Leishmania enriettii (Muniz & Medina, 1948): A highly diverse parasite is here to stay

Leishmaniases are a spectrum of diseases caused by protozoans from the genus Leishmania(Kinetoplastida: Trypanosomatidae) and are divided into 2 main clinical forms: tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL). Transmission occurs after the bite of sandfly vectors (Diptera: Phlebotominae) when females take a blood meal from the vertebrate host [1]. 
In the New World, several species of Leishmania (~20) cause disease to man, the symptoms and epidemiology of which vary depending on species. However, there are species that are nonpathogenic to humans, such as L. enriettii. In 1946, Medina observed ear lesions in 2 farm-reared guinea pigs (Cavia porcellus [Rodentia: Cavida]) from the neighboring state of Sao Paulo. After lesion analysis, Leishmania was confirmed as the pathogen. The complete L. enriettii description was published by Muniz and Medina in 1948 at the Federal University of Parana, Brazil [2]. Although this species has been used as a model for cutaneous leishmaniasis (CL), many aspects of its biology remain unknown. In the past 6 years, an increased interest has emerged after the finding of a similar isolate in the red kangaroo (Macrofus rufus) in Australia [3]. This article aims to summarize some of the most important publications on this unique pathogen. It demonstrates a high phenotypic plasticity, being able to infect different vertebrate hosts and vectors. It also discusses recent human and veterinary infections due to other L. enriettii complex members.

should be prospected. A recent interesting study on Leishmania detection in road-killed wild animals in São Paulo found 1 C. aperea (1/4) infected with Leishmania. Although this study did not type the species, it provided some evidence that sylvatic C. aperea could harbor Leishmania [6]. Although the above-mentioned rodents have been considered the most common reservoirs for L. enriettii in Brazil, the finding of a putative similar isolate in captive red kangaroos (M. rufus), northern wallaroos (Macropus robustus woodwardii), black wallaroos (M. bernardus), and agile wallabies (M. agilis) was a landmark in the leishmaniasis epidemiology in Australia. The complete identification at species level only came in 2011 [3], classifying this isolate as a member of the L. enriettii complex [7].

L. enriettii: Possible invertebrate vectors
Only 20 years after the description of L. enriettii (1967), evidence appeared regarding its invertebrate host [8]. A survey of phlebotomine fauna in Paraná state identified Lutzomyia monticola and Lutzomyia correalimai in the vicinities where the guinea pigs were infected. Experimental sandfly infections on those guinea pigs were performed where 60% (6/10) of L. monticula exhibited a very high infection in their midguts. For this reason, L. enriettii was considered a suprapylarian species and included in the subgenus Leishmania. The authors recovered those parasites from sandflies and inoculated naive guinea pigs. However, no development of infection was observed, and transmission through L. monticola bite has yet to be determined.
Since no L. monticola laboratory colony is available, transmission experiments are difficult to perform. For this reason, it is a challenge to ascertain its current status as a L. enriettii vector. This vector is widely spread from southern to northern Brazilian states. Although L. monticola has no human medical importance, it is very anthropophilic and is often captured together with vectors of CL and VL. More importantly, its distribution overlaps with domestic C. porcellus and wild C. aperea, reinforcing further studies with this vector.
In spite of having many sandfly species in Australia, the first evidence that midges from the subgenus Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae) could be an alternative vector of Leishmania has emerged. Although this vector fell into most of the Killick-Kendrick criteria, transmission still needs to be demonstrated after its colonization [3]. It is important to mention that this work triggered a recent increase in studying L. enriettii by many groups. Recently, 2 other ceratopogonids (Culicoides nubeculosus and Culicoides sonorensis) and Lutzomyia longipalpis were tested for L. enriettii infection using 2 strains (Brazilian and Australian) [9]. Only C. sonorensis was able to sustain infection, whereas L. longipalpis (permissive vector) developed moderate infections. Those data remarkably demonstrated L. enriettii's ability to sustain and develop infection in different invertebrate hosts. Also, those studies stimulate further epidemiological investigations to identify other potential/alternative non-sandfly vectors.

The L. enriettii complex members in human and veterinary infections
A detailed and historical review on Leishmania and sandflies was recently reported. However, a universal consensus regarding Leishmania classification is yet to be achieved [1], especially for the "L. enrietti complex." In addition to L. enriettii, it may include Leishmania martiniquensis [10], "Leishmania siamensis" [11], and the Australian isolate [12]. In spite of the molecular techniques in establishing phylogenetic relationships among Leishmania species, a few studies have included L. enriettii complex members. The L. martiniquensis (strain MAR1) was isolated in Martinique Island, French Antilles, in 1995, causing diffuse CL in an HIV-infected patient [13]. A similar human isolate of L. martiniquensis causing VL was also reported from Thailand [14,15]. Finally, "L. siamensis" was first reported in Thailand as causing VL [16] and disseminated CL/VL in a patient with HIV. The isolate from this patient was named Trang strain and had 100% identity with L. enrietti after molecular analysis [11]. More recently, some isolates of L. enriettii complex members, probably "L. siamensis" and L. martiniquensis, were isolated from CL lesions in Ghana [15]. Detection of this species was also reported from a low number of cases in horses from Florida (2) [17] and Central Europe (6) [18] and in 1 cow from Switzerland [19]. However, as mentioned by Kwakye-Nuako et al. [15], although it appeared in the literature several times, the species "L. siamensis" was not formally described, and its name should be used in quotation marks.
The above-mentioned data suggest that members of L. enriettii complex are able to infect a wide range of different hosts, from rodents to humans, and to cause many clinical manifestations. In Table 1, we summarized some of the main isolates and reference strains of the L. enriettii complex members. Therefore, an international consortium for whole genome sequencing could be useful to ascertain the taxonomic status of this complex using those strains. This is of importance since the real status of "L. siamensis" and L. martiniquensis are still the subject of debate.

Concluding remarks
Since its discovery in the 1940s, L. enriettii studies have been occurring in pulses. In the 1950s and 1960s, most of the studies were focused on its biology, transmission, and epidemiology. Later on, in the 1970s, some immunopathological and immunological studies appeared. In the 1990s, its use as a model for molecular biology was successfully employed. More recently, the findings of other putative L. enriettii complex members such as L. martiniquensis and "L. siamensis" have generated great interest in understanding the taxonomical relationships among those different isolates. Whole genome sequencing would be a very important tool to investigate such relationships and help to establish their real species status. The species of this complex exhibit a high phenotypic plasticity in being able to infect a wide range of vertebrate hosts, including humans and other vectors. They may also cause different symptoms, ranging from CL to VL. However, many aspects of their epidemiology are still unknown in the geographic areas where those parasites were isolated. Although there is strong evidence of some suspected vectors, another important gap yet to be demonstrated is vectorial transmission by either ceratopogonids or L. monticula.
In this context, almost 70 years after L. enriettii discovery, many questions and uncertainties about its biology, epidemiology, classification, and immunology remain unanswered. The finding of members of this complex on different continents and its veterinary and human medical importance, especially in patients with HIV, reinforces the need for more studies. Several groups in the world are now investigating those parasites, and an increase in the published papers in the past years has demonstrated that this parasite is here to stay.