**4. Discussion**

In this chapter, we present data on human leishmaniasis cases, CanL cases, as well as the presence of important leishmania Phlebotomine fly vectors in Slovenia. Among the five collected species that were found in the region, two of them *P. neglectus* and *P. perniciosus* are well known and proven vectors of *L. infantum* in the Mediterranean and two others *P. papatasii* and *P. mascitii* are known vectors of several phleboviruses [16]. Leishmania parasites were not isolated from Phlebotomine flies that were caught and tested in Slovenia. Nevertheless, since one autochthonous CanL case and at least one autochthonous human case were reported, we presume that there is a natural life‐cycle of *Leishmania* sp. in the coastal region of Slovenia. Although other infection routes are proved in dogs (placental route, by sexual intercourse, by blood transfusion, and by infected flea bites) [17–20] they seem unlikely in our CanL case. The female dog was born in the region and had never traveled outside the country. It never got blood transfusion. It regularly slept outdoors. Clinical state of the mother and the spouse of the female dog (it got litter once), concerning leishmaniasis, was unknown but it is very likely that the female dog has been infected by Phlebotomine fly bite. The presence of infected vectors in Southeastern part of Slovenia is therefore highly probable.

Concerning the Mediterranean region, Phlebotomine fly's distribution pattern is quite bimo‐ dal. Western population was and it still is under direct influence of the African lineages. On the other hand, Eastern population is under the influence of both African and Asian continents. Northern part of the Mediterranean land mass is characterized by two narrow passages which directly influence spreading of Phlebotomine flies species. First one is restricted by Maritime Alps in the Southwestern part of the Alps, on the border between France and Italy. The second one and much more important for the Phlebotomine fly distribution is Slovenian Littoral, the westernmost part of Slovenia, bordering with the Italian region of Friuli‐Venezia Giulia, true contact zone between Eastern and Western populations. It is about 13 km wide, karst plateau, a unique geological formation displaying distinctive surface features, containing cenotes, sinkholes, or dolines [21].

As a bridge between East and West, the region of Slovenian Littoral hosts unknown number of Phlebotomine fly species. Until now we have found some of the most important vectors of *Leishmania* parasites in the Mediterranean, including *Phlebotomus neglectus*, *P. perniciosus*, and *P. perfiliewi*, species that already have penetrated this narrow land bridge (**Figure 1**). Never‐ theless, due to both environmental transformations and human activities there could be some other, more neglected ones such as *P. kandelaki* proven vector of *Leishmania* parasites in the Middle East.

Generally, most of the Phlebotomine fly species belonging to the subgenus Larroussius are potential vectors of *L. infantum*, and evidently due to rapid climatic changes, two of these are showing rapid aerial expansion. In 2003, the westernmost point of the range of *P. kandelaki* was in Montenegro [22], while in 2008, it was collected further to the west, at the coast of Croatia in Krk island (V. Ivović, unpublished data). Another fast spreading species and even more important vector, *P. neglectus* regularly situated in the Balkans, was recently found near Budapest (Hungary) [23]. Thanks to the attention recently drawn to the formerly neglected discipline of medical entomology, the latest results show the widening of the Phlebotomine fly distribution range to regions where they have never been found before.

Five human cases (two women and three men, 24–55 years old) were reported during the

During the period from 2013 to 2015, five species of Phlebotomine flies were collected and

In this chapter, we present data on human leishmaniasis cases, CanL cases, as well as the presence of important leishmania Phlebotomine fly vectors in Slovenia. Among the five collected species that were found in the region, two of them *P. neglectus* and *P. perniciosus* are well known and proven vectors of *L. infantum* in the Mediterranean and two others *P. papatasii* and *P. mascitii* are known vectors of several phleboviruses [16]. Leishmania parasites were not isolated from Phlebotomine flies that were caught and tested in Slovenia. Nevertheless, since one autochthonous CanL case and at least one autochthonous human case were reported, we presume that there is a natural life‐cycle of *Leishmania* sp. in the coastal region of Slovenia. Although other infection routes are proved in dogs (placental route, by sexual intercourse, by blood transfusion, and by infected flea bites) [17–20] they seem unlikely in our CanL case. The female dog was born in the region and had never traveled outside the country. It never got blood transfusion. It regularly slept outdoors. Clinical state of the mother and the spouse of the female dog (it got litter once), concerning leishmaniasis, was unknown but it is very likely that the female dog has been infected by Phlebotomine fly bite. The presence of infected vectors

Concerning the Mediterranean region, Phlebotomine fly's distribution pattern is quite bimo‐ dal. Western population was and it still is under direct influence of the African lineages. On the other hand, Eastern population is under the influence of both African and Asian continents. Northern part of the Mediterranean land mass is characterized by two narrow passages which directly influence spreading of Phlebotomine flies species. First one is restricted by Maritime Alps in the Southwestern part of the Alps, on the border between France and Italy. The second one and much more important for the Phlebotomine fly distribution is Slovenian Littoral, the westernmost part of Slovenia, bordering with the Italian region of Friuli‐Venezia Giulia, true contact zone between Eastern and Western populations. It is about 13 km wide, karst plateau, a unique geological formation displaying distinctive surface features, containing cenotes,

As a bridge between East and West, the region of Slovenian Littoral hosts unknown number of Phlebotomine fly species. Until now we have found some of the most important vectors of *Leishmania* parasites in the Mediterranean, including *Phlebotomus neglectus*, *P. perniciosus*, and *P. perfiliewi*, species that already have penetrated this narrow land bridge (**Figure 1**). Never‐ theless, due to both environmental transformations and human activities there could be some other, more neglected ones such as *P. kandelaki* proven vector of *Leishmania* parasites in the

period from 1997 to 2016, two of them in 2015, and one of them in 2016.

in Southeastern part of Slovenia is therefore highly probable.

identified (**Table 2**).

8 The Epidemiology and Ecology of Leishmaniasis

**4. Discussion**

sinkholes, or dolines [21].

Middle East.

Sixteen symptomatic dogs in Slovenia in the population of 220,700 officially registered dogs at the end of 2015 seem to be a small number. Nevertheless, point of view turns different taking into consideration that majority of the infected dogs never developed clinical symptoms. Incidence rate in endemic regions is usually less than 10% while seroprevalence can be as high as 90% [24]. We can speculate that the number of infected dogs in Slovenia is already higher in the moment. As many as 65% of asymptomatic dogs can harbor circulating parasites in their blood and as high as 93% of asymptomatic dogs are competent to transmit Leishmania to the vector, therefore these dogs allow transmission and spread of the disease [25, 26]. Beside dogs, other animals such as rats, cats, horses, rabbits, foxes, and jackals can be infected and may serve as a reservoir [12, 24, 27, 28]. Import of infected dogs to nonendemic regions creates one of the main risk factors for the spread of the disease. Many infected stray dogs have been brought to the north of Europe by compassionate tourists as well as social rescuing societies. There is an estimate that about 20,000 dogs infected with Leishmania presently live in Ger‐ many [29]. From the Crete and Cyprus example we can learn and predict increase of seropos‐ itive dogs in the Slovenian population. Seroepidemiological studies in dogs on island Crete, during the last 25 years, showed that the number of seropositive animals has been increasing [30]: from 0.27% in 1990 (data of the Greek Ministry of Agriculture) to 2.9% in 1994 and 19.8% in 2009. Same happened in Cyprus with a ninefold increase of seropositivity in dogs in the last 10 years [8]. This may be explained by the fact that dogs are brought into the island from mainland, especially from Attica, where leishmaniasis is endemic. The number of seropositive dogs in Crete continues to increase every year [30]. Another example is a situation that recently emerged in Australia. Before 2004, Antarctica and Australia were the only continents in the world that were free of leishmaniasis. In 2006, Biosecurity Australia ordered mandatory serological testing of dogs stationed in quarantine prior to importation. Few positive cases have been recognized until now, but there is concern of possibility of higher numbers [31]. Incubation time in leishmaniasis may be quite prolonged and clinical signs are not specific; therefore, veterinarians in nonendemic countries might overlook it.

Seroprevalence in people and dogs in Slovenia has not been estimated yet. High seroprevalence ratios in dogs were recently found in neighboring countries such as Italy and Croatian region of Dalmatia (21% and 42.85%, respectively) [32, 33]. Unfortunately, no information on the prevalence of healthy human inhabitants from Dalmatia is available. Recent data on seropo‐ sitivity of human population residing in the Istrian region nearby the southern Slovenian border confirm the possibility of spreading the disease toward the north of Europe, including Slovenia [34]. Moreover, seropositivity of Austrian inhabitants, living nearby the northern Slovenian border [35], indicates that the spread might have already happened. A potential vector *Phlebotomus mascitii* was found in this region [36]. Although *P. mascitii* is only an assumed vector of *Leishmania* spp.—data on its experimental transmission capacity are still lacking the wide distribution of Phlebotomine flies in Austria, a country thought to be free of these insects, further supports a potential emergence of endemic leishmaniasis in Central Europe. Studies from France showed that *P. perniciosus*, species that was present in Slovenia too, is the most common leishmania vector in regions at low altitudes (less than 600 m above sea level). This suits to the spread of the disease in the Slovenian coastal region. Another species *P. ariasi* was found in France as the main vector at the attitudes between 200 and 1400 m above sea level [6]. If this species would spread to Slovenia, it looks that mountain region would perfectly suit it. Until now the presence of this species in Slovenia has not been proved.

Epidemiological studies show that incidence of human infections are directly related to the number of infective dogs and the presence of suitable vectors in the region. Control of reservoirs by dog culling is, apart from being expensive and time consuming, also not efficient. Because the breeding sites of Phlebotomine flies are unknown, control measures are focused mostly on adults [37]. There are several strategies targeting adult vectors. Nevertheless, applying environmental changes such as trimming trees and shrubs and cleaning and reorganizing in and around human dwellings and animal shelters can prevent favorable conditions for the development of Phlebotomine fly larvae. In some regions with high Phlebotomine flies control efforts have focused on the use of chemical insecticides, mostly on synthetic pyrethroids. Unfortunately, these measures, although initially attractive, are gener‐ ally not permanent but are still most frequently used in controlling adult vectors. More advanced and sophisticated methods in Phlebotomine control includes planting of different plant species rich in phytochemicals that have a toxic effect against adult insects and larvae and use of bioinsecticides, particularly entomopathogenic against Phlebotomine flies [38].

Visceral leishmaniasis in people and dogs show similar clinical presentation involving intermittent pyrexia, lymphadenopathy, malaise, anemia, cachexia, hypergammaglobuline‐ mia, and hepatosplenomegaly. Beside these dogs develop skin changes, mainly in the form of exfoliative and nodular dermatitis [39]. Hyperglobulinemia is thought to be related to a Th2‐ dominated immune response resulting in a marked humoral response and increased gamma globulin production [31].

Unfortunately, chemotherapy is not a successful measure in control of canine visceral leish‐ maniasis. Relapsing cases are common and drugs do not lead to the inhibition of infectivity to Phlebotomine flies [1]. Parasites were proved even from healthy looking skin of infected dogs. Unlike dogs, people cured of leishmania infections usually develop lifelong immunity. Treatment of VL in people is dependent on chemotherapy that should cure the patient and reduce the risk for relapse. The first‐line drug for VL treatment should be liposomal ampho‐ tericin B or alternatively pentavalent antimonials and amphotericin B deoxycholate [40]. Miltefosine is being used on a compassionate basis in several European AIDS coinfected patients unresponsive to amphotericin B or pentavalent antimonials. Recently, this drug has been launched in the market for canine leishmaniasis treatment in Portugal, Spain, Italy, Greece, and Cyprus. Because dogs are never cured parasitologically and given the long half‐ life of the drug, the lack of European policy might contribute to the emergence of parasites resistant to miltefosine. Indeed, while this drug is successfully used in CanL treatment, there are reports on increasing incidences of relapse in humans on this treatment [41]. The same may happen with the use of ambisome in domestic pets that might produce spreading of resistant strains [3]. Like the Leish Vet group (international group of veterinary experts dealing with CanL), human experts conclude that chemotherapy alone would probably not be sufficient to eliminate the disease. Therefore, an effective vaccine should be developed and used in animals as well as in humans [40].

Slovenia [34]. Moreover, seropositivity of Austrian inhabitants, living nearby the northern Slovenian border [35], indicates that the spread might have already happened. A potential vector *Phlebotomus mascitii* was found in this region [36]. Although *P. mascitii* is only an assumed vector of *Leishmania* spp.—data on its experimental transmission capacity are still lacking the wide distribution of Phlebotomine flies in Austria, a country thought to be free of these insects, further supports a potential emergence of endemic leishmaniasis in Central Europe. Studies from France showed that *P. perniciosus*, species that was present in Slovenia too, is the most common leishmania vector in regions at low altitudes (less than 600 m above sea level). This suits to the spread of the disease in the Slovenian coastal region. Another species *P. ariasi* was found in France as the main vector at the attitudes between 200 and 1400 m above sea level [6]. If this species would spread to Slovenia, it looks that mountain region would perfectly suit

Epidemiological studies show that incidence of human infections are directly related to the number of infective dogs and the presence of suitable vectors in the region. Control of reservoirs by dog culling is, apart from being expensive and time consuming, also not efficient. Because the breeding sites of Phlebotomine flies are unknown, control measures are focused mostly on adults [37]. There are several strategies targeting adult vectors. Nevertheless, applying environmental changes such as trimming trees and shrubs and cleaning and reorganizing in and around human dwellings and animal shelters can prevent favorable conditions for the development of Phlebotomine fly larvae. In some regions with high Phlebotomine flies control efforts have focused on the use of chemical insecticides, mostly on synthetic pyrethroids. Unfortunately, these measures, although initially attractive, are gener‐ ally not permanent but are still most frequently used in controlling adult vectors. More advanced and sophisticated methods in Phlebotomine control includes planting of different plant species rich in phytochemicals that have a toxic effect against adult insects and larvae and use of bioinsecticides, particularly entomopathogenic against Phlebotomine flies [38].

Visceral leishmaniasis in people and dogs show similar clinical presentation involving intermittent pyrexia, lymphadenopathy, malaise, anemia, cachexia, hypergammaglobuline‐ mia, and hepatosplenomegaly. Beside these dogs develop skin changes, mainly in the form of exfoliative and nodular dermatitis [39]. Hyperglobulinemia is thought to be related to a Th2‐ dominated immune response resulting in a marked humoral response and increased gamma

Unfortunately, chemotherapy is not a successful measure in control of canine visceral leish‐ maniasis. Relapsing cases are common and drugs do not lead to the inhibition of infectivity to Phlebotomine flies [1]. Parasites were proved even from healthy looking skin of infected dogs. Unlike dogs, people cured of leishmania infections usually develop lifelong immunity. Treatment of VL in people is dependent on chemotherapy that should cure the patient and reduce the risk for relapse. The first‐line drug for VL treatment should be liposomal ampho‐ tericin B or alternatively pentavalent antimonials and amphotericin B deoxycholate [40]. Miltefosine is being used on a compassionate basis in several European AIDS coinfected patients unresponsive to amphotericin B or pentavalent antimonials. Recently, this drug has been launched in the market for canine leishmaniasis treatment in Portugal, Spain, Italy,

globulin production [31].

10 The Epidemiology and Ecology of Leishmaniasis

it. Until now the presence of this species in Slovenia has not been proved.

In the past, an effective vaccine against human leishmaniasis has already been used. This involved inoculation with live, virulent parasites in a process called leishmanization. It was practiced successfully in the former Soviet Union, the Middle East, and Israel. However, it was abandoned in most countries because of logistical problems and safety concerns, as some individuals developed nonhealing lesions and immune suppression [40, 42]. According to the authors' knowledge, no vaccine for routine use in people has been produced yet. The reason is that good understanding of immunity generated against pathogens is surely important for developing an effective vaccine. Current understanding of human immune responses gener‐ ated against Leishmania parasites is mainly based on the studies in animal models and this cannot be simply extrapolated to humans [40]. While interferon‐γ seems protective in mice and people, there have been enough of differences to prove that more studies in humans are needed. Patients developing visceral or diffuse cutaneous disease exhibit helper T‐cell subtype 2 cytokine profile [13]. Genetically modified Leishmania parasites lacking essential genes such as dihydrofolate reductase, biopterin reductase, or cystine proteases have been shown to stimulate protection against challenge with virulent parasite strains in people. The main problem is the concern relating to safety and feasibility for large‐scale use in the field [40]. Veterinary medicine probably gained an advantage in development of vaccines against leishmaniasis. A saponin formulation of fucose mannose ligand that is expressed throughout the lifecycle of a parasite was found to be safe, protective, and immunogenic and has become the Leishmune veterinary vaccine, licensed after a series of canine VL field studies [40]. In Europe, a vaccine based on the secreted‐excreted antigen of *L. infantum* (CaniLeish, Virbac Animal Health) has been recently licensed, and has been available in some European countries since 2011. Some studies show good immunogenicity of this vaccine, although large‐scale field studies are missing [43]. CanL vaccines proved to be efficacious not only in prevention of the disease but also in prophylactic manner, converting immune status of the infected dogs to more efficacious cell‐mediated immunity, that is able to prevent visceral leishmaniasis [43]. We therefore agree with the authors Foroughi‐Parvar and Hatam stating in their review article that the only efficacious method for control of CanL might be a vaccine [44].

The issue of notification of leishmaniasis differs in EU countries but even where notification is compulsory (i.e., Italy and Spain), notification of CanL cases is not a common practice. Generally, notification is compulsory in southern EU countries (Bulgaria, Greece, Italy, Portugal, and parts of Spain) but not in the part of EU, traditionally considered as nonendemic (France, Netherlands, etc.) [6, 45]. In Slovenia, notification of human case is mandatory in 3 days after the diagnosis [46] and record of CanL case should be reported to regional veterinary administration as well as to regional public health service as soon as confirmed by laboratory tests. These data are entered in the computer system of the Veterinary Administration of Republic of Slovenia monthly and reported twice annually to the World Organisation for Animal Health (OIE) via World Animal Health Information System (WAHIS). Notification of CanL cases in Slovenia is mandatory since 2014 but no CanL cases have been reported. Our data confirm that underreporting is taking place in Slovenia too, similarly to other border countries. Unrecognizing and underreporting of human and animal leishmaniasis in nonen‐ demic countries can have wide‐ranging consequences. Long reporting delay may happen even in endemic region like it was the case during recent community outbreak in Madrid (median of 151 days—41 days for visceral leishmaniasis and 183 days for cutaneous leishmaniasis). The delay arises from a number of factors that may be related to the patient (delay in seeking care) or the healthcare system (delay in diagnosis and reporting) [47].

Number of notified human cases in Slovenia at this moment is low. Interviews of these patients unfortunately were not done therefore no data were collected on clinical presentation nor traveling abroad. We even do not know whether reported cases were VL cases or cutaneous leishmaniasis cases. Nevertheless, having increased number of infected dogs and proven Phlebotomine fly vectors at the same time in climatically suitable region may lead to endemic spread of the disease [9]. That is a situation calling for governmental regulation. According to WHO's recommendations for epidemic‐prone areas, basic preparedness and rapid response mechanisms should be in place. Leishmaniasis cases should be detected early and reaction should be quick. In epidemic‐prone areas and before the anticipated outbreak season, the responsibilities of the outbreak task force members should be defined; the necessary needs for response, surveillance, and control should be assessed; the surveillance system should be reinforced; criteria for epidemic alert should be set up; and all health facilities should be provided with minimum stocks of basic diagnostic and treatment supplies [2].

Successful preventive measures in Slovenia and Slovenia‐like border countries should include regular veterinary checks of all imported dogs and dogs traveling outside the country, vector control, combined to use of effective repellents, and sleeping indoors with nets on the windows, and importantly, antileishmanial vaccination of dogs.
