Galán‑Puchades et al. Parasites & Vectors https://doi.org/10.1186/s13071-022-05309-4 (2022) 15:211 Parasites & Vectors SHORT REPORT Open Access Molecular detection of Leishmania infantum in rats and sand flies in the urban sewers of Barcelona, Spain María Teresa Galán‑Puchades1* , Jennifer Solano2, Gloria González2, Antonio Osuna2, Jordi Pascual3, Rubén Bueno‑Marí1,4, Sandra Franco3, Víctor Peracho3, Tomás Montalvo3,5 and Màrius V. Fuentes1 Abstract Background: Classically, dogs have been considered to be the only reservoir of leishmaniasis in urban areas. How‑ ever, in a previous study, we found a 33.3% prevalence of Leishmania infantum in the spleens of Norway rats (Rattus norvegicus) sampled in the underground sewer system of the city of Barcelona (Spain). The aim of the present study was to verify, using molecular methods, the potential reservoir role of these rats in the same sewer system. Methods: A sensitive real-time PCR (qPCR) assay, DNA sequencing and phylogenetic analysis were carried out to identify and quantify the presence of L. infantum DNA in sand fly individuals captured in the same underground sewer system of Barcelona as in our previous study and in the spleens and ears of rats captured in the same sewer system. Results: Leishmania infantum DNA was found in 14 of the 27 (51.9%) sand flies identified as Phlebotomus perniciosus, and 10 of the 24 (41.7%) rats studied were infected. Leishmania infantum was found in the spleens (70%) and in the ears (40%) of the infected rats. Quantitative results revealed the presence of high loads of L. infantum in the rats stud‑ ied (> 3 × ­106 parasites/g ear tissue) and among the sand flies (> 34 × ­106 parasites in 1 individual). Conclusions: The molecular methods used in this study demonstrated a high prevalence of L. infantum in the under‑ groundsewer populations of both R. norvegicus and P. perniciosus. These results suggest that sewer rats, in addition to dogs, are likely to act as reservoirs of leishmaniasis in cities, where sewer systems seem to offer the ideal scenario for the transmission of leishmaniasis. Therefore, to achieve the WHO 2030 target on the elimination of leishmaniasis as a public health problem successfully, an efficient control strategy against leishmaniasis in rats and sand flies should be implemented, particularly in the sewer systems of urban areas of endemic countries. Keywords: Leishmania infantum, Phlebotomus perniciosus, Rattus norvegicus, Barcelona sewer system, Underground leishmaniasis Background The road map for neglected tropical diseases (NTDs) 2021–2030, elaborated by the WHO and endorsed by the Seventy-third World Health Assembly in Novem- ber 2020, sets global targets and milestones to prevent, *Correspondence: mteresa.galan@uv.es control, eliminate or eradicate 20 diseases. These 20 dis- 1 Parasite and Health Research Group, Department of Pharmacy, eases and disease groups, classified as NTDs, have one Pharmaceutical Technology and Parasitology, Faculty of Pharmacy, University feature in common: their devastating impact on impov- of Valencia, 46100 Burjassot, Valencia, Spain erished communities [1]. Leishmaniasis, a vector-borne Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Galán‑Puchades et al. Parasites & Vectors (2022) 15:211 Page 2 of 8 disease transmitted by the bite of phlebotomine females, host-sand fly contact is necessary; Leishmania prevalence is caused by 20 Phlebotomus species pathogenic to should be > 20%; the infection should be non-pathogenic humans, and the disease ranks among the top ten NTDs and persistent; and the parasite should be available in worldwide [2]. About 350 million people are at risk of blood or skin. Norway rats meet most of these criteria acquiring the disease in 97 endemic countries. In terms [12]. However, the host-sand fly contact, which can be of disease burden, in 2019, 277,224 new cases of cuta- proved by collecting infected vectors in sewers, and the neous leishmaniasis (CL) and 13,814 of visceral leish- presence of L. infantum in the skin of the rats, were not maniasis (VL) (including 491 deaths) were reported in proved in our previous study. endemic countries [1]. In the Mediterranean basin, of In the present article, we report evidence showing high the four Leishmania species present (Leishmania infan- levels of L. infantum in sand flies as well as in the skin tum, L. major, L. tropica and L. donovani), L. infantum of the rats by qPCR, in specimens captured in the sewer is predominant and the causative agent of CL, VL and system of Barcelona. We also demonstrate the pres- mucocutaneous leishmaniasis [3]. A dozen Phlebotomus ence of high L. infantum loads among sand fly and rat species have been implicated in the transmission of Med- individuals. iterranean L. infantum, of which eight have been impli- cated as vectors, i.e. Phlebotomus ariasi, P. balcanicus, P. Methods kandelakii, P. langeroni, P. neglectus, P. perfiliewi, P. perni- A total of 609 sand fly individuals were captured with ciosus and P. tobbi [4]. sticky traps in 66 out of the 167 different sections pros- In the context of animal reservoirs of L. infantum, dogs pected in the underground sewer system of Barcelona are classically considered to be the only vertebrate res- (unpublished data). Sampling was mainly carried out at ervoir of leishmaniasis in urban areas in Mediterranean those points in the sewer system where Leishmania para- countries. Consequently, dogs are the only target for dis- site prevalences were highest according to our previous ease surveillance and control [5, 6]. study [9]. A total of 25 points was sampled on a weekly Spain is one of the endemic countries of leishmaniasis basis in August/September 2018. A4-sheets impregnated in the Mediterranean basin [7], with P. perniciosus and with castor oil [13] were placed in each sewer section, P. ariasi being the main vectors of L. infantum, which is one at the top of the section and the other at the bot- endemic in the Iberian Peninsula [4]. Studies in different tom, in order to capture the maximum number of speci- areas of Spain have reported seroprevalences of canine mens (Fig. 1). Once the traps had been collected, they leishmaniasis (CanL) in dogs varying from 0 to 57.1% [8]. were kept at 4º C until treated to separate and identify Specifically in Barcelona province, the seroprevalence of the phlebotomine sand flies. Individuals separated from CanL has been reported to range from 14.5 to 16.7% [8]. the traps by means of a paintbrush were placed in abso- As part of the multifocal project “BCN Rats” program lute ethanol to remove the castor oil and then stored in developed in Barcelona (Spain), we investigated and 70% ethanol until their subsequent assembly and identi- quantified the presence of L. infantum in the spleens of fication. Twenty-seven randomly selected females of the an urban population of the Norway rat (Rattus norvegi- trapped sand flies were identified by means of morpho- cus), in a sampled population of 98 individuals, using a logical keys [14–16]. highly sensitive real-time PCR (qPCR) method for Leish- New individuals of R. norvegicus were also captured mania DNA detection. Only one out of 14 (7.1%) rats with snap traps in the sewage system as part of the rodent captured in parks tested positive for L. infantum. How- surveillance and control program in Barcelona. ever, the prevalence of Leishmania DNA in rats captured For the molecular detection of Leishmania spp. in the from the underground sewage system was 33.3% (28/84 female sand flies, genomic DNA was extracted from rats) [9]. This was the first time that rats trapped in sew- individual phlebotomines by incubation overnight in ers were analyzed from a parasitological point of view. 500 µl of TESCa + pK buffer (30 mM Tris–HCl, pH 8, Thus, when only sewer rats are considered—and not 10 mM EDTA, 1% SDS, 5 mM ­CaCl2 and 0.42 µg/µl those living above ground—a 0.13 rat-per-person sce- proteinase K). After incubation, an equal volume of a nario is suspected in Barcelona [10]. Based on this ratio, mixture of chloroform:isoamyl alcohol (24:1) was added there could be more than 72,000 L. infantum-positive to the incubation medium, followed by a vortexing step rats living in the underground sewers in Barcelona, a city and centrifugation at room temperature for 5 min at with 1,664,182 inhabitants in 2020, representing a major 14,000 rpm. The top aqueous layer was transferred to public health concern as a potential reservoir. a different tube and the DNA was precipitated in a 2.5 The criteria to define a reservoir in the case of leishma- volume of absolute ethanol. All pellets were dissolved niasis were established by WHO in 2010 [11]. Specifically, in 10 μl of nuclease-free water [17]. The DNA concen- the reservoir must be abundant and long lived; an intense tration and the absorbance ratio at 260 and 280 nm Galán‑Puchades et al. Parasites & Vectors (2022) 15:211 Page 3 of 8 Germany), according to the manufacturer’s instruc- tions. Each DNA sample was eluted in 200 μl of nucle- ase-free water and frozen at − 20 °C until use. The concentration and quality of the DNA obtained from these tissues were determined on a NanoDrop OneC spectrophotometer (Thermo Fisher Scientific). The primer pair ACTBF 5ʹ-TCC​ATC​ATG​AAG​TGT​GAC​ GT-3ʹ and ACTBR 5ʹGAG​C AA​TGA​TCT​TG ATC​T TC​ AT-3ʹ was used to detect the beta-actin gene as a sys- tem control check; the presence of a 150-bp band con- firmed that the extracted DNA was of appropriate PCR amplifiable quality [19]. Leishmania spp. DNA was detected by qPCR using the primers LEISH-1 (5-AAC​TTT​TCT​GGT​CCT​CCG​ GGTAG-3), LEISH-2 (5-ACC​CCC​AGT​TTC​CCGC C-3) and the TaqMan-MGB probe (FAM-5-AAA​ AAT​ GGG​TGC​AGA​AAT​-3-non-fluorescent quencher-MGB) [20]. Each PCR reaction volume contained 1 µl of tem- plate DNA and a reaction master mix [2× SsoAdvanced Universal Probes Supermix (Bio-Rad Laboratories, Her- cules, CA, USA), 0.9 µM of each primer and 0.2 µM of probe]. All qPCR assays were run with appropriate con- trols, including the non-template control (NTC) and positive control (DNA obtained from L. major Friedlin strain MHOM/IL/80/Friedlin; V1). Thermocycling con- ditions included an initial denaturation step of 4 min at 94 ºC, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The qPCR was carried out in the CFX96 real-time PCR detection system (Bio-Rad Laboratories). Thresh- old cycle (Ct) values were calculated automatically by CFX Manager 3.1 software (Bio-Rad Laboratories) using default parameters. The number of parasites was calcu- Fig. 1 Sticky traps placed in the Barcelona sewer system lated using 123-bp fragments amplified by the LEISH 1 and LEISH 2 primers and based on the mass of the Leish- mania genome (71.8 fg) and the number of copies of the histone H2B gene (1000 copies). Standard curves were (A260/280 ratio) were evaluated using a NanoDrop constructed in triplicate by serial dilutions of genomic OneC spectrophotometer (Thermo Fisher Scientific, DNA from L. infantum promastigotes (reference strain: Waltham, MA, USA). DNA extraction quality was eval- MHOM/BR/1974/PP75) with the dilution factor increas- uated with an internal control PCR, using the primers ­ 0–7 (1.10 × ­107 parasites). The parasite num- ing by 10 to 1 LCO1490 (5ʹ-GGT​C AA​C AA​ATC​ATA​AAG​ATA​T TG​ ber was determined by interpolating the mean of Ct) G-3ʹ) and HCO2198 (5ʹ-TAA​ACT​TCA​G GG​TGA​CCA​ values of the samples in the standard curves. AAA​AAT​C A-3ʹ), which amplify a cytochrome c oxi- Some results were repeated three more times for vali- dase subunit 1 (COI) gene [18]. The presence of a 600- dation purposes. The mean and standard deviation of Ct bp band visualized by electrophoresis on a 1% agarose values of each standard curve dilution were calculated gel confirmed that the extracted DNA was of appropri- using the GraphPad Instat software package (GraphPad ate PCR amplifiable quality. Software Inc., San Diego, CA, USA). For the molecular detection of Leishmania spp. in Leishmania species were identified by amplifying the the tissues (ears and spleens) of the Norway rat, total coding region of heat shock protein 70 (HSP70) using genomic DNA was extracted from approximately the 70-IR-D (5ʹ-CCA​AGG​TCG​AGG​AGG​TCG​AC 150 mg of ear and 100 mg of spleen samples using TA-3ʹ) and 70-IR-M (5’-ACG​GGT​AGG​GGG​AGG​AAA​ the QIAamp Blood and Tissue Kit (QIAGEN, Hilden, GA-3ʹ) primers [21]. The PCR products that generated Galán‑Puchades et al. Parasites & Vectors (2022) 15:211 Page 4 of 8 amplification bands of approximately 700 bp were exam- Table 1 Real-time PCR results for the 14 Leishmania infantum- ined by electrophoresis in agarose gels and purified positive Phlebotomus perniciosus individuals captured in the using NucleoSpin Gel and PCR Clean-up procedure Barcelona underground sewer system (MACCHEREY-NAGEL, Düren, Germany), accord- P. perniciosus qPCR ­resultsa Estimated n of Standard deviation ing to the manufacturer’s instructions. DNA sequencing ID numbers Ct parasites was carried out in the Genomics Unit IPBLN of López Neyra Institute, CSIC, Granada. DNA sequencing of the 1 32.10 157.18 17.88 amplified product was submitted to the BLAST (NIH) 2 31.62 220.37 40.32 database. 3 30.81 371.93 24.20 4 32.35 134.98 32.22 Results and discussion 5 29.38 979.45 65.34 The selected sand fly females were identified as Phlebot- 6 33.97 65.57 28.05 omus perniciosus, the most abundant sand fly vector in 7 18.31 1,528,989.74 446,327.06 Spain [8], which, in addition to P. ariasi, has been cred- 8 13.84 34,005,842.84 6,927,767.90 ited as a competent vector of L. infantum in the Iberian 9 32.10 157.18 17.88 Peninsula [22]. The presence of these two main leishma- 10 31.62 220.37 40.32 niasis vectors is known due to the monitoring of the city 11 30.81 371.93 24.20 of Barcelona by ASPB (Agència de Salut Pública de Bar- 12 32.35 134.98 32.22 celona), the institution responsible for vector surveillance 13 29.38 979.45 65.34 and control in the city [23]. 14 33.97 65.57 28.06 The sequence of the 3’-untranslated region (UTR) Cut-off established for a positive result: Ct values < 36 is shown in Additional file 1: (Figure S1). The results of a Values are the mean of the three replicates of each sample the NCBI BLAST analysis of this sequence verified that the amplicon corresponded to a non-transcribed region of the UTR of HSP70 (700 bp) corresponding to chro- infantum was also found in the ears of four rats (i.e. mosome 28 of Leishmania infantum, with a 61% cover- 40% of the Leishmania-positive ones). Table 2 shows age and an E of 2.00E−156 and a percentage of identity the respective qPCR quantitative results in R. norvegi- of 89.86%. Similar values were obtained for Leishmania cus obtained in the spleens and ears of the Leishmania- chagasi. positive individuals (Additional file 4: Figures S5, S6; A phylogenetic tree was created with the same program Additional file 5: Figures S7, S8). One of the rats was (Additional file 2: Figure S2), showing how the amplified determined to have an estimated load of > than 430,000 region corresponds to L. infantum /L. chagasi with a high parasites per 150 mg ear tissue (R. norvegicus no. 8 in identity. Table 2). Leishmania infantum DNA was detected in 14 out of In six Norway rats the protozoan was present only in the 27 (51.9%) sand fly individuals analyzed. The quan- the spleen (rat ID numbers 2–7 in Table 2). One rat pre- titative results obtained by the qPCR method for the sented Leishmania in the spleen and the ear (rat num- infected P. perniciosus are shown in Table 1. Regard- ber 1 in Table 2), and three only in the ears (rat numbers ing parasite loads, sand flies captured in a leishmaniasis 8–10 in Table 2). focus near Madrid (Spain) were classified into different Despite the limitations of this study, the most impor- groups [24]. According to our results, most of the sand tant of which are the low number of sand flies/rats flies (85.7%) presented moderate loads (between 10 and analyzed and the limited period of time in which the sam- 1000 parasites) [24]. However, in the Madrid focus, the pling was carried out, the results obtained are relevant. highest parasite load found, considered to be a very Taking into account both the high percentage of L. infan- high load, was about 100,000 parasites. In our study, we tum-infected rats in the Barcelona underground sewer detected an individual of P. perniciosus with a parasite system [9] and the suitability of the sewers as a breeding load of > 1,500,000 and another one with an extraordi- site for sand flies [4], we expected a positive result for L. nary parasite load of > 34,000,000 (Table 1) (Additional infantum in the phlebotomines studied, although not at file 3: Figures S3, S4). such a high rate (51.9%). Leishmania infantum infection was also investigated in Data on the prevalence of L. infantum in sand flies in 24 individuals of R. norvegicus. L. infantum was found to urban areas of the Mediterranean basin are scarce. In be present in 10 out of the 24 rats (41.7%) studied. The Spain, entomological surveys carried out in four munici- protozoan was found in the spleens of seven of the sam- palities in the province of Madrid (considered a focus of pled rats (i.e. in 70% of the infected rats). Leishmania human leishmaniasis) revealed a P. perniciosus prevalence Galán‑Puchades et al. Parasites & Vectors (2022) 15:211 Page 5 of 8 Table 2 Real Time PCR results for the spleens and ears of the 10 Leishmania infantum-positive Rattus norvegicus captured in the Barcelona sewer system Rattus norvegicus ID numbers qPCR results a R. norvegicus spleens Ct Estimated n of parasites/100 mg Standard deviation 1 35.45 6.71 2.34 2 35.24 8.43 2.22 3 28.06 794.89 74.66 4 35.24 8.43 2.22 5 35.07 29.43 3.49 6 23.10 20,156.21 1,987.96 7 30.06 445.04 38.93 R. norvegicus ears Ct Estimated n of parasites/150 mg tissue Standard deviation 1 29.82 1222.35 242.37 8 18.09 430,119.63 86,890 9 34.87 10.66 2.32 10 33.86 84.33 37.63 Cut-off established for a positive result: Ct values < 36 a Mean values of the three replicates of each sample of 8.9% over a 7-year period, with the highest infection of 1,528,989.74 ± 446,327.06 in a sand fly (num- rate, 15.2%, in Leganés, one of the towns included in the ber 7, Table 1) and an astounding parasite load of study [25]. Likewise, in the cities of Marseille (France) 34,005,842.84 ± 6,927,767.90 in another P. perniciosus and Catania (Sicily, Italy), a prevalence of 5% and 11% individual (number 8, Table 1). Leishmania DNA was found in 72 and 99 P. perniciosus, Regarding the results obtained in the rats studied, respectively [26, 27]. A prevalence of 58.5% was found in although the leishmaniasis prevalence was higher than P. perniciosus in a peri-urban area of Madrid, related to that obtained in our previous study, the difference is not the human leishmaniasis focus mentioned above [28]. statistically significant (41.7 vs. 33.3%; χ2 = 0.262, df = 1, Most of the remaining data on L. infantum prevalences P = 0.4748). It is worth mentioning that, in addition to in Phlebotomus spp. in Spain were obtained in rural the spleens, L. infantum was also detected in the ears of areas. For example, 12 out of 31 P. perniciosus (38.7%) 40% of the infected rats. One of the rats studied harbored were found to be infected by L. infantum in a study con- a load of 3,089,047 ± 579,268 parasites/g ear tissue (num- ducted in Catalonia [29]. A prevalence of 12.5% (8 posi- ber 8, Table 2). Ear skin, in addition to blood, is directly tive individuals from 64 P. perniciosus analyzed) was accessible to sand flies, which preferentially feed on cer- found in a recent study in south-eastern Spain [30]. How- tain body parts, such as the ears and nose [38]. An exper- ever, these values are notably higher (up to 58.5%) when imental infection revealed persistent parasite depots at studies focus on areas of Leishmania outbreaks [22]. bite sites in dog skins [38]. The large number of parasites Prevalences found in rural areas of other countries of the we found in the ear of the rat probably represents one of Mediterranean basin are low, varying between 0.1% and these depots in a naturally infected R. norvegicus. 15% [31–33]. The prevalences are also higher and vary CanL has always been considered to be a public health between 37.6% and 60.5% in high endemic areas of leish- problem in endemic countries based on evidence that maniasis in Tunisia and Italy [34–36]. cases of CanL are known to precede human cases of the The high prevalence of L. infantum found in the sand disease, and it is well known that dogs act as a reser- flies from the underground Barcelona sewers in our study voir [3, 4]. Therefore, leishmaniasis in dogs undoubtedly is consistent with those found in active leishmaniasis foci plays an important role in the maintenance of transmis- in the Mediterranean basin. sion levels and the spread of the disease, specifically in Regarding the quantitative results, to our knowl- urban areas [39, 40]. In fact, culling dogs, although con- edge, the highest L. infantum parasite load detected troversial, seems to be the only effective reservoir con- in previous studies in naturally infected sand flies trol method available to date [41]. However, the control was 2,820,246 ± 106,072 in an individual of Lutzo- measures taken specifically in urban areas that have myia longipalpis in Brazil [37]. We detected a load involved the euthanasia of seropositive dogs [39, 42] did Galán‑Puchades et al. Parasites & Vectors (2022) 15:211 Page 6 of 8 not modify the incidence of leishmaniasis in humans and leishmaniasis control programs [47, 48], an increase in dogs, suggesting the possible involvement of other reser- rat populations has been observed in several cities due to voirs of infection [43]. Regarding sand flies, urban envi- lockdowns and the interruption of control measures [49, ronments in Spain had previously not been considered 50]. suitable for P. perniciosus [22]. Our studies clearly dem- onstrate the importance of the trapping site for finding a Conclusions large number of Leishmania-infected sand flies and rats. This is the first report of high molecular levels of leish- To our knowledge, the present study is the first and only maniasis in sand flies captured in urban sewers and the study to date to assess the presence of L. infantum in rats first time L. infantum has been detected, in addition to in living in underground sewage systems of urban areas, the spleen, also in the ears of infected rats captured in the where intense contacts between rat and sand fly individu- same underground sewer system of the city of Barcelona. als have apparently led to a high prevalence of leishmani- We found, among both sand flies and rats, high parasite asis, in turn potentiated by the presence of heavy parasite loads in certain individuals. As a result, Norway rats, in loads in certain hosts. addition to dogs, are likely to act as reservoirs of leish- The identification of L. infantum in naturally infected maniasis in cities where the sewer systems seem to offer sand flies is important for predicting the risk and expan- the ideal scenario for leishmaniasis transmission. There- sion of leishmaniasis in endemic areas [29]. The high fore, to achieve the WHO 2030 target on the elimination prevalence of natural L. infantum infection in a small of leishmaniasis as a public health problem successfully, sample of P. perniciosus trapped in sewers, as detailed in an efficient control strategy against leishmaniasis in rats the present study, represents a clear risk of transmission, and sand flies should be implemented, particularly in the not only to dogs, but also to the human population. It is sewer systems of urban areas of endemic countries. important to note that sand flies can find suitable ways to move from sewers to urban surfaces, since they are able Supplementary Information to use open structures that connect both types of envi- The online version contains supplementary material available at https://​doi.​ ronments, such as catch basins or storm drains, among org/​10.​1186/​s13071-​022-​05309-4. others. Once sand flies arrive at the surface, they can also bite humans/dogs close to urban green areas, as oppor- Additional file 1: Sequence of the 3’-UTR region. Figure S1. The 3’-UTR region of the HSP70 gene was amplified with primers 70-IR-D (5’- CCA​AGG​ tunistic blood feeding patterns have been demonstrated TCG​AGG​AGG​AGG​TCGAC TA-3’) and 70-IR-M (5’-ACG​GGT​AGG​GGG​GGA​GGA​ in P. perniciosus, including anthropophilic behavior [25]. AAGA-3’). Miró and López-Vélez [5] predicted in 2018 that “In the Additional file 2: Phylogenetic analysis. Figure S2. Phylogenetic tree near future, we are likely to find new animal reservoirs to using 3’-UTR sequences of the HSP70 gene. which special attention will have to be paid as these could Additional file 3: Sand flies. Figure S3. Standard curve of L. infantum even further complicate the epidemiological situation of DNA. Standard curve obtained from serial dilutions of L. infantum DNA ­(108 to ­101 parasites). Each point was tested in triplicate. Slope = — 3.70; human and animal leishmaniosis”. Recent epidemiologi- efficacy = 98 %; R2 = 0.991. Figure S4. Amplification curves. The plot cal studies of leishmaniasis carried out in the city of Bar- showing the dilution of DNA concentrations (8 to 8 × ­10-7 ng). celona showed that autochthonous transmission occurs Additional file 4: Rat spleens. Figure S5. Standard curve of L. infantum and is even on the increase [4, 44]. According to the DNA. Standard curve obtained from a series of dilutions of L. infantum DNA ­(108 to ­101 parasites). Each point was tested in triplicate. Slope = results of the present study, the Norway rat R. norvegicus -3.367; efficacy = 98.2%; R2 = 0.998. Figure S6. Amplification curves. The is likely to be this new reservoir, as we already indicated plot showing the dilution of DNA concentrations (8–8 × ­10-7 ng). in our previous study [9], and, in addition to dogs, could Additional file 5: Rat ears. Figure S7. Standard curve of L. infantum DNA. be sustaining leishmaniasis levels in urban areas. Standard curve obtained from a series of dilutions of L. infantum DNA (­ 108 Sewer rat populations are important to humans since to ­101 parasites). Each point was tested in triplicate. Slope = -3.47; efficacy = 94.1%; R2 = 0.998. Figure S8. Amplification curves. The plot showing they could act both as a reservoir of individuals to re- the dilution of DNA concentrations (8 to 8 × ­10-7 ng). colonize the surface after successful control actions and as reservoirs, not only of leishmaniasis but also of other Author contributions parasitic zoonoses [10, 45, 46]. In fact, the rat harboring MTGP, AO and MVF designed the study. JP, RBM, SF, VP and TM conducted the the highest L. infantum load was also infected by three field work. JS, GG, AO, MVF conducted the laboratory work. MTGP prepared the manuscript. AO, MVF, RBM, JP, VP, TM contributed to the discussion and other parasitic zoonotic agents (Hymenolepis nana, H. improvement of the manuscript. All authors read and approved the final diminuta and Taenia taeniaeformis larvae) (unpublished manuscript. data). Availability of data and materials The obvious complexity of L. infantum control in Materials and data supporting our findings and conclusions are included rats represents a challenge, particularly in the COVID- in the article. The GenBank accession number of the submitted nucleotide 19 scenario in which, in addition to a disruption in the sequence is ON364134. Galán‑Puchades et al. Parasites & Vectors (2022) 15:211 Page 7 of 8 Declarations 11. WHO. Control of the Leishmaniases. WHO Technical Report Series no. 949. 2010. https://​apps.​who.​int/​iris/​bitst​ream/​handle/​10665/​44412/​WHO_​ Ethics approval and consent to participate TRS_​949_​eng.​pdf;​jsess​ionid=​DFE10​7FC21​06DCA​57A9B​F8054​3F920​5C?​ Permission to carry out the study was granted by the Department of Territory seque​nce=1. Accessed Oct 2021. and Sustainability of the regional government of Catalonia (reference number: 12. Galán-Puchades MT, Fuentes MV. On the reservoirs of Leishmania infan‑ SF/044). Rats were treated according to Directive 2010/63/EU of the European tum in urban areas. Vet Parasitol. 2021;293:109408. https://​doi.​org/​10.​ Parliament and Council decision of 22nd September 2010 on the protection 1016/j.​vetpar.​2021.​109408. of animals used for scientific purposes. 13. Arnedo A, Bellido J, González F, Arias A, Calvo C, Safont L, et al. Leishma‑ niasis en Castellón: Estudio epidemiológico de los casos humanos, vector Consent for publication y reservorio canino. Rev Sanidad Hig Pública. 1994;68:481–91 (in Spanish). Not applicable. 14. Gil Collado J, Morillas F, Sanchís MC. Los flebotomos en España. Rev Sanid Hig Pública. 1989;1989:15–34. Competing interests 15. Gállego-Berenguer J, Botet-Fregola J, Gállego-Culleré M, Portús-Vinyeta The authors declare that they have no competing interests. M. Los flebotomos de la España peninsular e Islas Baleares: identificación y corología: comentarios sobre los métodos de captura. In: Hernández S, Author details editor. 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Silgado A, Armas M, Sánchez-Montalvá A, Goterris L, Ubals M, Temprana- • support for research data, including large and complex data types Salvador J, et al. Changes in the microbiological diagnosis and epidemiol‑ ogy of cutaneous leishmaniasis in real-time PCR era: a six-year experience • gold Open Access which fosters wider collaboration and increased citations in a referral center in Barcelona. PLoS Negl Trop Dis. 2021;15:e0009884. • maximum visibility for your research: over 100M website views per year https://​doi.​org/​10.​1371/​journ​al.​pntd.​00098​84. 45. Galán-Puchades MT, Sanxis-Furio J, Pascual JM, Bueno-Marí R, Franco At BMC, research is always in progress. S, Peracho V, et al. First survey on zoonotic helminthiases in urban Norway rats (Rattus norvegicus) in Spain and associated public health Learn more biomedcentral.com/submissions