<table><caption>**Table 8.** List of *Leptospira* human covariates identified from the selected studies in decreasing order according to the number of references</caption><thead><tr><td>Human drivers</td><td>n. of papers# (n. 30)</td><td>% of impact* (n. 119)</td></tr></thead><tbody><tr><td>Infection (incidence rates or presence of antibodies)</td><td>65</td><td>54.6%</td></tr><tr><td>Age (prevalence or titre of seropositive response by age)</td><td>19</td><td>15.9%</td></tr><tr><td>Gender (correlation of incidence with gender)</td><td>16</td><td>13.4%</td></tr><tr><td>Socio economic (professional soldiers or civilians)</td><td>7</td><td>5.9%</td></tr><tr><td>Clinical outcomes (hospitalization, mortality)</td><td>5</td><td>4.2%</td></tr><tr><td>Population (human density)</td><td>4</td><td>3.3%</td></tr><tr><td>Mortality</td><td>2</td><td>1.7%</td></tr></tbody></table>

<figure><img src="image_32.png" /><figcaption>**Figure 3.** Distribution of *Leptospira* incidence by gender and age group (data from TESSy-ECDC)</figcaption></figure>

# Diagnostic procedures and notification systems used at local, national and European scale

## Diagnosis

Because the clinical manifestations of leptospirosis are nonspecific and common many other febrile diseases, the combination of exposure history and symptoms should prompt confirmatory testing. In general, a definitive diagnosis of leptospirosis can be made via either traditional

<footnote># number of papers extracted-the number of references per covariate is higher because from one paper more covariates could have been extracted) *% calculated on the total number of references</footnote>
microbiological methods (direct detection, culture) or serology. Culture of *Leptospira* from patient samples is challenging: the organisms typically take 1-2 weeks to grow but may take over a month, and specific growth media is required; blood and CSF cultures are most useful during the first ten days of illness (leptospiremic phase), when organisms are spreading hematogenously (43). After the second week of the disease, urine cultures for *Leptospira* are more likely to be positive due to the organism's tendency for renal tubule deposition. They may remain positive for up to 30 days after the resolution of symptoms. Serological methods are the most commonly used for confirming a diagnosis of leptospirosis. The "gold standard" is the microscopic agglutination test (MAT), in which acute and convalescent sera from a suspected case are mixed with a panel of live antigens from different serogroups of *Leptospira* organisms and examined for agglutination (12). Though test characteristics are overall superior to culture and microscopy (90% sensitivity, > 90% specificity), this method has several limitations: i) the test requires a panel of live organisms specific to the area the patient is suspected of having acquired the infection, ii) specialized lab expertise, iii) there is significant cross-reactivity both between different serogroups of *Leptospira*, as well as with other spirochetes (Treponema and Borrelia species) and iv) antibody response required for MAT testing is often insufficient for detection until the second week of disease (when the immune phase begins). As both culture and serological methods are limited in early detection, newer molecular methods have been developed to facilitate early detection. Both conventional and real-time PCR techniques are highly sensitive, even at early stage of the disease, before the development of antibody response (44). Because this period correlates with the leptospiremic phase, blood is the best sample for detecting leptospiral nucleic acid. However, urine, CSF, or tissue may also have detectable levels later in the disease. Of note, because PCR detects nucleic acid and is not dependent on the presence of live organisms, this technique can be used even after empiric therapy with antibiotics.

Infrastructure capacity to identify pathogens
for each Member State

According to the Commission Implementing Decision (EU) 2018/945, leptospirosis is among the communicable diseases that are covered by epidemiological surveillance. It means that EU Member States must establish the national capacity to detect and report human cases.

The decision provides a case definition and laboratory criteria for case confirmation, which are at least one of the following four: 1) isolation of *Leptospira interrogans* or any other pathogenic *Leptospira* spp. from a clinical specimen; 2) detection of *L. interrogans* or any other pathogenic *Leptospira* spp. nucleic acid in a clinical specimen; 3) demonstration of *L. interrogans* or any other pathogenic *Leptospira* spp. by immunofluorescence in a clinical specimen; 4) *Leptospira interrogans* or any other pathogenic *Leptospira* spp. specific antibody response. Clinical microbiology laboratories routinely make the diagnosis.

However, most EU countries do not have a national reference laboratory, and there is not a European network of laboratories. In animals, the disease is not reportable at the EU level – Regulation (EU) 2016/429 and Commission Implementing Regulation (EU) 2020/2002. Yet, it is reportable internationally to the World Organization for Animal Health (WOAH). As for human surveillance, veterinary microbiology laboratories routinely make the diagnosis, and there is no European-wide reference laboratory network or national laboratories in most EU countries.
# Estimated influence of environmental change on the disease future trends

The potential for leptospirosis to spread to new territories, either through the reappearance of sylvatic transmission in rural territories or through urbanization, is highlighted. Furthermore, some risk scenarios highlight that the leptospirosis burden in Europe might increase in the coming years due to several factors: (i) alterations in climate (current global warming and/or heavy rainfalls with flooding), (ii) the increasing population of urban rodents in European cities in close contact with human beings and with associated high leptospirosis carriage rates, (iii) human population growth and subsequent urbanization of affected rural territories, and (iv) the increase in intercontinental travels. Based on these aspects, it can be assumed that leptospirosis will be a growing public health problem in Europe, particularly in urban settings (45).

## References

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17. Pappas G, Papadimitriou P, Siozopoulou V, et al. The globalization of leptospirosis: worldwide incidence trends. *Int J Infect Dis.* 2008;12:351-357.

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27. Hathaway SC, Ellis WA, Little TW, et al. Leptospira interrogans serovar hardjo in pigs: a new host-parasite relationship in the United Kingdom. *Vet Rec.* 1983;113:153-154.

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29. Oliveira SJ de, Bortolanza F, Passos DT, et al. Diagnóstico molecular de Leptospira spp em matrizes suínas descartadas. *Braz J Vet Res Anim Sci.* 2007;44:18.

30. Griffith ME, Hospenthal DR, Murray CK. Antimicrobial therapy of leptospirosis: *Curr Opin Infect Dis.* 2006;19:533-537.

31. Lane AB, Dore MM. Leptospirosis: a clinical review of evidence based diagnosis, treatment and prevention. *World J Clin Infect Dis.* 2016;6:61.

32. Khattak MB, Khan SJ, Khan A. Leptospirosis: a disease with global prevalence. *J Microbiol Exp.* 2018;6(5):219-221. doi:10.15406/jmen.2018.06.00218.

33. Cortese VS, Behan S, Galvin JE, et al. Evaluation of two antimicrobial therapies in the treatment of Leptospira borgpetersenii serovar hardjo infection in experimentally infected cattle. *Vet Ther Res Appl Vet Med.* 2007;8:201-208.

34. Vernel-Pauillac F, Werts C. Recent findings related to immune responses against leptospirosis and novel strategies to prevent infection. *Microbes Infect.* 2018;20:578-588.
35. Adler B, de la Peña Moctezuma A. *Leptospira and leptospirosis*. *Vet Microbiol*. 2010;140:287-296.

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37. Matsunaga J, Barocchi MA, Croda J, et al. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily: Leptospiral Ig-like protein family. *Mol Microbiol*. 2003;49:929-946.

38. Monaris D, Sbrogio-Almeida ME, Dib CC, et al. Protective immunity and reduced renal colonization induced by vaccines containing recombinant Leptospira interrogans outer membrane proteins and flagellin adjuvant. *Clin Vaccine Immunol*. 2015;22:965-973.

39. Desai S, van Treeck U, Lierz M, et al. Resurgence of field fever in a temperate country: an epidemic of leptospirosis among seasonal strawberry harvesters in Germany in 2007. *Clin Infect Dis*. 2009;48(6):691-697. doi:10.1086/597036.

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# LYME BORRELIOSIS

Isabelle Lebert (a, b), Francesca Dagostin (c), Luca Busani (d), Valentina Tagliapietra (c), Annapaola Rizzoli (c), Luciano Toma (e), Xavier Bailly (a, b), Magalie René-Martellet (a, b)

(a) Université de Lyon, INRAE, VetAgro Sup, UMR EPIA, Marcy l'Etoile

(b) Université Clermont Auvergne, INRAE, VetAgro Sup, UMR EPIA, Saint-Genès-Champanelle

(c) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

(d) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(e) Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome

## Biological, ecological and molecular features
of the causative agent

### Disease name

Lyme Disease, Lyme Borreliosis (LB).

### Disease agent

#### Common, scientific and Latin name

Bacteria of the *Borrelia burgdorferi* sensu lato (Bbsl) species complex.

#### Taxonomy

Phylum: Spirochaetes; Order: Spirochaetales; Family: *Borreliaceae*; Genus: *Borrelia/Borreliella*

The etiologic agents of human Lyme Borreliosis (LB) belong to the phylum of Spirochaetes, the order of Spirochaetales and the family of *Borreliaceae*. Within this family, DNA sequences analyses illustrated that diversity is clustered in two major clades of related species, i.e. species complexes, that respectively include: i) the etiologic agents of human relapsing fever; and ii) the agents of human LB. The classification of these two groups in different genus is the subject of a lively scientific controversy (1-4). Depending on defended positions, the species complex including Lyme disease agents is assigned to a specific genus called *Borreliella*, or remains in its historical genus *Borrelia*. We will hereafter use *Borrelia burgdorferi* sensu lato (Bbsl) to describe the species complex that contains all agents responsible for human LB as in the latest version of the Bergey's manual of systematic bacteriology (5), the reference text book on the subject (6), and the latest publication in the *International Journal of Systematic and Evolutionary Microbiology* (4) corresponding to the official publication of the International Committee on Systematics of Prokaryotes.

The Bbsl complex of bacteria comprises at least 21 genospecies worldwide (7). Among the nine genospecies present in Europe, five have confirmed human pathogenicity (*Borrelia afzelii*, *B. garinii*, *B. burgdorferi* sensu stricto, *B. bavariensis*, *B. spielmanii*) – with *B. afzelii* and *B. garinii* considered as the major cause of human illness – and four are considered to have potentially or unknown human pathogenicity (*B. bissettiae*, *B. lusitaniae*, *B. valaisiana* and *B. turdi*) (7, 8).
## Disease agent characteristics

Bbsl bacteria are helical in shape, comprising several non-cohesive coils. They range in size from 3 to 30 µm long and from 0.2 to 0.5 µm wide. They are surrounded by a surface layer, an outer membrane, periplasmic flagella, and a protoplasmic cylinder and are motile by rotation and translational movement. The protoplasmic cylinder consists of a peptidoglycan layer and an inner membrane which encloses the internal components of the cells.

Because of its clinical relevance, a reference genome of the Bbsl complex was obtained before the emergence of high-throughput sequencing, based on *B. burgdorferi* sensu strictu strain B31 (9). This reference genome illustrated the genome structure of the species complex, characterized by a linear chromosome and a large set of linear and circular plasmids, called lp and cp respectively. The genomic diversity of the species complex has then been explored, benefiting from the progress of sequencing techniques, either based on the sequencing of isolated strains (10, 11) or the sequence capture of genome components from infected material (12). Completed assemblies of Bbsl result in chromosomes from 850 kb to 910 kb long. The organization of the chromosome is similar within the whole species complex. Homologous recombination of chromosomal fragments occurs both within and among the chromosomes of species of the complex Bbsl. However, association measures among polymorphisms suggest that homologous recombination has a limited impact on the distribution of variability within species and modelling suggests that chromosomal homologous recombination rate is lower between species than within species (11).

Most of the 30 plasmid types of the species complex are distributed in different species, but show an extensive presence/absence polymorphism, with rearrangement occurring especially among linear plasmids (13). The most frequent plasmids are lp17, lp28-4, lp36, lp54 and cp26, followed by lp28-3, lp38 and cp32-5. The critical role of some of these plasmids in the process of vertebrate host infection has been documented (14). Most of the acquisition of new genes occurs on plasmids, at a regular rate, mostly through duplication of lipoprotein genes rather than by the import of heterologous material (15). The dynamics of plasmid evolution and exchange result in non-systematic but significant associations between chromosomal and plasmid diversity patterns (16).

Due to the relative consistency of polymorphism patterns, multilocus sequence typing (17) and eventually single gene barcoding still represent common methods to characterize the diversity of the Bbsl complex from either bacterial isolates or infected material (18). This is particularly convenient as Bbsl isolation is difficult. In recent years, the use of high-throughput sequencing, either through amplicon sequencing (19, 20) or sequence capture (21), proved useful to resolve and study co-infections by different Bbsl genotypes, which occur both in ticks and vertebrate hosts.

## Physiochemical properties

Bbsl isolates are usually grown at an optimal temperature between 30 and 37°C. Isolates that have been grown *in vitro* are microaerophilic and have complex nutritional requirements usually satisfied by the Barbour-Stoenner-Kelly (BSK II) medium (22) and later derivatives (23). They grow slowly, typically dividing every 8-12 h during the exponential growth phase *in vitro*. Culture-adapted isolates can usually reach cell densities of 10⁷ to 10⁸ per mL after *in vitro* cultivation for 5-7 days.
Priority level for EU

In 2018, the European Centre for Disease Control (ECDC) has added the neurological form of LB called Lyme NeuroBorreliosis (LNB) to the list of communicable diseases (24). This is a first step for uniformization of data retrieval from EU countries surveillance systems and will allow comparison of incidence data at EU level.

In 2021, notification of LB human cases was mandatory in 19 European countries: Bulgaria, Croatia, Czech Republic, Denmark, Estonia, Finland, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Norway, Poland, Portugal, Romania, Slovakia and Slovenia (25). However, case definitions, reporting and surveillance systems differ from one country to the other.

Distribution of the pathogen

The pathogen is widely distributed in Europe (Figure 1), where it varies spatially, but also temporally (26-28). The distribution of the pathogen is highly dependent on the distribution of infected *Ixodes* spp. ticks (Figure 2a and 3) and hosts (Figure 2b).

<figure><img src="image_33.png" /><figcaption>Figure 1. European Union and European Economic Area countries that reported at least one locally-acquired human Lyme borreliosis (hLB) case (including all clinical forms of the disease and all Bbsl pathogenic strains): hLB information was extracted from the TESSy-ECDC; Lyme neuroborreliosis (cases from 2017 to 2021) and from other sources (all clinical forms of hLB), such as national surveillance systems and published literature</figcaption></figure>
<figure><img src="image_34.png" /><figcaption>Figure 2. Distribution of Bbsl in questing *Ixodes* spp. ticks (a) and animal hosts (b) (derived from a literature review updated to 2023)</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>Figure 3. Distribution of: *B. afzelii*, *B. burgdorferi* s.s., *B. garinii*, *B. valaisiana* present in each country (a); *B. lusitaniae* (b); *B. bavariensis* (c); and *B. spielmanii* (d) in questing *Ixodes* spp. ticks (derived from a literature review updated to 2023)</figcaption></figure>
## Ecology and transmission routes

Bbsl transmission occurs mainly during a tick bite, either from an infected tick to its host, or from the infected host to the tick. Transmission can also take place by cofeeding (29). Transovarian transmission (vertical transmission from adult female to larvae) seems absent or negligible in *Ixodes* tick (30). The occasional detection of bacteria in larvae can result either from an infection during a first blood meal attempt on an infected host or by cofeeding. In fact, bacteria can be detected in the larvae as early as 24 hours after the bite on an infected host (31, 32).

Vectors: Ticks of the *Ixodes* genus are the only confirmed vectors of Bbsl spirochetes. Among them, three species were experimentally confirmed to transmit Bbsl spirochetes in Europe (Table 1). However, several other species of this genus are suspected vectors of these bacteria based on epidemiological evidence (7, 8, 33). The main vector of the bacteria of the Bbsl complex in Europe is the hard tick *I. ricinus* (Figure 4).

<table><caption>Table 1. Tick species that are experimentally confirmed vectors of *Borrelia burgdorferi* s.l.</caption><thead><tr><td>Genus</td><td>Species</td><td>Distribution</td><td>Bbsl strains</td><td>References</td></tr></thead><tbody><tr><td>Ixodes</td><td>ricinus</td><td>Widely distributed in Europe except under Mediterranean climate</td><td>B. burgdorferi s.s; B. afzelii; B. garinii</td><td>34-41</td></tr><tr><td>Ixodes</td><td>persulcatus</td><td>Widely distributed from Poland to Japan</td><td>B. garinii</td><td>42,43</td></tr><tr><td>Ixodes</td><td>hexagonus</td><td>Reported from most European countries</td><td>B. burgdorferi s.s.</td><td>44</td></tr></tbody></table>

<figure><img src="image_36.png" /><figcaption>Figure 4. Current 1-km probability of presence of *Ixodes ricinus* across Europe, produced using random forest and boosted regression trees based spatial modelling techniques (source ERGO group)</figcaption></figure>
*Ixodes ricinus* is the most common and most widely distributed tick species in Europe. It is an exophilic tick capable of feeding on a wide number of vertebrate species including the main reservoirs of Bbsl spirochetes (birds and rodents) and humans.

*Ixodes ricinus* is vulnerable to desiccation at all life stages and needs to return regularly to the moist litter layer to rehydrate (45). Thus, tick development and questing depend strongly on local environmental conditions related to climate, meteorology, seasonality, abundance of hosts. These features highly affect the duration of tick life cycle but also the timing of its activity and distribution (46).

*Ixodes ricinus*, like all other Ixodidae, goes through three life stages: larva, nymph, and adult (Figure 5). Each stage feeds on vertebrate hosts for an average duration of seven days. During this long engorgement period, some hosts might be able to move over large distances, thus enabling the dissemination of ticks. After feeding, a tick falls from the host and moults to the next stage. Larvae and nymphs mainly feed on small hosts such as birds, small mammals, and rodents, while adult ticks generally feed on large mammals such as wild ungulates (47,48). Mating generally occurs on the host. The engorged female subsequently falls to the ground and lays between 2000 and 3000 eggs, leading to local multiplication of ticks. The life cycle of the tick thus alternates between several steps: i) resting and developing on the ground; ii) exophilic questing; and iii) blood feeding on a host. All steps are essential for the maintenance of the life cycle.

<figure><img src="image_37.png" /><figcaption>**Figure 5.** The *Ixodes ricinus* life cycle: A. development on the ground, B. questing activity, C. feeding on a vertebrate host, D. host-mediated dispersal</figcaption></figure>

*Ixodes persulcatus* is another confirmed vector of Bbsl spirochetes in Eastern Europe. The species is present in at least 14 countries from Poland to Japan and, as *I. ricinus*, is an exophilic species capable of engorging on a high number of different hosts increasing its capacity of pathogen transmission (49).

The vector competence of *I. hexagonus* for Bbsl spirochetes was also confirmed in the laboratory (44). *I. hexagonus* is an endophilic species reported from most European countries. Their main hosts are hedgehogs and carnivorous mammals (50).
Nevertheless, other species of ticks of the genus *Ixodes* are suspected vectors of Bbsl. Even if these tick species rarely bite humans, they can contribute to maintaining the zoonotic cycle of bacteria and therefore influence their prevalence and spatial distribution. These species are *I. acuminatus* (51), *I. arboricola* (52, 53), *I. frontalis* (52, 54), *I. trianguliceps* (55) and *I. uriae* (56). Hosts: The main reservoir hosts of *B. afzelii*, *B. bavariensis* and *B. spielmanii* are small mammals (57-61), those of *B. garinii* and *B. valaisiana* are birds (57, 58, 62), those of *B. lusitaniae* are lizards (62-64) and those of *B. burgdorferi* s.s. are small mammals and birds (58, 65, 66). Other vertebrate species can be reservoirs of one or more *Borrelia* species (33). For example, *B. afzelii* and *B. valaisiana* have been detected in the badger, *Meles meles* (67), *B. spielmanii* seems mainly restricted to glirid rodents, namely the garden dormouse, *Eliomys quercinus* and the hazel dormouse, *Muscardinus avellanarius*, but not the European edible dormouse, *Glis glis* (60). Similarly, *B. afzelii* and *B. burgdorferi* s.s. can be found in the red squirrel, *Sciurus vulgaris* and the Siberian chipmunk, *Tamias sibiricus barberi* (65).

Deer are not proved to be reservoirs and are considered incompetent hosts for *Borrelia* genospecies (68, 69), even if positive serologies were observed (70, 71).

# Drivers of disease emergence and spread

## Ecological drivers

### Drivers responsible for tick multiplication and questing activity

Disease presence, emergence and spread are highly related to *Ixodes* spp. tick distribution and questing activity. Many papers presented results of longitudinal studies performed in order to model spatial and temporal variations in tick abundance (27, 28, 46, 72-77). They provided observational data useful for the comprehension of the main ecological drivers that might contribute to disease emergence and spread:

- Climate: *Ixodes ricinus* ticks are able to develop in a wide range of climates if local conditions, in particular humidity and temperature, are suitable (78). To date, the species has been reported from Northern Africa to Northern Russia (79) and was recently detected in Iceland (80). A wide range of territories is thus suitable for their development in Europe. An exception is in the area around the Mediterranean basin, where the hot and dry conditions are generally considered unfavourable for the species (81, 82).

- Seasons: Due to their high sensitivity to desiccation tick questing activity highly depend on meteorological conditions and seasons. In most of European countries, density of questing ticks shows a high seasonality pattern with a peak of activity between April and August depending on the latitudes of the countries (83-85).

- Altitude: Despite many studies have shown that tick densities decrease with increasing altitude (with a sharper decrease above 1,000 m asl) (77, 86-89) since 2003, expansions in the distribution of ticks have been observed up to 1,100 m asl in the Czech Republic and Switzerland (90, 91), up to 1,250 m asl in Slovakia and the Czech Republic (77, 87), and up to 1,700-1,800 m asl in the Northern Alps (92) and the French Pyrenees (93).

- Land cover: *Ixodes ricinus* can be encountered in many types of vegetation, but forests and, in general, wooded areas offer better conditions for its development (94-96). In European countries, tick abundance was found higher in deciduous such as oaks and beech forests than in coniferous ones (92, 94, 97-99). The species has also been detected in suburban forests and public parks, as well as in private gardens (97, 100-102).
- Hosts: The development and local abundance of *I. ricinus* ticks is linked with the density and the wide variety of vertebrate hosts on which they feed. Local densities of rodents and other small mammals play an important role in sustaining populations of larvae and nymphs (103-105). Abundance of rodents is modulated by food availability, such as acorn density that can be used as a proxy for rodent density (76). The density of wild ungulates has been found to affect the local densities of *I. ricinus* and the risk of tick-borne disease transmission (97,106-108). Deer specifically, play an important role in the developmental cycle of ticks, with several studies demonstrating a significant correlation between deer population density and tick abundance (109-112). Birds are thought to play a relatively small role in maintaining tick populations, but an important role in tick infections and tick dissemination, through short or long-distance flights and migratory behaviour (113).

Many other key drivers may influence the local abundance of *I. ricinus*, in particular local weather conditions, soil composition, thickness of the litter layer, vegetation period, habitat structure and human management of wildlife and forests (78, 97, 111, 114, 115).

## Drivers of tick infections: reservoirs of Bbsl

As competent reservoirs of Bbsl, rodents and birds highly contribute to tick infections. However, it was shown that the influence of local rodent populations on tick abundance and local infection is difficult to estimate due to their rapid development and pronounced seasonal variations (116). Introduced chipmunks and lizards were also proved to be competent reservoirs of Bbsl pathogens (28, 117).

On the contrary, deer are considered incompetent reservoirs of Bbsl therefore playing a complex role in its circulation (118, 119).

The dilution effect of biodiversity on Bbsl prevalence in ticks and reservoirs, defined as “the effect that occurs when the diversity of an ecological community reduces the transmission of a pathogen” is discussed in many articles (120-124). The concept is debated and continues to be questioned (120-122, 125). In North America, where the only species of *Borrelia* infecting humans is *B. burgdorferi* s.s., the tick vector *I. scapularis* and the main hosts are the white-tailed deer and the white-footed mice, potential dilution effects have been observed (121, 123, 126). In Europe, due to the presence of several *Borrelia* genospecies with different reservoir hosts, the dilution effect remains uncertain and more difficult to evaluate (118, 124).

## Other drivers

Studies on demographic and geographic drivers of LB emergence in Europe are lacking. In a study performed in Wales and England, people with Lyme disease were from areas with higher socioeconomic status (127). New studies should be performed to better understand the consequences of such drivers on LB incidence in humans.

# Natural history of disease in humans and animals, including symptoms, morbidity and mortality

## Brief history of the pathogen and disease

The first descriptions of clinical manifestations of Lyme disease were performed between 1883 and 1909 by Buchwald and Afzelius (128-131). Afzelius described the cutaneous lesion now known as *erythema migrans* (EM) and suggested the role of the tick in the disease. In 1922, Garin
and Bujadoux reported the first case of neuroborreliosis in France confirming the role of the tick and the infectious nature of the disease (132).

Finally, in the mid-1970s, several patients, mainly children presented oligoarthritis with, for approximately ¼ of them, an expanding, annular skin lesion suggesting EM, before the onset of arthritis. All cases clustered around the town of Lyme in Southeastern Connecticut, USA, giving the name of Lyme disease (133). The aetiological agent was then identified first in the North American deer tick, *I. scapularis* (previously named *I. dammini*), by Willy Burgdorfer in 1982 (134). Molecular studies revealed the isolate to be a new species within the genus *Borrelia*, and the species was subsequently named *B. burgdorferi* (108).

## Disease in humans

In human health, the risk of infection is linked to the risk of exposure, defined as the product of the abundance of infected questing ticks and the human exposure to ticks. Transmission to humans is incidental and generally occurs during outdoor activities in a suitable habitat for ticks. The probability of pathogen transmission is considered low when the tick is removed within 24 hours (135).

However, the earliest the infected tick is removed, the lower the probability of *Borrelia* transmission is (136, 137). Humans are more commonly bitten by *I. ricinus* nymphs (70%) (138). Due to their small size, nymphs are sometimes only discovered late, typically few hours or days after infestation. Some patients may not remember any tick bite despite confirmed Bbsl infection (139). Thus, it is considered that the nymphal stage is the riskier for *Borrelia* transmission. In large surveys performed in humans, the seroconversion rate after a Bbsl-infected tick bite varied between 1.8% and 4.2% and the rate of clinical LB ranged between 0.7% to 4.0% (137,140,141).

Bbsl bacteria is mainly responsible for cutaneous, neurological and musculo-skeletal symptoms. Erythema migrans is the earliest and most common manifestation of LB and is also defined as the early localized stage of LB. It occurs in the first weeks after a tick bite. Systemic flu-like symptoms may accompany EM. If left untreated, the spirochetes can disseminate and cause early disseminated LB (weeks to months after tick bite) or late disseminated LB (from months to several years after tick bite) (142,143).

Three types of cutaneous lesions are usually observed:

- Erythema migrans (reported in 70 to 95% of cases in Europe depending on countries and studies). The classical form of erythema migrans corresponds to a pinkish to reddish colour cutaneous lesion, oval-shaped, with a central clearing, a regular growth, a centrifugal extension that appear in most cases at the site of the tick bite (144).

- Acrodermatitis Chronica Atrophicans (reported up to 10% of cases in Europe) corresponds to a macule or a plaque on a limb segment, of varying colour, more visible at bone tips, with progression from an initial oedematous stage to atrophy of the skin (145,146).

- Borrelial lymphocytoma is the rarest cutaneous manifestation of Lyme disease. It generally corresponds to a single lesion, very slow-growing, with varying colours, rarely pruritic with unusual localizations (ear lobe in children, breast in adults, exceptionally on the face, thorax or limbs) (133, 147).

Neurological manifestations can appear either in the early stages of the disease (more than 90% of cases) (148) or in the later stages (approximately 10%) and follow skin manifestations. Prevalence varies from 6 to 23% in European patients (133, 147) and affects mainly children/adolescents and adults over 50 years. LNB is generally due to infections by *B. garinii*
(the most frequent, 2/3 of cases) and *B. afzelii* and mainly manifests as meningoradiculitis and cranial nerve palsy (facial nerve, mainly in children). Other neurological manifestations such as clinical meningitis, acute myelitis, and encephalitis are less frequent. Cognitive disorders and dementia are rarely linked with LB infections but should be investigated in the absence of other aetiology (133, 147).

Arthralgia monoarthritis is a common sign of LB at the early stages and generally appears in an average time of 6 months (few weeks to two years) after an infected tick bite (133, 147). Without antibiotic therapy the affection can progress towards a chronic presentation. In Europe, the proportion of patients with Lyme arthritis with no EM or no tick bite recall seems higher than in the USA. Chronic myalgia has been associated with DNA detection of Bbsl in muscle samples (149). Many other atypical musculo-skeletal manifestations have been reported with low levels of scientific evidence.

Cardiac manifestations are reported in up to 4% of cases of LB. Outcome is favourable if correctly treated, but conduction disorders may require temporary cardiac pacing (147). Myocarditis, left ventricular dysfunction or cardiac failure are very rarely associated with LB (150, 151).

Several cases of patients with ophthalmological disorders and a positive serology for LB were reported. However, the link between symptoms and infection with Bbsl remains uncertain (147).

Other clinical manifestations are attributed to infection with the agents of LB with sometimes poor scientific evidence of their implication. Cases of death are rarely reported in patients with LB. Obel *et al*. did not show any significant differences in mortality rates between patients with LNB and the global population (152).

## Disease in animals

As for humans, the risk of transmission to animals is proportional to their probability to encounter infected ticks in a suitable habitat (153-157).

### Lyme disease in dogs and cats

Knowledge on consequences of Bbsl infection in domestic carnivores is incomplete. Most Bbsl-seropositive dogs and cats show no clinical signs of illness, neither after experimental nor after field infections. Koch's postulates have only been fulfilled experimentally for a clinical picture of transient fever, anorexia and clinical arthritis with lameness as well as reluctance to move, which was detected in puppies but not in dogs > 6 months. In contrast to humans, no EM has been observed in dogs (158), but synovial lesions were significant in infected dogs in all canine experimental studies (158-160).

When clinical signs compatible with Lyme disease are observed in dogs in the field, confirmation of the role of Lyme disease agents in the clinical outcome is difficult because diagnosis is based mainly on serology and no DNA of viable agents is detected (159). A small subset of infected dogs (<5%) presents transient signs of Lyme arthritis – corresponding to acute monoarticular or polyarticular lameness with joint swelling, fever, lethargy, and mild local lymphadenopathy – that generally responds well to an adapted antibiotic treatment. Nephritis (immune-complex glomerulonephritis) associated with Lyme infection is another supposed consequence of Bbsl infection described in dogs, less frequent but with the most serious impact on health. Other forms of infections described in humans are rare or not well documented in dogs (159). A predisposition of infection of Bernese Mountain Dogs was suggested due to a higher rate of seropositivity in this species in Central Europe but no clear consequences on health was found (no more risk of getting sick than other dog species) (161).
Vertical transmission and other non-vector borne modes of transmission (via semen, urine or blood) of *Borrelia* spp. are considered unlikely in dogs under natural conditions (160).

Seropositivity for Bbsl was detected in cats in Europe. However, no clear evidence of the consequence of infection with Bbsl on cats' health was demonstrated (159).

## Lyme disease in horses

Horses are often exposed to *I. ricinus* ticks in Europe and seroprevalence of Bbsl infections in this group was evaluated from 12.4% to 48.4% depending on the studies and the countries (162, 163). Contrary to the high seroprevalence observed in the field, the paucity of documented cases of Lyme disease has made Bbsl infection and Lyme disease a controversial topic in equine practice (164). In horses, a broad spectrum of clinical manifestations, including arthritis, lameness, anterior uveitis, encephalitis and abortion, has been attributed to Bbsl infections (163). However, several other frequently described tick-borne pathogens – in particular *A. phagocytophilum*, *Babesia caballi* and *Theileria equi* – infect horses in Europe with sometimes similar clinical manifestations (162). Experimental inoculations in ponies led to systemic infection but did not induce any clinical signs nor histopathological alterations, except for skin lesions.

The association of Bbsl infection with other clinical signs is not well documented (164). It remains unclear if European Bbsl isolates are capable of causing such clinical manifestations in horses.

## Lyme disease in cattle

As grazing vertebrates, cattle are often exposed to Bbsl (as well as *A. phagocytophilum* and *Babesia*) infected ticks in Europe (165). However, clinical relevance of Bbsl for ruminants is questionable. Active *B. burgdorferi* s.s., and *B. afzelii* infections with associated symptoms (skin erythema, fever, acute lameness due to arthritis) have been described in cattle in rare cases. Other studies suggested a link between serological evidence of Bbsl infection and clinical signs but causality remain uncertain. Experimental infections of cattle with *B. burgdorferi* s.s., *B. garinii*, and *B. afzelii* produced no clinical signs in cattle and in sheep. Neither clinical cases nor infection experiments have been published in goats to date (163).

# Availability of preventive, therapeutic and control measures, including licensed or pipelines vaccines

## Therapy in humans

*Borrelia* species are susceptible to several classes of antimicrobial agents, including penicillins, second- and third-generation cephalosporins, tetracyclines, macrolides and glycopeptides. On the other side they are relatively resistant to aminoglycosides, trimethoprim, rifampicin and quinolones (166,167). Treatment of human cases of LB is based on the use of a targeted antibiotic therapy. Use of first line antibiotics depends on the age of the patient and the clinical manifestation of the disease (168).
## Therapy in animals

Doxycycline 10 mg/kg SID or BID is the first line antibiotic recommended for use in dogs with suspected LB because of its efficacy on *Borrelia* and on other tick-borne pathogens such as *Ehrlichia* and *Anaplasma* bacteria and purported antiarthritic and anti-inflammatory properties. β-lactams and macrolids are also antibiotic families that have proven their efficacy on the bacteria. A rapid response to the treatment occurs generally within 1-2 days, but the treatment is recommended for 4 weeks. As a matter of fact, in some cases, 4 weeks of high dose treatment (10 mg/kg doxycycline q12h) is not sufficient to clear all organisms in dogs. Relapse may be caused by coinfections or reinfections (159). Additional symptomatic treatments are recommended depending on the clinical forms observed (arthritis or nephritis).

Consistent with human guidelines, tetracyclines and β-lactam drugs are most commonly used to treat equine Lyme disease (164). However, the ideal treatment regimen for equine Lyme disease is unknown and further studies are necessary to prove the pertinence of use of those antibiotics in European horses.

## Licensed or pipelined vaccines

There is actually no licensed vaccine for human prevention of LB in Europe.

An anti-*Borrelia* vaccine (Merilym 3 ND) using inactivated *B. burgdorferi* s.s., *B. garinii* and *B. afzelii* strains is available for use in dogs in Europe. The vaccine reduces the risk of skin infection by *Borrelia*, however the efficacy against the disease in dogs was not studied. This anti-*Borrelia* vaccination of dogs is actually the subject of controversies in particular because the disease remains poorly characterised in the species and infected dogs seem to respond readily to a correctly administered antibiotic treatment (159, 160, 169).

In North America, the off-label use of anti-*Borrelia* dog vaccine in horses is described. However, duration of immunity with dog vaccine used in horses seems short (170).

An oral reservoir-targeted vaccine using an OspA protein was developed and gave promising results in the USA in protecting uninfected mice from infection and reducing transmission of Bbsl bacteria (171). However, the feasibility of the use of such a vaccine as a long-term strategy to reduce hLB risk in Europe (by reducing infection of rodent reservoirs) remains uncertain taking into account the diversity of Bbsl strains, the role of birds in the epidemiological cycle and the rate of renewal of wild rodents in the field.

## Other prevention measures

### Prevention of tick bites in humans and domestic animals

Prevention of tick-borne diseases essentially relies on avoiding tick bite and early tick removal (147, 172-175).

Personal protective measures (wearing long trousers tucked into socks, white clothes, etc.) and use of repellents are effective to prevent tick bites. The main repellents recommended for ticks prevention in humans include DEET (N,N-diethyl-mtoluamide), IR3535 (ethyl butylacetyl-aminopropionate) or picardin (1-piperidinecarboxylic acid) (176). The use of permethrin for impregnation of clothes is no longer recommended, even for brief use in high exposure situations, due to skin irritation. It is also recommended to carefully check the body for ticks after coming back from outdoor activities (177). In case of bite, mechanical removal of attached tick and disinfection with antiseptics, followed by one month of observation in order to see if EM occurs are the recommendations.
In domestic animals, prevention is essentially based on tick prophylaxis i.e. acaricidal/repellent use. A high number of acaricidal specialities, developed to prevent tick infestations in domestic animals, are now available in the market of veterinary drugs. However, as 100% protection is not guaranteed, domestic animals should also be regularly checked and ticks removed in case of exposure.

## Reduction of tick population or Bbsl infection, using acaricidal treatments

Many studies tested the effects of acaricidal treatments of the environment and/or wild animals (rodents, deer) on the reduction of tick density or of *Borrelia* infection (178-182). Even if an effectiveness on the reduction of the density of ticks has been demonstrated experimentally, consequences of these treatments on the environment and their acceptability by the society should be widely studied before implementation in the field (149,183).

## Reduction of tick population or Bbsl infection, using hosts control

Host population control can be easily managed on domestic animals but is more complex on wild animals.

Several studies assessed the effects of ungulate management (culling or fencing, effects of deer density, habitat types, sizes of enclosed or unenclosed areas) on tick abundance (107, 110, 118, 184). *Ixodes ricinus* density tended to be lower in plots without deer than in those with deer suggesting a positive correlation between deer and tick densities (110). However, small enclosures with high deer densities were associated with reduction in tick densities as well as rodent numbers probably linked with the effect of high deer density on vegetation height (110, 118).

In Denmark, the reduction of roe deer for several years led to a reduction in LNB with a lag of 1 to 3 years, leading to the hypothesis of a reduction in the total population of ticks (Andersen *et al.*, 2018). However, in the United States, deer culling or reduction had a limited effect on tick abundance and no evidence of an effect on human disease risk reduction (126). Predators such as red fox (*Vulpes vulpes*) and stone marten (*Martes foina*) or assemblage of predators (foxes, raccoons and bobcat) were suggested as possible tick and Bbsl control measure (76, 185). They may act indirectly on Lyme disease risk and disease transmission by lowering the density of reservoir-competent mammalian hosts, such as rodents. It was also suggested that predation can change host behaviour in limiting their movement and consequently their contacts with ticks (185-187).

## Effects of landscape modifications on tick population or Bbsl infection

Many methods using landscape management such as burning, mowing, leaf litter removal, herbicide treatment or desiccation were tested to reduce tick density and prevalence of Bbsl (188). Mowing (vegetation management) seems to have a reduction effect (189) while cattle grazing in forested areas was not shown to have any effect on infected nymph densities (190).

In urban areas, restoration of green areas is increasing in order to maintain human health and well-being (188). Consequences of this revegetation on vectors dynamic remain poorly understood. As people are encouraged to move around and engage in outdoor physical activities, a global study should consider the risk of tick-borne pathogen infections and implement risk prevention communication campaigns in these specific areas (191).

To implement effective control measures, it is essential to have a clear understanding of all factors that contribute to the risk of tick multiplication and Lyme disease. In the one hand, factors that may favour ticks and wild animal presence and, on the other hand, factors that could prevent
tick bites and reduce human and pet exposure (drawing channel visitor routes, developing leisure areas) should be further explored (192).

Attention should be paid to the effects of management methods on biodiversity and environment as well as the consequences on the abundance of hosts and ticks and the risk of Lyme disease (187). It would be useful to experiment the effects of all these factors with new integrated management strategies through consortia involving decision-makers, managers, scientists and citizens (183).

## Disease specific recommendations

Sexual and mother-to-foetus transmission have been suggested, but never demonstrated. Pregnant women should thus be treated as the general population taking into account their status for the prescription of treatments (173). Similarly, there are actually no confirmed reported cases of *Borrelia* transmission to humans through blood transfusions or grafts (193).

## Epidemiological situation at different spatial scales: past and current trends

The incidence of LB is difficult to estimate and to compare between years and countries because of a marked heterogeneity in surveillance systems, monitoring scale, case definitions, and testing methods.

In 2011, the European Concerted Action on Lyme Borreliosis (EUCALB) published a series of LB case definitions that incorporate clinical findings and essential laboratory evidence (194); however, implementation remains inconsistent in European countries. In 2023, only two countries (France and Poland) use the EUCALB cases definitions in the context their national surveillance system (195).

LNB was finally considered as the most specific form of the disease, easier to standardized and was thus chosen for future estimation of LB cases in Europe. Since 2019, ECDC added LNB to the communicable disease list with a standardized case definition. In 2023, only two countries (Bulgaria and Romania) use the ECDC LNB case definition in the context their national surveillance system of LB (195).

## Past trends – results of Lyme borreliosis surveillance from 2005 to 2020

Burn *et al.* recently reviewed incidence data of LB in European countries from either national surveillance systems or systematic review for the period 2005-2020 (195,196). Taking into account all surveillance systems and considering any case definition, Burn *et al.* estimated an annual average of LB case number of 128,888 in Europe (195).

Comparison of incidences suggests (i) an increase of LB incidence at the national level in some countries of Northern and Eastern Europe from 2005 to 2020, while it remains globally constant in the majority of other countries during the same period (195,196).

Other studies suggest evidence of a geographical expansion of the disease in previously non-endemic areas as it was shown in the province of Verone, Northern Italy, using a five-years (2015-2019) sentinel surveillance (143).

Comparison of incidences between countries is difficult due to the lack of standardized surveillance system at the European level. Analysis of results of incidence data from surveillance
systems shows high reporting variations between countries. In particular, during the 2005-2020 period, Estonia, Lithuania, Slovenia, and Switzerland reported more than 100 cases/100,000 Population Per Year (PPY), France and Poland from 40-80/100,000 PPY, Finland and Latvia from 20-40/100,000 PPY while Belgium, Bulgaria, Croatia, England, Hungary, Ireland, Norway, Portugal, Romania, Scotland, and Serbia reported less than 20/100,000 cases PPY. However, at the subnational level, hotspots of infections (>100 cases/100,000 PPY) were found in some areas in particular in Belgium, Czech Republic, France, Germany, and Poland confirming the high possible variations in incidence within countries (79, 80).

## Current trends – results of Lyme Neuroborreliosis surveillance according to the ECDC definition of case from 2019-2021

Since 2019, the ECDC has added the neurological form of LB called Lyme NeuroBorreliosis (LNB) to the list of communicable diseases (24).

To estimate the current epidemiological trend, we included all cases of LNB reported to the European Surveillance System Database (TESSy) during the years 2019-2021. Population denominator data were provided by the Statistical Office of the EU (Eurostat) for calculating incidence rates.

Over the 2019-2021 period, 13 EU/EEA (European Union/European Economic Area) countries reported 2645 cases of Lyme neuroborreliosis.

Analyses of the European LNB surveillance data showed that:

- The number of reported LNB cases varied considerably from one country to the other from 2019 to 2021 (Table 2) with the higher number of LNB cases reported in Czechia and Norway, followed by Poland, Italy, Denmark and Slovakia. All other countries that participate to the surveillance reported less than 40 cases during the 2019-2021 period.

- A higher number of LNB cases was reported in 2019 compared to 2020 and 2021 (Figure 6). However, the short period of surveillance and the limited number of reported cases do not allow to bring out any epidemiological trend.

- A peak of LNB cases was reached each year in the summer months (from June to September) (Figures 7 and 8).

- Two age groups – i.e., young people <20 years old (39% of cases) and adults between 50 and 79 years old (40% of cases) – represent 79% of LNB reported cases during the reporting period (Table 3, Figure 9).

The recent implementation of LNB records at the European level should lead to a cautious interpretation of the results presented here. As a matter of fact, due to the short period of reporting and the inconstant participation of European countries, it is possible that epidemiological trends in particular incidences data are underestimated in several countries.

The use of the ECDC LNB case definition in national surveillance systems in all European countries could help clarify these epidemiological trends in the future.
<table><caption>**Table 2.** Number of reported cases of *Lyme neuroborreliosis* and incidence rate (number of cases reported per country per 100,000 inhabitants) in 13 countries of the European Union and European Economic Area, 2019-2021 (data from TESSy-ECDC)</caption><thead><tr><td>Country</td><td>N. cases</td><td>Mean incidence</td><td>Imported cases</td></tr></thead><tbody><tr><td>Czechia</td><td>818</td><td>2,6</td><td>0</td></tr><tr><td>Denmark</td><td>157</td><td>0,9</td><td>0</td></tr><tr><td>Hungary</td><td>0</td><td>0,0</td><td>0</td></tr><tr><td>Ireland</td><td>24</td><td>0,2</td><td>2 (8%)</td></tr><tr><td>Italy</td><td>247</td><td>0,2</td><td>0</td></tr><tr><td>Lithuania</td><td>5</td><td>0,1</td><td>0</td></tr><tr><td>Luxembourg</td><td>2</td><td>0,1</td><td>0</td></tr><tr><td>Norway</td><td>806</td><td>5,0</td><td>7 (0,9%)</td></tr><tr><td>Poland</td><td>434</td><td>0,4</td><td>1 (0,0%)</td></tr><tr><td>Portugal</td><td>4</td><td>0,0</td><td>0</td></tr><tr><td>Romania</td><td>2</td><td>0,0</td><td>0</td></tr><tr><td>Slovakia</td><td>108</td><td>0,6</td><td>0</td></tr><tr><td>Slovenia</td><td>38</td><td>0,6</td><td>0</td></tr></tbody></table>

<figure><img src="image_38.png" /><figcaption>**Figure 6.** Total number of *Lyme neuroborreliosis* cases notified to ECDC during the period 2019-2021 (data from TESSy-ECDC)</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>**Figure 7.** Number of reported *Lyme neuroborreliosis* cases by month of onset, 2019-2021 (data from TESSy-ECDC)</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>Figure 8. Number of Lyme neuroborreliosis cases by week of onset in the declaring European Union and European Economic Area countries, 2019–2021 (data from TESSy-ECDC)</figcaption></figure>

<table><caption>Table 3. Main characteristics of reported cases of Lyme neuroborreliosis, European Union and European Economic Area countries, 2019–2021 (n = 2645) (data from TESSy-ECDC)</caption><thead><tr><th rowspan="2">Characteristics</th><th colspan="2">Cases</th><th rowspan="2">Incidence<br>per 100,000 inhabitants</th></tr><tr><th>Number</th><th>%</th></tr></thead><tbody><tr><td>Total</td><td>2645</td><td>100</td><td>0,50</td></tr><tr><td>Age group (years)</td><td></td><td></td><td></td></tr><tr><td>0-9</td><td>738</td><td>28</td><td>NA</td></tr><tr><td>10-19</td><td>303</td><td>11</td><td>NA</td></tr><tr><td>20-29</td><td>111</td><td>4</td><td>NA</td></tr><tr><td>30-39</td><td>166</td><td>6</td><td>NA</td></tr><tr><td>40-49</td><td>213</td><td>8</td><td>NA</td></tr><tr><td>50-59</td><td>338</td><td>13</td><td>NA</td></tr><tr><td>60-69</td><td>395</td><td>15</td><td>NA</td></tr><tr><td>70-79</td><td>305</td><td>12</td><td>NA</td></tr><tr><td>≥ 80</td><td>76</td><td>3</td><td>NA</td></tr><tr><td>Unknown</td><td>0</td><td>NA</td><td>NA</td></tr><tr><td>Sex</td><td></td><td></td><td></td></tr><tr><td>Female</td><td>1160</td><td>44</td><td>NA</td></tr><tr><td>Male</td><td>1483</td><td>56</td><td>NA</td></tr><tr><td>Unknown</td><td>2</td><td>NA</td><td>NA</td></tr><tr><td>Importation status</td><td></td><td></td><td></td></tr><tr><td>Imported</td><td>10</td><td>0</td><td>NA</td></tr><tr><td>Locally acquired</td><td>2367</td><td>89</td><td>NA</td></tr><tr><td>Unknown</td><td>268</td><td>10</td><td>NA</td></tr></tbody></table>

NA: not available
<figure><img src="image_41.png" /><figcaption>Figure 9. Number of Lyme neuroborreliosis (LNB) cases, by gender and age group in the reporting countries of European Union and European Economic Area, 2019-2021 (data from TESSy-ECDC)</figcaption></figure>

## Sociological and demographical dimension affecting susceptibility and exposure, including gender

Many patients with confirmed LB were exposed to ticks during leisure (142, 197) or outdoor professional activities. Main categories at risk include: farmers, forestry workers, horticulturists, hunters, rangers and veterinarians (198-209). In Poland, the seroprevalence of Bbsl infection in people with occupational risk of tick bites and forestry workers was higher than those in the control group composed of blood donors (203, 210). In Germany, military personnel showed a low but relevant risk of exposure to tick bites (211), while seroprevalence and incidence of Bbsl in football players were low (212). Recently, in Europe, an enhanced threat of exposure to tick bites has been observed in urban and peri-urban areas, green spaces and recreational parks. Infected ticks can be present in these areas and show a high risk of infection due to high density and activity of humans (213).

Several studies attempted to evaluate the clinical costs and economic outcomes of Lyme disease (214, 215). Many of these were conducted in the USA (68%) followed by Canada (9%) and the Netherlands (9%) and only one study in Sweden, Scotland and Germany (215). Lohr et al. estimated that the median medical cost for the hospitalization of a Lyme disease case ranged
from 2843 € for adults to 3917 € for adolescents between 2008-2011 (216). In Germany, using extrapolations, the direct costs of the disease were estimated at 23 million € annually for patient treatments and indirect costs such as loss of productivity and absence from work at 7 million €. In the Netherlands, Van den Wijngaard *et al*. estimated a mean cost (including direct and indirect costs) of about 5700 € per patient annually in 2014, resulting in a total cost of 23.5 million €/ per year (217).

Several studies reported that the general population and professionals (health care, exposed workers) have an incomplete knowledge of tick ecology and Lyme disease. Sometimes prevention practices are not adopted and knowledge of the recommendations is weak (198, 218-220). Improvement of knowledge and implementation of communication and health education actions for the public are essential, especially towards children (221-224). Leaflets, movies (225, 226), posters, information on the Internet (227), information at the entrance to a forest and mobile applications are all information and preventive tools widely used. As few studies dealt with the perception of risk by humans (218, 228, 229), new sociological studies should be performed in the future to ensure the feasibility and the acceptability of preventive measures proposed.

# Diagnostic procedures and notification systems used at local, national and European scale

## Diagnosis

There is currently no widely adopted standardized case definition for LB in Europe. Case definitions used for routine surveillance of LB vary by country (history of a tick bite, spectrum of clinical signs and laboratory tests used).

In laboratory LB diagnosis relies first on a suggesting clinical presentation and a history of tick exposure. Laboratory tests are often necessary to confirm infection. Culture of spirochaetes is considered as the gold-standard for confirmation of bacterial infections however it is often difficult to apply in case of LB due to the low number of viable spirochaetes usually present in patient samples and the difficulties to culture them. Thus, culture of spirochaetes is generally used only to confirm uncertain cases.

Serology is often used as a first line tool for the diagnosis of LB with an acceptable sensitivity and specificity in a majority of cases (194). However, the limits of serology should be known in particular: i) in case of negative serology in the early stages of the disease when the immune response has not yet been established; and ii) in case of positive serology in patients with successful antibiotic treatment due to the long persistence of antibodies despite recovery.

In Europe, recommended serology diagnosis relies on a two-step approach involving an initial screening test (usually ELISA, Enzyme-Linked Immunosorbent Assay), followed by a western blot for reactive and equivocal samples (168, 194). Nucleic acid amplification on infected tissues can help confirm doubtful cases. However, methods are not standardized, so test results obtained by different laboratories may show significant variability. In case of LNB, CerebroSpinal Fluid (CSF) examination is recommended.

In 2011, the EUCALB published a series of LB case definitions (Table 4) that incorporate clinical findings and essential laboratory evidence.
<table><caption>Table 4. Summary of clinical case definitions for Lyme borreliosis (194)</caption><thead><tr><th>Term</th><th>Clinical case definition</th><th>Laboratory evidence: essential</th><th>Laboratory/clinical evidence: supporting</th></tr></thead><tbody><tr><td>Erythema migrans</td><td>Expanding red or bluish-red patch (≥5 cm in diameter)<sup>a</sup>, with or without central clearing. Advancing edge typically distinct, often intensely coloured, not markedly elevated.</td><td>None</td><td>Detection of Bbsl by culture and/or PCR from skin biopsy.</td></tr><tr><td>Borrelial lymphocytoma (rare)</td><td>Painless bluish-red nodule or plaque, usually on ear lobe, ear helix, nipple or scrotum; more frequent in children (especially on ear) than in adults.</td><td>Seroconversion or positive serology<sup>b</sup><br>Histology in unclear cases</td><td>Histology.<br>Detection of Bbsl by culture and/or PCR from skin biopsy.<br>Recent or concomitant EM.</td></tr><tr><td>Acrodermatitis chronica atrophicans</td><td>Long-standing red or bluish-red lesions, usually on the extensor surfaces of extremities. Initial doughy swelling. Lesions eventually become atrophic. Possible skin induration and fibroid nodules over bony prominences.</td><td>High level of specific serum IgG antibodies</td><td>Histology.<br>Detection of Bbsl by culture and/or PCR from skin biopsy.</td></tr><tr><td>Lyme neuroborreliosis</td><td>In adults mainly meningo-radiculitis, meningitis; rarely encephalitis, myelitis; very rarely cerebral vasculitis. In children mainly meningitis and facial palsy.</td><td>Pleocytosis and demonstration of intrathecal specific antibody synthesis<sup>c</sup></td><td>Detection of Bbsl by culture and/or PCR from CSF.<br>Intrathecal synthesis of total IgM, and/or IgG and/or IgA.<br>Specific serum antibodies.<br>Recent or concomitant EM.</td></tr><tr><td>Lyme arthritis</td><td>Recurrent attacks or persisting objective joint swelling in one or a few large joints. Alternative explanations must be excluded.</td><td>Specific serum IgG antibodies, usually in high concentrations</td><td>Synovial fluid analysis.<br>Detection of Bbsl by PCR and/or culture from synovial fluid and/or tissue.</td></tr><tr><td>Lyme carditis (rare)</td><td>Acute onset of atrio-ventricular (I-III) conduction disturbances, rhythm disturbances, sometimes myocarditis or pancarditis. Alternative explanations must be excluded</td><td>Specific serum antibodies</td><td>Detection of Bbsl by culture and/or PCR from endomyocardial biopsy.<br>Recent or concomitant erythema migrans and/or neurologic disorders.</td></tr><tr><td>Ocular manifestations (rare)</td><td>Conjunctivitis, uveitis, papillitis, episcleritis, keratitis.</td><td>Specific serum antibodies</td><td>Recent or concomitant Lyme borreliosis manifestations.<br>Detection of Bbsl by culture and/or PCR from ocular fluid.</td></tr></tbody></table>

<footnote><sup>a</sup> If <5 cm in diameter, a history of tick-bite, a delay in appearance (after the tick bite) of at least 2 days and an expanding rash at the site of the tick-bite is required.</footnote>

<footnote><sup>b</sup> As a rule, initial and follow up samples have to be tested in parallel in order to avoid changes by inter-assay variation.</footnote>

<footnote><sup>c</sup> In early cases intrathecally produced specific antibodies may still be absent.</footnote>
For LNB, case definition has been specified in 2018 by ECDC (24) as follows:

- Confirmed case

Neurological symptoms suggestive of LNB (according to European Federation of Neurological Societies (EFNS) suggested case definition) without other obvious reasons
AND

- Pleocytosis in cerebrospinal fluid, AND

- Evidence of intrathecal production of LB antibodies, OR

- Bbsl isolation, OR

- Nucleic acid detection in cerebrospinal fluid

OR

- Detection of IgG LB antibodies in blood specimen only for children (age under 18) with facial palsy or other cranial neuritis and a recent (< 2 months) history of EM

- Probable case

Neurological symptoms suggestive of LNB (according to European Federation of Neurological Societies (EFNS) suggested case definition) without other obvious reasons
AND

- Pleocytosis in cerebrospinal fluid, AND positive LB serology in cerebrospinal fluid OR

- Specific intrathecal LB antibody production

## Infrastructure capacity to identify pathogens for each Member State

Infrastructures to implement serological surveys in the general population allowed the publication of scientific articles describing the distribution of LB seroprevalence in 22 European countries, though only 14 studies are considered representative at the national scale (231). There was substantial heterogeneity among studies, which limits cross-study comparisons. Differences among studies were observed in terms of design, cohort types, periods sampled, sample sizes, and diagnostic methods (with a minority of studies using the recommended two-step serological approach described above).

More generally, LB incidence data are available for 25 European countries based on heterogeneous surveillance systems (196). Depending on the countries, these surveillance systems can be based on: i) passive or mandatory data collection; ii) defined sentinel sites or deployed at a national scale; iii) case definitions (clinical, laboratory, or both) and if relevant testing methods. Twenty-one countries for which statistics were available (84%) implemented passive surveillance. Among those different countries, only four used standardized case definitions recommended by European public health institutions.

## Estimated influence of environmental change on the disease future trends

Tick population, abundance and activities are influenced by biotic and abiotic environmental factors (232). Knowledge of the ecology and epidemiology of *Ixodes* ticks and Bbsl remains a current issue to improve the control and prevention of LB (233, 234).
With climate change and global warming, *Ixodes* ticks are now reported in countries where they were not before (79,80) as in higher altitude in mountains (92,93). Global changes are susceptible to modify several key drivers that influence *Ixodes* ticks distribution, activity and survival as well as human exposure to tick bite. In particular, climate change and warming may impact: i) the life cycle of *I. ricinus*, the duration of interstadial development and mortality rates; ii) the composition and distribution of hosts on which *I. ricinus* feeds; and iii) human and animal exposures to ticks, due to changes in the spatial and temporal distributions of ticks and modifications of activities.

Several longitudinal studies were conducted in order to understand how ticks respond to environmental changes in particular weather, host density and human practices (27, 28, 46, 72-77). These studies allowed collection of data and development of models of tick activity (46, 235). In order to project future climate changes on tick population density, temperature, relative humidity or precipitation, data covering a reference period (1971-2000) were compared to similar variable in future periods (236). The future climate scenarios were applied to the near future (2012 to 2040/2050), the mid-term future (2050-2070) and the far future (2071-2100) taking into account different Representative Climate Pathways (RCP) i.e. low to extreme RCP scenarios (46,237).

In France, if the future climate followed the RCP8.5 trajectory, approximately 65% of the French surface area would have a Mediterranean climate, unfavourable for *I. ricinus* ticks by 2100 (46). In Germany, climate projections suggested that the peak of questing nymphs would shift towards the first seasons of the year and that a greater spatial heterogeneity of nymph densities would occur throughout the Germany (237). All of these projections describe situations in the near or distant future and should be taken with caution. However, recent studies have already reported perceptible changes in tick phenology in particular the presence of active ticks in winter in places where they were not before during this season (84, 238).

Anticipation of such changes in tick distribution and activity is essential since it could modify the epidemiology, seasonality and distribution of hLB cases in the future.

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# TICK-BORNE ENCEPHALITIS

Valentina Tagliapietra (a), Annapaola Rizzoli (a), Maria Bellenghi (b), Claudia Cataldo (b), Francesca Dagostin (a), Simon Dellicour (c, d), Timothee Dub (e), Henna Mäkelä (e), Giovanni Marini (a), Luciano Toma (f), Maria Fernanda Vincenti González (d), William Wint (g), Luca Busani (b)

(a) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

(b) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(c) Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven

(d) Spatial Epidemiology Lab (SpELL), Université Libre de Bruxelles (ULB), Brussels

(e) Department of Health Security, Finnish Institute for Health and Welfare, Helsinki

(f) Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome

(g) Department of Biology, Environmental Research Group Oxford Ltd, Oxford

## Biological, ecological and molecular features of the causative agent

### Disease name

Tick-Borne Encephalitis (TBE), Russian Spring and Summer Encephalitis (RSSE), Central European Encephalitis (CEE).

### Disease agent

#### Common, scientific and Latin name

Tick-Borne Encephalitis Virus (TBEV), *Flavivirus tick-borne encephalitis*.

#### Taxonomy

Family: *Flaviviridae*; Genus: *Flavivirus*; Species: *tick-borne encephalitis*

#### Disease agent characteristics

TBE mature virions are smooth spherical particles, 50 nm in diameter. The core consists of a Nucleocapsid (NC) approximately 30 nm in diameter, surrounded by a host-derived lipid bilayer membrane in which the viral envelope (E) and membrane (M) proteins are embedded. The NC is a positive single-stranded RNA molecule approximately 11 kb in length. It shows no discernible symmetry, but the C protein surrounds the viral genome like a cage (1). The Open Reading Frame of the genome, flanked by 5' and 3' untranslated regions, encodes one large polyprotein of approximately 3400 amino acids which is processed into 3 structural (Capsid,C, precursor of the membrane protein M,prM and Envelope, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (2). Among the structural proteins, E is the major target for neutralizing antibodies and induces protective immunity. It is responsible for specific binding to a cellular receptor and penetration of the virus into the host cell and is also believed to be a determinant of TBEV virulence (3). In particular, amino acid substitutions in E protein often cause decrease in neuroinvasiveness, but not neurovirulence (4). The C protein represents a structural component of the NC with a low sequence homology between different flaviviruses. This protein accumulates on the surface of endoplasmic
reticulum-driven organelles named lipid droplets that may play multiple roles in the viral life-cycle (5). The prM protein is the precursor of the membrane protein and has a major role in virus maturation, assembly and secretion (6). Structural proteins have been proved to be the primary viral determinants of non-viral transmission between ticks (7). Non-structural proteins have multi functions such as virus-host interaction (NS1), viral replication (NS1, NS2A, NS4A and NS4B), translation and virion production, stress response (NS4B), interference with interferon signalling (NS2A, NS4B, and NS5), membrane re-arrangements (NS4A) and serve as RNA-dependent RNA polymerase (NS5). Moreover, NS determines the extent of the cytopathic effect in cell culture (7).

## Physiochemical properties

TBEV is readily inactivated by organic solvents and detergents. Non-ionic detergents, such as Triton X, solubilize the entire envelope, releasing M and E proteins; whereas sodium deoxycholate appears to remove only E, leaving M associated with the nucleocapsid. Mature virions sediment at about 200S and have a buoyant density of about 1.19 g cm⁻³ in sucrose (8). Flaviviruses are stable at slightly alkaline pH (8.0) and low temperatures (especially at -60°C or below), but TBEV has been reported to preserve at least residual infectivity over the broader pH range 1.42-9.19. Total inactivation of virus suspended in blood or other protein solutions occurs within 30 min at 56°C. Flaviviruses are stable for at least 6 h in liquid aerosol suspension at room temperature and 23-80% humidity. On the other hand, ultra-low temperatures preserve infectivity almost indefinitely and once freeze-dried they survive almost indefinitely at room temperature. Flaviviruses are inactivated by ultraviolet light, gamma-irradiation, and disinfectants, including 3-8% formaldehyde, 2% glutaraldehyde, 2-3% hydrogen peroxide, 500-5000-ppm available chlorine, alcohol, 1% iodine, and phenol iodophors (9).

## Priority level for EU

In 2011, the first attempt to collect TBE surveillance data at the EU/EEA (European Union/European Economic Area) level underlined the need for an agreed case definition and systematic data collection. Therefore, in 2012, the European Commission included TBE in the list of notifiable diseases in the EU/EEA (10). The European Centre for Disease Prevention and Control (ECDC) annually collects data from 28 countries plus Iceland and Norway, based on the EU case-definition.

## Distribution of the pathogen

Tick-Borne Encephalitis Virus (TBEV) has expanded its distributional range during the last decades and shows an irregular distribution over a large geographical range with a patchy occurrence in restricted foci of limited size where it circulates among vertebrate hosts and ticks. At continental scale TBEV has a Palearctic distribution, along the 8°C isotherm, in Eurasia and Northern Africa (Tunisia). At the European scale, TBEV is reported in 25 countries, specifically 22 EEA countries, 2 EFTA (European Free Trade Association) countries, 1 EU candidate countries and United Kingdom, with a distribution range from Spain and the Netherlands to the West, the Baltic countries to the east, the Southern coastal areas of Scandinavia and UK to the north and Greece to the south (Figure 1).
<figure><img src="image_42.png" /><figcaption>**Figure 1.** European Union and European Economic Area countries that reported at least one locally acquired TBE human case in the period 2010-2021. Data from TESSy-ECDC, or other sources such as national surveillance systems (Switzerland, Serbia) and published literature (24-26)</figcaption></figure>

TBEV currently includes five recognized main subtypes or lineages (Figure 2):

- *Western European subtype* (TBEV-Eu)

It is endemic in rural and forest areas mainly of central, eastern and Northern Europe including the European part of Russia and South Korea and has *Ixodes ricinus* as its principal tick vector. Recently, it has been reported also in Northern Africa (Tunisia) (11). Infections are typically biphasic and are characterized by a viraemic phase with fever, malaise, headache, myalgia, leukocytopenia, thrombocytopenia and elevated liver enzymes; after a 1-week latency period, 25% of patients develop clinical signs of neurological involvement. Residual sequelae are observed in 25–50% of patients, but < 2% of cases are fatal (12).

- *Far Eastern subtype* (TBEV-FE)

It is endemic in far-eastern Russia and in forest regions in China and Japan and has *I. persulcatus* as its principal tick vector (13,14). Cases infected with TBEV-FE were reported in Estonia and Latvia (15). This subtype causes the most severe central nervous system disorder with a development of focal meningoencephalitis or polyencephalitis accompanied by loss of consciousness and fatigue during recovery. Major damage to neurons in different parts of the brain and spinal cord is possible. A fatality rate of 20-60% and an absence of chronic forms are reported.

- *Siberian subtype* (TBEV-Sib)

It is endemic in the Ural region, Siberia, far-eastern Russia and some areas in north-eastern Europe (Estonia and Finland) and has *Ixodes persulcatus* as its principal tick vector (16).
TBEV infections in Siberian-Ural regions present as a less severe disease with a high prevalence of the non-paralytic febrile form of encephalitis, while chronic forms seem to be more frequent. Case fatality rates rarely exceed 6-8%.

- *Baikalian subtype (TBEV-Bkl)*

This subtype was detected in the Irkutsk region (Ekhirit-Bulagatskiy district), Buryat Republic (Bichurskiy and Barguzinskiy district) and Transbaikalia (National Park Alkhanay, Duldurginskiy district and Krasnochikoiskiy district). The amino acid sequences of 886-84 subtypes confirmed that its genetic structure is a unique mixture of the three common subtypes. Thirty unique substitutions were detected. Genetic difference from the three common subtypes (TBEV-Eu, TBEV-FE, and TBEV-Sib) is more than 12%. The pathogenic potential is high (17-19).

- *Himalayan subtype (TBEV-Him)*

The strains (Himalaya-1 and Himalaya-2) have been detected in *Marmota himalayana* in Qinghai-Tibet Plateau, China (20). Phylogenetic analysis demonstrated that this could be considered as a new member of TBEV-FE group. At amino acid level, the diversity of the E protein was less than 2.2% within the subtype and 3.6-5.6% between the subtypes. TBEV-Him differed by 5.0-7.3% from the other subtypes, while polyprotein diverged of 4.8-7.4%. Him-TBEV displayed 69 amino acid substitutions in complete polyprotein of which 36 were unique. The profile of pathogenic associated amino acid substitution of Him-TBEV is similar to low virulence strain Oshima (5-10) (TBEV-FE).

<figure><img src="image_43.png" /><figcaption>**Figure 2.** Eurasian distribution and abundance of the main TBEV subtypes sequences (number of sequences used= 1928) (data sources: https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/ and https://www.bv-brc.org/).</figcaption></figure>
Other TBEV subtypes differing from the main ones namely Sallandse strain from the Netherlands (21) and 178-179 single strain from Irkutsk (22) are still under review.

In some countries, two or three subtypes co-circulate (e.g., Baltic States, Siberia, Ukraine) (23) (see Figure 2).

## Ecology and transmission routes

TBEV circulates in natural cycles involving several vertebrate hosts and vectors. A map showing evidence of TBE virus occurrence in vector or host is provided in Figure 3. Ticks are both vectors and main reservoirs of the virus remaining infected throughout their life cycle due to the transstadial and transovarial transmission. The tick life-cycle needs three blood meals to be completed and the same number of hosts. The larvae mainly feed on small mammals, nymphs feed on small- and medium-sized mammals, birds, and reptiles, and adults on large animals such as ungulates.

Uninfected ticks can also acquire the infection while feeding on a viraemic competent host (systemic transmission). However, especially in case of TBEV-Eu, the most effective amplification route is through co-feeding where viral transmission takes place when naïve ticks at different developmental stages (particularly nymphs and larvae) co-feed with infected ticks on the same animal host. According to this mechanism, the virus is transmitted non-systemically, even in an immune host, to the next tick generation (27).

For an effective TBEV-Eu transmission and amplification, synchronous activity of larvae and nymphs supported by specific climatic conditions (autumnal cooling and spring warming) are needed (28, 29). However, in areas with ineffective co-feeding transmission, such as in the northern distribution areas (30) and at high altitudes, other mechanisms for maintaining virus circulation, as the transmission from infected host to ticks, in natural foci become relatively more important.

<figure><img src="image_44.png" /><figcaption>**Figure 3.** Distribution of TBEV presence in vectors (a) and vertebrate hosts (b) (derived from a literature review updated to 2021)</figcaption></figure>
## Vectors

Ticks are the main vectors and reservoirs of TBEV, although it has also been isolated from mosquitoes (TBEV-FE in *Aedes vexans*) (31). All the tick species involved in the epidemiological cycle of TBEV belong to the *Ixodidae* family. The most common and widespread species are *Ixodes ricinus* (also main vector for the TBEV-EU subtype) and *I. persulcatus*. *I. ricinus* is widely distributed throughout Europe, West to East from Ireland to the Urals, and North to South from Northern Sweden to North Africa (Figure 4a), while *I. persulcatus* is predominant in Northern Europe (Figure 4b). The 14 species of ticks listed in Table 1 are proven to transmit TBEV.

<figure><img src="image_45.png" /><figcaption>Figure 4. Current 1-km ecological suitability estimates for *I. ricinus* and *I. persulcatus* across Europe, produced using random forest and boosted regression trees analyses (source: ERGO group)</figcaption></figure>

<table><caption>Table 1. List of *Ixodidae* species that transmit TBEV</caption><thead><tr><td>Genus</td><td>Species</td><td>Reference</td></tr></thead><tbody><tr><td>Ixodes</td><td>ricinus</td><td>(32,33)</td></tr><tr><td>Ixodes</td><td>arboricola</td><td>(34)</td></tr><tr><td>Ixodes</td><td>hexagonus</td><td>(35,36)</td></tr><tr><td>Ixodes</td><td>trianguliceps</td><td>(37)</td></tr><tr><td>Ixodes</td><td>persulcatus</td><td>(33)</td></tr><tr><td>Ixodes</td><td>ovatus</td><td>(38)</td></tr><tr><td>Dermacentor</td><td>marginatus</td><td>(39-41)</td></tr><tr><td>Dermacentor</td><td>reticulatus</td><td>(39, 40, 42, 43)</td></tr><tr><td>Haemaphysalis</td><td>concinna</td><td>(44)</td></tr><tr><td>Haemaphysalis</td><td>inermis</td><td>(40)</td></tr><tr><td>Haemaphysalis</td><td>punctata</td><td>(41)</td></tr><tr><td>Haemaphysalis</td><td>longicornis</td><td>(45)</td></tr><tr><td>Haemaphysalis</td><td>flava</td><td>(45)</td></tr><tr><td>Haemaphysalis</td><td>nipponensis</td><td>(45)</td></tr></tbody></table>
## Hosts

Various animal species are susceptible and can be infected with TBEV.

However, their ecological role in viral transmission can vary. They can act as dead-end hosts or maintain and amplify the virus (reservoir hosts), with or without evident symptoms of the disease.

In some species, the viremia is followed by viral shedding in urine and milk, therefore enhancing the risk of transmission through milk and milk products.

In other cases, although being incompetent as reservoir for the virus, they participate in maintaining or amplifying the tick population and therefore affecting indirectly the viral transmission.

Rodents and insectivores are considered the main reservoir hosts for TBEV maintenance and circulation. Once infected with TBEV, they are supposed to develop a chronic infection, although the duration of viremia and thus their infectivity to ticks are commonly considered short (two to nine days).

A recent experimental study in bank voles (*Myodes glareolus*) showed that viremia might last up to 28 days, but infectivity to ticks was not tested (46). Also, the possibility exists that low viral replication in latently infected rodent hosts may occur under the influence of fluctuations in stress of sexual hormone levels during their life cycle (30, 47, 48). In laboratory mice, sexual transmission has also been reported (49).

Domestic ruminants, such as goats, sheep and cattle, rarely show any clinical symptom, although they develop a detectable viremia that can be a source of infection either directly or indirectly (milk, milk products and blood), although not sufficient to be uptaken by feeding ticks (50).

The role of birds as TBEV reservoirs still needs to be demonstrated, although they contribute to the dispersal of the virus including the introduction into new areas by carrying infected ticks.

TBEV was isolated occasionally from *Turdus pilaris*, *T. iliacus*, other *Turdus* spp., *Corvus monedula*, *C. corone*, *Pica pica*, *Sturnus vulgaris*, *Lanius collurio*, *Fringilla montifringilla*, *F. coelebs*, *Loxia curvirostra*, *Carduelis flammea*, *Anthus trivialis*, *Erithacus rubecula*, *Motacilla alba*, *M. flava*, *Emberiza* spp., *Jynx torquilla*, *Bonasa bonasia*, *Crex crex*, *Scolopax rusticola*, *Clangula hyemalis*, *Melanitta fusca*, *Anas querquedula*, *Fulica atra*.

TBEV-infected larvae were collected from *Anthus trivialis*, *Phoenicurus phoenicurus*, *Erithacus rubecula* and *Turdus philomelos* (51), while a potential for transovarial transmission by isolation of the virus from the eggs, was demonstrated in *T. iliacus*, *T. pilaris*, *T. ruficollis*, *T. pallidus*, *Lanius cristatus*, *Emberiza fucata*, *Troglodytes troglodytes*, *Accipiter gentilis* (52).

Wild ungulates are important hosts for the maintenance of tick populations, by providing a sufficient blood meal to adult stages, although they are not competent for viral transmission (53).

Carnivores can be locally important, specifically, the role of red foxes (*Vulpes vulpes*) deserves more investigations as they are exposed to TBEV both from infected tick bites and from eating TBE-infected preys (54-56).

In Table 2 the vertebrate species for which TBEV symptoms or viremia or both are reported.
<table><caption>Table 2. List of vertebrate species that reported TBE symptoms and/or developed viremia</caption><thead><tr><td>Species (Latin name)</td><td>Symptoms (ref.)</td><td>Viremia</td></tr></thead><tbody><tr><td>Goat (Capra hircus)</td><td>No</td><td>Yes</td></tr><tr><td>Sheep (Ovis aries)</td><td>Yes (57)</td><td>Yes</td></tr><tr><td>Cow (Bos taurus)</td><td>No</td><td>Yes</td></tr><tr><td>Dog (Canis lupus familiaris)</td><td>Yes (58)</td><td>Yes</td></tr><tr><td>Horse (Equus caballus)</td><td>Yes (59, 60)</td><td>Yes</td></tr><tr><td>Roe deer (Capreolus capreolus)</td><td>Yes (61)</td><td>Yes</td></tr><tr><td>Red deer (Cervus elaphus)</td><td>No</td><td>Yes</td></tr><tr><td>Fallow deer (Dama dama)</td><td>No</td><td>Yes</td></tr><tr><td>Wild boar (Sus scrofa)</td><td>No</td><td>Yes</td></tr><tr><td>White-tailed deer (Odocoileus virginianus)</td><td>No</td><td>Yes</td></tr><tr><td>Moose (Alces alces)</td><td>No</td><td>Yes</td></tr><tr><td>Mouflon (Ovis ammon musimon)</td><td>Yes (62)</td><td>Yes</td></tr><tr><td>European bison (Bison bonasus bonasus)</td><td>No</td><td>Yes</td></tr><tr><td>Red fox (Vulpes vulpes)</td><td>No</td><td>Yes</td></tr><tr><td>Raccoon dog (Nyctereutes procyonoides)</td><td>No</td><td>Yes</td></tr><tr><td>Mountain hare (Lepus timidus)</td><td>No</td><td>Yes</td></tr><tr><td>European hare (Lepus europaeus)</td><td>No</td><td>Yes</td></tr><tr><td>Bank vole (Myodes glareolus)</td><td>No</td><td>Yes</td></tr><tr><td>Grey-sided vole (Myodes rufocanus)</td><td>No</td><td>Yes</td></tr><tr><td>Northern red-backed vole (Myodes rutilus)</td><td>No</td><td>Yes</td></tr><tr><td>Field vole (Microtus agrestis)</td><td>No</td><td>Yes</td></tr><tr><td>Common vole (Microtus arvalis)</td><td>No</td><td>Yes</td></tr><tr><td>Hedgehog (Erinaceus europaeus)</td><td>Yes (63)</td><td>Yes</td></tr><tr><td>Northern white-breasted hedgehog (Erinaceus roumanicus)</td><td>No</td><td>Yes</td></tr><tr><td>Eastern hedgehog (Erinaceus concolor)</td><td>No</td><td>Yes</td></tr><tr><td>Yellow-necked mouse (Apodemus flavicollis)</td><td>No</td><td>Yes</td></tr><tr><td>Wood mouse (Apodemus sylvaticus)</td><td>No</td><td>Yes</td></tr><tr><td>Striped field mouse (Apodemus agrarius)</td><td>No</td><td>Yes</td></tr><tr><td>Large japanese field mouse (Apodemus speciosus)</td><td>No</td><td>Yes</td></tr><tr><td>Small japanese field mouse (Apodemus argenteus)</td><td>No</td><td>Yes</td></tr><tr><td>Red squirrel (Sciurus vulgaris)</td><td>No</td><td>Yes</td></tr><tr><td>Eastern grey squirrel (Sciurus carolinensis)</td><td>No</td><td>Yes</td></tr><tr><td>Brown rat (Rattus norvegicus)</td><td>No</td><td>Yes</td></tr><tr><td>Northern birch mouse (Sicista betulina)</td><td>No</td><td>Yes</td></tr><tr><td>European mole (Talpa europaea)</td><td>No</td><td>Yes</td></tr><tr><td>European polecat (Mustela putorius)</td><td>No</td><td>Yes</td></tr><tr><td>Common shrew (Sorex araneus)</td><td>No</td><td>Yes</td></tr><tr><td>Barbary macaque (Macaca sylvanus)</td><td>Yes (64)</td><td>Yes</td></tr></tbody></table>

# Drivers of disease emergence and spread

## Ecological drivers

TBE epidemiology typically shows a profound fluctuation in the annual cases reported and a patchy distribution. Explanation could be found in the complex interplay of biotic and abiotic factors which characterize the transmission chain of the TBE virus (65).

In Table 3, the ecological and environmental covariates included in the sixty-two studies selected are reported.
<table><caption>**Table 3.** List of ecological and environmental covariates included in the selected studies in decreasing order according to the number of studies including them</caption><thead><tr><td>Environmental drivers</td><td>N. of papers (n. 62)</td><td>% of impact</td></tr></thead><tbody><tr><td>Temperature (average daily, monthly seasonal and annual temperature, autumnal cooling rate, spring warming rate, mean diurnal temperature range)</td><td>40</td><td>65%</td></tr><tr><td>Land use (forest, agriculture, vegetation cover)</td><td>19</td><td>31%</td></tr><tr><td>Precipitation (total daily, monthly, seasonal and annual precipitation)</td><td>15</td><td>24%</td></tr><tr><td>Deers (roe deer, red deer, fallow deer, moose, white-tailed deer abundance or density)</td><td>13</td><td>21%</td></tr><tr><td>Vegetation (Mean NDVI, EVI, Beech fructification index)</td><td>12</td><td>19%</td></tr><tr><td>Humidity (daily, monthly, annual mean relative humidity)</td><td>11</td><td>18%</td></tr><tr><td>Rodents (yellow-necked mouse, bank vole abundance or density)</td><td>10</td><td>16%</td></tr><tr><td>Topography (distance from forest or meadow, distance to sea or water course, latitude, exposition, slope)</td><td>9</td><td>15%</td></tr><tr><td>Altitude</td><td>8</td><td>13%</td></tr><tr><td>Feeding ticks (number or density of feeding and co-feeding ticks)</td><td>8</td><td>13%</td></tr><tr><td>Time (year and season)</td><td>7</td><td>11%</td></tr><tr><td>Questing ticks (density of questing larvae and nymphs)</td><td>6</td><td>10%</td></tr><tr><td>Foxes (abundance or density)</td><td>5</td><td>8%</td></tr><tr><td>Atmosphere (Scandinavian Index, NAO Index)</td><td>5</td><td>8%</td></tr><tr><td>Hares (European hare, mountain hare abundance or density)</td><td>5</td><td>8%</td></tr><tr><td>Wild boars (abundance or density)</td><td>4</td><td>6%</td></tr><tr><td>Soil (mean soil temperature and humidity, soil types)</td><td>4</td><td>6%</td></tr><tr><td>Saturation deficit</td><td>4</td><td>6%</td></tr><tr><td>Snow cover</td><td>3</td><td>5%</td></tr><tr><td>Other hosts (lynx, racoon abundance or density)</td><td>3</td><td>5%</td></tr><tr><td>Tick ecology (birth and mortality rate, average egg production.)</td><td>2</td><td>3%</td></tr><tr><td>Light (daily, annual sunshine hours)</td><td>2</td><td>3%</td></tr></tbody></table>

NDVI: Normalized Difference Vegetation Index; EVI: Enhanced Vegetation Index; NAO: North Atlantic Oscillation

Climate change is generally considered a primary driver of TBE trend and temporal fluctuations, although its complex role in affecting the zoonotic system or human behaviour is still debated (66). One of the most efficient mechanisms of TBEV transmission involves the co-feeding of larvae and nymphs through the seasonal synchrony of the different tick life stages that is in turn affected by climatic variables. In particular, it has been shown that a high rate of autumnal cooling (i.e., a rapid decrease in late summer temperatures) induces a behavioural diapause in larvae and nymphs therefore reducing the delay between nymphal and larval activity during the following spring (28, 29, 67-70). Similarly, rapidly rising temperatures in spring, referred to as “spring warming rate”, cause larvae to become active at the same time as nymphs (71, 72).

Abiotic factors directly affect off-host stages of the ticks, in particular temperature and relative humidity. The known temperature threshold values for tick activity range between 5 and 10 °C (73-76), with significant differences found for larvae (6°C) and for nymphs and adults (5°C) (77). Regarding Relative air Humidity (RH), a peak in activity for larvae has been reported at 75-85% RH and for nymphs and adults between 50-100% RH (77). It has been demonstrated that tick abundance is positively correlated with temperature and relative humidity, which affects the hydration of tick's body (78,79), but negatively with saturation deficit (an index combining temperature and relative humidity) (72,80). The effect of severe winters on annual nymphal
density (81) and on subsequent TBE incidence (82) has also been proved. The latitudinal and altitudinal expansion of ticks and their increase of the activity range have consequences on increased probability of contacts between humans and parasites.

Density of animal host's population and/or their tick load are key drivers for TBEV circulation. Small rodents (mainly *Apodemus* spp. and *Myodes* spp.) play a crucial role both as a source of bloodmeal for immature ticks and as bridge hosts for the virus (83). The TBE incidence was repeatedly demonstrated to be correlated with their 1-year lagged population density (84). Among medium-sized and large mammals, deer and hares are reported to affect the presence of TBE foci. In particular, ungulates are able to greatly amplify tick abundances, but at the same time divert tick bites from competent hosts, and thus may cause a dilution effect of TBEV prevalence in ticks above certain threshold densities (85, 86). Hares and red foxes might also contribute to such dilution effect, as high host species diversity and high population numbers weaken the incidence of TBE-infected ticks on any given host (87). A similar relationship has been found between the number of co-feeding tick groups and *A. flavicollis* density, with a decline in the number of co-feeding ticks after a threshold of approximately 10 mice/ha (70). Sylvatic carnivores may have an indirect effect on TBEV incidence through predation of deer and small mammals. Other hosts such as ruminants are considered non-competent, but due to their long-term persistent viremia and antibody response, they are often used as sentinels. Birds, especially migratory, contribute to the dispersal of the ticks over long distances.

Regarding the environment, forests, especially well-connected coniferous, broad-leaf and mixed forest patches with open areas, provide suitable habitat and resources for ungulates, rodents, and ticks, thus favouring their encounter and boosting the risk of occurrence of TBE human cases (88-93). Evidence has been found for a significant correlation between TBE incidence and an increasing ratio of high stand forest to coppice cover, which corresponds to an increase in habitat suitability for small mammals, especially *Apodemus* spp. (94). Landscape characteristics also affect human risk to tick exposure, as people engaged in recreational or occupational outdoor activities in a risk area are at increased risk of tick bites. Therefore, proxies for forest accessibility, such as forest road length (95, 96) and the maximum distance from forests or meadows (97) are associated with higher TBE risk. Greener and healthier vegetation, which can be evaluated by the satellite derived Normalized Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI), promotes tick presence, availability of food resources for primary consumers and their coexistence, in turn reflecting in a higher TBE suitability (93, 97).

In the current context of global biodiversity loss, recent efforts (mostly focused on Lyme disease) were made to understand the effect of biodiversity on vector-borne diseases transmission, with contrasting conclusions. Species diversity might be important in the ecology of infectious diseases, particularly vector-borne zoonosis: on one hand, high species diversity in vertebrate hosts may play a beneficial role by acting as competent or not competent reservoirs of the pathogen (98); on the other hand, high diversity of vertebrate hosts may help generalist vectors or pathogens to avoid local extinction and therefore may play a role in increasing the disease risk to humans. At broad spatial scales, habitat alteration can also influence disease risk (99, 100): habitat destruction and the fragmentation of landscapes into small, isolated units are known to cause reduction or elimination of some vertebrate species and therefore diversity (101, 102). Often, species that occupy high trophic levels are the most sensitive to such habitat destruction. Loss of these species, which are generally non competent reservoirs for vector-borne zoonosis, may increase disease risk both via reduction of diverted blood meals from these incompetent hosts and via the loss of a regulatory "predator" effect on typically more reservoir-competent hosts.

Environmental variables are not the only ones that can explain the spatio-temporal distribution of TBE cases. Human activity and behaviour can act in synergy with them by increasing the chances of coming in contact with infected ticks. Low economic status has been identified as a
significant risk factor for TBE infection in some parts of Europe, forcing people to exploit the rural and natural habitat in search for food products or wood. For example, for eight central and eastern European countries, the differential degree of TBE upsurge in the early 1990s was significantly positively correlated with contemporary poverty indicators, including the percentage of total household expenditure spent on food (103). An increase in TBE incidence has been also reported in Europe in 2020. A possible explanation could be an enhanced exposure to people to infected ticks as a consequence of the post lock-down human behaviour.

# Natural history of disease in humans and animals, including symptoms, morbidity and mortality

## Brief history of the pathogen and disease

In the early 1920s, residents and workers of villages in the Taiga Forest from the far Eastern region of Russia, reported a severe form of brain disease. An expedition led by Prof. Lev A. Zilber was sent in 1937 to Khabarovsk Territory to investigate the origins and causes of this disease. Despite extremely difficult conditions of the remote taiga location and the absence of infrastructures, the expedition successfully isolated the virus from patients, vertebrates and ticks. Furthermore, the pioneer researchers expounded on basic eco-epidemiology of the disease and provided some useful prophylactic information on how to avoid infection (104). The first cases in China were reported in 1943 and virus isolation from patients in 1944 (105). The first documented TBEV isolation in Europe was made from *I. ricinus* ticks in Belarus in 1939 (strain 256), while the second was reported in former Czechoslovakia in 1948 (strain Hanzalova) from both patients and ticks in Beroun near Prague and in Moravia (106). The alimentary route of infection was first identified in the European part of Russia between 1947 and 1951 (9). In the early 1990's, non-viraemic transmission of TBEV between co-feeding *I. ricinus* ticks was proved by prof. Labuda and collaborators from the Institute of Zoology SAS (Slovakia) in collaboration with the Institute of Virology NERC (Oxford, UK), using ticks and TBE virus strain from Central Bohemia (107). The European TBE vaccine was made available in 1976 (108).

## Disease in humans

Humans are dead-end accidental hosts for TBEV. The infection can be primarily contracted through the TBEV-infected saliva injected from the bite of a tick. Among the most relevant developmental stages involved in human transmission, nymphs of *I. ricinus* are the most abundant, aggressive and less host specific, while for *I. persulcatus* the adult tick's stage is considered the most important. It is assumed that dairy unpasteurized products from infected animals (cattle, sheep, goats) stand for 1% of all TBE cases and has been reported mostly in eastern Europe and the Balkans; however, small outbreaks of foodborne TBE have also been reported in central and western Europe and Russia (109-113) (Figure 5). Other non-vectorial routes of infection include: single cases of tick-borne encephalitis after slaughter of probably viraemic goats, blood transfusions, solid organ transplantation (114), breastfeeding and laboratory investigations (115, 116).
<figure><img src="image_46.png" /><figcaption>**Figure 5.** European countries with reported cases of TBE foodborne infections
(literature review updated to 2021)</figcaption></figure>

Most of the infections (between 70 and 98%) with TBE virus strains of the European subtype are either sub-clinical or asymptomatic. Clinically, the course of infection may depend on the virus subtype, the inoculation dose, the age and immune status of the host. The European subtype typically take a biphasic course (about two-thirds of patients), characterized by a first stage with non-specific symptoms and a second one with additional neurological involvement. Symptomatic infection without central nervous system (CNS) involvement is defined as “abortive TBE”. The incubation period ranges from 2 to 28 days and is usually 7-14 days. Foodborne TBEV transmission has a shorter incubation period (3 to 4 days) and exhibits a monophasic course of the disease in 50% of recorded cases.

When symptoms of TBE infection progress in two phases, the initial one correlates with viremia and usually presents with non-specific symptoms such as moderate fever, headache, body pain (myalgia and arthralgia), fatigue, general malaise, anorexia, nausea, and others. This phase lasts for 2-7 days and is followed by amelioration or even an asymptomatic interval that usually lasts for about 1 week (range 1-21 days). If a second phase appears, it presents as meningitis in
approximately 50% of adult patients, as meningoencephalitis in about 40%, and in meningoencephalomyelitis in around 10%. Meningitis typically manifests with high fever, headache, nausea, and vomiting; many patients have photophobia, and some vertigo. Encephalitis can be manifested by impaired consciousness ranging from somnolence to stupor and, in rare cases, coma. Other manifestations comprise personality changes, behavioural disorders, concentration and cognitive function disturbances, tongue fasciculations and tremor of extremities; very rarely focal or generalized seizures, delirium and psychosis develop. Flaccid paresis, which are a typical characteristic of meningoencephalomyelitis, usually arise during the febrile phase of the disease, and are occasionally preceded by severe pain in the affected muscle groups.

Viral genotypes or the apparent virulence of a particular TBEV subtype and infection dose can determine different degrees of disease severity. TBEV-Eu infection usually results in a rather mild form of TBE. TBEV-Sib infection generally results in a mild illness associated with a non-paralytic febrile form of encephalitis (9), with a tendency towards persistent TBE caused by chronic viral infection. TBEV-FE infection is believed to cause the most severe forms of TBE (117). In addition, in Russia, haemorrhagic forms of TBE and chronic (or progressive) forms have been reported (9, 117-119). Other factors affecting the severity of the symptoms include age (TBE tends to be more severe in elderly people); presence of comorbidities; immunosuppressed individuals and co-infection with other tick-borne pathogens (120-127). Genetic predisposition is currently under study, polymorphisms in genes such as CCR5D32, TLR3, OAS2, OAS3, IL28B, IL10, CD209 and MMP-9 are associated with more severe forms. Finally, the monophasic course of the infection is usually more severe.

Many patients with neurologic forms of TBE are left with chronic sequelae of infection with significant consequences for daily activities and the quality of life. The most frequently reported signs are neuropsychiatric disorders, such as apathy, irritability, memory and concentration dysfunction, sensory disturbances, persistent flaccid paresis, or paralysis and altered sleep patterns. A particular form following infection by TBEV-FE and -Sib is reported in Russia. Some patients may develop a chronic (progressive) TBE that manifests after several months or years under certain circumstances (hypothermia, physical or psychological trauma, overwhelming physical labor, alcohol intoxication, abortion, labour or even physiotherapy) or as a continuously progressive form with an increase in focal CNS lesions, leading to patient death after 9-20 years post infection (23).

Studies on the three main TBEV genotypes showed the following mortality rates: TBEV-Eu <2%; TBEV-Sib 3-6%; TBEV-FE 20-40%.

## Disease in animals

Animals can get TBEV from the bite of an infected tick and develop neurological symptoms similarly to humans. Usually, the veterinary aspects of this disease are poorly recognized even in domestic animals:

- *TBE in dogs*

Canine TBEV infection is possible in endemic areas and seroprevalence varies from country to country ranging from 0.1% in Belgium to 53.6% in Germany. Results are of difficult comparison due to different diagnostic factors considered and type of tests performed. In general, infection can have an acute course with complete remission within 1-2 weeks (31-59%), although a longer period is possible (12-25%). Late sequelae like paresis, muscle atrophy, epileptic seizures or blindness are described. A definite diagnosis is rarely achieved due to low sensitive PCRs availability. Virus detection is very difficult,
due to the fast immunological virus clearance from the brain and CSF or the quick fatal outcome (58).

- TBE in horses

Horses are prone to TBE infection although they remain mostly asymptomatic. Few cases have been reported so far (59,60,128,129) and the clinical signs mainly depend on the affected brain region(s). The outcome displays a broad spectrum of neurological symptoms such as ataxia, tonic-clonic seizures, apathy, and stupor, inappetence, mydriasis, convulsions of the legs, skittishness, bruxism and altered reaction to environmental stimuli.

- TBE in ruminants (sheep, roe deer and mouflon)

Domestic and wild ruminants are important amplifier hosts for tick populations and capable of developing a detectable viremia in the blood that is not transmissible to feeding ticks although it can pass on the milk and eventually milk products. Few cases of sub-clinical and clinical infections have been reported, all with neurological signs and fatal outcomes. A lamb and a roe deer suffered from ataxia, torticollis, tremor, nystagmus, hypersalivation and finally somnolence with inappetence and recumbency, while a muflon was found moribund before suppression (57,61,62).

- TBE in other animals

Insectivores are known to be reservoirs of TBEV, but records of clinical signs are rare. A study screening the presence of TBEV in several European hedgehogs, detected antibodies and virus RNA from an animal that contemporarily showed neurological symptoms (63). Monkeys have been used as animal models to test the efficacy of vaccines. For viruses such as TBEV, clinical manifestation and pathomorphological lesions in the Central Nervous System in the monkeys are similar to humans (64).

# Availability of preventive, therapeutic and control measures, including licensed or pipeline vaccines

## Therapy in humans

No specific antivirals are approved for the treatment of TBE in Europe (23), and patient care is mainly symptomatic and supportive, including intensive care interventions in severe cases. Specific anti-TBEV immunoglobulins, nonspecific immunoglobulins or recombinant anti-TBEV immunoglobulins are used or tested for prophylaxis or treatment. Vaccines can also be used in a therapeutic context as well. Specific small molecule antivirals are not yet recognized or widely used.

## Therapy in animals

Dogs and horses develop similar clinical symptoms as humans. There is no licensed anti-TBE vaccine for dogs. Symptomatic therapy is strongly recommended for dogs with TBE. Sedation and relaxation are necessary in the case of seizures. Steroid use is controversial, as immunosuppression may prolong the presence of the virus, although in dogs with marked cerebrospinal fluid pleocytosis, steroids seem to be mandatory to effectively protect the brain tissue from further fulminant immune response.
In cases of muscle atrophy and paresis, physiotherapy as early as possible has been shown to improve the general outcome and shorten the time of rehabilitation. TBE in horses is a rare event. Regarding therapeutic options and prognosis, a horse with recumbent status due to TBE has a poor prognosis if it is not possible to force the horse to stand up again.

## Licensed or pipelined vaccines

Currently, all six licensed vaccines (two from Europe, three from Russia and one from China) are based on inactivated whole viruses containing various strains of the European or Far-Eastern TBEV subtype. The two European vaccines are based on the Austrian isolate Neudoerfl (FSME-IMMUN®, Pfizer, USA) approved in 1976 (130) and the German isolate K23 (Encepur®, GlaxoSmithKline) approved in 1991 (131, 132). Sucrose is used as a stabilizer in Encepur®, whereas human serum albumin in FSME-IMMUN®. Both vaccines have the pediatric formulations namely FSME-IMMUN® (Junior) and Encepur-K®. Data from clinical studies and post-marketing surveillance show that FSME-IMMUN® and Encepur® are safe, efficacious (with seroconversion rates reaching 92-100% after complete vaccination) and interchangeable (133-135). Three vaccines are produced in the Russian Federation: TBE vaccine Moscow and Tick-E-Vac (Klesch-E-Vac) are both produced by the Chumakov FSC R&D IBP RAS in Moscow and are based on the Sofjin strain of TBEV-FE; EnceVir® vaccine is produced by Microgen in Tomsk using strain 205 of TBEV-FE (23, 136) (Table 4). In China, SenTaiBao based on the Chinese TBEV-FE strain Sen-Zhang is approved as a TBEV vaccine (137).

<table><caption>Table 4. TBE vaccines licensed in Europe and Russia (23)</caption><thead><tr><td>Name</td><td>Strain</td><td>Amount of antigen</td></tr></thead><tbody><tr><td>FSME-IMMUN® (Pfizer)</td><td>Neudoerfl (TBEV-Eu)</td><td>2.4 µg (adults)/1.2 µg (children)</td></tr><tr><td>Encepur® (GSK)</td><td>K23 (TBEV-Eu)</td><td>1.5 µg (adults)/0.75 µg (children)</td></tr><tr><td>TBE Moscow (FSBSI “Chumakov FSC R&D IBP RAS”)</td><td>Sofjin (TBEV-FE)</td><td>1.0 ± 0.5 µg/mL (dose 0.5 mL for children from 3 years old and adults)</td></tr><tr><td>Tick-E-Vac/Klesch-E-Vac (FSBSI “Chumakov FSC R&D IBP RAS”)</td><td>Sofjin (TBEV-FE)</td><td>1.0 ± 0.5 µg/mL (dose 0.25 mL for children 1-13 years old; 0.5 mL for adults)</td></tr><tr><td>EnceVir® (Microgen)</td><td>205 (TBEV-FE)</td><td>2.0-2.5 µg</td></tr></tbody></table>

## Other prevention measures

Other than active immunization as a protective measure against infection with TBEV, those preventing tick bites are also very important. These include: the use of chemical repellents with DEET or picaridin; wear light-coloured protective clothing; tuck pant legs into socks; avoid tick-infested areas and most importantly careful body check after visiting tick infested areas. The virus in tick saliva increases ten to 100-fold during feeding, therefore early removal of ticks does not prevent disease.
## Disease specific recommendations

TBEV is transiently present in the blood, therefore it could be hypothetically transmitted through blood transfusion or organ transplantation. A cluster with a fatal outcome has been recently reported (114) recognising the fragility of transplant recipients and the need to screen organ donors who live or have recently visited TBE-endemic areas.

The course of acute TBEV infections during pregnancy has not yet been investigated systematically. Moreover, the transmission and impact on foetal development is not fully understood. According to the few cases reported from literature, intrauterine transmitted infection doesn't occur (138), while trans placental transmission of IGg maternal antibodies has been detected with decreasing titres over time, where they no longer could be found after nine months of the infants (139). Finally, also breast-feeding transmission should be carefully considered especially in areas with high TBEV-endemicity (140).

## Epidemiological situation at different spatial scales:
past and current trends

Since 2012, the ECDC requires all 27 EU Member States, plus Iceland and Norway, to annually report their TBE data to TESSy repository. More detailed information on surveillance systems is available in the ECDC Annual Epidemiological Report (AER) (141).

### Past trends

The number of TBE cases reported in Europe, excluding Russia, increased over the years 1990-1994, probably reflecting the start of surveillance in many countries (33). Over the following 15 years (1995-2009), the trend was stable with an annual number of TBE cases fluctuating between 2,000 and 4,000 cases. Peaks occurred when a set of countries reported unusually high numbers of TBE cases, e.g. 2006 and 2009 (33). An analysis carried out in eight European countries suggested that human behaviour in response to good weather conditions, e.g. increased outdoor recreational activities, was the main explanation for the 2006 spike rather than tick abundance (142).

### Current trends

In 2012, TBE became a notifiable disease in Europe and uniform clinical and laboratory data were adopted as case definition. To analyse the current epidemiological trend, we included all cases reported to the TESSy during the years 2012-2020. Population denominator data were provided by the Statistical Office of the EU (Eurostat) for calculating incidence rates. Annual rates of change and their 95% confidence intervals (CI) were estimated using a log-linear regression, while the goodness of fit was assessed using F statistics, according to (15). We used RStudio software (R Core Team 2021) for all data management and statistical analyses.

Over the 2012-2020 period, 25 EU/EEA countries reported 25,825 TBE cases, of which 24,088 (93.0%) were confirmed cases and 1737 (7.0%) probable cases (Table 5). We excluded 59 cases with unknown classification. Cyprus, Iceland, Malta, and Portugal had no TBE surveillance. In the same period, the annual incidence increased with an annual variation of 5.4% (p = 0.05) (Table 5).
<table><caption>**Table 5.** Number of reported cases of tick-borne encephalitis, incidence (number of cases per 100,000 inhabitants), imported cases and trend, in 25 countries of the European Union and European Economic Area, 2012-2020 (data from TESSy-ECDC)</caption><thead><tr><th rowspan="2">Country</th><th colspan="3">2012-2020</th><th colspan="2">Trend</th></tr><tr><th>N. cases</th><th>Mean incidence</th><th>Imported cases*</th><th>Annual variation (%)</th><th>95% CI</th></tr></thead><tbody><tr><td>Austria</td><td>1043</td><td>1.33</td><td>13 (1%)</td><td>15.3</td><td>6 to 25</td></tr><tr><td>Belgium</td><td>2</td><td>0.00</td><td>2 (100%)</td><td>NA</td><td>NA</td></tr><tr><td>Bulgaria</td><td>6</td><td>0.01</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Croatia</td><td>63</td><td>0.17</td><td>NA</td><td>8.74</td><td>-75 to 93</td></tr><tr><td>Czechia</td><td>5552</td><td>5.83</td><td>29 (1%)</td><td>6.5</td><td>-1 to 14</td></tr><tr><td>Denmark</td><td>4</td><td>0.01</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Estonia</td><td>897</td><td>7.55</td><td>6 (1%)</td><td>-8.3</td><td>-14 to -3</td></tr><tr><td>Finland</td><td>574</td><td>1.16</td><td>NA</td><td>10.4</td><td>6 to 15</td></tr><tr><td>France</td><td>121</td><td>0.02</td><td>20 (17%)</td><td>29.3</td><td>-4 to 63</td></tr><tr><td>Germany</td><td>3670</td><td>0.50</td><td>139 (4%)</td><td>12.3</td><td>4 to 21</td></tr><tr><td>Greece</td><td>4</td><td>0.00</td><td>2 (50%)</td><td>19.1</td><td>-39 to 77</td></tr><tr><td>Hungary</td><td>255</td><td>0.29</td><td>4 (2%)</td><td>-11.7</td><td>-21 to -3</td></tr><tr><td>Ireland</td><td>1</td><td>0.00</td><td>1 (100%)</td><td>NA</td><td>NA</td></tr><tr><td>Italy</td><td>210</td><td>0.04</td><td>6 (3%)</td><td>34.3</td><td>-12 to 81</td></tr><tr><td>Latvia</td><td>1692</td><td>9.53</td><td>0</td><td>-0.8</td><td>-7 to 6</td></tr><tr><td>Lithuania</td><td>4551</td><td>17.59</td><td>42 (1%)</td><td>5.9</td><td>-2 to 14</td></tr><tr><td>Luxembourg</td><td>1</td><td>0.02</td><td>1 (100%)</td><td>NA</td><td>NA</td></tr><tr><td>Netherlands</td><td>21</td><td>0.01</td><td>9 (43%)</td><td>3.8</td><td>-31 to 39</td></tr><tr><td>Norway</td><td>165</td><td>0.35</td><td>16 (10%)</td><td>23.0</td><td>16 to 30</td></tr><tr><td>Poland</td><td>1943</td><td>0.57</td><td>8 (%)</td><td>0.8</td><td>-7 to 9</td></tr><tr><td>Romania</td><td>12</td><td>0.01</td><td>3 (25%)</td><td>-1.8</td><td>-49 to 45</td></tr><tr><td>Slovakia</td><td>1215</td><td>2.49</td><td>6 (%)</td><td>4.6</td><td>-6 to 15</td></tr><tr><td>Slovenia</td><td>1270</td><td>6.82</td><td>1 (%)</td><td>-2.2</td><td>-18 to 13</td></tr><tr><td>Spain</td><td>1</td><td>0.00</td><td>1 (100%)</td><td>NA</td><td>NA</td></tr><tr><td>Sweden</td><td>2552</td><td>2.86</td><td>43 (2%)</td><td>4.0</td><td>-3 to 11</td></tr></tbody><tfoot><tr><td>EU/EEA</td><td>25825</td><td>0.65</td><td>352 (11%)</td><td>5.4</td><td>0 to 11</td></tr></tfoot></table>

Such a trend is mainly driven by a constant and significant increase in TBE mean annual incidence, which increased of 33% between the period 2012-2016 to 2017-2020 from 0.75/100,000 to 0.56/100,000, respectively 24,355 cases reported the date of onset of symptoms. Among these, 22,601 (93%) were recorded between April-October, with a peak in June and July, while the minimum (290 cases; 1.2%) in December-March (Figure 6).

The weekly distribution of the cases over the whole period reveals the seasonal intra and inter-annual heterogeneity of this disease (Figure 7).

In 2020, 3,808 TBE cases were reported to TESSy from 17 EU/EEA countries with an incidence rate of 0.72 cases/100,000. Top five incidence rate countries were: Lithuania 25.7/100,000, Slovenia 8.5/100,000, Czechia with 7.9/100,000, Latvia 7.2/100,000 and Estonia 5.1/100,000. In 2019, the average mortality rate in Europe was 0.7%.

From the total cases recorded, 72.9% had a known vaccination status. Among these 18,821 cases, 17,881 (95%) were not vaccinated, 392 received one or two doses (2%), 411 (2.2%) three doses or more and 137 (0.7%) an unknown number of doses (Table 6).

In conclusion, the European TBE surveillance data highlighted an increasing trend over the years since the disease became notifiable. This is mainly driven by a few countries reporting the majority of cases. For example, three countries (Czech Republic, Germany, and Lithuania) accounted for 53% of all reported TBE cases, while two countries (Estonia, Hungary), which accounted for 4% of all reported TBE cases, showed a significant negative trend in the period 2012-2020.
<figure><img src="image_47.png" /><figcaption>Figure 6. Number of reported tick-borne encephalitis cases by month of onset, 2012-2020 (data from TESSy-ECDC)</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>Figure 7. Number of reported tick-borne encephalitis cases by week of onset, and 52-week moving average in the European Union and European Economic Area countries, 2012-2020 (data from TESSy-ECDC)</figcaption></figure>
<table><caption>Table 6. Main characteristics of reported cases of tick-borne encephalitis, European Union and European Economic Area countries, 2012–2020 (n = 25825) (data from TESSy-ECDC)</caption><thead><tr><th>Characteristics</th><th>N. cases</th><th>%</th><th>Incidence rate per 100,000 persons</th></tr></thead><tbody><tr><td>Total</td><td>25,825</td><td>100</td><td>5.81</td></tr><tr><td>Age group (years)</td><td></td><td></td><td></td></tr><tr><td>&lt;20</td><td>3,043</td><td>12%</td><td>0.68</td></tr><tr><td>20-29</td><td>2,332</td><td>9%</td><td>0.52</td></tr><tr><td>30-39</td><td>3,271</td><td>13%</td><td>0.74</td></tr><tr><td>40-49</td><td>4,344</td><td>17%</td><td>0.98</td></tr><tr><td>50-59</td><td>5,140</td><td>20%</td><td>1.16</td></tr><tr><td>60-69</td><td>4,470</td><td>17%</td><td>1.01</td></tr><tr><td>70-79</td><td>2,589</td><td>10%</td><td>0.58</td></tr><tr><td>≥ 80</td><td>629</td><td>2%</td><td>0.14</td></tr><tr><td>Unknown</td><td>7</td><td>NA</td><td>NA</td></tr><tr><td>Sex</td><td></td><td></td><td></td></tr><tr><td>Female</td><td>10,439</td><td>40%</td><td>0.00</td></tr><tr><td>Male</td><td>15,375</td><td>60%</td><td>0.00</td></tr><tr><td>Unknown</td><td>11</td><td>NA</td><td>NA</td></tr><tr><td>Importation status</td><td></td><td></td><td></td></tr><tr><td>Imported</td><td>352</td><td>1%</td><td>NA</td></tr><tr><td>Locally acquired</td><td>24,320</td><td>94%</td><td>NA</td></tr><tr><td>Unknown</td><td>1153</td><td>4%</td><td>NA</td></tr><tr><td>Vaccination status</td><td></td><td></td><td></td></tr><tr><td>Four doses</td><td>185</td><td>1%</td><td>NA</td></tr><tr><td>Three doses</td><td>226</td><td>1%</td><td>NA</td></tr><tr><td>Two doses</td><td>192</td><td>1%</td><td>NA</td></tr><tr><td>One dose</td><td>200</td><td>1%</td><td>NA</td></tr><tr><td>Vaccinated unknown doses</td><td>137</td><td>1%</td><td>NA</td></tr><tr><td>Not vaccinated</td><td>17,881</td><td>69%</td><td>NA</td></tr><tr><td>Unknown</td><td>7,004</td><td>27%</td><td>NA</td></tr></tbody></table>

The reported TBE cases followed a pronounced seasonality with most cases occurring during the warmer months May-October, which correspond to the period when people tend to spend a greater amount of time outdoors (48) and tick populations are more active.

No vaccination data are available at European level.

Finland

In Finland, TBE is endemic, with a mean of 64 cases reported annually and an average incidence of 1.16 per 100,000 inhabitants over the period 2012-2020 (Figure 8a).

The total number of cases reported is 574. The trend is generally increasing. TBE cases affect mainly males both in adult and older age (Figure 8b).

The place of infection was reported to TESSy from 2017 onwards. Since then, the most cases were reported from the Åland Islands (Figure 9).
a

<figure><img src="image_49.png" /></figure>

b

<figure><img src="image_50.png" /><figcaption>Figure 8. FINLAND - TBE cases (n. 574, period 2012-2020): yearly number of cases and incidence per 100,000 inhabitants (a) and distribution of TBE incidence by sex and age group (b) (data from TESSy-ECDC)</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Figure 9. FINLAND - TBE average incidence rate per 100,000 inhabitants by NUTS3 (n. 257, period 2017-2020) (data from TESSy-ECDC)</figcaption></figure>
France

A total of 121 cases of TBE are reported in France from 2012 to 2020, with a mean of 15 cases reported annually and an average incidence of 0.02 per 100 000 inhabitants (Figure 10a). Age and gender distribution includes mainly males from 50 to 69, but also categories from 30 to 49 are affected (Figure 10b).

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /><figcaption>**Figure 10.** FRANCE - TBE cases (n. 121, period 2012-2020): yearly number of cases and incidence per 100,000 inhabitants (a) and distribution of TBE incidence by sex and age group (b) (data from TESSy-ECDC)</figcaption></figure>

The place of infection was reported to TESSy from 2017 onwards. In 2020 there was a steep increase in the number of reported cases (n. 46) with respect to the previous year (n. 4), mostly due to an alimentary outbreak located in the eastern part of France (Figure 11) (143).
<figure><img src="image_54.png" /><figcaption>Figure 11. FRANCE - TBE average annual incidence rate per 100 000 inhabitants by NUTS2 (n. 64, period 2017-2020) (data from TESSy-ECDC)</figcaption></figure>

## Italy

The total number of cases reported from Italy over the period 2012-2020 is 210, with a mean of 35 cases reported annually and an average incidence of 0.04 per 100 000 inhabitants (Figure 12a). It involves mainly males included in the 40-49 to 70-79 age groups (Figure 12b).

The place of infection was reported to TESSy from 2017 onwards, evidencing that TBE incidence is mainly located in the north-eastern part of the country (Figure 13).

## Spain and Serbia

At the current date there is no evidence of locally acquired TBE human cases in Spain, as during the period 2012-2020 Spain reported only 1 imported case.

Information about TBE human infections in Serbia were provided by the Department for Communicable Diseases Prevention and Control of National Public Health Institute "Dr Milan Jovanovic-Batut". Data are available for the years 2017-2018 only, with a total of 18 reported cases.
<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /><figcaption>Figure 12. ITALY - TBE cases (n. 210, period 2012-2020): a) Yearly number of cases (bars) and incidence per 100 000 inhabitants (line) and b) distribution of TBE incidence by gender and age group (data from TESSy-ECDC)</figcaption></figure>
<figure><img src="image_57.png" /><figcaption>Figure 13. ITALY - TBE average annual incidence rate per 100 000 inhabitants by NUTS3 (n. 123, period 2017-2020) (data from TESSy-ECDC)</figcaption></figure>

## Sociological and demographical dimension affecting susceptibility and exposure, including gender

Human infections arise principally through tick bites to which people are exposed as they enter the forests for occupation and recreation. Socio-economic conditions have been identified as part of a network of independent but synergistic factors significantly correlating with TBE incidence. These conditions may operate with different force and at different time-scales depending on the cultural, societal and political contexts characteristic of each country. Abrupt socio-economic transitions could explain the TBE upsurge in ex-communist countries, while slower increases in western countries may have happened such as more retired people engaging in outdoor activities. Furthermore, changes in public health services (144) and the recent economic crisis (2009) (145) have been shown to explain annual spikes in incidence via their effects on human behaviour.

TBE has been observed with a higher incidence among risk groups such as: agricultural and forest workers, hunters, and in general people spending time outdoors such as hikers, bikers, foragers of mushrooms and berries, etc. In Europe 25,818 cases recorded in TESSy between 2012 and 2020 had information on sex (see Table 6). TBE was more common in males, with a male-to-female ratio of 1.5:1 (see Table 6). This proportion could be explained by the fact that men are less prone to use protective measures and have a lower risk perception (146-148) than women. Moreover, they present a 5-fold higher risk of neurological/neurocognitive sequelae than women (OR: 5017, 95%CI: 1199-20,987) (149).
With respect to the demographic distribution, 25,814 cases between 2012 and 2020 reported information on age (see Table 6) with 13,954 cases (54%) in the 40-69 years old group. TBE incidence increased with age in both sexes, peaking at 0.66 cases per 100,000 population in males aged 50-59 years, and then decreasing in older age groups (Figure 14).

<figure><img src="image_58.png" /><figcaption>Figure 14. Incidence of tick-borne encephalitis per 100,000 population, by gender and age group in the European Union and European Economic Area countries, 2012-2020 (data from TESSy-ECDC)</figcaption></figure>

The burden of TBE is mostly represented by its incidence and is generally not expressed in Disability-Adjusted Life Years (DALYs) in Global Burden Diseases studies, nonetheless it's long-term sequelae. DALYs measures the burden of diseases combining information on incidence, mortality and sequelae associated with them. As far as we know, only Slovenia performed such analysis revealing the importance of this approach in terms of vaccination policy (150, 151).

# Diagnostic procedures and notification systems used at local, national and European scale

## Diagnosis

ECDC publishes weekly and annual epidemiological reports regarding communicable diseases of concern to the EU. Data provided by EU Member States are uploaded into TESSy which is a flexible database for collection, validation, cleaning, analysis and dissemination of data for public health actions. All European Union Member States (28) and EEA countries (3) report their available data on communicable diseases as described in Decision No 2119/98/EC, to the system. TBE reporting and data retrieving is yet not homogeneous (Table 7). For 2020, 24 EU/EEA
countries reported data on TBE, among these 19 used various versions of the EU case definition for TBE (2008, 2012 and 2018), three reported using another case definition and two did not specify which case definition was used. 19 countries over 24 compulsory report TBE.

Cases are notified on a case-based level for all countries except Belgium and Bulgaria, and different administrative geographical units (NUTS) are used.

TBE is a notifiable disease in European Union since 2012 (152). The criteria considered are:

- **Case definition**

A confirmed case is defined as any person meeting the clinical i.e. symptoms of inflammation of the central nervous system AND laboratory criteria i.e. at least one of the following five:

1. TBE specific IgM AND IgG antibodies in blood;

2. TBE specific IgM antibodies in CSF;

3. Seroconversion or four-fold increase of TBE-specific antibodies in paired serum samples;

4. Detection of TBE viral nucleic acid in a clinical specimen;

5. Isolation of TBE virus from clinical specimen.

A probable case is defined as any person meeting the clinical criteria and the laboratory criteria for a probable case i.e. detection of TBE-specific IgM-antibodies in a unique serum sample OR any person meeting the clinical criteria with exposure to a common source (unpasteurised dairy products).

- **Anamnestic**

Patient medical records should include: age, gender, vaccination coverage for TBEV, consciousness of tick bite, Charlson Comorbidity Index (CCI), type of course (biphasic or monophasic), clinical manifestations, and professional exposure.

- **Clinical**

Regarding the TBE European subtype, a series of symptoms are possible including: fever, headache, asthenia, arthromyalgia, nausea, vomiting, ataxia, constipation, thremor, diarrhea, abdominal pain, visual disorders, limb palsy, disorientation, paresthesia, ideomotor slowing, hearing disorders, syncope, lymphadenopathy, urinary retention, sore throat, aphasia, dizziness, stupor, and amnesia.

- **Laboratory**

TBEV can be detected in blood samples during the first febrile phase and in brain tissue during the phase involving CNS symptoms. Therefore RT-PCR is a valuable diagnostic tool when there's the need to confirm an infection of a febrile illness following a tick bite or as a confirmation in fatal cases or in immunosuppressed patients. RT-PCR can detect viral RNA in urine samples for up to 19 days after the start of neurological symptoms. During the initial phase, leukopenia as well as thrombocytopenia are found in about 70% of patients (153). During the second phase, elevated white blood cell count may be present, C-reactive protein (CRP) concentration and erythrocyte sedimentation rate (ESR) may be elevated, especially in long-lasting severe cases. Analysis of cerebrospinal fluid (CSF) usually shows pleocytosis and a moderately raised protein level (153).

Changes in blood cells count during clinical manifestations include mostly neutropenia, lymphocytopenia, monocytosis, leukocytosis and thrombocytopenia. C-reactive protein (CRP) concentration and Erythrocyte Sedimentation Rate (ESR) are elevated. Pleocytosis and raised protein levels are found in cerebrospinal fluid. Other laboratory findings are: high levels of bilirubin, Gamma-GlutamylTransferase (GGT) or transaminase level, electrolyte disorders, increase of fibrinogen and amylase, creatine phosphokinase and lactate dehydrogenase (149). Computed Tomography (CT), Magnetic Resonance Imaging (MRI) and ElectroEncephaloGram (EEG) abnormalities could be found.
<table><caption>Table 7. Surveillance system overview for tick-borne encephalitis</caption><thead><tr><th rowspan="2">Country</th><th rowspan="2">Data source</th><th colspan="4">Type of surveillance</th><th colspan="4">Data reported by</th><th rowspan="2">Case definition used</th></tr><tr><th>Cp, V, O</th><th>A, P</th><th>C, A</th><th>Laboratories</th><th>Physicians</th><th>Hospitals</th><th>Others</th></tr></thead><tbody><tr><td>Austria</td><td>AT-Epidemiegesetz</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>EU-2012</td></tr><tr><td>Belgium</td><td>BE-REFLAB</td><td>V</td><td>P</td><td>A</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>EU-2018</td></tr><tr><td>Bulgaria</td><td>BG-NATIONAL_SURVEILLANCE</td><td>Cp</td><td>P</td><td>A</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>EU-2018</td></tr><tr><td>Croatia</td><td>HR-CNIPH</td><td></td><td></td><td>C</td><td></td><td></td><td></td><td></td><td>Not specified/unknown</td></tr><tr><td>Czechia</td><td>CZ-ISIN</td><td>Cp</td><td>A</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2008</td></tr><tr><td>Estonia</td><td>EE-NAKIS</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2012</td></tr><tr><td>Finland</td><td>FI-NIDR</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>EU-2012</td></tr><tr><td>France</td><td>FR-NATIONAL_REFERENCE_CENTRES</td><td>V</td><td>P</td><td>C</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>EU-2012</td></tr><tr><td>Germany</td><td>DE-SURVNET@RKI-7.1/6</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Other</td></tr><tr><td>Greece</td><td>EL-NOTIFIABLE_DISEASES</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td></td><td>EU-2018</td></tr><tr><td>Hungary</td><td>HU-EFRIR</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2012</td></tr><tr><td>Ireland</td><td>IE-CIDR</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2012</td></tr><tr><td>Italy</td><td>IT-ARBO</td><td>Cp</td><td>P</td><td>C</td><td>N</td><td>Y</td><td>Y</td><td></td><td>Other</td></tr><tr><td>Latvia</td><td>LV-BSN</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2012</td></tr><tr><td>Lithuania</td><td>LT-COMMUNICABLE_DISEASES</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>EU-2018</td></tr><tr><td>Luxembourg</td><td>LU-SYSTEM1</td><td>V</td><td></td><td>C</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Not specified/unknown</td></tr><tr><td>Netherlands</td><td>NL-TBE</td><td>V</td><td>P</td><td>C</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>EU-2012</td></tr><tr><td>Norway</td><td>NO-MSIS_A</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2012</td></tr><tr><td>Poland</td><td>PL-NATIONAL_SURVEILLANCE</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2012</td></tr><tr><td>Romania</td><td>RO-RNSSy</td><td>Cp</td><td>P</td><td>C</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>EU-2018</td></tr><tr><td>Slovakia</td><td>SK-EPIS</td><td>Cp</td><td>A</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2012</td></tr><tr><td>Slovenia</td><td>SI-SURVIVAL</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2008</td></tr><tr><td>Spain</td><td>ES-STATUTORY_DISEASES</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>EU-2018</td></tr><tr><td>Sweden</td><td>SE-SMINET</td><td>Cp</td><td>P</td><td>C</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>Other</td></tr></tbody></table>

(from: https://www.ecdc.europa.eu/en/publications-data/surveillance-systems-overview-2020).

Cp Compulsory; V voluntary, O other

A Active, P Passive

C Case-based; A Aggregated
Infrastructure capacity to identify pathogens
for each Member State

In 2007, the EU-funded dedicated surveillance network for enteric pathogens subsequently broadened to cover 21 food- and waterborne diseases and zoonoses, the European Food- and Waterborne Diseases and Zoonoses Network (FWD-Net). FWD-Net is coordinated by ECDC with the support of a coordination committee (CC). Each Member States nominated an Institute to be referred to as National Focal Point (Table 8).

<table><caption>Table 8. List of National Focal Points of the European Food- and Waterborne Diseases and Zoonoses Network (FWD-Net – ECDC) (link to websites included)</caption><thead><tr><td>Country</td><td>Institute</td><td>Address</td></tr></thead><tbody><tr><td>Austria</td><td>Austrian Agency for Health and Food Safety</td><td>Beethovenstraße 6, AT-8010 Graz</td></tr><tr><td>Belgium</td><td>Sciensano</td><td>Rue Juliette Wytsmanstraat 14, 1050 Brussels</td></tr><tr><td>Bulgaria</td><td>National Centre of Infectious and Parasitic Diseases</td><td>26 Yanko Sakazov Blvd, 1504 Sofia</td></tr><tr><td>Croatia</td><td>Croatian National Institute of Public Health</td><td>Rockefellerova 7, 10000 Zagreb</td></tr><tr><td>Cyprus</td><td>Directorate of Medical and Public Health Services</td><td>1, Prodromou str, CY-1448 Nicosia</td></tr><tr><td>Czech Republic</td><td>National Institute of Public Health/ Regional Public Health Authority Moravian-Silesian Region in Ostrava</td><td>Šrobárova 48, 100 42 Praha 10/ Na Belidle 7, 702 00 Ostrava</td></tr><tr><td>Denmark</td><td>Statens Serum Institut</td><td>5 Artillerivej, DK-2300 Copenhagen S</td></tr><tr><td>Estonia</td><td>Health Board</td><td>81 Paldiski Mnt, 10617 Tallinn</td></tr><tr><td>Finland</td><td>National Institute for Health and Welfare</td><td>Mannerheimintie 166, (00)271 Helsinki</td></tr><tr><td>France</td><td>Institute for Public Health Surveillance</td><td>12 rue du Val d'Osne, FR-94410 Saint-Maurice cedex</td></tr><tr><td>Germany</td><td>Robert Koch Institute</td><td>Seestraße 10, 13353 Berlin</td></tr><tr><td>Greece</td><td>National Public Health Organization</td><td>3-5 Agrafon St., EL-15123 Athens</td></tr><tr><td>Hungary</td><td>National Center for Epidemiology</td><td>Gyali ut 2-6, Budapest</td></tr><tr><td>Iceland</td><td>Centre of Health Security and Communicable Disease Prevention</td><td>Barónsstíg 47, IS - 101 Reykjavík</td></tr><tr><td>Ireland</td><td>Health Protection Surveillance Centre</td><td>25-27 Middle Gardiner Street, IR-1 Dublin</td></tr><tr><td>Italy</td><td>National Institute of Health</td><td>Viale Regina Elena 299, 00161 Rome</td></tr><tr><td>Latvia</td><td>Centre for Disease Prevention and Control</td><td>Duntes 22, 1005 Riga</td></tr><tr><td>Liechtenstein</td><td>Principality of Liechtenstein</td><td>Äulestrasse 51, 9490 Vaduz</td></tr><tr><td>Lithuania</td><td>Centre for Communicable Diseases and AIDS</td><td>Nugaletoju st. 14D, LT-10105 Vilnius</td></tr><tr><td>Luxembourg</td><td>Ministry of Health</td><td>1, rue Charles Darwin L-1433 Luxembourg</td></tr><tr><td>Malta</td><td>Superintendence of Public Health</td><td>37-39 Rue D'Argens, MT-5 Msida MSD</td></tr></tbody></table>