Genomic and epidemiological evidence for the emergence of a L. infantum/L. donovani hybrid with unusual epidemiology in northern Italy

| Gene ics and Molecula Biology | Resea ch A icle Genomic and epidemiological e idence o he eme gence o a L. in an um/L. dono ani hyb id wi h unusual epidemiology in no he n I aly F. B uno,1 G. Cas elli,1 B. Li,2 S. Reale,1 E. Ca a,3 F. Vi ale,1 S. Scibe a,1 M. Calzola i,3 S. Va ani,4 M. O alli,4,5 E. F anceschini,6 W. Genna i,7 G. Rugna,3 G. F. Spä h8 AUTHOR AFFILIATIONS See a ilia ion lis on p. 16. ABSTRACT Leishmania (L.) in an um is one o he main causa i e agen s o animal and human leishmaniasis ac oss many endemic a eas in Sou h Ame ica, Eu ope, No h A ica, and Asia. Despi e i s clinical signi icance, li le is known abou he gene ic di e si y o L. in an um ci cula ing in a gi en endemic a ea. He e, we in es iga e his impo an open ques ion by applying a compa a i e genomics app oach o se en L. in an um isola es om di e en hos s and I alian egions, including he no he n pa o he coun y (Emilia-Romagna, RER), Sicily, and Sa dinia, as an ini ial a emp o explo e he b ead h o pa asi e gene ic he e ogenei y in I aly. Addi ionally, mic osa elli e analysis was ca ied ou o compa e he isola es om RER wi h o he 70 L. in an um s ains om he same egion as well as 65 s ains belonging o he L. dono ani complex om o he coun ies. We e ealed impo an ka yo ypic ins abili y and iden i ied s ain-speci ic changes in gene dosage, which a ec ed impo an i ulence ac o s such as amas ins and su ace an igen-like p o eins. Single nucleo ide polymo phism-based clus e ing analysis o hese genomes oge he wi h o e 80 publicly a ailable L. in an um and L. dono ani genomes placed he I alian isola es in o h ee geog aphically dis inc clus e s wi hin he Medi e a nean basin and unco e ed h ee isola es clus e ing wi h pu a i e L. in an um/L. dono ani hyb ids isola ed in Cyp us. As judged by mic osa elli e p o iling, hese hyb id isola es a e ep esen a i e o a sub-popula ion o pa asi es ci cula ing in no he n I aly ha p e e en ially in ec humans bu no dogs. Ou esul s place I aly a he c oss oads o L. in an um in ec ion in he Medi e anean and call a en ion o he public heal h isk ep esen ed by he in oduc ion o non-Eu opean Leishmania species. IMPORTANCE This s udy closes impo an knowledge gaps wi h espec o Leishmania (L.) in an um gene ic he e ogenei y in a gi en endemic coun y, as exempli ied he e o I aly, and e eals gene ic hyb idiza ion as a main cause o e-eme ging human leishmaniasis in no he n I aly. The obse ed high di e si y o Leishmania pa asi es on he I alian peninsula sugges s di e en geog aphical o igins, wi h genomic adap a ion o a ious ecologies a ec ing bo h pa hogenici y and ansmission po en ial. This is documen ed by he disco e y o a pu a i e L. in an um/L. dono ani hyb id s ain, which has been shown o p e e en ially in ec humans bu no dogs. Ou esul s p o ide impo an in o ma ion o heal h au ho i ies, which need o conside he public heal h isk ep esen ed by he in oduc ion o new Leishmania species in o EU coun ies due o popula ion displacemen o a el om coun ies whe e exo ic/alloch honous pa asi e species a e endemic. KEYWORDS Leishmania, compa a i e genomics, gene ic hyb id, isce al leishmaniasis July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 1 In i ed Edi o Da id L. Sacks, NIH, NIAID, Be hesda, Ma yland, USA Edi o L. Da id Sibley, Washing on Uni e si y in S . Louis School o Medicine, S . Louis, Missou i, USA Add ess co espondence o G. F. Spä h, ge ald.spae h@pas eu . , o G. Rugna, gianluca. ugna@izsle .i . F. B uno and G. Cas elli con ibu ed equally o his a icle. Au ho o de was de e mined based on alphabe ical o de . The au ho s decla e no con lic o in e es . See he unding able on p. 16. Recei ed 4 Ap il 2024 Accep ed 30 Ap il 2024 Published 4 June 2024 Copy igh © 2024 B uno e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional license. Leishmaniases a e a g oup o sand ly- ansmi ed pa asi ic diseases ha can a ec mammals, including humans. In humans, hese diseases a e cha ac e ized by cu aneous, muco-cu aneous, o isce al issue damage, he la e o en being a al i le un ea ed. The pa hogenesis and clinical ou come o Leishmania in ec ion ely on a se ies o pa asi e-speci ic ai s ha ensu e p oli e a ion and i ness gain in di e en hos sys ems. Fi s , hese pa asi es ha e e ol ed di e en li e cycle s ages, including mo ile p omas igo e o ms ha a e adap ed o su i al inside he midgu o phlebo omine sand lies and in acellula amas igo es ha in ec immune cells o he e iculoendo he lial sys em inside he mammalian hos . Second, aside s age di e en ia ion, Leishmania can u he adap o en i onmen al a ia ions encoun e ed inside bo h insec and e eb a e hos s; his phenomenon has been linked o he in insic plas ici y o he Leishmania genome and he gene ic ecombina ion ha can ollow he o ma ion o pa asi e hyb ids be ween s ains and species inside he insec ec o . In he wake o nex -gene a ion sequencing and he de elopmen o powe ul compu a ional pipelines o sequencing da a analysis (1), compa a i e genomics app oaches ha e p o ided impo an insigh in o he epidemiology o Leishmania in ec ion in he ield o pa asi e i ness gain in expe imen al se ings. Fo example, compa a i e genomics has in o med on (i) he mechanisms unde lying d ug esis ance and pa asi e e olu ion in ea ed pa ien s (2), (ii) he o igin, popula ion s uc u e, and phylogene ic ela ionship o clinical isola es, (iii) hyb idiza ion e en s and hei e ec on issue opism o clinical ou come o in ec ion (3, 4), (i ) he ole o dynamic gene dosage changes in pa asi e i ness gain (5), o ( ) he genomics o geog aphic adap a ion, which is he scope o he cu en s udy. Leishmania in an um causes leishmaniasis in Medi e anean Eu ope, wi h signi ican eme gence o human cases obse ed in localized a eas o Spain (6) and I aly (7). I aly is hypo-endemic o human leishmaniasis, wi h less han 100 cases o cu aneous and isce al leishmaniasis (VL) epo ed o he WHO in 2020 (8), while dogs a e consid e ed he main ese oi hos s. His o ically, leishmaniasis in I aly was es ic ed o he Ty henian li o al, he sou he n peninsula, and he islands. Howe e , in he las 20 yea s, sand ly ec o s as well as human and canine Leishmania in ec ions ha e been de ec ed in no he n I aly, adi ionally classi ied as a cold a ea unsui able o sand ly su i al (9). In he las decade, a su ge o human leishmaniasis cases has been obse ed in he Emilia-Romagna egion (RER), no he n I aly (7). Howe e , he e was no concu en inc ease in he p e alence o canine leishmaniasis in he same pe iod. In addi ion, molecula yping s udies in RER showed ha he Leishmania s ains ci cula ing in dogs belonged o a di e en popula ion compa ed o s ains isola ed om human VL cases and sand lies (10). Fu he mo e, he bi ing p e e ence o Phlebo omus (Ph) pe iliewi, he suspec ed ec o esponsible o he inc ease o VL cases in RER, sugges ed he p esence o a pe i-u ban o syl a ic ese oi o he han dogs, which is capable o sp eading he in ec ion in such a eas (11). Consis en wi h his obse a ion, a high equency o Leishmania in ec ion was ecen ly iden i ied in o he po en ial ese oi hos s, including oe dee , ha es, wol es, o ed oxes (12). While some s udies ha e e alua ed he epidemiology o VL in I aly, gene ic cha ac e i za ion o L. in an um ac oss di e en egions is lacking. He e, we combined compa a i e genomics and mic osa elli e p o iling app oaches on L. in an um ield isola es ha we e ob ained in I aly o shed ligh on pa asi e gene ic he e ogenei y. We p o ide e idence o a ema kable gene ic di e si y o L. in an um ac oss he I alian peninsula and iden i y he s ains om RER as pu a i e L. in an um/L. dono ani hyb ids, which a e closely ela ed o Cyp io s ains, hus co ela ing hei unusual epidemiology and ansmission pa e n o hei unique gene ic cons i u ion. Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 2 RESULTS Cul u ed L. in an um isola es show s ain-speci ic and con e gen aneuploi dies We applied a compa a i e genomics app oach o gain insigh in o he gene ic di e si y o L. in an um ield isola es in I aly. We delibe a ely chose ini ially i e isola es om di e en hos s (human, dog, ca , and ma en) and geog aphic egions (RER in no he n I aly, Sicily, and Sa dinia) o maximize he assessmen o pa asi e gene ic he e ogenei y (Table 1; Fig. 1A). Following cul u e expansion, DNA ex ac ion, and Illumina sho ead sequencing, we applied ou genome ins abili y pipeline (GIP) (1) o map he eads on he JPCM5 L. in an um e e ence genome (13) and assess ead dep h a ia ions o each indi idual sample. We hen compa ed ch omosome ead dep h a ia ions be ween samples using pe ch omosome boxplo s o no malized co e age (somy sco e) in 300 bp genomic bins (see Ma e ials and Me hods). As judged by he changes in he somy sco e, all samples showed impo an ka yo ypic a ia ions, hus con i ming he in insic ins abili y o he L. in an um genome as p e iously epo ed (2) (Fig. S1). As expec ed om p e ious s udies, all samples we e e asomic o ch omosome (ch ) 31 (14) while displaying unique ka yo ypic p o iles conside ing he o he ch omosomes. This di e si y may be la gely a ibu ed o he s ain-speci ic selec ion o di e en aneuploidies du ing cul u e adap a ion, which we p e iously linked o in i o i ness gain (5). The hea map shown in Fig. 1B e eals leish4, a s ain isola ed om a Sicilian dog om Pale mo (Table 1), as he ka yo ypically mos s able sample, wi h mos ch omosomes showing a median somy sco e be ween 2 and 2.5. All o he samples showed mo e impo an somy a ia ions, including ull isomies o highe , mosaic aneuplodies. Ou da a e ealed pen asomies o ch 8 and 23 in he leish16 sample. Gi en ha his sample ep esen s he i s L. in an um genome sequenced om a eline isola e, we in es iga ed i his unusual ampli ica ion pa e n may be selec ed in his pa icula hos . This possibili y was uled ou by he quan i ica ion o ch 8 copy numbe by quan i a i e PCR (qPCR) analysis o h ee independen eline isola es, which showed a disomic ampli ica ion sco e compa able o disomic ch 28 included as a con ol (Fig. 1C). Thus, he pen asomy o ch 8 may ei he ha e been uniquely selec ed in leish16 du ing eline in ec ion o unde wen impo an ampli ica ion du ing he 16 cul u e passages o his sample (see Table 1). O no e, ampli ica ion o ch 8 and 23 is qui e common in cul u ed p omas igo es o o he species. They ha e been desc ibed as among he mos equen polysomal ch omosomes in L. dono ani isola es (15) and ha e been shown o a ise du ing L. majo sel -hyb idiza ion (16). Thus, he I alian L. in an um ield isola es show impo an somy a ia ions in cul u e compa ed o he haploid e e ence genome. Con e gen ampli ica ion o a small numbe o ch omosomes in mos o all s ains sugges s ha hese aneuploidies a e a po en ial TABLE 1 O e iew o he L. in an um isola es ha we e analyzed in his s udy by whole-genome sequencing Lab ID In e na ional code Hos /loca ion (p o ince)/da e/ cul u e passage (p) In o ma ion Sequencing eads: numbe /% mapped leish3 MCAN /IT/2021/51327 Dog/Sa dinia (Caglia i)/2021/p8 Glucan ime esis an 27,739,245/0.98 leish4 MCAN/IT/2020/1265 Dog/Sicily (Pale mo)/2020/p8 –a25,856,894/0.91 leish5 MMST/IT/2006/V2921 Ma en/Sicily (Pale mo)/2006/p15 – 21,183,149/0.98 leish7 MHOM/IT/2014/IZSLER-MO22 Human/Emilia-Romagna (Modena)/ 2014/p8 VL case; como bidi y: solid neoplasia 26,421,239/0.98 leish16 MFEL/IT/2018/10816 Ca /Sicily (Pale mo)/2 018/p16 – 25,578,672/0.81 leishMO23 MHOM/IT/2014/IZSLER-MO23 Human/Emilia-Romagna (Modena)/ 2014/p8 VL case; como bidi y: hema ologic diso de 22,041,069/0.98 leishMO38 MHOM/IT/2016/IZSLER-MO38 Human/Emilia-Romagna (Bologna)/ 2016/p8 VL case; como bidi y: HIV posi i e 20,902,208/0.92 a"–" indica es ha in o ma ion is no a ailable. Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 3 d i e o L. in an um i ness gain in cul u e. Su p isingly, simila ch omosomes we e linked o cul u e adap a ion o L. dono ani isola es om he Indian sub-con inen and Sudan (e.g., ch 5, 20, and 26) (5), sugges ing conse ed i ness mechanisms be ween bo h pa asi e species when selec ed o g ow h in cul u e. Aside om hese con e gen aneuploidies, we u he obse ed s ain-speci ic, ka yo ypic changes ha may ine- une adap a ion in esponse o o he gene ic di e ences be ween he s ains, which we u he analyzed in he ollowing sec ion. FIG 1 Ka yo ypic analyses o he L. in an um isola es (n = 5). (A) Map indica ing he geog aphic loca ion and o igin o he L. in an um isola es in es iga ed in his s udy. (B) Hea map showing he median somy sco e o he di e en s ains ac oss he 36 ch omosomes. Da ke onali ies e lec highe somy alues. (C) Quan i a i e PCR analysis wi h SYBR G een assay o quan i y ch 8 copy numbe in eline Leishmania s ains. CT alues a e shown o he his one deace ylase gene on ch 8, and he glyce ophospho yl dies e phosphodies e ase gene on disomic ch 28 used as a con ol. Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 4 Gene copy numbe a ia ions in he L. in an um isola es a ec known Leishmania i ulence genes We nex examined he gene-le el co e age in o ma ion gene a ed by GIP o assess s ain-speci ic and con e gen gene dosage changes wi h espec o he JPCM5 e e ence genome (supplemen al ma e ial; Fig. S2; and Da a Se S1A). Unlike ka yo ypic changes ha a e apidly selec ed du ing sho - e m cul u e adap a ion (5–20 passages), gene copy numbe a ia ions (CNVs) a e much less dynamic and hus can e eal gene dosage changes ha we e selec ed in he ield. Plo ing he no malized ead dep h co e age pe gene, we obse ed a wa y pa e n o all isola es caused by mino ead dep h a ia ions ac oss he ch omosomes (Fig. 2A; supplemen al ma e ial; Fig. S2). A se ies o gene CNVs we e obse ed, which co espond o he ampli ica ion o deple ion o gene copies wi h espec o he e e ence genome, bu also in be ween he i e samples. Signi ican ly, hese a ia ions we e no sca e ed ac oss he genome bu localized in de ined genomic egions ha we e simila in all samples, hus e ealing possible ho spo s o gene dosage changes (Fig. 2A). Close inspec ion o he 50 mos signi ican gene CNVs iden i ied bo h single gene and sub-ch omosomal ampli ica ions, many o which ha e been p e iously linked o in ec i i y, including amas ins (17), su ace an igen-like p o eins (18), o genes implica ed in he syn hesis o phosphogly can i ulence ac o s (19), such as phosphoglycan be a 1–3 galac osyl ans e ase genes (LINF_020006900 and LINF_020007000) o ppg3 (LINF_350010100 and LINF_350010200) encoding ilamen ous p o eophosphoglycan (20) (supplemen al ma e ial; Fig. S3A; Da a Se S1B). Signi ican ly, mos gene ampli ica ions a e obse ed ac oss se e al isola es. Such con e gence is no only obse ed a he gene le el bu also a he le el o gene unc ion, wi h ampli ied genes o en sha ing he same anno a ion e en hough hey a e on di e en ch omosomes and encoding di e en p o eins, as shown, o example, o he genes encoding su ace an igen p o eins and amas ins (supplemen al ma e ial; Fig. S3B). We also iden i ied 116 gene deple ions (no malized mean co e age < 0.5) and dele ions (no malized mean co e age = 0) ha we e sha ed o s ain-speci ic in he i e L. in an um isola es (Fig. 2B). Mos o hese changes occu ed in mul i-copy gene a ays (Da a Se S1C), whe e ecombina ion e en s be ween iden ical sequences allow o dynamic gene copy numbe changes. In pa icula , such dynamic a ia ions be ween he s ains we e obse ed o (i) i e gene copies on ch 9 encoding o he au ophagy gene ATG8, which has been implica ed in Leishmania s ess esponse and in ec i i y (21), (ii) six gene copies on ch 22 encoding o an uncha ac e ized, hypo he ical p o ein, (iii) a clus e o nine amas in genes encoded on ch 34 co esponding o wo mul i-copy gene a ays (Fig. S3B) and ha we e p e iously classi ied as δ-amas ins by Jackson (22) known o con ol amas igo e in ec i i y (17), (i ) 10 HSP70 gene copies encoded on ch 28 ha we p e iously e ealed as a po en ial ho spo o en i onmen -geno ype in e ac ion in L. dono ani ield isola es (23), and ( ) 10 genes on ch 34 encoding o pa a lagella od p o ein (Fig. 2C). The educed ead dep h ha we obse ed o indi idual gene copies could indica e mosaic gene CNVs, i.e., mixed popula ions o wild- ype and dele ed geno ypes, o ep esen a popula ing-wide, he e ozygous s a e. Only a ew genes show ue dele ions as judged by he absence o eads, including LINF_270010900 likely encoding o a calpain-like p o ease as sugges ed by a BLAST sea ch (da a no shown), which is dele ed in leish16, leish3, leish5, and leish7, o he ATG8 gene LINF_190013600 ha is dele ed in leish5 (Fig. 2C). The p esence o hese dele ions wi hin non-clonal, he e ogeneous pa asi e popula ions sugges s selec ion agains hese genes, e en hough loss due o gene ic d i canno be uled ou . Disco e y o leish7 as a highly di e gen L. in an um s ain We nex assessed he e olu iona y ela ionship be ween ou i e L. in an um isola es by compa ing single nucleo ide polymo phism (SNP) numbe s, posi ions, and equencies om he GIP ou pu . Based on he numbe o al e na i e (al ) alleles, leish16 is he mos Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 5 FIG 2 Gene copy numbe analyses o he L. in an um isola es (n = 5). (A) Sca e plo showing he no malized mean co e age (y-axis) o each gene ac oss he 36 ch omosomes (x-axis). Di e en s ains a e shown in di e en colo s (see legend in he igu e). (B) Hea map o no malized mean co e age. The plo shows gene deple ions de ined by a no malized mean co e age < 0.5 (g een, da k g een co esponding o dele ions wi h ead dep h = 0) and gene ampli ica ions de ined by a no malized mean co e age > 1.5 ( ed). The column on he le indica es he MAPQ sco e ( ed, MAPQ < 40; black, MAPQ > 40). (C) Rada plo s o he no malized mean co e age alues o he genes indica ed. Di e en s ains a e shown in di e en colo s (see legend in he igu e). The expec ed no malized co e age wi h espec o he e e ence genome is 1. Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 6 FIG 3 Genome-wide SNP analyses o he L. in an um isola es (n = 5). (A) Upse plo o SNPs iden i ied in he di e en s ains. The ba s isualize he numbe o SNPs common o a gi en combina ion o samples. The numbe s in he ba plo indica e sha ed SNPs o he gi en combina ion. The numbe s on he le side o he s ain ID indica e he o al numbe o SNPs in each isola e. (B) Violin plo ep esen ing densi y es ima es o he dis ibu ion o al -allele equencies o he indica ed samples. S ip plo s a e supe imposed on he iolin plo s in o de o show indi idual loci a in e media e equencies. Only hose loci whe e a leas one sample had an al -allele equency be ween 0.2 and 0.8 a e ep esen ed. (C) Clus e analysis based on pai wise Euclidean dis ances be ween al -allele equencies ac oss SNPs. The ee is cons uc ed om hese dis ances using he UPGMA me hod. (D) The sca e plo s display indi idual SNPs as do s. The x-axis indica es he ela i e posi ion o each SNP along he genome, while he y-axis indica es he a ian allele equency (see Fig. S4 o indi idual panels). The di e en ch omosomes a e displayed one a e he o he , and hei bounda ies a e isualized as e ical lines. The indi idual SNPs a e colo ed acco ding o he samples as indica ed by he legend in he g aph. The dense supe posi ion o do s co esponding o SNPs a equency 0 o 1 esul s in blu y lines a y = 0 and y = 1. The e a e no SNP in he (0, 0.1) equency in e al due o he il e ing applied du ing he SNP calling s ep o he GIP pipeline ( he co esponding equency is se o 0 when da a pe aining o a ious samples a e me ged oge he ). Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 7 simila o he JPCM5 e e ence genome wi h only 451 SNPs iden i ied, while leish7 is he mos di e gen wi h 49,053 SNPs (Fig. 3A and B; Da a Se S1D). O no e, including Illumina eads o he JPCM5 e e ence s ain i sel (24) e ealed a o al o 387 SNPs, o which 267 we e unique, while 120 we e sha ed wi h one o mo e o he isola es. Clus e analysis based on hese a ian s e ealed he highes simila i ies be ween JPCM5, leish4, and leish16 on one hand, and leish3 and leish5 on he o he hand, while leish7 ep esen ed i s own b anch, wi h simila dis ance om ei he o he wo o he clus e s (Fig. 3C). Plo ing equency agains loca ion e ealed de ined pa ches o he e ozygous SNPs a a equency o abou 50% in leish7, sugges ing he p esence o dis inc haplo ypes ha may be emnan s o o me hyb idiza ion e en s (Fig. 3D, see also indi idual plo s shown in Fig. S4). In es iga ing he possible o igin o he leish7 pu a i e hyb id We nex es ablished he genome sequence o wo addi ional isola es (leishMO23 and leishMO38) ob ained om human VL cases om RER, which ha e been p e iously shown o clus e wi h leish7 (o iginally named MO22) (10). Clus e analyses e ealed hei close ela ionship wi h leish7, con i ming he ansmission o a dis inc pa asi e sub-popula ion—likely a hyb id—in RER (Fig. 4A, op igh inse , and Da a Se S1D). We u he expanded he clus e analysis including 11 genomes ha we p e iously gene a ed as pa o he LeiSHield p ojec (www.leishield.o g) (2) and 63 genomes analyzed by F anssen e al. (15). While ou o ou I alian L. in an um s ains clus e ed wi h isola es om Tunisia (leish3 and leish5) and Spain (leish4 and leish16), he h ee RER hyb id- like s ains (leish7, leishMO23, and leishMO38) clus e ed wi h he known L. in an um/L. dono ani hyb ids i s desc ibed in Cyp us (Lin _CH33, 35, 36 and Ldo_CH33). Compa i son o SNPs be ween hese hyb ids e ealed o e 34,000 sha ed SNPs (Fig. 4B), indica ing ha hese geog aphically dis inc hyb ids a e likely he esul o simila hyb idiza ion e en s be ween L. in an um and L. dono ani pa en al s ains. This common hyb id na u e is u he suppo ed using he K aken so wa e, a sequence classi ica ion ool de eloped o me agenomic analyses, which assigns axonomic labels o DNA sequences using he exac alignmen o k-me s (25). This app oach allowed us o classi y all s ains (including he I alian and Cyp io hyb ids) as belonging o he L. dono ani complex, wi h only he hyb ids e ealing a signi ican ma ch a he species le el o L. dono ani genomes (Fig. 4C). Howe e , whe he he RER and Cyp io hyb ids a e o common o igin emains o be es ablished. Popula ion s uc u e and phylogene ic analysis o pu a i e hyb id s ains om no he n I aly In o de o be e de ine he p e alence o he leish7 hyb id-like geno ype in no he n I aly, mic osa elli e analysis was pe o med on 73 L. in an um s ains, including 22 s ains ob ained om human VL cases, 8 s ains ob ained om sand lies, and 40 s ains ob ained om canine cases om a ious a eas o RER (Fig. 5). Bayesian clus e ing as implemen ed in STRUCTURE assigned he 73 mul ilocus mic osa elli e yping (MLMT) p o iles om hese L. in an um s ains o wo main popula ions ( he highes alue o ΔK was o K = 2; Fig. S5), namely, popula ion A (PopA) ha consis ed o canine L. in an um s ains only and popula ion B (PopB) ha consis ed o all he L. in an um s ains om VL (including leish7, leishMO23, and leishMO38) and sand lies (Fig. 5). A mino ΔK peak a K = 5 (Fig. S5) indica ed a subs uc u e in he whole da a se , which was i s explo ed by inc easing he numbe o popula ions om K = 3 o K = 5: PopA exhibi ed subdi ision in o wo g oups a K = 3 and h ee g oups a K = 4, while PopB spli in o wo g oups a K = 5 (Fig. S6). Subsequen ly, we analyzed each o he wo main popula ions (PopA and PopB) sepa a ely; he calcula ion o ΔK alues (Fig. 5) showed he p esence o ou subpopula ions: PopA1, PopA2, PopB1 (including leishMO38), and PopB2 (including leish7 and leishMO23), while a i h sub-clus e ha was indica ed by he mino peak a K = 5 in he i s STRUCTURE analysis (all s ains) was no con i med. Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 8 The spa ial dis ibu ion o he gene ic g oups showed ha he wo main popula ions (PopA and PopB) o e lapped (Fig. 5), while pa ial o e lap was obse ed o he ou subpopula ions: PopA1 was widesp ead all o e RER, PopB1 was p e alen in he cen al-wes e n a eas, PopB2 was dis ibu ed in cen al-eas e n a eas, and PopA2 was limi ed o an eas e n a ea o RER (Fig. S7). The measu es o gene ic di e si y o each main popula ion de ined by STRUCTURE a e summa ized in Table 2. PopA and PopB showed a low expec ed he e ozygosi y (mean He = 0.258), which was highe han he obse ed he e ozygosi y (mean Ho = 0.056). Fu he mo e, he inb eeding coe icien s (Fis) we e e y high (mean Fis = 0.807). The global phylogene ic analysis showing he posi ion o human and sand ly s ains om RER wi hin he L. dono ani complex is ep esen ed in Fig. 6. Canine L. in an um s ains om RER belonging o he o iginal PopA we e g ouped in a monophyle ic clade oge he wi h VL s ains om o he I alian egions and Medi e anean human and canine s ains belonging o L. in an um zymodemes MON-1, MON-72, and MON-98. On he con a y, L. in an um s ains om VL pa ien s and sand lies om RER belonging o PopB g ouped in a monophyle ic clade ha was in e media e be ween L. in an um and L. dono ani clus e s. In addi ion, he PopB clade was close o he clus e , which included wo s ains om Cyp us, namely, CH35 and CD44cl.1, bo h classi ied as L. dono ani MON-37 (26). DISCUSSION The p esen s udy combines compa a i e genomics wi h molecula epidemiology app oaches o deli e a comp ehensi e, high- esolu ion in es iga ion o in aspeci ic gene ic di e si y and he e ozygosi y wi hin he L. in an um species in I aly, one o he mos impo an Leishmania species wo ldwide om bo h a e e ina y and public heal h pe spec i e. We e ealed a ema kable he e ogenei y in I alian L. in an um isola es ha a e ela ed o a ious pa asi e geno ypes o di e en geog aphic o igins, including a hyb id-like geno ype i s desc ibed in isola es om Cyp us. Ou esul s place I aly a he c oss oads o L. in an um in ec ion in he Medi e a nean and in o m on he possible o igin o egional sub-popula ions and hei local ansmission cycles. We iden i ied a se ies o L. in an um s ains om Sa dinia and Sicily ha a e gene ically close o hose om Spain (sou hwes e n[SW] Eu ope) and Tunisia (No h A ica), highligh ing I aly’s cen al loca ion in he Medi e anean Basin and i s his o ical and cu en ole as one o he main ou es o human mig a ion. Based on gene ic simila i y, he s ains leish3 and leish5 likely belong o he No h A ican MON-1 popula ion endemic in sou he n and eas e n Medi e anean egions. This popula ion is gene ically di e en om he Eu opean MON-1 popula ion ound in SW Eu ope and Sou h Ame ica, which co esponds o he s ains leish4 and leish16 and includes he JPCM5 s ain used as a e e ence in his s udy. The complex Leishmania epidemiology is he esul o he pa asi e’s ansmission dynamics, he dis ibu ion and geog aphic abundance o compe en ec o s, pas and cu en exposu e o human popula ions o he pa asi e, en i onmen al pa ame e s ha impac he seasonal ac i i y and dis ibu ion o sand lies (e.g., ai empe a u e, ela i e humidi y, g ound co e , al i ude, and ege a ion), and he p esence o ese oi hos s. In I aly, as in he whole Medi e anean basin, dogs (Canis lupus amilia is) a e inc imi na ed as he p ima y ese oi hos s o zoono ic human leishmaniasis, e en i o he domes ic and wild mammals in ec ed wi h L. in an um could play a possible ole in he biological cycle o he pa asi e (27). When compa ed o L. dono ani species, L. in an um MON-1, he mos common zymodeme in dogs and humans, has been conside ed a genomically a he homogeneous popula ion ega dless o geog aphic dis ibu ion (15). The pa asi e’s adap a ion o dogs may ha e been an e olu iona y ad an age, and he equen mobili y o he canine ese oi may ha e con ibu ed o he sp ead o he L. in an um MON-1 g oup (28). Howe e , mic osa elli e analyses and WGS showed ha he MON-1 “co e” L. in an um clade displays a ce ain deg ee o isola ion by dis ance. Th ee dis inc popula ions ha e been shown o ci cula e wi hin he Medi e anean Region, Resea ch A icle mBio July 2024 Volume 15 Issue 7 10.1128/mbio.00995-24 9 AUTHOR AFFILIATIONS 1WOAH Leishmania Re e ence Labo a o y, Is i u o Zoop o ila ico Spe imen ale della Sicilia, Cen o di Re e enza Nazionale pe le Leishmaniosi (C.Re.Na.L.), Pale mo, I aly 2Bioin o ma ics and Bios a is ics Hub, Ins i u Pas eu , Uni e si é Pa is Ci é, Pa is, F ance 3Is i u o Zoop o ila ico Spe imen ale della Lomba dia e dell'Emilia Romagna "B. Ube ini", B escia, I aly 4Depa men o Medical and Su gical Sciences, Uni e si y o Bologna, Bologna, I aly 5IRCCS Azienda Ospedalie o-Uni e si a ia di Bologna, Bologna, I aly 6In ec ious Disease Uni , Azienda Ospedalie a Uni e si a ia di Modena, Modena, I aly 7Vi ology and Molecula Mic obiology Uni , Uni e si y Hospi al o Modena, Modena, I aly 8Uni é de Pa asi ologie moléculai e e Signalisa ion, INSERM U1201, Ins i u Pas eu , Uni e si é Pa is Ci é, Pa is, F ance AUTHOR ORCIDs M. Calzola i h p://o cid.o g/0000-0001-7489-2556 G. Rugna h p://o cid.o g/0000-0001-7437-1929 G. F. Spä h h p://o cid.o g/0000-0002-0256-2029 FUNDING Funde G an (s) Au ho (s) EC | Ho izon 2020 F amewo k P og amme (H2020) LeiSHield-MATI-REP-778298-1 G. F. Spä h Minis e o della Salu e (I aly Minis y o Heal h) E54I19002870001 G. Rugna Minis e o della Salu e (I aly Minis y o Heal h) IZS SI RC SI 04/21 F. B uno Minis e o della Salu e (I aly Minis y o Heal h) GR‐ 2019‐ 12369134 G. Cas elli Eu opean Commission (EC) P ojec no. PE00000007 INF-ACT S. Va ani DATA AVAILABILITY All ele an da a a e wi hin he pape and i s supplemen al iles. Genome sequence da a ha e been deposi ed in BioP ojec a PRJNA1008390. ETHICS APPROVAL Samples om human cases we e collec ed o diagnos ic pu poses. S o ing and molecula e alua ion o e ospec i ely collec ed samples ecei ed app o als om bo h he E hics Commi ee o he S . O sola-Malpighi Uni e si y Hospi al, Bologna, I aly (p o ocol No. 3729/2017, las amendmen app o ed: EM414-2023 97/2017/O/Tess/ AOUBO) and he Modena E hics Commi ee, Modena, I aly (p o ocol No. 239/13). Samples we e coded and anonymized. All animal samples we e ob ained o diagnos ic pu poses wi h no unnecessa y in asi e p ocedu es. E alua ion o hese samples did no equi e e hical app o al acco ding o he Eu opean Di ec i e 2010/63/EU. ADDITIONAL FILES The ollowing ma e ial is a ailable online. Supplemen al Ma e ial Da a Se S1 (mBio00995-24-s0001.xlsx). 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