**2.3. Transmission**

VL caused by *L. infantum* is a zoonosis and can display either wild or domestic transmission cycles depending on the eco-epidemiological conditions.

Leishmaniasis starts from the wild cycle, involving reservoirs such as wild canids and marsupials. As for the domestic cycle, the main reservoir is the domestic dog (Canis lupus familiaris) [7]. The link between wild and domestic cycles occurs when humans install dwellings on forest banks [14] and, most likely, because some wild reservoirs have synanthropic habits (**Figure 3**) [15].

Canine visceral leishmaniasis (CVL) is crucial from an epidemiological point of view, as it is more prevalent than human VL. Dogs exhibit high levels of subcutaneous parasites and high sensitivity to vectorial infection [12]. Additionally, between 50 and 60% of infected animals are asymptomatic [16]. It is estimated that three out of five asymptomatic positive dogs transmit the parasite to sand flies, and this transmission rate does not significantly change among symptomatic and asymptomatic groups of animals [17].

Other animals have been pointed as possible reservoirs, such as rodents [14] and cats, or accidental hosts, such as horses [18]. Adaptation of vectors to different animal species would be a favorable factor for VL transmission [19].

**Figure 3.** Eco-epidemiology of visceral leishmaniasis in Pará, Brazil*. L. infantum* parasite is maintained by a sand fly population (*Lutzomyia longipalpis*) and wild reservoirs (1). House invasion by sand flies near forests allows canine and human infection (2 and 3), where domestic dogs become the main source of the parasite. The same occurs in the urban areas. Solid lines indicate defined routes of transmission, and dashed lines represent possible transmission routes with other wildlife, and possibly with man, serving as a source of infection for sand flies [14].

There are other transmission forms of less epidemiological importance, such as blood transfusion and venereal transmission, apart from vertical transmission, which can be transplacental or transmammary [20–23]. Direct dog-to-dog transmission through bites or wounds has been suggested as responsible for sporadic CVL transmission as well [24, 25].

## **2.4. Geographic distribution and prevalence**

which will then multiply by binary division and differentiate into promastigotes. Promastigotes bind to the epithelium of the sand fly's esophagus or pharynx by the flagellum and then differentiate into metacyclic promastigotes, which is the infectious stage, unable to divide or

Transmission occurs when the infected vector does a new blood meal and inoculates the infective form of *Leishmania* in the host. The sand fly regurgitates around 1000 metacyclics into the wound caused on the skin of a mammal [9]. When inoculated into the host organism, the parasite is phagocytized by the cells of mononuclear phagocytic system and loses the flagella. Next, it multiplies intensively by binary division, to the point of rupturing the host cell and releasing amastigotes, which then will be phagocytized by other cells and spread via blood

VL vectors belong to the order Diptera, family Psychodidae, subfamily Phlebotominae, genus *Lutzomyia* in the new world, and *Phlebotomus* in the Old World. They can be found in tropical

Sand flies are small-sized, light brown-colored insects, with a coat of hair over their body. They measure between 2 and 3 mm [11]. Their flight range reaches about 150–300 m (diameter of 300–600 m), but may be longer depending on the species. They usually fly in small jumps and

Hematophagy is a unique habit of females, who require blood for ovary maturation. Hence, they are able to transmit the disease. When females feed on blood, they hold their wings upright. A variety of animals has been identified as dietary hosts of sand flies, which have very

VL caused by *L. infantum* is a zoonosis and can display either wild or domestic transmission

Leishmaniasis starts from the wild cycle, involving reservoirs such as wild canids and marsupials. As for the domestic cycle, the main reservoir is the domestic dog (Canis lupus familiaris) [7]. The link between wild and domestic cycles occurs when humans install dwellings on forest banks [14] and, most likely, because some wild reservoirs have synan-

Canine visceral leishmaniasis (CVL) is crucial from an epidemiological point of view, as it is more prevalent than human VL. Dogs exhibit high levels of subcutaneous parasites and high sensitivity to vectorial infection [12]. Additionally, between 50 and 60% of infected animals are asymptomatic [16]. It is estimated that three out of five asymptomatic positive dogs transmit the parasite to sand flies, and this transmission rate does not significantly change among

and subtropical regions of the planet and are popularly known as sand flies [10, 11].

eclectic feeding habits. Females lay eggs in moist soil, rich in organic matter [13].

bind to the midgut, remaining free in the digestive tract lumen [5, 7, 8].

and lymph to other tissues and organs [5, 7, 8].

22 Canine Medicine - Recent Topics and Advanced Research

have crepuscular and nocturnal activity [12].

cycles depending on the eco-epidemiological conditions.

symptomatic and asymptomatic groups of animals [17].

**2.2. Vector**

**2.3. Transmission**

thropic habits (**Figure 3**) [15].

In recent years, endemic regions have widened, and there has been a sharp increase in number of recorded cases of the disease. Still, it is believed that the impact of leishmaniasis on public health has been underestimated, since its notification is only mandatory in 32 of the 88 affected countries [2]. CVL is endemic in over 70 countries and occurs mainly in the Mediterranean region and South America [26]. Moreno and Alvar concluded that at least 2.5 million dogs are infected in only four countries in Southeastern portion of Western Europe, representing 16.7% of the canine population [27].

Franco et al. [28] developed a database of publications on the prevalence of LVC in Europe between the years 1971 and 2006. They found an overall prevalence of 23.2% and average of 10%, the highest taking place in Italy (17.7%), followed by France (8%), Portugal (7.3%), and Spain (5.9%) [28]. In another study, in 18 Portuguese cities with 3974 dogs, the overall prevalence of CVL was of 6.31% and ranged from 0.88% to 16.16% among cities, with high prevalence in inland regions [29].

In Croatia, seroprevalence of CVL ranges from 0 to 42.85% depending on the studied region [30]. In Cyprus, the seroprevalence of the disease in dogs had a ninefold increase compared to 10 years before, reaching 14.9% in 2010 [31]. The average seropositivity of dogs in Greece is 22.1%, and positive animals were found in 43 of 54 cities [32].

Seroprevalence of CVL in Southern Europe ranges from less than 5% to over 50% depending on the geographic region [33]. However, the prevalence of infection is significantly higher than both seroprevalence and apparent disease, due to sensitivity of serological techniques and because clinical signs usually only appear in less than half of the population, as in all endemic areas of the world [34].

Recent expansion to areas not previously endemic has been recorded in some parts of Europe, as northern Italy [35], in Germany [36], and in northern Spain [37]. The expansion of the CVL is associated with adjacent territories and often with global warming, which favors vector transmission, or with import of infected dogs to non-endemic areas, such as the United Kingdom and Poland [26, 33].

In Africa, VL transmission areas are located near forests [12]. Most of the information published in Africa have often been reports of human cases during epidemic situations, but information on the reservoirs are scarce [38]. In Algeria, the number of cases of CVL is on the rise, with a frequency of 35%, with 25% of positive dogs being asymptomatic [39]. Coastal regions of Northern Morocco are known to be endemic for canine and human VL. Moreover, several cases of CVL caused by *L. tropica* have been reported in areas where it is normally caused by *L*. *infantum* [40].

CVL exists in some Asian countries. It is endemic in Iran, with an average prevalence of 14.2%, and variations according to the region: 18.2% in northeast, 12.3% in the central region, and 4.4% in Southeastern Iran [41]. Another study, restricted to southwestern Iran, found a prevalence of 15.4% [42]. In China, VL is an anthropozoonosis which completes its life cycle in dogs, raccoons, coatis, and children [12]. The presence of *L. infantum* DNA in dogs, in Southwest China, ranged from 23.5 to 28.2% [43]. In the Western region, 51.9% of the dogs were positive in PCR, but only 36.8% of the dogs reacted in serology [44].

CVL has been expanding over the Americas. It currently occurs from southern Canada [45] and the United States [46, 47] to northern Argentina [48, 49]. In North America, it was first reported on hunting kennels of Foxhound dogs in New York, in 1999 [46]. Since then, CVL has been spreading and has so far been diagnosed in 18 North American and two Canadian states, totaling 58 kennels with positive Foxhounds, but with no reports of human cases. Isozyme characterization showed that the isolated agents from 46 Foxhounds are *Leishmania infantum* (MON1) [45]. High mortality and transmissibility rates associate with these foci in North America. It is believed that the disease was imported from Southern Europe due to dog travel history between these regions [50]. Direct transmission forms are associated with these outbreaks: from dog to dog, via placenta or during intercourse [22]. *L. infantum*, in these cases, remains capable of infecting sand flies and subsequently moving into a vertebrate host, which highlights the risk of vector-based transmission to humans [47].

prevalence of CVL was of 6.31% and ranged from 0.88% to 16.16% among cities, with high

In Croatia, seroprevalence of CVL ranges from 0 to 42.85% depending on the studied region [30]. In Cyprus, the seroprevalence of the disease in dogs had a ninefold increase compared to 10 years before, reaching 14.9% in 2010 [31]. The average seropositivity of dogs in Greece is

Seroprevalence of CVL in Southern Europe ranges from less than 5% to over 50% depending on the geographic region [33]. However, the prevalence of infection is significantly higher than both seroprevalence and apparent disease, due to sensitivity of serological techniques and because clinical signs usually only appear in less than half of the population, as in all endemic

Recent expansion to areas not previously endemic has been recorded in some parts of Europe, as northern Italy [35], in Germany [36], and in northern Spain [37]. The expansion of the CVL is associated with adjacent territories and often with global warming, which favors vector transmission, or with import of infected dogs to non-endemic areas, such as the United

In Africa, VL transmission areas are located near forests [12]. Most of the information published in Africa have often been reports of human cases during epidemic situations, but information on the reservoirs are scarce [38]. In Algeria, the number of cases of CVL is on the rise, with a frequency of 35%, with 25% of positive dogs being asymptomatic [39]. Coastal regions of Northern Morocco are known to be endemic for canine and human VL. Moreover, several cases of CVL caused by *L. tropica* have been reported in areas where it is normally caused by *L*.

CVL exists in some Asian countries. It is endemic in Iran, with an average prevalence of 14.2%, and variations according to the region: 18.2% in northeast, 12.3% in the central region, and 4.4% in Southeastern Iran [41]. Another study, restricted to southwestern Iran, found a prevalence of 15.4% [42]. In China, VL is an anthropozoonosis which completes its life cycle in dogs, raccoons, coatis, and children [12]. The presence of *L. infantum* DNA in dogs, in Southwest China, ranged from 23.5 to 28.2% [43]. In the Western region, 51.9% of the dogs were

CVL has been expanding over the Americas. It currently occurs from southern Canada [45] and the United States [46, 47] to northern Argentina [48, 49]. In North America, it was first reported on hunting kennels of Foxhound dogs in New York, in 1999 [46]. Since then, CVL has been spreading and has so far been diagnosed in 18 North American and two Canadian states, totaling 58 kennels with positive Foxhounds, but with no reports of human cases. Isozyme characterization showed that the isolated agents from 46 Foxhounds are *Leishmania infantum* (MON1) [45]. High mortality and transmissibility rates associate with these foci in North America. It is believed that the disease was imported from Southern Europe due to dog travel history between these regions [50]. Direct transmission forms are associated with these outbreaks: from dog to dog, via placenta or during intercourse [22]. *L. infantum*, in these cases,

positive in PCR, but only 36.8% of the dogs reacted in serology [44].

prevalence in inland regions [29].

24 Canine Medicine - Recent Topics and Advanced Research

areas of the world [34].

Kingdom and Poland [26, 33].

*infantum* [40].

22.1%, and positive animals were found in 43 of 54 cities [32].

There are few studies on CVL in Mexico, but human VL cases are constant. Rosete-Ortíz et al. analyzed skin lesions of 25 dogs by immunohistochemistry and PCR in a region where *Lutzomyia longipalpis* has been described and found *Leishmania* sp. in 60% of samples [51].

In Latin America, VL occurs in 12 countries, with 90% of cases concentrated in Brazil [11], distributed in all states of the Northeastern, Midwestern, and Southeastern regions, plus in Roraima, Tocantins, and Pará states, in the Northern region [14]. Among Brazilian cities and states, variation in prevalence of CVL is huge, ranging from 0.7% in Salvador, in the state of Bahia [52], to 51.6% in São Luis Island, Maranhão state [53], both in Northeast, where the disease is most prevalent.

CVL urbanization correlates with increasing global mobility [54] associated with demographic and ecological factors. In Latin America, especially in Brazil, Colombia, and Venezuela, migration and urbanization have contributed to the increase in American VL. In Brazil, one example is the rural exodus from the Northeast fields, causing thousands of people to migrate to cities like Fortaleza, Jacobina, João Pessoa, Natal, Petrolina, St. Louis, Sobral, Teresina, and Salvador. They then proceed to live in suburban areas with unsanitary conditions and malnutrition. Migrants often bring along their dogs and raise chicken and pigs around their homes, all ultimately serving as a feed source for the vector. According to Moreno et al., in urban areas, some factors favor the spread of *L. infantum*, such as the presence of waste and degraded areas around houses, lack of vector recognition by locals, and increased exposure to sand fly, especially in the early evening [55]. Thus, "ruralization" of the outskirts of the Northeastern cities has been identified as the cause of epidemics detected in the region [56], although this same process may not be necessary for entry of visceral leishmaniasis in other cities, as happened in the Southeast of the country.

The state of Rio Grande do Sul, Brazil, was considered CVL-free until 2008, when a case of an autochthonous canine was reported in São Borja [57]. In the same year, *Lutzomyia longipalpis* was found in urban areas of San Borja, a species not previously reported in the Southern region [58]. Since then, new cases were reported: 24 in dogs and 10 in humans between 2008 and 2014. Of these 34 reports, 20 were autochthonous, showing that it is an emerging zoonosis in the state [59]. It is believed that the disease has entered the state via Santo Tomé city in Argentina, which borders San Borja and already had reports of the disease [57, 59].

Prevalence of CVL in the world varies widely, and such variation also applies to different locations within the same city, suggesting that different ecosystems favor maintenance of vectors in different manners [60]. As noted by Azevedo et al. [61] and Belo et al. [62], results of studies on prevalence are influenced by various factors such as the region and population studied, the diagnostic method, as well as the sample used.

Migration of humans and their pets, disorderly occupation, poor living conditions, deforestation, and climate change associated with vector-adaptive capacity are some of the causes of the global urbanization of leishmaniasis [54].

The best example of the phenomenon of urbanization of zoonotic visceral leishmaniasis is happening in Brazil [54, 63]. VL has invaded urban centers and large capitals with no previous record of autochthonous cases [12]. Epidemiological data show the suburbanization and urbanization of visceral leishmaniasis, highlighting the outbreaks in Rio de Janeiro (RJ), Belo Horizonte (MG), Aracatuba (SP), Santarém (PA), Corumbá (MS), Teresina (PI), Natal (RN), São Luís (MA), Fortaleza (CE), Camaçari (BA), and more recently, occurrence of epidemics in the municipalities of Três Lagoas (MS), Campo Grande (MS), and Palmas (TO) [11].

The prevalence of human VL caused by *L. infantum* has decreased where living standards improved [2], showing that one of the most important interventions may be socioeconomic development and improved nutrition of children [8], but CVL cases have increased.
