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 [10-13]. 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 [14-17]. 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.

Methods

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′56″N 12°34′23″E) is 20.2 km2 and has a population of 6,000 inhabitants, whereas Linosa is 5.4 km2 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 [21-23], 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 anti-human IgG-conjugate coupled with the horseradish peroxidase was performed. Finally, the substrate was added, and the reaction was blocked with H2S04. 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 real-time 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).

Statistical analysis

Exact binomial 95% confidence intervals (CI) were established for proportions. The chi-square was used to compare proportions with a probability p value < 0.05 regarded as statistically significant. Agreements between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were evaluated using Cohen’s kappa statistic (κ) as follows: no agreement (κ < 0), slight agreement (0 < κ < 0.20), fair agreement (0.21< κ < 0.40), moderate agreement (0.41 < κ < 0.60), substantial agreement (0.61 < κ < 0.8) and almost perfect agreement (κ > 0.81). Analyses were done using the GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, CA, USA)

Results

Overall, 41 out of 346 human samples (11.8%, 95% CI: 8.9–15.7) were positive to L. infantum (n = 18; 5.2%, 95% CI: 3.3–8.1), L. tarentolae (n = 18; 5.2%, 95% CI: 3.3–8.1) and to both species (n = 5; 1.4%, 95% CI: 0.6–3.3) by serology and/or molecular test.

Out of 23 patients positive to L. infantum (including 18 + 5 patients exposed only to L. infantum and to both Leishmania spp., respectively; 6.6%, 95% CI: 4.5–9.8), 17 (4.9%, 95% CI: 3.1–7.7) tested positive by IFAT with antibodies titres of 1:80 (n = 10), 1:160 (n = 4), 1:320 (n = 1) and 1:1280 (n = 2), 21 (6.1%, 95% CI: 4.0–9.1) by ELISA and 16 (4.6%, 95% CI: 2.9–7.4) by WB (). Circulating L. infantum kDNA was detected in blood of 2 patients (0.6%, 95% CI: 0.2–2.1) seropositive to both L. infantum and L. tarentolae with IgG titers of 1:1280 and 1:320, respectively. The two individuals above showed simultaneous positivity to L. infantum by all three serological (IFAT, ELISA and WB) and molecular testing (qPCR and/or dqPCR). Kappa agreement between serological tests was almost perfect between WB and ELISA results (κ = 0.86), and substantial between IFAT and WB (κ = 0.78) and IFAT and ELISA (κ = 0.80). Whereas the agreement between different combination of the serological and molecular tests for the detection of both Leishmania spp. was slight (k < 0.20).

Out of 23 human samples (6.6%, 95% CI: 4.5–9.8) positive to L. tarentolae, 12 (3.5%, 95% CI: 2–6) were positive by IFAT with antibodies titres of 1:80 (n = 6), 1:160 (n = 4), and 1:320 (n = 2) and 11 (3.2%, 95% CI: 1.9–5.6) by dqPCR ().

Out of 13 Leishmania spp. dq/qPCR positive samples (i.e., 2 to L. infantum and 11 to L. tarentolae), 5 specimens were successfully amplified by cPCR targeting the rRNA ITS1 gene, confirming L. tarentolae (n = 4) and L. infantum (n = 1) species identification with a nucleotide identity of 99–100% with the L. tarentolae reference sequence MT416142 and of 100% with L. infantum sequence MN648764 available in the GenBank database. Representative sequences of pathogens detected in this study were deposited in the GenBank database (L. tarentolae 278bp, 276bp with accession numbers MW930736, MW930737; L. infantum 265bp with accession number MW930738).

None of the individuals (i.e., 162 males and 184 females; median age, 49 years; age range, 21 to 87 years) included in the study had a medical history of visceral or cutaneous leishmaniasis.

Out of 149 dogs, 59 (39.6%; 95% CI: 32.1–47.6) were exposed to Leishmania spp. of which two, seropositive to both Leishmania spp., scored positive by qPCR to L. infantum (.

Twenty-three seropositive dogs showed antibodies titers against L. infantum by IFAT of 1:80 (n = 12), 1:160 (n = 6), 1:320 (n = 4) and 1:640 (n = 1). Two dogs were seropositive to anti-L. tarentolae with titers of 1:80 and 1:160. Thirty-four dogs tested positive to IgG against both species, had titers ranging from 1:160 to 1:2560 for L. infantum and from 1:80 to 1:1280 for L. tarentolae. Two animals positive to L. infantum DNA by qPCR were also seropositive to both species. The prevalence of dogs living in Lampedusa and exposed only to L. infantum or L. tarentolae or to both species (53.5%, 95% CI: 43.8–63) was statistically higher than that recorded in those from Linosa (12%, 95% CI: 5.6–23.8) (χ2 = 23.9, df = 1, p < 0.00001).

As far as cats from Lampedusa, 14 out of 32 (43.7%, 95% CI: 28.2–60.7) were positive to L. infantum (n = 4; 12.5%, CI: 5–28.1), L. tarentolae (N = 3; 9.4%, 95% CI: 3.2–24.2) and to both species (n = 6; 18.2%, 95% CI: 8.9–35.3) by IFAT and one seronegative was positive to L. infantum (3.1%, 95% CI: 0.5–1.6) by qPCR. Ten cats seropositive to L. infantum had antibodies titers of 1:80 (n = 7) and 1:160 (n = 3), whereas 9 seropositive to L. tarentolae of 1:80 (n = 7) and 1:160 (n = 2). The three cats from Linosa scored negative by IFAT as well as by dp/qPCR. The K agreement between IFAT and the molecular tests in dogs and cats was slight (k < 0.20).

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 [34-36]. 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 18th 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.

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24 Aug 2021

Dear Dr. Otranto,

Thank you very much for submitting your manuscript "Leishmania tarentolae and Leishmania infantum in humans, dogs and cats in the Pelagie archipelago, southern Italy" for consideration at PLOS Neglected Tropical Diseases. Apologies for the delay in sending a decision, but we had a delay finding suitable reviewers for your work. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: All clear.

Reviewer #2: It is not clear how the sizes of human, canine and feline populations were determined.

Reviewer #3: In this manuscript authors show interesting results of a potentially new Leishmania transmission scenario, involving a parasite from the subgenus Sauroleishmania. However, in order to strengthen the findings, more robust methods should be used.

Some relevant questions:

Can you perform an identification of the L. tarentolae strain (TAR and LEM) to provide further evidence of the infection potential to human hosts?

How specific are the serological tests used for L. tarentolae screening?

Can you include other Leishmania species in your IFATS to account for inespecificities?

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Lines 208-210: were these the same samples? meaning, were the serologically positive individuals also PCR-positive?

lines 217-219: please, also give the number of base pairs (lengths of the respective fragments)

line 240: why 53.5%? how does this correlate to data given in table 2?

Reviewer #2: Comparison between serological and molecular results could have been made.

Reviewer #3: ITS based analyses are characterized by a short fragment, which can lead to inespecificities. To strengthen the results, authors could include a marker with a longer fragment for the detection of L. tarentolae. In this way, they could perform comparative analysis, even including other Leishmania species. HSP70 has already been used in L. tarentolae and it generates larger fragments, so there are good reference bases. Also including a tree would be very helpful.

Regarding serological tests as the basis for the diagnosis of these two Leishmania species, the specificity of the test against L. tarentolae is not clear, again, the use of other Leishmania species as reference would be desirable.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: line 259: except those with detectable DNA (how does this correlate to antibody titers and any known immunodeficiency?).

line 268: how does your study prove long-lasting contact with reservoirs and why is this important?

line 283: rephrase, as this would imply that DNA detection was possible already in the 18th century.

line 326: this "potential beneficial role" should be explained and discussed in more detail in the Discussion.

Reviewer #2: Conclusions are those of a preliminary study. Cross-reactivity of the serological tests could have been addressed.

Reviewer #3: Although authors are conscious of the limitations of their study, evidence for public health relevance is missing and could benefit from additional experiments and analysis.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Numerous minor grammatical errors and typos, some examples are given below.

lines 31-32: "by multiple serological and molecular testing" should go behind the bracket in line 32.

line 35: should be: sera, and: tested for

line 75: this should be "immunocompromised", I assume?

line 82: should be: Sergentomyia and perniciosus

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is an interesting study with several novel aspects (detection of L. tarentolae and corresponding antibody response in humans and pets), but it could easily be condensed to a Short Report.

Reviewer #2: Line 30 – change to: Leishmania tarentolae WAS detected

Line 42 – Leishmania spp. – change accordingly throughout the manuscript

Line 52 – change to: This species, non-pathogenic to mammals, has recently been…

Line 62 – change to: With over 20 Leishmania spp. as causative agents, THE leishmaniases are neglected tropical diseases…

Line 67 – add comma: most important species of zoonotic concern, with domestic dogs

Line 68 – change to: The Mediterranean basin is A hyperendemic area

Line 70 – change to: immunocompetent hosts, ARE 5–10 times greater

Line 77 – change to: Tyrrhenian and the low Adriatic Coasts and ON the islands, according to the distribution of the sand

Line 79 – centre

Line 81 – parasites (instead of parasite)

Line 90 – change to: In addition, this species, non-pathogenic to humans, has…

Lines 93-96 – sampling of dogs and cats should also be mentioned

Line 122 – which criteria (statistical, etc.) have determined these samples sizes?

Line 123 – change to: and n=101 from Linosa) without any specific sign of leishmaniasis, were COLLECTED and sent to the

Line 126 – were purposely collected samples (under the frame of this study, for health check analyses) all from Linosa? Please, discriminate how many were from the analyses lab and purposely collected

Lines 127-128 – which criteria (statistical, etc.) have determined these samples sizes?

Line 130 – delete “anamnestic” and change to: … sampling animals were clinically examined and data (i.e., age, sex, breed, clinical…

Line 140 – change to: L. tarentolae by IFAT, whereas CANINE and FELINE sera to both Leishmania spp. WERE TESTED only by IFAT.

Line 179 – change to: … 1 (ITS1, ~300bp) and AMPLIFICATION RUN AS DESCRIBED ELSEWHERE [26].

Line 186 – use CI (instead of CIs)

Line 199 – to both Leishmania spp.

Are there any results regarding agreement between serology and PCR for humans? The same question for dogs and cats

Line 216 – insert comma after gene,

Line 261 – Are these seronegative patients? (The detection of parasite’s DNA in few patients suggests that the parasitemia is very low or…)

Line 263 – which of the protozoa?

Line 264 – replace individuals with “humans” (in order to avoid confusion with dogs and cats)

Line 277 – could not this agreement be due to a lack of specificity of the serological tests between L. infantum and L. tarentolae?

Line 280 – Leishmania sp.

Line 281 – remove comma: … (i.e., 3.2%)

Line 284 – insert comma: … of a Brazilian mummy,

Line 291 – change to: may cause transient infections IN humans

Line 302 – delete: which had

Line 304 – change to: As far as the dog and cat populations living on the islands,

Line 307 – replace positive with “seropositive”

This seropositivity to both L. infantum and L. tarentolae should not be compared to seropositivity to only L. infantum

Line 316 – “canine leishmaniosis” – is this just disease or disease plus subclinical infection? Please report in the main text

Line 318 – the meaning of “firstly retrieved” is not clear

Line 323 – Leishmania spp.

Line 324 – replace charming with “fascinating”

Reviewer #3: Although the work and the research question are very interesting, the methods used seem insufficient to demonstrate the hypotheses that authors wish to test. The fact that in cell cultures L. tarentolae can transform into amastigotes, doesn’t imply that the parasite can effectively replicate in macrophages and sustain infection in mammals. The existing evidence points rather to the transient presence of the parasite in mammals. Without this evidence, it is very risky to suggest that the results obtained through serology are indicative of immunity, much less cross-immunity.

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27 Aug 2021

Submitted filename: Authors response.docx

Click here for additional data file.

16 Sep 2021

Dear Dr. Otranto,

We are pleased to inform you that your manuscript 'Leishmania tarentolae and Leishmania infantum in humans, dogs and cats in the Pelagie archipelago, southern Italy' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Yara M. Traub-Csekö

Associate Editor

PLOS Neglected Tropical Diseases

Alvaro Acosta-Serrano

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Results

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-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Summary and General Comments

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Reviewer #1: The authors have satisfyingly addressed all my comments.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

20 Sep 2021

Dear Prof. Otranto,

We are delighted to inform you that your manuscript, "Leishmania tarentolae and Leishmania infantum in humans, dogs and cats in the Pelagie archipelago, southern Italy," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Table 1

Number and prevalence of humans exposed to or infected by L. infantum (Li) and L. tarentolae (Lt) based on the serological and molecular diagnostic tests and geographical origin.

Test	IFAT		ELISA	WB	dq/qPCR
	Li	Lt	Li	Li	Li	Lt
Lampedusa (n = 245)	10 (4.1%)	10 (4.1%)	12 (4.9%)	8 (3.3%)	2 (0.8%)	7 (2.9%)
Linosa (n = 101)	7 (6.9%)	2 (2.0%)	9 (8.9%)	8 (6.9%)	0	4 (4.0%)
TOT (n = 346)	17 (4.9%)	12 (3.5%)	21 (6.1%)	16 (4.6%)	2 (0.6%)	11 (3.2%)

Table 2

Number and prevalence of dogs positive to L. infantum (Li) and/or L. tarentolae (Lt) by IFAT and qPCR based on the geographical origin.

Test	IFAT			qPCR
	Li	Lt	Li + Lt	Li
Lampedusa (n = 99)	19 (19.2%)	2 (2.0%)	32 (32.3%)	1 (1.0%)
Linosa (n = 50)	4 (8.0%)	0	2 (4.0%)	1 (2.0%)
TOT (n = 149)	23 (15.4%)	2 (1.3%)	34 (22.8%)	2 (1.3%)