Leishmania tarentolae and Leishmania infantum in humans, dogs and cats in the Pelagie archipelago, southern Italy

Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean basin with most of the infected human patients remaining asymptomatic. Recently, the saurian-associated Leishmania tarentolae was detected in human blood donors and in sheltered dogs. The circulation of L. infantum and L. tarentolae was investigated in humans, dogs and cats living in the Pelagie islands (Sicily, Italy) by multiple serological and molecular testing. Human serum samples (n = 346) were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT. Meanwhile, sera from dogs (n = 149) and cats (n = 32) were tested for both Leishmania species by IFAT and all blood samples, including those of humans, by specific sets of real time-PCR for L. infantum and L. tarentolae. The agreement between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were also assessed. Overall, 41 human samples (11.8%, 95% CI: 8.9–15.7) were positive to L. infantum (5.2%, 95% CI: 3.3–8.1), L. tarentolae (5.2%, 95% CI: 3.3–8.1) and to both species (1.4%, 95% CI: 0.6–3.3) by serology and/or molecular tests. A good agreement among the serological tests was determined. Both Leishmania spp. were serologically and/or molecularly detected in 39.6% dogs and 43.7% cats. In addition to L. infantum, also L. tarentolae circulates in human and animal populations, raising relevant public health implications. Further studies should investigate the potential beneficial effects of L. tarentolae in the protection against L. infantum infection.


Introduction
With over 20 Leishmania spp. as causative agents, the leishmaniases are neglected tropical diseases inducing cutaneous, mucocutaneous and, the most severe, visceral form. Visceral leishmaniasis (VL) is mainly caused by Leishmania donovani and Leishmania infantum, both transmitted by phlebotomine sand fly bites and distributed worldwide, being endemic in over 98 countries across Asia, East Africa, South America and the Mediterranean regions [1]. In particular, L. infantum is the most important species of zoonotic concern, with domestic dogs as the main reservoirs and phlebotomine sand flies as vectors. The Mediterranean basin is a hyperendemic area for VL since cases account for up to 6% of the global burden [2]. However, asymptomatic Leishmania infections in immunocompetent hosts, are 5-10 times greater than the number of clinically apparent disease cases [3]. Although large outbreaks of VL are uncommon, especially in European countries [4], an epidemic of leishmaniasis started in June 2009, occurred in an urban area of Madrid (Spain) with more than 700 cases recorded [5]. In this scenario, hares (Lepus granatensis) were the sylvatic reservoir of L. infantum, with a minor role played by dogs [6], and humans presented mainly cutaneous forms, with visceral manifestations in immunocompromised patients [7].
In Italy, VL is typical of rural and peri-urban areas and is present in patchy spots along areas on the Tyrrhenian and the low Adriatic Coasts and on the islands, according to the distribution of the sand fly vectors and of the main dog reservoirs. Sicily, one of the two major Italian islands located in the centre of the Mediterranean Sea, is highly endemic for VL [8,9] along with canine and feline leishmaniosis, given the temperate climate that facilitates the vector circulation and the spread of the parasites [10][11][12][13]. A recent surveillance on phlebotomine sand flies conducted in Sicily reported a high abundance of Sergentomyia minuta and Phlebotomus perniciosus, both competent vectors of Leishmania tarentolae and L. infantum in reptiles and mammalian hosts, respectively [14]. Unexpectedly, an increasing number of scientific evidence suggests that S. minuta feeds also on humans [14][15][16][17]. The first evidence of L. tarentolae DNA in a human being was from bone marrow and intestines of a 300-year-old Brazilian mummy [18]. Only recently the parasite DNA has been detected in the blood of human donors [17] and in sheltered dogs [16] in central and southern Italy, respectively. In addition, experimental studies demonstrated that L. tarentolae may infect human phagocytic cells, differentiate into amastigote-like forms, therefore indicating it may transiently infect human hosts [18,19]. In addition, this species, non-pathogenic to humans, has aroused great interest in the scientific community given its application as a promising biotechnological expression host and immunotherapy agent for human leishmaniasis treatment [20].
In this context, a comprehensive survey on the occurrence of L. infantum and L. tarentolae infection in asymptomatic humans, in dogs and cats living in VL endemic area was carried out by performing molecular and serological tests, in order to assess the circulation of these protozoa in a close environment where many interactions among humans, dogs, cats and reptiles occur.

Ethics statement
The study on humans was conducted in accordance with ethical principles (Declaration of Helsinki) and the research protocol was approved by the Ethical Committee of the University Hospital of Palermo, Italy (n. 8/2020). Written informed consent was obtained from each participant.
The animals were handled and sampled following the approval of the Ethical Committee of animal experiments of the Department of Veterinary Medicine, University of Bari, Italy (Approval Number 01/2021).

Study site
The study was carried out on Lampedusa and Linosa, two small islands of the Pelagie archipelago which is the southernmost Italian territory (205 km from Sicily) and the nearest to North Africa (113 km). The island of Lampedusa (35˚30 0 56@N 12˚34 0 23@E) is 20.2 km 2 and has a population of 6,000 inhabitants, whereas Linosa is 5.4 km 2 with about 300 inhabitants, with a significant increase of population during the summer season due to the arrival of tourists. Lampedusa geologically belongs to the African continent, affecting the vegetation and animal population, whereas Linosa is of volcanic origins and is the closest to Sicily (175 Km). The Pelagie Islands are characterized by a semi-arid and windy climate, moderate rainfall during the mild winters and hot and humid summers, generally with temperature reaching up to 30˚C during the period from April to October.
Data available from Regional Pet Animal Database Sicilian Region (February 2021) has censused a total of 999 dogs (i.e., 794 owned and 205 stray) and 301 cats (i.e., 16 owned and 285 from colony) in both islands belonging to the same municipality.

Study population and sample collection
From July to October 2020, serum and blood samples of 346 volunteer patients (n = 245 from Lampedusa and n = 101 from Linosa) without any specific sign of leishmaniasis, were originally received from human analysis laboratory for health check analyses (Lampedusa) or purposedly collected (Linosa) under the frame of this study, and subsequently sent to the Parasitology Unit of the Department of Veterinary Medicine, University of Bari (Italy) for serological and molecular testing. During the same period, 99 dogs and 29 cats from Lampedusa and 50 dogs and 3 cats from Linosa were blood sampled. All animals were autochthonous and owned, and an informed consent was signed by the owner before sampling. Before sampling, animals were clinically examined and data (e.g., age, sex, breed, clinical signs) and previous antiparasitic treatments were recorded in each animal's file. Whole blood was collected from each animal in vacuum containers with EDTA (i.e., human = 5mL; dog = 2 mL; cat = 1 mL) and serum collection tubes with clot activator (i.e., human = 5 mL; dog = 2.5 mL; cat = 2.5 mL). Blood and serum samples were tested for L. infantum and L. tarentolae by molecular and/or serological methods.

Serological testing
Human serum samples were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT, whereas canine and feline sera to both Leishmania spp. were tested only by IFAT.
The detection of IgG anti-L. infantum in humans, dogs and cats was performed as previously described [21][22][23], whereas for antibodies against L. tarentolae in humans, the IFAT was performed by using as antigen whole promastigotes of L. tarentolae (strain RTAR/IT/81/ ISS21-G.6c/LEM124) following the same procedure for L. infantum IFAT using fluoresceinated rabbit anti-human immunoglobulin G (Sigma-Aldrich, Germany) diluted 1:50 as conjugate. Serum samples from each specific host (i.e., human, dog and cat) positive for L. infantum by cytological and molecular analyses or WB for human, and a healthy host negative for L. infantum, were used as positive and negative controls, respectively in each IFAT. Samples were considered as positive when they produced a clear cytoplasmic and membrane fluorescence of promastigotes from a cut-off dilution of 1:80. Positive sera were titrated by serial dilutions until negative results were obtained.
The detection of antibodies against L. infantum in human sera was also performed using a commercial enzyme immunoassay following the manufacturer's instructions (Leishmania Ab, Cypress Diagnostics, BE). Briefly, 100μl of serum sample diluted 1:20 in the buffer supplied by the kit, were added to each microwell coated with inactivated Leishmania antigens and incubated for 10 min at room temperature. After washing, a second incubation with 100μl of antihuman IgG-conjugate coupled with the horseradish peroxidase was performed. Finally, the substrate was added, and the reaction was blocked with H 2 S0 4 . The absorbance was measured in a microplate reader (Biorad, model 680) at 450 nm. Sera with an absorbance value greater than 1.0 were considered as positive. The human serum samples positive to L. infantum by ELISA and/or IFAT were further confirmed by immunoblot assay (Leishmania Western Blot IgG, LDBIO Diagnostics, Lyon, FR). A volume of 50μl of serum 1:48 diluted in the buffer supplied by the kit, was distributed to nitrocellulose membranes previously bounded with L. infantum antigens. The binding of the alkaline phosphatase-anti human IgG conjugate with the immunocomplex was revealed by the presence on the strip of specific 14 and 16 kDa antigenic bands. Positive and negative controls supplied by the kit were included in both the serological assays.

Molecular testing
Genomic DNA (gDNA) was extracted from human, dog and cat blood samples by using a commercial GenUPBlood DNA kit (Biotechrabbit GmbH, Hennigsdorf, Germany) respectively, according to the manufacturer's instructions. All samples were tested by duplex realtime PCR (dqPCR) for the detection of partial region of the internal transcribed spacer 1 (ITS1) gene of L. infantum and L. tarentolae as described in Latrofa et al. [24] and of L. infantum kDNA minicircle (120 bp) by real time-PCR (qPCR), using primers, probes and protocol described elsewhere [25]. Genomic DNA from L. infantum isolate cultured in Tobie-Evans medium from a leishmaniotic dog living in Italy (zymodeme MON-1) and L. tarentolae (strain RTAR/IT/81/ISS21-G.6c/LEM124) promastigotes were used as positive controls, whereas gDNA extracted from blood sample of a healthy dog and negative to L. infantum was used as negative control. The DNA samples positive to Leishmania by dqPCR were successively amplified by conventional PCR (cPCR) using primers L5.8S/LITSR targeting partial region of the ITS1 (~300bp) and amplification run as described elsewhere [26]. Amplicons were purified and sequenced in both directions using the same primers as for PCR, employing the Big Dye Terminator v.3.1 chemistry in an automated sequencer (3130 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA). All sequences were aligned using the ClustalW program [27] and compared with those available in GenBank using the BLASTn tool (http://blast.ncbi.nlm. nih.gov/Blast.cgi).

Discussion
Findings of this survey indicate that either L. infantum and/or L. tarentolae may infect humans living in the Pelagie archipelago with the same prevalence (i.e., 6.6%). Although asymptomatic L. infantum infection is expected in a geographical area highly endemic for VL, the detection of antibodies against L. tarentolae and of circulating DNA in the blood samples of the screened population is of major interest and opens new perspectives into the study of human leishmaniasis. Indeed, the presence of specific antibodies anti-L. infantum revealed by IFAT, ELISA and WB in 23 patients (6.6%) and the detection of circulating L. infantum kDNA (0.6%) indicates an important cumulative exposure to or infection by the parasite in the examined populations in the investigated area. Meanwhile, the low IgG titers (up to 1:320) against L. infantum detected by IFAT in patients molecularly negative may suggest an exposure to the pathogen. Contrarily, the detection of L. infantum DNA only in two patients with high antibody titers (1:1280) may suggest the occurrence of subclinical infection or a recent exposure to the protozoan infection. Though the prevalence of L. infantum infection in asymptomatic humans, including blood donors, is variable (0.6-71%), depending on the diagnostic test and the geographical area [28], prevalence of positivity herein recorded are overall consistent with those recorded in healthy subjects in other European countries such as southern France [29], southern Spain [30], the Balearic Islands [31] and Italy [32,33]. In endemic areas, most Leishmania spp. infections remain asymptomatic, indeed the ratio of subclinical to clinical cases of VL has been estimated to be up to 50:1 based on parasite virulence and host susceptibility [34][35][36]. Asymptomatically infected hosts can harbor viable parasites throughout life and may develop the clinical disease if immunosuppression occurs thereafter [37]. Moreover, the parasite transmission may be maintained by asymptomatically infected hosts exposed to bites of competent sand fly species [38]. The comparison of the serological tests for the detection of antibodies against L. infantum revealed a substantial agreement between them, with a slightly higher score among ELISA and WB suggesting a higher specificity of these tests than IFAT. Therefore, IFAT and ELISA are recommended for routine screening while WB as confirmatory test in the diagnosis of VL [33]. The slight agreement between serological and molecular tests for the detection of antibodies or DNA of both Leishmania spp. was expected because of the smaller number of molecularly positive samples from humans than those seropositive.
Strikingly, the human population under investigation turned out to be also exposed to or infected by L. tarentolae, which has been historically considered a saurian-associated Leishmania spp.. This data is of importance considering that the serological positivity (i.e., 3.2%) has been reported herein for the first time. Such seropositivity overlaps that recorded by molecular testing (3.1%). The first evidence of L. tarentolae DNA in humans was in soft and hard tissues, including bone marrow, of a Brazilian mummy dating to the end of 18 th century [18], and recently in blood donors from central Italy [17]. Both findings suggest the occurrence of parasite systemic spreading in humans. In addition, L. tarentolae is experimentally capable of infecting human phagocytic cells and differentiating into amastigote-like forms [19]. Therefore, these findings along with our results suggest that L. tarentolae may naturally infect and circulate in human hosts. Indeed, L. tarentolae is commonly regarded infecting geckoes (i.e., Tarentola annularis and T. mauritanica) and recently also lacertid lizards (Podarcis siculus) [16]. Nonetheless, some strain (i.e., LEM-125) of L. tarentolae and other species belonging to the subgenus Sauroleishmania, such as Leishmania adleri, may cause transient infections in humans [19,39].
Although the accuracy of the IFAT for the detection of antibodies anti-L. tarentolae has not been investigated and cross-reactions with highly similar species of Leishmania may occur, the presence of L. tarentolae DNA in the blood of 11 patients suggests that this protozoan may infect humans. Therefore, the sensitivity and specificity of this serological method, should be evaluated by comparing it with a more accurate test (i.e., WB) as reported for the diagnosis of VL.
To date, L. tarentolae is considered a promising parasite for the expression of human recombinant proteins [20,40], immunotherapy agent for human leishmaniasis treatment as demonstrated in the murine model [41] and vaccine candidate [42], thus suggesting a potential protective and beneficial effect against L. infantum in hosts naturally infected by the parasite. Moreover, considering that L. tarentolae DNA has been detected in P. perniciosus and Phlebotomus perfiliewi, which usually feed on mammals, as well as in S. minuta fed on humans [16,17], the epidemiology of this parasite requires careful and thorough evaluation.
As far as the dog and cat populations living on the Islands, the overall exposure to L. infantum is high in both dogs (37.6%) and cats (28.1%), with higher seroprevalence (i.e., 51.5%) and antibody titers in dogs from Lampedusa than from Linosa. The higher statistically significant prevalence of seropositive dogs from Lampedusa (53.5%) than Linosa (12%) may be due mainly to the rainy and windy conditions in the latter island which are unfavorable for phlebotomine sand flies. Overall, data overlap those of a previous survey where more than 50% of dogs scored positive to the parasite and where a high abundance of P. perniciosus (67.7%) and S. minuta (28.5%) was detected [10]. A similar proportion of canine (41.8%) and feline (25.7%) infection by L. infantum was reported in a study conducted on the Aeolian Islands (Sicily) [11], demonstrating that humans and animal populations, in such confined environment, have a high risk of Leishmania infection.
The positivity of dogs to L. tarentolae by serological (24.2%) and molecular testing (1.3%), was recently reported in shelter dogs (5% and 1%, respectively) in southern Italy where a high incidence of L. infantum subclinical infection (10%) was also recorded along with a high abundance of S. minuta [16]. These findings along with the serological positivity to L. tarentolae, firstly retrieved also in cats, suggest the circulation of both Leishmania species in human and animal populations.

Conclusions
Humans, dogs, reptiles, and sand flies living in the same confined environment in small islands in the middle of the Mediterranean Sea may share Leishmania spp. including the non-pathogenic L. tarentolae. The presence of L. tarentolae in humans and dogs, undoubtedly raises many scientific questions about the potential beneficial effects this species of Leishmania may have in cross-protecting hosts infected by pathogenic Leishmania species. Under the above circumstances, more comprehensive studies on the occurrence of this parasite need to be addressed and its usefulness in public health evaluated. For example, the expression of recombinant antigens in live vectors (e.g., L. tarentolae) has been investigated for its protective effect for preparing vaccines against L. infantum [20,42]. As future perspectives, studies focus on the validation of more accurate tests (e.g., WB) for a proper serological diagnosis of L. tarentolae infection in humans and animals along with experimental infection studies in mammals would be advised to establish whether the parasite effectively replicates in macrophages. This could eventually provide important knowledge on the infection establishment in the host and in the stimulation of the animal immune response.