the EURL for Rabies. References [1] Tasioudi KE, Iliadou P, Agianniotaki EI, Robardet E, Liandris E, Doudounakis S, et al. Recurrence of animal rabies, Greece, 2012. Emerg Infect Dis 2014;20:326–8. [2] Cvetkovikj A, Radeski M, Mrenoshki S, Kirandjiski T, Krstevski K, Dzhadzhovski I, et al. Monitoring bait uptake through tetracycline presence and age structure of foxes in oral vaccination against rabies campaigns in R. Macedonia. In: Days of veterinary medicine 2012 3rd international scientific meeting. 2012. [3] Historical Perspective of Rabies in Europe and the Mediterranean Basin A testament to rabies by Dr Arthur A. King. Paris: World Organisation for Animal Health; 2004. [4] WHO Expert Consultation on Rabies Second report. Geneva: World Health Organization; 2013. [5] Rupprecht CE, Barrett J, Briggs D, Cliquet F, Fooks AR, Lumlertdacha B, et al. Can rabies be eradicated. Dev Biol 2008;131:95–121. [6] Vitasek J. A review of rabies elimination in Europe. Vet Med Czech 2004;49:171–85. [7] Muller T, Freuling CM, Wysocki P, Roumiantzeff M, Freney J, Mettenleiter TC, et al. Terrestrial rabies control in the European Union: historical achievements and challenges ahead. Vet J 2015;203:10–7. [8] Wasniewski M, Guiot AL, Schereffer JL, Tribout L, Mähar K, Cliquet F. Evaluation of an ELISA to detect rabies antibodies in orally vaccinated foxes and raccoon dogs sampled in the field. J Virol Methods 2013;187:264–70. [9] Capello K, Mulatti P, Comin A, Gagliazzo L, Guberti V, Citterio C, et al. Impact of emergency oral rabies vaccination of foxes in northeastern Italy, 28 December 2009–20 January 2010: preliminary evaluation. Euro Surveill 2010;15:19617. [10] Cliquet F, Combes B, Barrat J. Means used for terrestrial rabies elimination in France and policy for rabies surveillance in case of re-emergence. Dev Biol 2006;125:119–26. [11] Mahl P, Cliquet F, Guiot AL, Niin E, Fournials E, Saint-Jean N, et al. Twenty year experience of the oral rabies vaccine SAG2 in wildlife: a global review. Vet Res 2014;45:77.

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