| 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.
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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.
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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.
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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
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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.
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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 ).
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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.
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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). WGS suppo ing in o ma ion.
Resea ch A icle mBio
July 2024 Volume 15 Issue 7 10.1128/mbio.00995-2416

Da a Se S2 (mBio00995-24-s0002.xlsx). MLMT suppo ing in o ma ion.
Supplemen al ma e ial (mBio00995-24-s0003.docx). Suppo ing me hods and
supplemen al igu es.
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