Received: 23 February 2021 Revised: 26 May 2021 Accepted: 2 June 2021

DOI: 10.1111/tbed.14179

ORIGINAL ARTICLE

Understanding the factors that determine the emergence of anthroponotic cutaneous leishmaniasis due to Leishmania tropica in Morocco: Density and mitochondrial lineage of Phlebotomus sergenti in endemic and free areas of leishmaniasis

Patricia Gijón-Robles1 Naima Abattouy2 Gemma Merino-Espinosa1 Nora El Khalfaoui3 Francisco Morillas-Márquez1 Victoriano Corpas-López1 Noureddine Jaaouani4 Victoriano Díaz-Sáez1 Myriam Riyad3 Joaquina Martín-Sánchez1

1 Department of Parasitology, Faculty of PharmacyUniversity of Granada, Spain Abstract 2 Higher Institute of Nursing and Health Anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica is spreading to Techniques, Laâyoune, Morocco new areas in Morocco. Exposure to the vector, Phlebotomus sergenti, is the only proven 3 Laboratory of Cellular and Molecular Pathology/Research Team on risk factor. Our objective was to compare the densities and genetic characteristics of Immunopathology of Infectious and Systemic P. sergenti populations in two nearby localities in Morocco, one in an ACL endemic area Diseases, Hassan II University of Casablanca, Morocco (El Borouj) and another in a nonendemic area (Sidi Hajjaj). P. sergenti density was sig- = 4 Délégation du Ministère de la Santé, province nificantly higher in the endemic area than in the nonendemic town (p 0.032). A dif- de Settat, Morocco ferent predominant P. sergenti mitochondrial lineage was evidenced in each one of the two localities, and for the first time, the P. sergenti lineage acting as a vector of L. tropica Correspondence Joaquina Martín-Sánchez, Faculty of Phar- has been identified. Bioclimatic differences were detected between both localities. In macy, Department of Parasitology, Campus conclusion we found differences in both the density and the mitochondrial lineage of Universitario de Cartuja, Granada, Spain. Email: [email protected] P. sergenti populations that may explain the different epidemiological situation. Given that the density of P. sergenti in the locality without ACL cases seems sufficient to allow Patricia Gijón-Robles is a PhD student of the doctoral programme in Pharmacy, University of transmission, the main factor that would justify its nonendemic character could be the Granada. absence of P. sergenti Lineage IV, which seems to prefer warmer and drier climates.

Funding information KEYWORDS University of Granada anthroponotic cutaneous leishmaniasis, Leishmania tropica, mitochondrial lineage, Morocco, Phlebotomus sergenti, vector density

1 INTRODUCTION botomus sergenti Parrot, 1917 (Diptera: Psychodidae) is its main vector (Guilvard et al., 1991; Schnur et al., 2004). For a long time, P. sergenti was Leishmania tropica (Kinetoplastida: Trypanosomatidae) is the major considered the sole vector of L. tropica (Al-Zahrani et al., 1988; Guil- cause of anthroponotic cutaneous Leishmaniasis (ACL) in the Middle vard et al., 1991) however, the vectorial capacity of Phlebotomus ara- East and some areas of North Africa (Pratlong et al., 2009)andPhle- bicus has been demonstrated in a focus in northern Israel (Svobodová

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH

Transbound Emerg Dis. 2021;1–10. wileyonlinelibrary.com/journal/tbed 1 2 GIJÓN-ROBLES ET AL. et al., 2006), and Phlebotomus similis is considered a probable vector on Borouj whereas no cases have been reported in Sidi Hajjaj (Ministry of the island of Crete (Ntais et al., 2014). Although P. sergenti is believed to Health, Morocco, 2016). have a marked preference for semi-arid habitats (Boussaa et al., 2009), this species exhibits a wide ecological plasticity in Morocco making increased vector surveillance essential to prevent and control leish- 2.2 Sand fly collection and species identification maniasis outbreaks. In emerging ACL Moroccan foci,P.sergentidensity varies from 4 to 16 specimens/m2 (Ramaoui et al., 2008)andrelative Sand flies were caught in both localities using CDC light traps inside abundance ranges from 12.8% to 76.7% (Boussaa et al., 2009). households and sticky papers outside dwellings, from June 20 to July The World Health Organization included Morocco among one of 10 and from September 20 to October 10, 2015. In El Borouj, houses the 12 high-burden countries for CL (WHO, 2016). There are three with and without ACL cases were sampled. One to two CDC traps were endemic Leishmania speciesinMorocco:Leishmania major, L. tropica, set in each selected house for one night under favourable weather con- (both dermotropic) and Leishmania infantum (mainly viscerotropic). ditions. Sticky traps consisted of 21 × 29.5 cm sheets of papers cov- Leishmania tropica has the widest geographic distribution (Ministry of ered in castor oil, 9–17 traps were set the same day in adult sand fly Health, Morocco, 2016; Mouttaki et al., 2014) but until 1989, ACL had resting places (holes in house walls and other nearby walls) and left been mainly reported in hypoendemic rural foci scattered around the for 4 days. The captured sand flies were stored in 70% alcohol. Males sub-arid area of central Morocco. Later, ACL emerged in several north- and females were separated and morphologically identified using tax- ern, central and southern provinces of the country, initially as new out- onomic keys (Benabdennbi et al., 1999; Berchi et al., 2007;ElSawaf breaks and then establishing endemic foci that highlighted the expan- et al., 1989; Gil Collado et al., 1989; Leger et al., 1983; Rioux & Golvan, sion of this Leishmania species (Ajaoud et al., 2013). The first CL case 1969; Rioux et al., 1978; Sáez et al., 2018). The specimens were placed in Settat province (central Morocco) was detected in El Borouj locality in Marc André solution and heated to boiling point, and finally mounted in 2006, preceding an epidemic outbreak and then becoming endemic on slides under a coverslip using Berlese solution. The genitalia of P. (Amarir et al., 2015; Gijón-Robles et al., 2018). Currently, El Borouj is sergenti specimens were individually removed and mounted on slides the only active CL focus in the province of Settat (Ministry of Health, under a coverslip for morphological identification whereas the rest of Morocco, 2016). the body was stored at −20◦C for DNA extraction. The identification of factors that determine the emergence and The gonotrophic cycle of the female sand flies was categorised as expansion of ACL is required to develop better interventions for this blood-fed, non-fed or gravid. Density (sand flies/trap), relative abun- largely neglected disease. We showed that differences in the exposure dance (% specimens of a given species/total sand flies) and frequency to the L. tropica vector, reflected by differences in P. sergenti density in (% positive sampling stations for a given species) data were estimated the households, was the only factor associated with CL cases in the El by species. Borouj focus (Gijón-Robles et al., 2018). On the other hand, three of the four mitochondrial (mt) lineages pre- viously reported within P. sergenti are present in Morocco (Barón et al., 2.3 Sand fly DNA extraction 2008; Merino-Espinosa et al., 2016; Yahia et al., 2004). Phenotypic dif- ferences of biomedical importance may exist between these mitochon- Genomic DNA was extracted from the head, thorax and attached drial lineages, thus population genetics could help to assess the threat anterior abdomen of individual P. sergenti males and females (Martín- of the geographical expansion of ACL. Sánchez et al., 2000). A commercially available kit was used [RealPure Therefore, our objective was to compare the densities and genetic kit from REAL (Ref. RBMEG01], according to the manufacturer instruc- characteristics of P. sergenti populations in two nearby localities in tions. Each sand fly was individually placed in a sterile 1.5 mL Eppen- Morocco, one within an ACL endemic area (El Borouj) and another in dorf tube and kept in liquid nitrogen for a few seconds to facilitate the a nonendemic region (Sidi Hajjaj). mechanic rupture of the tissues using a pestle. The DNA was resus- pended in 20 µL of bidistilled water and kept at −20◦C until use.

2 MATERIAL AND METHODS 2.4 Mitochondrial lineage determination by mt 2.1 Study area DNACytbPCR-RFLP

El Borouj (coordinates 07◦36’W-32◦29’N) and Sidi Hajjaj (07◦24’W- Polymerase chain reaction (PCR) was used to amplify a 550-bp frag- 33◦06’43’’N) are situated at an altitude of 410 and 547 m above sea ment containing the 3′ end of the Cytochrome b mitochondrial gene level respectively, in the Settat province, central Morocco. Both local- (mt DNA Cyt b) following the methodology described by Esseghir et al. ities are separated by only 51 km and have common rural features (1997). and an economic activity mainly based on agriculture and animal hus- Restriction fragment length polymorphism (RFLP) was carried out bandry. In both towns, the population is around 20,000 inhabitants and through the digestion of a 550-bp mtDNA Cyt b fragment with Hae the average growth rate is close to 2%. ACL by L. tropica is endemic in El III (Thermo Scientific, Germany). The reaction was performed at 37◦C GIJÓN-ROBLES ET AL. 3 for 10 minutes in a 20 µL total volume, containing 16 µLofPCRprod- Average values (95% confidence interval) and minimum and maxi- uct, 2 µL of enzyme (10 U/µL) and 2 µL of standard buffer (10×). The mum values of each of the bioclimatic variables from El Borouj and Sidi digested samples were separated by electrophoresis in a 3% agarose Hajjaj are shown as supplementary data. gel and their sizes determined by comparison with HyperLadder V (Bio- line, UK) leading to a characteristic banding pattern for each of the four 3 RESULTS mitochondrial lineages: Lineage I, two fragments (290 and 220 bp); Lin- eage II, two fragments (290 and 260 bp); Lineage III, three fragments 3.1 Sand fly fauna and abundance by species (290, 140 and 110 bp) and Lineage IV, two fragments (330 and 220 bp) (Merino-Espinosa et al., 2016). Species distribution by sex, sampling method, sampling period and site are shown in Table 1. In total 5674 sand fly specimens, 2215 females (39.0%) and 3459 males (61.0%), were collected. The mean density 2.5 Detection of Leishmania tropica DNA values in El Borouj were 14.9 sand flies/CDCtrap/night and 26.1 sand flies/m2 whereas in Sidi Hajjaj were 8.1 sand flies/CDCtrap/night and The presence of L. tropica DNA was investigated in P. sergenti females 14.8 sand flies/m2. Thirteen sand fly species were present (9 from Phle- captured in Sidi Hajjaj, using Granaleish Multiplex qPCR (University botomus genus and 4 from Sergentomyia genus) and differences in den- of Granada, Spain, Trade Mark Number 3667362/5). This PCR tech- sity, relative abundance and frequency were observed between species nique can differentiate between L. infantum, L. tropica and L. major and localities (Table 1). Phlebotomus chabaudi, Phlebotomus alexandri, and allows quantification of the parasite load (Merino-Espinosa et al., Phlebotomus duboscqi, Sergentomyia antenata and Sergentomyia dreyfussi 2018). Primers F, R and the 3 Taqman probes were provided by the were caught in low numbers in El Borouj and were absent in Sidi Hajjaj. manufacturer. The following thermal profile was used: 10 minutes at In both localities, Sergentomyia minuta was the most abundant species 95◦C, then 36 cycles of 30 seconds at 95◦C and 60 seconds at 60◦C. in the captures with adhesive traps whereas Phlebotomus sergenti was the most abundant species in the intra-household captures collected with CDC traps (Table 1). 2.6 Bioclimatic differences between El Borouj Sand fly density was higher in El Borouj and significant differ- and Sidi Hajjaj ences were found in captures carried out peridomestically in June (p < 0.0001) and intradomiciliary in October (p = 0.066). Phleboto- In order to investigate the possible association between bioclimatic mus sergenti density was also higher in this locality and significant characteristics and the presence/absence of ACL in the two stud- differences were detected in peridomiciliary captures performed in ied localities, a logistic regression analysis was carried out includ- June (p = 0.007), intradomiciliary captures in October (p = 0.007), ing the locality as a dependent variable and each bioclimatic variable in global intradomiciliary collections (p = 0.032) and peridomestically under study as an independent variable. The bioclimatic data analysed (p = 0.022) (Table 1). Regarding differences between capture periods were as follows: mean monthly average temperature (from January to the intradomiciliary density of P. sergenti was significantly higher in December), maximum monthly average temperature (from January to June in El Borouj (p = 0.012) and in Sidi Hajjaj (p = 0.020). December), minimum monthly average temperature (from January to Blood-fed, non-fed and gravid P. sergenti females were found both in December), maximum annual average temperature, minimum annual intradomiciliary and peridomiciliary captures in both localities; density average temperature, monthly precipitation (from January to Decem- values were higher in El Borouj (Table 2). ber), annual mean temperature (BIO1), mean diurnal range (mean of monthly – max temp – min temp–)(BIO2), isothermality (BIO2/BIO7) × 100 (BIO3), temperature seasonality (standard deviation × 100) 3.2 Leishmania infection rate in the vector (BIO4), max temperature of warmest month (BIO5), min tempera- ture of coldest month (BIO6), temperature annual range (BIO5–BIO6) L. tropica DNA was not detected in any of the 84 female P. sergenti cap- (BIO7), mean temperature of wettest quarter (BIO8), mean tempera- tured in Sidi Hajjaj. ture of driest quarter (BIO9), mean temperature of warmest quarter (BIO10), mean temperature of coldest quarter (BIO11), annual precip- 3.3 Mitochondrial lineage determination by itation (BIO12), precipitation of wettest month (BIO13), precipitation mtDNA Cyt b PCR-RFLP of driest month (BIO14), precipitation seasonality (coefficient of varia- tion) (BIO15), precipitation of wettest quarter (BIO16), precipitation of The mitochondrial lineage was identified in 81 male and female P. ser- driest quarter (BIO17), precipitation of warmest quarter (BIO18) and genti, 41 from El Borouj and 40 from Sidi Hajjaj. The results are shown precipitation of coldest quarter (BIO19). This information was taken in Table 3. from the WorldClim global climate data (www.worldclim.org/) consid- The females captured in El Borouj in which L. tropica DNA was ering the average values from 2007 to 2015. Data were analysed with detected in a previous study (Gijón-Robles et al., 2018) were identified IBM SPSS Statistics 20.0 for Windows (IBM Corp., Armonk, NY, USA). as lineage IV. 4 GIJÓN-ROBLES ET AL. ) 2 * * *** 9 88 61 91 28 1.66 0.36 0.57 7.53 (8/1) 1.60 1.11 11.23 (84/4) 0.16 (38/23) (81/10) 442.8 ST (54.9 m (Continues) ) 2 *** *** 51 86 91 18 1.33 2.35 0.56 0.67 6.49 (8/1) 11.55 (82/4) (32/19) (81/10) 309.8 ST (38.4 m tober 2015, both in ) 2 ** * 0 0 0.23 2 09 0 0 0 10 10 0.40 (6/4) (2/0) 45.45 0.61 (16.5 m Sidi Hajjaj * * 2 8 31 13 112 0.11 1.30 0.62 0.15 (1/1) (7/1) 5.89 0.42 72.73 (51/61) (13/18) * ** 7 1 7 9 11 0.09 3.70 0.44 0.11 (4/3) (1/0) (5/6) (6/1) 25.93 0.64 11 CDC 19 CDC 133 ST 1 4 20 traps 105 2.50 1.00 1.63 0.12 2.21 0.13 0.64 0.13 0.79 1.00 0.25 (0/1) (1/0) (47/58) 13.13 82.68 (8/12) 8CDC ) 2 * * 134) *** + 66 79 0 736 151 1.13 4.35 0.75 0.39 0.50 5.53 21.18 (60/6) (134/17) 1074.9 ST (133.2 m (602 ) 2 *** *** 73 21 44 274 5.79 1.54 4.05 0.64 0.34 15.21 (21/0) (62/11) 381.6 ST (232/42) (47.3 m ) 2 * ** 00 00 01 78 45 35 462 4.66 0.89 0.46 0.91 27.62 5.38 (72/6) (39/6) (370/92) (85.9 m El Borouj * * 5 68 53 583 471 0.83 0.82 5.51 0.30 (5/0) 7.02 0.06 47.21 (52/16) (297/286) (279/192) 83 CDC traps 693.2 ST * ** 1 25 56 46 traps 231 0.83 0.36 6.23 0.26 (1/0) 57.61 3.35 (21/4) (22/34) 69 CDC (108/123) Capture intradomiciliary Capture peridomiciliary Capture intradomiciliary Capture peridomiciliary JO G J O O G G JOGJ 4 7 43 traps 352 415 0.71 0.37 0.42 0.310.480.29 0.20 0.25 0.02 0.25 0.40 0.06 0.75 0 0.56 0 0.62 0 0.10 0 0.67 0 0 0.46 0.79 0.25 25.93 0.22 5.19 0.23 0 0 1.15 0.33 1.11 0.21 3.07 5.16 0.86 0.57 0.29 0.01 (4/0) 49.76 13.97 38.14 2.69 1.17 1.90 15.75 40.74 20.13 9.09 10.91 10.86 25.14 42.21 29.64 0.81 5.67 0.52 0.44 (31/12) (189/163) (257/158) A A A A D D D D N N Fr Fr Fr Fr (M/F) (M/F) (M/F) (M/F) Comparison of species and densities of sand flies captured in El Borouj (ACL endemic) and Sidi Hajjaj (ACL free) in the two sampling periods, June and Oc . P P. sergenti perniciosus P. langeroni N P. longicuspis N Number of stations Number of traps 14 CDC TABLE 1 intradomiciliary with CDC light traps and peridomiciliary with sticky papers GIJÓN-ROBLES ET AL. 5 *** 9 1 3 810 548 0.16 0.12 0.02 0.04 0.07 0.37 (2/7) (0/1) (2/1) 0.05 (536/274) (321/227) ** ** 1 3 788 538 0.03 0.13 0.06 0.11 0.38 (0/1) (2/1) (2/7) 14.02 9.99 20.48 14.77 0.23 0.08 (525/263) (318/220) *** *** ** ** global results. Total number of sand flies 0 0 0 0 0 0 0 22 10 0 0 = (3/7) 001. (11/11) < p Sidi Hajjaj *** 01, ** October, G 1 0 0 0 0 0 0 0 0 < 0 154 relative abundance (representative percentage of each = (1/0) p (73/81) ** = A ), 2 June, O * = 0 0 0 0 0 0 0 0 0 27 (1/0) 0.05 level, (17/10) < p * * 0 0 0 000 0 9 01 0 0 0 0 0 0 127 (56/71) 3 ** *** 7 828 337 0.20 0.05 0.05 0.61 (2/5) 1345 23.83 3475 6.22 (270/67) (462/366) (627/718) (2162/1313) ** 4 538 218 674 4.61 2.53 0.22 0.08 0.02 0.59 (0/4) 1802 14.25 10.10 0 0.09 0.0538.11 0.61 26.09 15.88 2.45 8.11 1.34 29.86 11.38 . Statistical differences were detected at (180/38) (264/274) (269/405) (1028/774) 3 *** ** ** *** S. antennata 3 ♂ 290 119 671 0.03 0.18 0.09 0.63 (2/1) 5 17.33 1.39 3.38 1673 3 (90/29) (198/92) ; (358/313) (1134/539) El Borouj S. dreyfussi ** 1, 2 ♂ 6 30 52 14 0.07 0.49 0.11 0.36 2.43 0.21 (0/6) (8/6) 0.63 density (no. of captured sandflies/CDC trap/night or no. captured sandflies/area, m (17/13) (29/23) 1235 (688/547) = and 3 frequency (presence of a given species in the stations sampled in each time period). J = Fr * 2 6 females, D 29 35 13 P. duboscqi 0.09 1.50 0.13 0.42 7.23 0.22 (0/6) (8/5) 401 0.51 = ♂ (17/12) (19/16) (197/204) ,1 Capture intradomiciliary Capture peridomiciliary Capture intradomiciliary Capture peridomiciliary males, F * 1 = 0 1 0 0 0 1 17 100 100 100 100 100 100 100 100 100 100 100 100 0.07 0.12 0.14 2.040.71 8.73 0.37 4.21 0.42 7.11 0.54 12.10 0.43 9.70 0.49 0 0 0 0 0 0 0 0 1.15 0.28 1.11 0.18 0.120.14 3.24 0.22 1.13 0.21 40.11 0.89 37.400.86 0.89 38.71 0.89 0.87 0 0.89 0 3.70 1.00 0.65 0.11 45.45 0.08 0.95 0.20 68.45 0.97 67.65 0.94 1.00 0.68 0.67 0.77 0.60 1.00 0.86 0.07 0.19 0.17 7.81 (0/1) (0/1) 834 1.21 P. chabaudi 59.57 5.81 14.88 19.48 (10/7) ♀ (491/343) 1 2 A A A A A D D D D D N N N Fr Fr Fr Fr Fr (M/F) (M/F) (M/F) (M/F) (M/F) P. alexandri; ♂ 1 (Continued) 1 Total S. fallax S. minuta N P. papatasi N P. bergeroti number of captured sandflies, M = TABLE 1 N also includes: species with respect to total captured in a given period), 6 GIJÓN-ROBLES ET AL. ; 3 8 ) 29 12 2 0.21 3.04 1.14 0.31 0.08 0.76 4.56 11.03 9 4 6 5 P. bergeroti 8, 9 4 18 6 0.16 2.28 0.13 0.10 1.52 0.47 1.90 6.84 ; 8 3 2 206 5.37 1.14 0.05 0.08 5.63 0.76 309.8 ST (38.4 m 216 78.33 82.13 4 0 1 3 P. bergeroti ) 1 2 5 0.06 9.09 0.00 0.18 0.00 0.24 27.27 36.36 1 0 0 1 11 263 El Borouj (ACL endemic) and Sidi 0.00 0.00 0.06 0.00 0.00 0.06 9.09 9.09 6 0 6 0 P. chabaudi; 133 ST (16.5 m 0.36 0.00 0.00 0.00 0.36 0.00 54.55 54.55 and 1 4 0 0 2 0.00 0.00 0.00 0.18 0.00 0.36 Sidi Hajjaj 20.00 40.00 3 0 0 1 7 P. bergeroti 0.00 0.00 0.09 0.00 0.00 0.27 1 10.00 30.00 4 11 CDC 7 0 0 0 3 0.00 0.00 0.00 0.00 0.00 0.27 0.00 30.00 relative abundance (representative percentage of each species of the = 0 0 P. bergeroti; 15 15 A 1 0.00 0.00 0.00 1.88 0.00 1.88 3 21.13 21.13 1 0 9 12 71 10 0.00 0.00 1.41 1.13 0.13 1.50 12.68 16.90 8CDC S. dreyfussi; 0 0 44 34 0.00 0.00 4.25 0.00 0.00 5.50 47.89 61.97 and 3 4 7 ) 38 11 2 0.15 0.90 0.52 0.23 0.08 0.80 1.42 4.91 in case of adhesive traps), 2 2 8 6 45 18 P. bergeroti 0.17 1.03 0.38 0.04 0.26 0.95 2.33 5.81 4 774 2 6 5 13 390 8.25 0.27 0.11 0.65 1.68 691 50.39 14.61 89.28 381.6 ST (47.3m 5 1 ) 22 24 2 P. alexandri; 59 0.26 4.08 0.28 0.01 0.19 0.69 4.45 1 10.95 1 5 1 40 11 18 0.13 2.04 0.21 0.01 0.19 0.47 3.34 7.42 539 4 50 440 280 3.26 0.58 0.05 0.74 5.12 9.28 693.3 ST (85.9 m 51.95 81.63 4 0 0 0 1 55 63 0.80 0.01 0.05 0.66 El Borouj 26.96 30.88 3 1 1 24 2, 3, 4 43 0.01 0.49 0.35 0.01 0.05 0.45 11.76 21.08 69 CDC 204 2 4 2 44 98 0.06 1.96 0.64 0.03 0.10 1.02 21.57 48.04 0 0 0 2 78 67 4.79 0.14 0.58 5.57 19.53 22.74 . 0 0 0 3 October. Total number of females also includes: 1 43 25 = JOJOJOJO density (no. of females/no. of CDC traps/night or no. of females/area in m Capture intradomiciliary Capture peridomiciliary Capture intradomiciliary Capture peridomiciliary 1.79 0.21 0.87 7.29 3.07 100 100 100 100 100 100 100 100 343 12.54 24.50 2.96 6.28 16.37 8.88 0.91 0.67 6.85 = 14 CDC D 1 0 0.140 0.36 0.58 0.13 1.46 0.07 4.41 0.03 2.45 0.21 0.98 0.07 3.34 0.06 1.11 0.36 0.93 0.27 2.20 0.17 1.68 0.00 1.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.00 0.00 0.76 0.13 0.00 1.90 1 7 025952186517138000000000205 1001110861526248000000000100 71 P. langeroni June, O 141134211032310001020222000121 5.07 0.50 2.04 0.07 0.29 0.070.29 0 0 0 0.16 0 0.01 5.39 0.49 0 1.00 0 0.01 15.96 0.19 0.06 0.93 5.54 33.85 0.08 0.52 0.17 1.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.38 0 0 0 0 1 222 9 20.70 15.86 64.72 10.07 0.9341.11 0.29 3.79 0.30 1.17 10.29 0.14 4.90 0.04 1.47 0.02 0.03 0.37 0.56 0.01 0.19 0 0 0 0.00 0 0.00 1.25 14.08 0.25 2.82 0.00 0.00 0.18 20.00 20.00 0.18 20.00 0.18 0.00 0.00 0.00 0.00 0.00 0.00 0.38 0.03 0.76 0.05 0.38 0.03 = D A D A D A NF D A D A D A NF D A NF NF D A NF P. bergeroti; 1 8 Number of nonfed (U), blood-fed (B), and gravid (G) female sand flies, captured in the two sampling periods (June and October 2015) in both localities, S. fallax NF S. minuta P. sergenti P. papatasi NF Number of traps P. longicuspis NF number of captured females, P. perniciosus Total females Number of stations 7 46 35 44 4 9 10 18 females = P. langeroni; Total U, B and G pce UBGUBGUBGUBGUBGUBGUBGUBG Species 1 TABLE 2 NF 7 Hajjaj (ACL free) total captured specimens). J GIJÓN-ROBLES ET AL. 7

TABLE 3 Number of Phlebotomus sergenti specimens from El Borouj (ACL endemic) and Siddi Hajjaj (ACL free) in captures made throughout 2015 analysed by mt DNA Cytb PCR-RFLP with Hae III restriction enzyme and the mitochondrial lineage identified

Mitochondrial lineage and Hae III RFLP banding pattern

Locality Number I (290 and 220 bp) II (290 and 260 bp) IV (330 and 220 bp) Sidi Hajjaj Male 26 4 22 0 Female 14 4 10 0 Total 40 8 (20%) 32 (80%) 0 (0%) El Borouj Male 21 0 0 21 Female 20 0 1 19 Total 41 0 (0%) 1 (2.4%) 40 (97.6%)

TABLE 4 Average values (95% confidence interval), and minimum and maximum values of each of the bioclimatic variables in El Borouj (ACL endemic) and Sidi Hajjaj (ACL free) for which differences were detected

Average value (95% confidence interval), and minimum and maximum values

Bioclimatic variable Siddi Hajjaj El Borouj p Value by logistic regression Maximum 34.74 (34.66–34.81) 36.42 (36.41–36.43) Absolute differences temperature July 34.6–34.9 ◦C 36.3–36.5 ◦C Rainfall September 11.14 (10.93–11.35) Constant value of 9 mm Absolute differences 11–12 mm Rainfall October Constant value of 37 mm Constant value of 34 mm Absolute differences BIO3 Constant value of 49 Constant value of 47 Absolute differences BIO15 68.4 (68.1–68.7) 68.1 (68.1–68.2) 0.009 68–69 68–69 (OR = 0.218 IC95% 0.07–0.68). BIO18 13.14 (12.93–13.35) Constant value of 11 mm Absolute differences 13–14 mm

BIO3 = isothermality; BIO15 = precipitation seasonality; BIO18 = precipitation of warmest quarter.

3.4 Bioclimatic differences between El Borouj even though the geographical extension of the vector is greater than and Sidi Hajjaj that of the parasitic protozoan, and the identification of factors for parasite expansion is essential for effective disease control. Phleboto- Bioclimatic data were collected from 199 georeferenced points, 185 mus sergenti density and genetic characteristics were investigated as houses with ACL cases and 14 houses in Sidi Hajjaj. Table 4 shows the determining factors for the existence of ACL transmission. Compara- average values (95% confidence interval), and minimum and maximum tive intradomiciliary and peridomiciliary sand fly captures in the ACL values of each of the bioclimatic variables in El Borouj and Sidi Haj- endemic locality of El Borouj and the non-endemic locality of Sidi Haj- jaj for which differences were detected. Significant differences were jaj were made using CDC light traps and sticky papers. detected at precipitation seasonality (BIO15, p = 0.009) which was Phlebotomus sergenti density in Sidi Hajjaj was lower than that of 78% lower in El Borouj [OR = 0.22 (IC95% 0.07–0.68)] whereas both the endemic locality, both in peridomiciliary settings and within house- localities showed absolute differences at maximum annual tempera- holds. Interestingly, P. sergenti was the most abundant and densest ture in July, rainfall in September and October, isothermality (BIO3) species within households in Sidi Hajjaj, however it was the fourth and precipitation of warmest quarter (BIO18). species outdoors, after S. minuta and the L. infantum vectors, P. per- niciosus and P. longicuspis (Table 1).The relative abundance of P. ser- genti males and females varied between the trapping methods since the 4 DISCUSSION males were more abundant in the sticky papers. In both localities, the density of P. sergenti females was higher in the intradomiciliary captures In Morocco, ACL due to L. tropica is transmitted by P. sergenti which carried out in June and non-fed, fed and gravid females, were found has a large geographic distribution probably related to the wide eco- (Table 2). logical plasticity of this vector (Boussaa et al., 2009; Ramaoui et al., Although to date no ACL cases have been diagnosed in Sidi Hajjaj, 2008; Rioux et al., 1986). CL due to L. tropica is an emerging disease these sand fly density figures seem sufficient for the maintenance of 8 GIJÓN-ROBLES ET AL.

L. tropica transmission (Barón et al., 2013; Ramaoui et al., 2008; Rioux tures of July. The coefficient of isothermality reflects well these differ- et al., 1986) and would make this locality susceptible to the establish- ences in temperature between both localities, being two points higher ment of an ACL transmission cycle. Over the last few decades, L. tropica in El Borouj. Environmental humidity and temperature are two deter- foci have spread to several regions of Morocco including those where mining factors in the distribution of P. sergenti (Barón et al., 2013)and CL caused by L. major or L. infantum has been reported, which shows within this species there appear to be lineages with different ranges the changing geographical patterns of this species (Baghad et al., 2020). of tolerance. Particularly in El Borouj, the presence of water wells The growing mobility of humans raises the possibility of new emerg- was independently associated with a greater P. sergenti density in the ing foci in areas where P. sergenti populations are well established. households (Gijón-Robles et al., 2018). Kholoud et al. (2020) suggested that ACL dissemination in Morocco is associated to an increase in human travel and local tourism linked to economic expansion and infrastructure development as shown by 5 CONCLUSION the synchronized occurrence of new ACL foci with the construction of new motorways. However, the factors underlying the spatio-temporal The density and genetic background of the L. tropica vector,P.sergenti, transmission dynamics of leishmaniasis are not well understood, and seem to play a significant role in the prevalence of ACL in Morocco. Dif- the epidemiological picture is not as simple as deduced from the previ- ferences in both the vector density and its main mitochondrial lineage ous statement. in two localities, one endemic and the other free of the disease, may The molecular characterization of P. sergenti populations in both explain the different epidemiological situation. Given that the P. sergenti localities using the PCR-RFLP technique of the cytochrome b mito- density in Sidi Hajjaj seems sufficient to allow transmission, the main chondrial gene, has allowed us to find that the lineage IV is the main factor that would explain the absence of ACL cases could be the lack of mitochondrial lineage of P. sergenti in El Borouj (97.6%) while the P. sergenti lineage IV, which seems to prefer warmer and drier climates. remaining 2.4% belong to lineage II. In contrast, lineage II was the most abundant in Sidi Hajjaj (80%) followed by the lineage I (20%). There- ACKNOWLEDGEMENT fore, a different main P. sergenti mitochondrial lineage was dominant in This study was funded by the University of Granada (Centro de Inicia- each locality under study. Phlebotomus sergenti is characterised by its tivas de Cooperación al Desarrollo, CICODE, 2013). Funding for open high genetic diversity and classified with at least 20 haplotypes in four access charge: Universidad de Granada/CBVA. mitochondrial lineages (Barón et al., 2008; Yahia et al., 2004). In El Borouj, L. tropica DNA was detected in 5 out of 184 (2.7%) P. ser- CONFLICT OF INTEREST genti females in captures made in 2014 and 2015 (Gijón-Robles et al., The authors declare no competing interests. 2018). The five infected females belong to lineage IV, the most preva- lent P. sergenti lineage in El Borouj. This is the first time that a mitochon- DATA AVAILABILITY STATEMENT drial lineage of P. sergenti is involved as a vector for L. tropica.Alocal The data that support the findings of this study are available from the increase in the abundance of this P. sergenti lineage that seems to trans- corresponding author upon reasonable request. mit L. tropica more efficiently, could explain the emergence of ACL in El Borouj and its absence in Sidi Hajjaj. ORCID Lineage I is over-represented in southwestern Europe (Merino- Joaquina Martín-Sánchez https://orcid.org/0000-0003-3981-9227 Espinosa et al., 2016) and this is the first time that its presence in Morocco is reported. No autochthonous ACL cases have been detected REFERENCES in the Iberian Peninsula, despite P. sergenti being commonly found at Ajaoud, M., Es-sette, N., Hamdi, S., El-Idrissi, L. A., Riyad, M. & Lemrani, M. sufficient densities to act as a vector, and the existence of 2 mitochon- (2013). Detection and molecular typing of Leishmania tropica from Phle- botomus sergenti and lesions of cutaneous leishmaniasis in an emerging drial lineages, one of them, held in common with Morocco (Lineage III) focus of Morocco. Parasite & Vectors, 6, 217. https://doi.org/10.1186/ (Barón et al., 2008, 2013 ; Merino-Espinosa et al., 2016). The existence 1756-3305-6-217 of differential ecological traits between P. sergenti mitochondrial lin- Al-Zahrani, M. A., Peters, W., Evans, P. A., Ching-Chin, I., Smith, V. & Lane, eages has been pointed out: Merino-Espinosa et al. (2016) found that R. P. (1988). Phlebotomus sergenti, a vector of Leishmania tropica in Saudi Arabia. Transactions of the Royal Society of Tropical Medicine and Hygiene, Lineage I appear to have adaptive advantages represented by a wider 82(3), 416. tolerance to temperature and altitude changes that would make it bet- Amarir, F., Sebti, F., Fellah, H., Pratlong, F., Dedet, J. P., El Mansouri, B., ter suited to lead a geographical expansion into Europe. Similarly, there Hmamouch, A., Delouane, B., Habbari, K., Sadak, A., Abassi, I. & Rhajaoui, are bioclimatic differences between El Borouj and Sidi Hajjaj (Table 4) M. (2015). Epidemiological characteristics of a new focus of cutaneous that could explain the over representation of lineage IV in El Borouj, leishmaniasis caused by Leishmania tropica in Settat, Morocco. Acta Trop- ica, 150, 116–121. https://doi.org/10.1016/j.actatropica.2015.07.020 which is warmer and drier, and its absence in Sidi Hajjaj. Baghad, B., Razanapinaritra, R., Maksouri, H., El Bouri, H., Outlioua, A., Fel- In general, rainfall is greater in Sidi Hajjaj than in El Borouj with a lah, H., Lemrani, M., Akarid, K., Martin-Sanchez, J., Chiheb, S. & Riyad, M. strong seasonality from July to October. In contrast, temperatures are (2020). Possible introduction of Leishmania tropica to urban areas deter- almost two degrees higher in El Borouj throughout the year; however mined by epidemiological and clinical profiles of patients with cutaneous the differences were only significant between the maximum tempera- GIJÓN-ROBLES ET AL. 9

leishmaniasis in Casablanca (Morocco). Parasite Epidemiology and Control, and International Health, 21(5), 630–641. https://doi.org/10.1111/tmi. 9, e00129. https://doi.org/10.1016/j.parepi.2019.e00129 12686 Barón, S., Martín-Sánchez, J., Gállego, M., Morales-Yuste, M., Boussaa, S. Merino-Espinosa, G., Rodríguez-Granger, J., Morillas-Márquez, F., Tercedor, & Morillas-Márquez, F. (2008). Intraspecific variability (rDNA ITS and J., Corpas-López, V., Chiheb, S., Alcalde-Alonso, M., Azaña-Defez, J. M., mtDNA Cyt b) of Phlebotomus sergenti in Spain and Morocco. Acta Trop- Riyad, M., Díaz-Sáez, V. & Martín-Sánchez, J. (2018). Comparison of PCR- ica, 107, 259–267. https://doi.org/10.1016/j.actatropica.2008.07.003 based methods for the diagnosis of cutaneous leishmaniasis in two dif- Barón, S. D., Morillas-Márquez, F., Morales-Yuste, M., Díaz-Sáez, V., Gál- ferent epidemiological scenarios: Spain and Morocco. Journal of the Euro- lego, M., Molina, R. & Martín-Sánchez, J. (2013). Predicting the risk of pean Academy of Dermatology and Venereology, 32(11), 1999–2003. https: an endemic focus of Leishmania tropica becoming established in South- //doi.org/10.1111/jdv.15034 Western Europe through the presence of its main vector, Phlebotomus Ministry of Health, Morocco (2016). Santé en chiffres 2015. Ministry of sergenti Parrot, 1917. Parasitology, 140, 1413–1421. https://doi.org/10. Health, Morocco, 168 pp. 1017/S0031182013000942 Mouttaki, T., Morales-Yuste, M., Merino-Espinosa, G., Chiheb, S., Fellah, H., Benabdennbi, I., Pesson, B., Cadi-Soussi, M. & Morillas-Márquez, F. (1999). Martín-Sánchez, J. & Riyad, M. (2014). Molecular diagnosis of cutaneous Morphological and isoenzymatic differentiation of sympatric popula- leishmaniasis and identification of the causative Leishmania species in tions of Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Morocco by using three PCR-based assays. Parasite & Vectors, 4(7), 420. Psychodidae) in northern Morocco. Journal of Medical Entomology, 36, https://doi.org/10.1186/1756-3305-7-420 116–120. https://doi.org/10.1093/jmedent/36.1.116 Ntais, P., Christodoulou, V., Tsirigotakis, N., Dokianakis, E., Dedet, J. P., Prat- Berchi, S., Bounamous, A., Louadi, K. & Pesson, B. (2007). Différenciation long, F. & Antoniou, M. (2014). Will the introduction of Leishmania tropica morphologique de deux espèces sympatriques: Phlebotomus perniciosus MON-58, in the island of Crete, lead to the settlement and spread of this Newstead 1911 et Phlebotomus longicuspis Nitzulescu 1930 (Diptera: rare zymodeme? Acta Tropica, 132, 125–130. https://doi.org/10.1016/j. Psychodidae). Annales de la Société Entomologique de France (n.s.), 43, 201– actatropica.2014.01.003 203. Pratlong, F., Dereure, J., Ravel, C., Lami, P., Balard, Y., Serres, G., Lanotte, Boussaa, S., Pesson, B. & Boumezzough, A. (2009). Faunistic study of the G., Rioux, J. A. & Dedet, J. P. (2009). Geographical distribution and epi- sandflies (Diptera: Psychodidae) in an emerging focus of cutaneous leish- demiological features of Old World cutaneous leishmaniasis foci, based maniasis in Al Haouz province, Morocco. Annals of Tropical Medicine and on the isoenzyme analysis of 1048 strains. Tropical Medicine and Interna- Parasitology, 103, 73–83. http://doi.org/10.1179/136485909X384910 tional Health, 14(9) 1071–1085. https://doi.org/10.1111/j.1365-3156 El Sawaf, B. M., Mansour, N. S., El Said, S. M., Daba, S., Youssef, F. G., Kenawy, Ramaoui, K., Guernaoui, S. & Boumezzough, A. (2008). Entomological and M. A. &. Beier, J. C. (1989). Feeding patterns of Phlebotomus papatasi and epidemiological study of a new focus of cutaneous leishmaniasis in Phlebotomus langeroni (Diptera: Psychodidae) in El Agamy, Egypt. Journal Morocco. Parasitology Research, 103, 859–63. https://doi.org/10.1007/ of Medical Entomology, 26 (5), 497–498. s00463-008-1068-3 Esseghir, S., Ready, P. D., Killick-Kendrick, R. & Ben-Ismail, R. (1997). Rioux, J. A. & Golvan, Y. J. (1969). Épidémiologie des leishmanioses dans le Mitochondrial haplotypes and geographical vicariance of Phle- sud de la France. Institut National de la Santé et de la Recherche Médicale. botomus vectors of Leishmania major. Insect Molecular Biology, 6, París (16e) 288-32-84. 211–225. Rioux, J. A., Lanotte, G., Petter, F., Dereure, J., Akalay, O., Pratlong, F., Gil Collado, J., Morillas Márquez, F. & Sanchís Marín, M. C. (1989). Los fle- Velez, I. D., Fikri, N. D., Maazoun, R., Denial, N., Jarry, D. M., Zahaf, A., botomos en España. Revista de Sanidad e Higiene Pública, 63, l5–34. Ashford, R. W., Cadi-Soussi, M., Killick-Kendrick, R., Benmansour, N., Gijón-Robles, P., Abattouy, N., Merino-Espinosa, G., El Khalfaoui, N., Moreno, G., Périères, J., Guilvard, E., ... Serres, E. (1986). Les leishman- Morillas-Márquez, F., Corpas-López, V., Porcel-Rodríguez, L., Jaaouani, ioses cutanées du bassin méditerranéen occidental; De l’identification N., Díaz-Sáez, V., Riyad, M. & Martín-Sánchez, J. (2018). Risk factors for enzymatique à l’analyse éco-épidémiologique; L’exemple de trois “foy- the expansion of cutaneous leishmaniasis by Leishmania tropica: Possible ers”, tunisien, marocain, et français. In Leishmania. Taxonomie et Phy- implications for control programmes. Transboundary and Emerging Dis- logenèse. Applications Eco-épidémiologiques, JA Rioux (ed.), pp. 365– eases, 65 (6), 1615–1626. https://doi.org/10.1111/tbed.12914 395. CNRS/INSERM/OMS, Institut Méditerranéen d’Etudes Epidémi- Guilvard, E., Rioux, J. A., Gállego, M., Pratlong, F., Mahjour, J., Martínez- ologiques et Ecologiques, Montpellier. Ortega, E., Dereure, J., Saddiki, A., & Martini, A., 1991. Leishmania tropica Rioux, J. A., Perieres, J., Killick-Kendrick, R., Maistre, M. & Bayar, N. (1978). au Maroc. III. Ròle vecteur de Phlebotomus sergenti. Annales de Parasitolo- Confirmation de lt’existence en Tunisie de Sergentomyia antennata (New- gie Humaine et Comparée, 66, 96–99. stead, 1912) (Diptera-Psychodidae). Annales de Parasitologie Humaine et Kholoud, K., Bounoua, L., Sereno, D., El Hidan, M. & Messouli, M. (2020). Comparée, 53(4), 431–435. Emerging and re-emerging Leishmaniases in the Mediterranean Area: Sáez, V. D., Morillas-Márquez, F., Merino-Espinosa, G., Corpas-López, What can be learned from a retrospective review analysis of the sit- V., Morales-Yuste, M., Pesson, B., Barón-López, S., Lucientes- uation in Morocco during 1990 to 2010? Microorganisms, 8(10), 1511. Curdi, J. & Martín-Sánchez, J. (2018). Phlebotomus langeroni https://doi.org/10.3390/microorganisms8101511 Nitzulescu (Diptera, Psychodidae) a new vector for Leishma- Léger, N., Pesson, B., Madulo-Leblond, G. & Abonnenc, E. (1983). [Differen- nia infantum in Europe. Parasitology Research, 117, 1105–1113. tiation of females of the subgenus Larroussius Nitzulescu 1931 (Diptera- https://doi.org/10.1007/s00436-018-5788-8 Phlebotomidae) of the Mediterranean region]. Annales de Parasitologie Schnur, L. F., Nasereddin, A., Eisenberger, C. L., Jaffe, C. L., El Fari, M., Azmi, Humaine et Comparée, 58(6), 611–623. K., Anders, G., Killick-Kendrick, M., Killick-Kendrick, R., Dedet, J. P., Prat- Martín-Sánchez, J., Gramiccia, M., Pesson, B. & Morillas-Márquez, F. (2000). long, F., Kanaan, M., Grossman, T., Jacobson, R. L., Schonian, G. & War- Genetic polymorphism in sympatric species of the genus Phlebotomus, burg, A. (2004). Multifarious characterization of Leishmania tropica from with special reference to Phlebotomus perniciosus and Phlebotomus longi- a Judean desert focus, exposing intraspecific diversity and incriminating cuspis (Diptera, Phlebotomidae). Parasite, 7, 247–254. https://doi.org/10. Phlebotomus sergenti as its vector. American Journal of Tropical Medicine 1051/parasite/2000074247 and Hygiene, 70, 364–372. Merino-Espinosa, G., Corpas-López, V., Callejón-Fernández, R., Porcel- Svobodova, M., Votypka, J., Peckova, J., Dvorak, V., Nasereddin, A., Baneth, Rodríguez, L., Díaz-Sáez, V., Gállego, M., Ballart, C., Molina, R., Jiménez, G., Sztern, J., Kravchenko, V., Orr, A., Meir, D., Schnur, L. F., Volf, P. & M., Morillas-Márquez, F. & Martín-Sánchez, J. (2016). Differential eco- Warburg, A. (2006). Distinct transmission cycles of Leishmania tropica in logical traits of two Phlebotomus sergenti mitochondrial lineages in south- 2 adjacent foci, Northern Israel. Emerging Infectious Diseases, 12, 1860– western Europe and their epidemiological implications. Tropical Medicine 1868. https://doi.org/10.3201/eid1212.060497 10 GIJÓN-ROBLES ET AL.

WHO (2016). Leishmaniasis in hight-burden countries: An epidemiological update based on data reported in 2014. Weekly Epidemiology Records, How to cite this article: Gijón-Robles, P., Abattouy, N., 91(22), 287–296. Merino-Espinosa, G., El Khalfaoui, N., Morillas-Márquez, F., Yahia, H., Ready, P.D., Hamdani, A., Testa,J. M. & Guessous-Idrissi, N. (2004). Corpas-López, V., Jaaouani, N., Díaz-Sáez, V., Riyad, M., Regional genetic differentiation of Phlebotomus sergenti in three Moroc- can foci of cutaneous leishmaniasis caused by Leishmania tropica. Parasite, Martín-Sánchez, J. (2021). Understanding the factors that 11, 189–199. determine the emergence of anthroponotic cutaneous leishmaniasis due to Leishmania tropica in Morocco: density and mitochondrial lineage of Phlebotomus sergenti in endemic and SUPPORTING INFORMATION free areas of leishmaniasis. Transboundary and Emerging Additional supporting information may be found online in the Support- Diseases. , 1–10. https://doi.org/10.1111/tbed.14179 ing Information section at the end of the article.