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Molecular detection and characterization of Leishmania infantum in free-ranging Egyptian mongoose (Herpestes ichneumon)
Journal Pre-proof Molecular detection and characterization of Leishmania infantum in free-ranging Egyptian mongoose (Herpestes ichneumon) Jacinto Gomes, Hugo Rocha, Carina Carvalho, Victor Bandeira, Carlos Fonseca, Luís Miguel Rosalino, Mónica V. Cunha PII:
S2213-2244(20)30013-4
DOI:
https://doi.org/10.1016/j.ijppaw.2020.02.001
Reference:
IJPPAW 452
To appear in:
International Journal for Parasitology: Parasites and Wildlife
Received Date: 15 December 2019 Revised Date:
2 February 2020
Accepted Date: 2 February 2020
Please cite this article as: Gomes, J., Rocha, H., Carvalho, C., Bandeira, V., Fonseca, C., Rosalino, L.M., Cunha, M.V., Molecular detection and characterization of Leishmania infantum in free-ranging Egyptian mongoose (Herpestes ichneumon), International Journal for Parasitology: Parasites and Wildlife, https://doi.org/10.1016/j.ijppaw.2020.02.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Author(s). Published by Elsevier Ltd on behalf of Australian Society for Parasitology.
N=132
MH799321 (Herpestes ichneumon) (2012)
45 39
JQ609530 (1998)(a) JQ609526 (1970)(b)
Spleen
AF103740 (1988) KX098512 AF169131 (1988)(c) JQ609527 (1998)(d) AJ275332 (1997)
Leishmania kDNA PCR
AF184044 (1980) JQ609524 (1980) 86
KX098510 AF169133 (1985) AF169140 AF169139 KX098513 KX098511
kDNA sequencing
93
AF103735 (1978) KX098514 100
AF167716 (1992) AF190476 (1984) AF190475 (1991)
0.020
1
Molecular detection and characterization of Leishmania infantum in free-ranging Egyptian mongoose
2
(Herpestes ichneumon)
3 4
Jacinto Gomes1,2, Hugo Rocha3, Carina Carvalho1,4, Victor Bandeira5, Carlos Fonseca5, Luís Miguel
5
Rosalino5, Mónica V. Cunha1,6,7
6 7
1
8
Edifício Principal, 2780 -157 Oeiras, Portugal.
9
2
National Institute for Agrarian and Veterinary Research (INIAV IP), Av. da República, Quinta do Marquês,
Interdisciplinary Centre of Research in Animal Health (CIISA), Faculdade de Medicina Veterinária,
10
Universidade de Lisboa, 1300-477, Lisboa, Portugal.
11
3
12
Lisboa, Portugal.
13
4
14
Technology ECT, University of Évora, Portugal.
15
5
16
193 Aveiro, Portugal.
17
6
18
de Lisboa, Edifício C2, 4º Piso, Campo Grande, 1749-016 Lisboa, Portugal.
19
7
20
Campo Grande, 1749-016 Lisboa, Portugal.
Divisão de Medicina Veterinária, Guarda Nacional Republicana, Tapada da Torre, Calçada da Ajuda,
Institute of Mediterranean Agricultural and Environmental Science (ICAAM), School of Science and
Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-
Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências da Universidade
Biosystems & Integrative Sciences Institute (BioISI), Faculdade de Ciências da Universidade de Lisboa,
21 22 23
Corresponding author: Mónica V. Cunha, INIAV, IP- National Institute for Agrarian and Veterinary
24
Research, Av. da República, Quinta do Marquês, Edifício Principal, 2780 -157 Oeiras, Portugal.
25 26 27
1
28
Abstract
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Wild mammals are susceptible to infection by Leishmania parasites. Although canine leishmaniasis is widely
30
distributed in mainland Portugal, the sylvatic cycle of the parasite remains poorly understood. In this study,
31
the occurrence of L. infantum in wild carnivores from Portugal was assessed by molecular screening of 132
32
hunted or accidentally road-killed animals. Spleen samples from Egyptian mongooses, red foxes, stone
33
marten, common genet and European badgers were tested by amplification of Leishmania kinetoplastid DNA
34
and ITS-1. Five egyptian mongoose were confirmed Leishmania DNA-positive by kDNA-PCR.
35
Phylogenetic analysis of a kDNA amplicon sequence clustered the strain with L. infantum sequences from
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Portugal. These results may suggest that L. infantum strains circulating in wild animals are genetically
37
related with strains from more humanized stettings. Exposure of wild carnivores to Leishmania infantum
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emphasizes the need of systematic studies to clarify the role of several taxa in the eco-epidemiology of
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leishmaniasis in Portugal, particularly in areas of carnivore species synanthropy and wherein disease control
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in the domestic population is inefficient or insufficient.
41 42
Keywords: Leishmania; mongoose; Herpestes ichneumon; wild carnivore; kDNA
2
43
1. Introduction
44
Leishmaniasis is a complex of diseases that affects humans, domestic and wild mammals worldwide. It is
45
caused by parasitic protozoans classified as Leishmania species (order Kinetoplastida, family
46
Trypanosomatidae). Natural transmission may be zoonotic with the involvement of reservoir hosts such as
47
rodents, marsupials, edentates, monkeys, domestic dogs, and wild canids, usually by the bite of a
48
phlebotomine sandfly species of the genera Phlebotomus or Lutzomyia. In a few cases and for particular
49
Leishmania species, the transmission is strictly anthroponotic, i.e., transmitted from human to human
50
(Ready, 2010; Millán, 2014; Quinnnell and Courtenay, 2009). Leishmania infantum infection is endemic in
51
southern Europe but animal cases and vector sand flies have also been detected in central Europe (Maroli et
52
al. 2008; Poeppl et al. 2013). This species is the agent of both cutaneous and visceral forms of human and
53
viscero-cutaneous canine leishmaniasis in Europe. Dogs are considered the main reservoir of L. infantum
54
infection in Mediterranean countries, with apparent prevalence rates ranging from 5% to 30%, depending on
55
the region (Millán, 2014). Wildlife monitoring programs and the increasing use of molecular techniques such
56
as PCR have enabled the identification of previously unreported infected species in Spain, Italy and France,
57
specially among carnivores. Serological or direct evidence of L. infantum infection in animals from the
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Canidae, Felidae, Mustelidae, Viverridae and Herpestidae families have been reported (reviewed in Maia et
59
al., 2018; Millán, 2014; Franco et al., 2011). Although there is no official strategy for the control of canine
60
leishmaniasis, an increasing access to vaccination, awareness of dog owners, use of repellent collars and the
61
treatment of animals have jointly contributed to decrease the number of clinical cases. Despite the
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prophylactic measures provided to the canine population, leishmaniasis remains widely distributed in
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Portugal and highly endemic in certain areas. Notwithstanding the major importance of leishmaniasis in the
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dog population, it is also important to understandthe contribution of wild carnivores to the epidemiology of
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this disease. This study is thus a preliminary work aiming at detecting natural infection of wild
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mesocarnivores from Portugal with Leishmania spp., particularly focusing on a species that has been under
67
expansion in the mainland territory for the last decades, the Egyptian mongoose (Herpestes ichneumon).
68
Although not considered threatened, some wild species included in this study are widely distributed in
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Portugal (Bencatel et al., 2017) and thus are candidates for further studies to confirm them as Leishmania
70
spp. suitable hosts.
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2. Materials and methods
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2.1. Sample collection
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One hundred and thirty-two wild carnivores belonging to Canidae, Mustelidae, Viverridae and Herpestidae
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taxonomic families (order Carnivora) were collected during 2011 and 2012, namely Egyptian mongoose
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(Herpestes ichneumon, n=106), red fox (Vulpes vulpes, n=18), stone marten (Martes foina, n=2), European
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badger (Meles meles, n=3) and common genet (Genetta genetta, n=3). Animal carcasses were collected in all
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five regions from Continental Portugal, originated from accidental road-kills or legal predator control actions
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(Egyptian mongoose and red fox) and were donated for scientific purposes (Fig. 1). All species listed as
3
79
game species, or targeted by predator control actions (the case of red fox and mongoose), by Portuguese
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legislation (Portaria n.º 142/2015 - Diário da República n.º 98/2015, Série I), can be legally hunted by the
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hunting associations and fellow credentiated hunters authorized by the National Institute for Nature
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Conservation and Forest (ICNF). After collection, all animals were preserved in sealed plastic bags,
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refrigerated and transported to a collection centre, under a license from ICNF – Licence no.
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222/2010/TRANS, where they were kept at -20ºC. The sex and age [determined by dental analysis- Bandeira
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et al. (2016)] of the specimens and the geo-coordinates of the collection sites were registered. Necropsy and
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subsequent spleen collection were performed by pathologists at the national reference laboratories for animal
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health, the National Institute for Agrarian and Veterinary Research (INIAV IP), in appropriate facilities.
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Spleen fragments were collected and stored at -20°C until further use.
89 90
2.2. DNA extraction and molecular analysis
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The study samples were screened for the presence of Leishmania spp. using molecular methods. Total DNA
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was extracted from 25 mg of spleen tissue using the High Pure PCR Template Preparation kit (Roche),
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according to the manufacturer’ instructions. Leishmania kinetoplast DNA (kDNA) minicircle was selected as
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the molecular target using primers MC1 (5’-GTTAGCCGATGGTGGTCTTG-3’) and MC2 (5’-
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CACCCATTTTTCCGATTTTG-3’), which hybridize with a hypervariable portion of kDNA minicircles
96
sequence (Cortes et al., 2004). Reaction and PCR conditions were as described by Cortes and collaborators
97
(2006) and, for positive samples, a 447 bp fragment amplicon was expected. As additional confirmation,
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amplification of internal transcribed spacer 1 (ITS1) region of the ribosomal RNA (rRNA) encoding gene
99
was performed. This second PCR enables the amplification of a 300-350 bp fragment corresponding to the
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non-coding spacer region ITS1 found in the ribosomal operon, located between the 18S rRNA and the 5.8S
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rRNA coding regions. Primers used were LITSR (5´-TGATACCACTTATCGCACTT-3´) and L5.8S (5´-
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CTGGATCATTTTCCGATG-3´) and reactions carried out according to El Tail et al. (2000). No-template
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(water) and positive controls (Leishmania infantum DNA from a smear-positive dog isolate) were used in
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each PCR batch. Amplification reaction products were analyzed by electrophoresis on 2.0% agarose gels
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stained with ethidium bromide.
106 107
2.3 Sequencing and Phylogenetic analysis
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Amplicons from kDNA positive samples were excised from agarose gel, purified using a commercial kit
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(QIAquick gel extraction kit, Qiagen or Gel Band Purification, GE Healthcare) and sequenced at a
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commercial company (StabVida, Portugal). The same pairs of primers used in the amplification of each
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fragment were used in sequencing. Original chromatogram files were inspected and manually reviewed using
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the ChromasPro® software (version 2.6.5).
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sequences available in public databases (http://www.ncbi.nlm.nih.gov) by BLASTn analysis and submitted
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to GenBank (http://www.ncbi.nlm.nih.gov).
Edited sequences were compared with similar reference
4
115
The phylogenetic relationships between the L. infantum sequence obtained during this study with other
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available sequences in GenBank were investigated by Maximum Likelihood (ML) using MEGA7 software
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(Kumar et al., 2016). The same software was used to perform the multiple sequence nucleotide alignments
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(msa) and determine the appropriate substitution model, using the model selection analysis. Robustness of
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the tree nodes was assessed by bootstrapping 1000 times. The graphical edition of the phylogenetic tree was
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performed resourcing tree explorer, MEGA7 software (Kumar et al., 2016).
121 122
3. Results
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Detection of Leishmania kDNA in the spleen samples of 18 red foxes, two stone martens, three European
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badgers, three common genets and 101 of the surveyed Egyptian mongooses was negative. However, a
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kDNA fragment of the expected size (approximately, 447 bp) was obtained in five Egyptian mongoose
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samples among the 132 DNA spleen extracts analysed. A second, independent PCR directed towards the
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ITS1 intergenic region was employed to test in parallel the 132 carnivore samples. Confirmation of the
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presence of Leishmania DNA by both techniques was attained in only one spleen sample. This specimen
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belonged to a female adult Egyptian mongoose collected in Beja district during 2011, southern Portugal. A
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kDNA sequence with 402 bp obtained from the amplicon of this positive Egyptian mongoose was deposited
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in GenBank under accession number MH799321 (on hold upon publication). Despite several attempts, it was
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not possible to sequence the amplicon obtained from amplification of the ITS1 fragment.
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Positive animals were detected in Beja (n=3), Portalegre (n=1) and Viseu (n=1) administrative units (Fig. 1).
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Using partial kinetoplastid minicircle nucleotide sequences, the relationship between this strain and other
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Leishmania infantum strains from nine countries, mostly from the Mediterranean basin (mainland Portugal,
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Spain, Morocco, Tunisia, Algeria and Italy) was investigated by Maximum Likelihood (ML) phylogenetic
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inference. The Hasegawa-Kishino-Yano (HKY) model (Hasegawa et al., 1985) with gamma distributed rate
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variation among sites (+G) (HKY+G) showed the lowest BIC (2456.521) and AICc (2147.195) values and
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was subsequently used to infer phylogenetic relationships using ML analysis. It is noteworthy that the
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T92+G model also displayed similar BIC (2456.564) and AICc (2161.279) values.
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Phylogenetic analysis suggests a higher resemblance of the L. infantum strain obtained from the Egyptian
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mongoose (MH799321) with other L. infantum strains obtained in mainland Portugal from 1970 to 2003,
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originating from dogs (Canis lupus familiaris) and humans (Homo sapiens), although the Bootstrap (Bs)
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values supporting those nodes are <70 (Figure 2). This closer genetic relatedness is also suggested by the
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high genetic similarity, with homologies ranging from 97,51% to 98,01% (Table 1, supplementary material),
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and low genetic distances, ranging from 0,01282 to 0,01807 (Table 2, supplementary material), found
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between strain MH799321 and the L. infantum strains from mainland Portugal.
148 149
4. Discussion
5
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Leishmaniasis is a parasitic disease of great importance in veterinary medicine, considering the high number
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of infected dogs, but also with a major impact on Public Health. Canine leishmaniasis is virtually present
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throughout Portugal, although prevalence rates vary among regions. In the present study, the confirmation of
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Leishmania infection in wild carnivores was based on the detection of Leishmania infantum nucleic acids in
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spleen samples. Using an end-point PCR for the detection of kinetoplastid DNA, it was possible to identify
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five positive animals among Egyptian mongoose. The kinetoplast DNA minicircle was selected as the
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molecular target as it is present in hundreds of copies in Leishmania, increasing the probability of detecting
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this protozoan in frozen samples in which microscopic analysis for detection of amastigotes or protozoan
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isolation could not be performed. The animal from whom kDNA was sequenced was an adult female and
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probably an asymptomatic carrier, since during the necropsy no evidence of clinical disease nor cutaneous or
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visceral lesions were apparent. This specimen was collected in Beja district, one of the southern regions with
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higher seroprevalence of canine leishmaniasis (12.12%) (Cortes et al., 2012). Sequencing analysis of the
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kinetoplastid DNA allowed the identification of L. infantum and phylogenetic inference by ML suggested a
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closer genetic relationship of this strain with other L. infantum strains from mainland Portugal, originating
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from dogs and humans. This geographic relationship of Leishmania sp. isolates and, particularly, the genetic
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relatedness between strains circulating in the wild and in more humanized environments emphasize that the
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interface between these two compartments at a local scale may be attenuated by the vector-borne nature of
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Leishmania transmission.
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The historical process that led to Egyptian mongoose colonization of Iberia is an issue that is still under
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debate. While Gaubert et al. (2011) proposed that mongooses reached Iberia through the Strait of Gibraltar
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during the Middle to Late Pleistocene, more recently Detry et al. (2018) collected data that suggested that
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this herpestid might have been introduced by the Romans, during their presence in Hispania. This African
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origin of mongoose could provide additional clues for an ancient Leishmania/mongoose relationship.
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Mongoose have underwent a tremendous natural expansion process in the last decades from southern to
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northern Portugal. And recently, the species also invaded the North-eastern areas of the country, from where
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it was absent in the beginning of twentieth century (Barros et al., 2015, 2016). The recent abandon of
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croplands, rural depopulation, prodigious bio-ecological adaptability, generalist opportunistic behaviour and
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lack of natural predators possibly cumulatively led to this expansion success (Barros et al., 2015). Some
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population biology aspects of this mesocarnivore species are still largely unknown, namely its health status,
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contribution to pathogen cross-species transmission and competence as Leishmania spp. reservoir. Although
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mongooses have distributions across several African regions and some SW European areas, Leishmania
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infection has only been reported in two mongooses from Sudan (Elnaiem et al., 2001). In the report from
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Elnaiem and collaborators (2001), DNA sequences were not publicaly available for phylogenetic
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comparison, but the authors reported that the species L. donovani was responsible for infection in mongoose.
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A recent report identified L. infantum DNA in rodents, lagomorphs and wild carnivores from Southeast
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Spain, including stone marten, common genet and red foxes (Risueño et al., 2018). The red fox was the
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second most tested species [13.6% (18/132)] in our study. Although it is frequently reported as an L.
6
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infantum carrier and a previous study performed in the 80’s in the Arrábida region (mainland Portugal)
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identified positive animals (Abranches et al., 1983), no kDNA-positive red fox could be confirmed among
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the population we surveyed. Other species, such as the stone marten, common genet and European badger
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have occasionally been found positive in other regions of the world, but we did not find any evidence of
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positive cases, which could also be related with the limited number of samples examined from these species.
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This study enabled the detection of L. infantum infection in wildlife from Western Iberia. Such data
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contributes to consolidate knowledge on the epidemiology of this pathogen in the Mediterranean basin and
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identifying, for the first time, Herpestes ichneumon as a possible carrier host in Portugal. Although the
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results obtained suggest that most mesocarnivore species are not particularly exposed to this parasite in
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Portugal, it is necessary to carry out further studies to better clarify the role of wild carnivores and taxa, such
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as leporids and rodents in the enzootic Leishmania transmission cycle and the potential consequences
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thereof. The limited number of positive cases in this study may also suggest that tissue tropism of
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Leishmania sp. may vary according to host species. To confirm this hypothesis, future studies should
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consider testing skin and other organs besides spleen. Furthermore, xenodiagnostic analyses could also bring
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some light into the role of this species as reservoir or as dead-end host. Anthropogenic changes of
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ecosystems resulting in the expansion or overabundance of particular wild species, such as the mongoose,
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may provide more opportunities for direct or indirect contact with domestic animals and humans,
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significantly affecting parasite distribution and risk.
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The surveillance programs directed towards wildlife or focused on particular pathogens should thus be
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adapted and extended to other animal taxa and regions of the national territory. Such programs should be a
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priority in areas where interaction between wild animals, domestic animals, and humans is pronounced, the
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prevalence in dogs is high and the control strategy is insufficient. Scrutiny in areas wherein natural and
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anthroponotic conditions propitiate proliferation of phlebotomine vectors should also be granted.
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5. Acknowledgments
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We are grateful to hunters and hunting organizations for animal carcasses donation. We acknowledge the
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public company Estradas de Portugal IP for contributing with cadavers from accidental road-kills. We thank
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Fernanda Simões (INIAV IP) for helpful suggestions. This work was partially funded by INIAV IP and by
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national funds through Fundação para a Ciência e a Tecnologia (FCT/MEC) in the frame of the strategic
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funding to cE3c and BioISI Research Units (UID/BIA/00329/2019 and UID/Multi/04046/2019). Financial
217
support from Programa de Desenvolvimento Rural, FEADER and P2020 are also gratefully acknowledged in
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the scope of project Alliance-i9-Caça (ref. PDR2020-2024-049959).
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Thanks are also due for the financial support to University of Aveiro (Department of Biology), to CESAM
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(UID/AMB/50017) from FCT/MEC through national funds and the co-funding by the FEDER, within the
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PT2020 Partnership Agreement and Compete 2020 by co-funding through the project "Genetic assessment of
7
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a successful invasion: Population genetics of the Egyptian mongoose (Herpestes ichneumon) in Portugal",
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PTDC/BIA-BEC/104401/2008.
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6. Conflict of Interest Statement
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On behalf of all authors, the corresponding author states that there is no conflict of interest.
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7. Captions
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Figure 1. Spatial distribution of wild carnivore samples in mainland Portugal. Administrative regions at the
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district level are indicated. The overall proportion of samples per district is indicated by the grey scale.
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The abreviatures of districts are as follows: Viana do Castelo (VC), Braga (BR), Vila Real (VR), Bragança
232
(BG), Porto (PT), Aveiro (AV) Viseu (VS), Guarda (GR), Coimbra (CM), Castelo Branco (CB), Leiria (LR),
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Santarém (SA), Portalegre (PA), Lisboa (Lx), Setúbal (ST), Évora (EV), Beja (BJ) and Faro (FR).
234
White circles with numbers in red specify the number and location of Egyptian mongooses that were kDNA-
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positive by PCR.
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Figure 2. Maximum Likelihood (ML) phylogenetic tree of 27 L. infantum nucleotide sequences (410 nt long
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in the final dataset, including gaps), obtained during this study (MH799321) and others available in
238
GenBank, based on the Hasegawa-Kishino-Yano model (HKY) (Hasegawa et al., 1985). The tree with the
239
highest log likelihood (-1026.87) is shown. Initial tree(s) for the heuristic search were obtained automatically
240
by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the
241
Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log
242
likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites
243
(5 categories (+G, parameter = 0.3289) (HKY+G). The tree was drawn to scale, with branch lengths
244
measured in the number of substitutions per site.
245
Robustness of the tree nodes was assessed by bootstrapping 1000 times. The graphical edition of the
246
phylogenetic tree was performed with tree explorer, MEGA7 software (Kumar et al., 2016).
247
Only bootstrap (BS) values equal or greater than 70 are shown on the tree, with the exception of the
248
MH799321 cluster wherein the Bs values, although <70, are displayed for the reader.
249
A 2-letter code and a specific colour (Top left) was attributed to each country for better identification of the
250
origin of each strain. Whenever possible, the host was identified by a specific shape (Top left), namely dog
251
(Canis lupus familiaris, star), human (Homo sapiens, triangle), Egyptian mongoose (Herpestes ichneumon,
252
square) and hoary fox (Lycalopex vetulus, diamond). Sampling dates are indicated, whenever available.
253
8
254
Table 1 (supplementary material). Percentage of similarity between nucleotide sequences from the
255
hypervariable region of Leishmania kinetoplast (mitochondrial) DNA (kDNA) minicircles obtained in
256
Portugal from 1970 to 2003.
257 258
Table 2 (supplementary material). Genetic distances (GD) matrix for the sequences used in this study,
259
including the Egyptian mongoose (Herpestes ichneumon) sequence MH799321, determined by the Tamura-
260
3-parameters model (T92) with discrete gama distribution (G). The GD between strain MH799321 and the
261
other strains used in the study is marked in bold.
262 263
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(2011). Comparative phylogeography of two African carnivorans presumably introduced into Europe:
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Hasegawa, M., Kishino H., and Yano T. (1985). Dating the human-ape split by a molecular clock of mitochondrial DNA. J Mol Evol 22:160-174 Kumar, S., Stecher G., and Tamura K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870-1874 Maia, C., Dantas-Torres F., Campino L. (2018). Parasite biology: the reservoir hosts. In: The Leishmaniases: old neglected tropical diseases, pp 79-106, Springer.
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Maroli, M., Rossi, L., Baldelli, R., Capelli, G., Ferroglio, E., Genchi, C., Gramiccia, M., Mortarino, M.,
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Pietrobelli, M., Gradoni, L. (2008). The northward spread of leishmaniasis in Italy: evidence from
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retrospective and ongoing studies on the canine reservoir and phlebotomine vectors. Trop Med Int Health
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13, 256–264
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Millán, J., Ferroglio, E., Solano-Gallego, L. (2014). Role of wildlife in the epidemiology of Leishmania infantum infection in Europe. Parasitol. Res. 113, 2005–2014
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Poeppl, W., Herkner, H., Tobudic, S., Faas, A., Auer, H., Mooseder, G., Burgmann, H., Walochnik, J.
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(2013). Seroprevalence and asymptomatic carriage of Leishmania spp. in Austria, a non-endemic
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European country. Clin Microbiol Infect. 19, 572–577
314 315
Quinnell R.J., Courtenay O. (2009). Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. Parasitol. 136, 1915-1934.
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Ready, P.D. (2010). Leishmaniasis emergence in Europe. Euro Surveill 15: 19505
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Risueño J., Ortuño M., Pérez-Cutillas P., Goyena E., Maia C., Cortes S., Campino L., Bernal L.J., Muñoz C.,
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Arcenillas I., Martínez-Rondán F.J., Gonzálvez M., Collantes F., Ortiz J., Martínez-Carrasco C., Berriatua
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E. (2018). Epidemiological and genetic studies suggest a common Leishmania infantum transmission
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cycle in wildlife, dogs and humans associated to vector abundance in Southeast Spain. Vet Parasitol.
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259:61-67. doi: 10.1016/j.vetpar.2018.05.012
10
322 323
Tamura, K (1992) Estimation of the number of nucleotide substitutions when there are strong transitiontransversion and G + C-content biases. Molecular Biology and Evolution 9:678-687.
11
1
1
3
Figure 2. TN (Tunisia) DZ (Algeria) MA (Moroco) FR (France) SU (Soviet Union) ES (Spain) PT (Portugal) IT (Italy) UK (United Kingdom)
Canis lupus familiaris Homo sapiens Herpestes ichneumon Lycalopex vetulus
MH799321 (Herpestes ichneumon) (2012)
45 39
JQ609530 (1998)(a) JQ609526 (1970)(b) AF103740 (1988) KX098512 AF169131 (1988)(c) JQ609527 (1998)(d) AJ275332 (1997) AF184044 (1980) JQ609524 (1980)
86
KX098510 AF169133 (1985) AF169140 AF169139 KX098513 KX098511 93
AF103735 (1978) KX098514 100
AF167716 (1992) AF190476 (1984) AF190475 (1991)
0.020 (a) Identical
to sequences to sequence (c) Identical to sequences (d) Identical to sequence (b) Identical
JQ609531 (1998) and JQ609533 (1997) JQ609528 (2000) KX098509 and AF103741 (1978) JQ609529 (2003)
Highlights •
Leishmania sp. occurrence in wild carnivores (n=132) from Portugal was assessed by PCR
•
Egyptian mongoose, red fox, stone marten, common genet and European badger were tested
•
Five Egyptian mongoose were confirmed Leishmania positive by kDNA-PCR
•
The 402-nt long sequence of mongoose kDNA amplicon displayed high similarity (>97,51%) with L. infantum from dogs and humans
•
Phylogenetic analysis by Maximum Likelihood clustered mongoose strain with L. infantum from Portugal
•
L. infantum strains circulating in sylvatic animals are genetically related with urban strains
Conflict of Interest Statement On behalf of all authors, the corresponding author states that there is no conflict of interest.
S2213-2244(20)30013-4
DOI:
https://doi.org/10.1016/j.ijppaw.2020.02.001
Reference:
IJPPAW 452
To appear in:
International Journal for Parasitology: Parasites and Wildlife
Received Date: 15 December 2019 Revised Date:
2 February 2020
Accepted Date: 2 February 2020
Please cite this article as: Gomes, J., Rocha, H., Carvalho, C., Bandeira, V., Fonseca, C., Rosalino, L.M., Cunha, M.V., Molecular detection and characterization of Leishmania infantum in free-ranging Egyptian mongoose (Herpestes ichneumon), International Journal for Parasitology: Parasites and Wildlife, https://doi.org/10.1016/j.ijppaw.2020.02.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Author(s). Published by Elsevier Ltd on behalf of Australian Society for Parasitology.
N=132
MH799321 (Herpestes ichneumon) (2012)
45 39
JQ609530 (1998)(a) JQ609526 (1970)(b)
Spleen
AF103740 (1988) KX098512 AF169131 (1988)(c) JQ609527 (1998)(d) AJ275332 (1997)
Leishmania kDNA PCR
AF184044 (1980) JQ609524 (1980) 86
KX098510 AF169133 (1985) AF169140 AF169139 KX098513 KX098511
kDNA sequencing
93
AF103735 (1978) KX098514 100
AF167716 (1992) AF190476 (1984) AF190475 (1991)
0.020
1
Molecular detection and characterization of Leishmania infantum in free-ranging Egyptian mongoose
2
(Herpestes ichneumon)
3 4
Jacinto Gomes1,2, Hugo Rocha3, Carina Carvalho1,4, Victor Bandeira5, Carlos Fonseca5, Luís Miguel
5
Rosalino5, Mónica V. Cunha1,6,7
6 7
1
8
Edifício Principal, 2780 -157 Oeiras, Portugal.
9
2
National Institute for Agrarian and Veterinary Research (INIAV IP), Av. da República, Quinta do Marquês,
Interdisciplinary Centre of Research in Animal Health (CIISA), Faculdade de Medicina Veterinária,
10
Universidade de Lisboa, 1300-477, Lisboa, Portugal.
11
3
12
Lisboa, Portugal.
13
4
14
Technology ECT, University of Évora, Portugal.
15
5
16
193 Aveiro, Portugal.
17
6
18
de Lisboa, Edifício C2, 4º Piso, Campo Grande, 1749-016 Lisboa, Portugal.
19
7
20
Campo Grande, 1749-016 Lisboa, Portugal.
Divisão de Medicina Veterinária, Guarda Nacional Republicana, Tapada da Torre, Calçada da Ajuda,
Institute of Mediterranean Agricultural and Environmental Science (ICAAM), School of Science and
Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-
Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências da Universidade
Biosystems & Integrative Sciences Institute (BioISI), Faculdade de Ciências da Universidade de Lisboa,
21 22 23
Corresponding author: Mónica V. Cunha, INIAV, IP- National Institute for Agrarian and Veterinary
24
Research, Av. da República, Quinta do Marquês, Edifício Principal, 2780 -157 Oeiras, Portugal.
25 26 27
1
28
Abstract
29
Wild mammals are susceptible to infection by Leishmania parasites. Although canine leishmaniasis is widely
30
distributed in mainland Portugal, the sylvatic cycle of the parasite remains poorly understood. In this study,
31
the occurrence of L. infantum in wild carnivores from Portugal was assessed by molecular screening of 132
32
hunted or accidentally road-killed animals. Spleen samples from Egyptian mongooses, red foxes, stone
33
marten, common genet and European badgers were tested by amplification of Leishmania kinetoplastid DNA
34
and ITS-1. Five egyptian mongoose were confirmed Leishmania DNA-positive by kDNA-PCR.
35
Phylogenetic analysis of a kDNA amplicon sequence clustered the strain with L. infantum sequences from
36
Portugal. These results may suggest that L. infantum strains circulating in wild animals are genetically
37
related with strains from more humanized stettings. Exposure of wild carnivores to Leishmania infantum
38
emphasizes the need of systematic studies to clarify the role of several taxa in the eco-epidemiology of
39
leishmaniasis in Portugal, particularly in areas of carnivore species synanthropy and wherein disease control
40
in the domestic population is inefficient or insufficient.
41 42
Keywords: Leishmania; mongoose; Herpestes ichneumon; wild carnivore; kDNA
2
43
1. Introduction
44
Leishmaniasis is a complex of diseases that affects humans, domestic and wild mammals worldwide. It is
45
caused by parasitic protozoans classified as Leishmania species (order Kinetoplastida, family
46
Trypanosomatidae). Natural transmission may be zoonotic with the involvement of reservoir hosts such as
47
rodents, marsupials, edentates, monkeys, domestic dogs, and wild canids, usually by the bite of a
48
phlebotomine sandfly species of the genera Phlebotomus or Lutzomyia. In a few cases and for particular
49
Leishmania species, the transmission is strictly anthroponotic, i.e., transmitted from human to human
50
(Ready, 2010; Millán, 2014; Quinnnell and Courtenay, 2009). Leishmania infantum infection is endemic in
51
southern Europe but animal cases and vector sand flies have also been detected in central Europe (Maroli et
52
al. 2008; Poeppl et al. 2013). This species is the agent of both cutaneous and visceral forms of human and
53
viscero-cutaneous canine leishmaniasis in Europe. Dogs are considered the main reservoir of L. infantum
54
infection in Mediterranean countries, with apparent prevalence rates ranging from 5% to 30%, depending on
55
the region (Millán, 2014). Wildlife monitoring programs and the increasing use of molecular techniques such
56
as PCR have enabled the identification of previously unreported infected species in Spain, Italy and France,
57
specially among carnivores. Serological or direct evidence of L. infantum infection in animals from the
58
Canidae, Felidae, Mustelidae, Viverridae and Herpestidae families have been reported (reviewed in Maia et
59
al., 2018; Millán, 2014; Franco et al., 2011). Although there is no official strategy for the control of canine
60
leishmaniasis, an increasing access to vaccination, awareness of dog owners, use of repellent collars and the
61
treatment of animals have jointly contributed to decrease the number of clinical cases. Despite the
62
prophylactic measures provided to the canine population, leishmaniasis remains widely distributed in
63
Portugal and highly endemic in certain areas. Notwithstanding the major importance of leishmaniasis in the
64
dog population, it is also important to understandthe contribution of wild carnivores to the epidemiology of
65
this disease. This study is thus a preliminary work aiming at detecting natural infection of wild
66
mesocarnivores from Portugal with Leishmania spp., particularly focusing on a species that has been under
67
expansion in the mainland territory for the last decades, the Egyptian mongoose (Herpestes ichneumon).
68
Although not considered threatened, some wild species included in this study are widely distributed in
69
Portugal (Bencatel et al., 2017) and thus are candidates for further studies to confirm them as Leishmania
70
spp. suitable hosts.
71
2. Materials and methods
72
2.1. Sample collection
73
One hundred and thirty-two wild carnivores belonging to Canidae, Mustelidae, Viverridae and Herpestidae
74
taxonomic families (order Carnivora) were collected during 2011 and 2012, namely Egyptian mongoose
75
(Herpestes ichneumon, n=106), red fox (Vulpes vulpes, n=18), stone marten (Martes foina, n=2), European
76
badger (Meles meles, n=3) and common genet (Genetta genetta, n=3). Animal carcasses were collected in all
77
five regions from Continental Portugal, originated from accidental road-kills or legal predator control actions
78
(Egyptian mongoose and red fox) and were donated for scientific purposes (Fig. 1). All species listed as
3
79
game species, or targeted by predator control actions (the case of red fox and mongoose), by Portuguese
80
legislation (Portaria n.º 142/2015 - Diário da República n.º 98/2015, Série I), can be legally hunted by the
81
hunting associations and fellow credentiated hunters authorized by the National Institute for Nature
82
Conservation and Forest (ICNF). After collection, all animals were preserved in sealed plastic bags,
83
refrigerated and transported to a collection centre, under a license from ICNF – Licence no.
84
222/2010/TRANS, where they were kept at -20ºC. The sex and age [determined by dental analysis- Bandeira
85
et al. (2016)] of the specimens and the geo-coordinates of the collection sites were registered. Necropsy and
86
subsequent spleen collection were performed by pathologists at the national reference laboratories for animal
87
health, the National Institute for Agrarian and Veterinary Research (INIAV IP), in appropriate facilities.
88
Spleen fragments were collected and stored at -20°C until further use.
89 90
2.2. DNA extraction and molecular analysis
91
The study samples were screened for the presence of Leishmania spp. using molecular methods. Total DNA
92
was extracted from 25 mg of spleen tissue using the High Pure PCR Template Preparation kit (Roche),
93
according to the manufacturer’ instructions. Leishmania kinetoplast DNA (kDNA) minicircle was selected as
94
the molecular target using primers MC1 (5’-GTTAGCCGATGGTGGTCTTG-3’) and MC2 (5’-
95
CACCCATTTTTCCGATTTTG-3’), which hybridize with a hypervariable portion of kDNA minicircles
96
sequence (Cortes et al., 2004). Reaction and PCR conditions were as described by Cortes and collaborators
97
(2006) and, for positive samples, a 447 bp fragment amplicon was expected. As additional confirmation,
98
amplification of internal transcribed spacer 1 (ITS1) region of the ribosomal RNA (rRNA) encoding gene
99
was performed. This second PCR enables the amplification of a 300-350 bp fragment corresponding to the
100
non-coding spacer region ITS1 found in the ribosomal operon, located between the 18S rRNA and the 5.8S
101
rRNA coding regions. Primers used were LITSR (5´-TGATACCACTTATCGCACTT-3´) and L5.8S (5´-
102
CTGGATCATTTTCCGATG-3´) and reactions carried out according to El Tail et al. (2000). No-template
103
(water) and positive controls (Leishmania infantum DNA from a smear-positive dog isolate) were used in
104
each PCR batch. Amplification reaction products were analyzed by electrophoresis on 2.0% agarose gels
105
stained with ethidium bromide.
106 107
2.3 Sequencing and Phylogenetic analysis
108
Amplicons from kDNA positive samples were excised from agarose gel, purified using a commercial kit
109
(QIAquick gel extraction kit, Qiagen or Gel Band Purification, GE Healthcare) and sequenced at a
110
commercial company (StabVida, Portugal). The same pairs of primers used in the amplification of each
111
fragment were used in sequencing. Original chromatogram files were inspected and manually reviewed using
112
the ChromasPro® software (version 2.6.5).
113
sequences available in public databases (http://www.ncbi.nlm.nih.gov) by BLASTn analysis and submitted
114
to GenBank (http://www.ncbi.nlm.nih.gov).
Edited sequences were compared with similar reference
4
115
The phylogenetic relationships between the L. infantum sequence obtained during this study with other
116
available sequences in GenBank were investigated by Maximum Likelihood (ML) using MEGA7 software
117
(Kumar et al., 2016). The same software was used to perform the multiple sequence nucleotide alignments
118
(msa) and determine the appropriate substitution model, using the model selection analysis. Robustness of
119
the tree nodes was assessed by bootstrapping 1000 times. The graphical edition of the phylogenetic tree was
120
performed resourcing tree explorer, MEGA7 software (Kumar et al., 2016).
121 122
3. Results
123
Detection of Leishmania kDNA in the spleen samples of 18 red foxes, two stone martens, three European
124
badgers, three common genets and 101 of the surveyed Egyptian mongooses was negative. However, a
125
kDNA fragment of the expected size (approximately, 447 bp) was obtained in five Egyptian mongoose
126
samples among the 132 DNA spleen extracts analysed. A second, independent PCR directed towards the
127
ITS1 intergenic region was employed to test in parallel the 132 carnivore samples. Confirmation of the
128
presence of Leishmania DNA by both techniques was attained in only one spleen sample. This specimen
129
belonged to a female adult Egyptian mongoose collected in Beja district during 2011, southern Portugal. A
130
kDNA sequence with 402 bp obtained from the amplicon of this positive Egyptian mongoose was deposited
131
in GenBank under accession number MH799321 (on hold upon publication). Despite several attempts, it was
132
not possible to sequence the amplicon obtained from amplification of the ITS1 fragment.
133
Positive animals were detected in Beja (n=3), Portalegre (n=1) and Viseu (n=1) administrative units (Fig. 1).
134
Using partial kinetoplastid minicircle nucleotide sequences, the relationship between this strain and other
135
Leishmania infantum strains from nine countries, mostly from the Mediterranean basin (mainland Portugal,
136
Spain, Morocco, Tunisia, Algeria and Italy) was investigated by Maximum Likelihood (ML) phylogenetic
137
inference. The Hasegawa-Kishino-Yano (HKY) model (Hasegawa et al., 1985) with gamma distributed rate
138
variation among sites (+G) (HKY+G) showed the lowest BIC (2456.521) and AICc (2147.195) values and
139
was subsequently used to infer phylogenetic relationships using ML analysis. It is noteworthy that the
140
T92+G model also displayed similar BIC (2456.564) and AICc (2161.279) values.
141
Phylogenetic analysis suggests a higher resemblance of the L. infantum strain obtained from the Egyptian
142
mongoose (MH799321) with other L. infantum strains obtained in mainland Portugal from 1970 to 2003,
143
originating from dogs (Canis lupus familiaris) and humans (Homo sapiens), although the Bootstrap (Bs)
144
values supporting those nodes are <70 (Figure 2). This closer genetic relatedness is also suggested by the
145
high genetic similarity, with homologies ranging from 97,51% to 98,01% (Table 1, supplementary material),
146
and low genetic distances, ranging from 0,01282 to 0,01807 (Table 2, supplementary material), found
147
between strain MH799321 and the L. infantum strains from mainland Portugal.
148 149
4. Discussion
5
150
Leishmaniasis is a parasitic disease of great importance in veterinary medicine, considering the high number
151
of infected dogs, but also with a major impact on Public Health. Canine leishmaniasis is virtually present
152
throughout Portugal, although prevalence rates vary among regions. In the present study, the confirmation of
153
Leishmania infection in wild carnivores was based on the detection of Leishmania infantum nucleic acids in
154
spleen samples. Using an end-point PCR for the detection of kinetoplastid DNA, it was possible to identify
155
five positive animals among Egyptian mongoose. The kinetoplast DNA minicircle was selected as the
156
molecular target as it is present in hundreds of copies in Leishmania, increasing the probability of detecting
157
this protozoan in frozen samples in which microscopic analysis for detection of amastigotes or protozoan
158
isolation could not be performed. The animal from whom kDNA was sequenced was an adult female and
159
probably an asymptomatic carrier, since during the necropsy no evidence of clinical disease nor cutaneous or
160
visceral lesions were apparent. This specimen was collected in Beja district, one of the southern regions with
161
higher seroprevalence of canine leishmaniasis (12.12%) (Cortes et al., 2012). Sequencing analysis of the
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kinetoplastid DNA allowed the identification of L. infantum and phylogenetic inference by ML suggested a
163
closer genetic relationship of this strain with other L. infantum strains from mainland Portugal, originating
164
from dogs and humans. This geographic relationship of Leishmania sp. isolates and, particularly, the genetic
165
relatedness between strains circulating in the wild and in more humanized environments emphasize that the
166
interface between these two compartments at a local scale may be attenuated by the vector-borne nature of
167
Leishmania transmission.
168
The historical process that led to Egyptian mongoose colonization of Iberia is an issue that is still under
169
debate. While Gaubert et al. (2011) proposed that mongooses reached Iberia through the Strait of Gibraltar
170
during the Middle to Late Pleistocene, more recently Detry et al. (2018) collected data that suggested that
171
this herpestid might have been introduced by the Romans, during their presence in Hispania. This African
172
origin of mongoose could provide additional clues for an ancient Leishmania/mongoose relationship.
173
Mongoose have underwent a tremendous natural expansion process in the last decades from southern to
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northern Portugal. And recently, the species also invaded the North-eastern areas of the country, from where
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it was absent in the beginning of twentieth century (Barros et al., 2015, 2016). The recent abandon of
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croplands, rural depopulation, prodigious bio-ecological adaptability, generalist opportunistic behaviour and
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lack of natural predators possibly cumulatively led to this expansion success (Barros et al., 2015). Some
178
population biology aspects of this mesocarnivore species are still largely unknown, namely its health status,
179
contribution to pathogen cross-species transmission and competence as Leishmania spp. reservoir. Although
180
mongooses have distributions across several African regions and some SW European areas, Leishmania
181
infection has only been reported in two mongooses from Sudan (Elnaiem et al., 2001). In the report from
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Elnaiem and collaborators (2001), DNA sequences were not publicaly available for phylogenetic
183
comparison, but the authors reported that the species L. donovani was responsible for infection in mongoose.
184
A recent report identified L. infantum DNA in rodents, lagomorphs and wild carnivores from Southeast
185
Spain, including stone marten, common genet and red foxes (Risueño et al., 2018). The red fox was the
186
second most tested species [13.6% (18/132)] in our study. Although it is frequently reported as an L.
6
187
infantum carrier and a previous study performed in the 80’s in the Arrábida region (mainland Portugal)
188
identified positive animals (Abranches et al., 1983), no kDNA-positive red fox could be confirmed among
189
the population we surveyed. Other species, such as the stone marten, common genet and European badger
190
have occasionally been found positive in other regions of the world, but we did not find any evidence of
191
positive cases, which could also be related with the limited number of samples examined from these species.
192
This study enabled the detection of L. infantum infection in wildlife from Western Iberia. Such data
193
contributes to consolidate knowledge on the epidemiology of this pathogen in the Mediterranean basin and
194
identifying, for the first time, Herpestes ichneumon as a possible carrier host in Portugal. Although the
195
results obtained suggest that most mesocarnivore species are not particularly exposed to this parasite in
196
Portugal, it is necessary to carry out further studies to better clarify the role of wild carnivores and taxa, such
197
as leporids and rodents in the enzootic Leishmania transmission cycle and the potential consequences
198
thereof. The limited number of positive cases in this study may also suggest that tissue tropism of
199
Leishmania sp. may vary according to host species. To confirm this hypothesis, future studies should
200
consider testing skin and other organs besides spleen. Furthermore, xenodiagnostic analyses could also bring
201
some light into the role of this species as reservoir or as dead-end host. Anthropogenic changes of
202
ecosystems resulting in the expansion or overabundance of particular wild species, such as the mongoose,
203
may provide more opportunities for direct or indirect contact with domestic animals and humans,
204
significantly affecting parasite distribution and risk.
205
The surveillance programs directed towards wildlife or focused on particular pathogens should thus be
206
adapted and extended to other animal taxa and regions of the national territory. Such programs should be a
207
priority in areas where interaction between wild animals, domestic animals, and humans is pronounced, the
208
prevalence in dogs is high and the control strategy is insufficient. Scrutiny in areas wherein natural and
209
anthroponotic conditions propitiate proliferation of phlebotomine vectors should also be granted.
210 211
5. Acknowledgments
212
We are grateful to hunters and hunting organizations for animal carcasses donation. We acknowledge the
213
public company Estradas de Portugal IP for contributing with cadavers from accidental road-kills. We thank
214
Fernanda Simões (INIAV IP) for helpful suggestions. This work was partially funded by INIAV IP and by
215
national funds through Fundação para a Ciência e a Tecnologia (FCT/MEC) in the frame of the strategic
216
funding to cE3c and BioISI Research Units (UID/BIA/00329/2019 and UID/Multi/04046/2019). Financial
217
support from Programa de Desenvolvimento Rural, FEADER and P2020 are also gratefully acknowledged in
218
the scope of project Alliance-i9-Caça (ref. PDR2020-2024-049959).
219
Thanks are also due for the financial support to University of Aveiro (Department of Biology), to CESAM
220
(UID/AMB/50017) from FCT/MEC through national funds and the co-funding by the FEDER, within the
221
PT2020 Partnership Agreement and Compete 2020 by co-funding through the project "Genetic assessment of
7
222
a successful invasion: Population genetics of the Egyptian mongoose (Herpestes ichneumon) in Portugal",
223
PTDC/BIA-BEC/104401/2008.
224 225
6. Conflict of Interest Statement
226
On behalf of all authors, the corresponding author states that there is no conflict of interest.
227 228
7. Captions
229
Figure 1. Spatial distribution of wild carnivore samples in mainland Portugal. Administrative regions at the
230
district level are indicated. The overall proportion of samples per district is indicated by the grey scale.
231
The abreviatures of districts are as follows: Viana do Castelo (VC), Braga (BR), Vila Real (VR), Bragança
232
(BG), Porto (PT), Aveiro (AV) Viseu (VS), Guarda (GR), Coimbra (CM), Castelo Branco (CB), Leiria (LR),
233
Santarém (SA), Portalegre (PA), Lisboa (Lx), Setúbal (ST), Évora (EV), Beja (BJ) and Faro (FR).
234
White circles with numbers in red specify the number and location of Egyptian mongooses that were kDNA-
235
positive by PCR.
236
Figure 2. Maximum Likelihood (ML) phylogenetic tree of 27 L. infantum nucleotide sequences (410 nt long
237
in the final dataset, including gaps), obtained during this study (MH799321) and others available in
238
GenBank, based on the Hasegawa-Kishino-Yano model (HKY) (Hasegawa et al., 1985). The tree with the
239
highest log likelihood (-1026.87) is shown. Initial tree(s) for the heuristic search were obtained automatically
240
by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the
241
Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log
242
likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites
243
(5 categories (+G, parameter = 0.3289) (HKY+G). The tree was drawn to scale, with branch lengths
244
measured in the number of substitutions per site.
245
Robustness of the tree nodes was assessed by bootstrapping 1000 times. The graphical edition of the
246
phylogenetic tree was performed with tree explorer, MEGA7 software (Kumar et al., 2016).
247
Only bootstrap (BS) values equal or greater than 70 are shown on the tree, with the exception of the
248
MH799321 cluster wherein the Bs values, although <70, are displayed for the reader.
249
A 2-letter code and a specific colour (Top left) was attributed to each country for better identification of the
250
origin of each strain. Whenever possible, the host was identified by a specific shape (Top left), namely dog
251
(Canis lupus familiaris, star), human (Homo sapiens, triangle), Egyptian mongoose (Herpestes ichneumon,
252
square) and hoary fox (Lycalopex vetulus, diamond). Sampling dates are indicated, whenever available.
253
8
254
Table 1 (supplementary material). Percentage of similarity between nucleotide sequences from the
255
hypervariable region of Leishmania kinetoplast (mitochondrial) DNA (kDNA) minicircles obtained in
256
Portugal from 1970 to 2003.
257 258
Table 2 (supplementary material). Genetic distances (GD) matrix for the sequences used in this study,
259
including the Egyptian mongoose (Herpestes ichneumon) sequence MH799321, determined by the Tamura-
260
3-parameters model (T92) with discrete gama distribution (G). The GD between strain MH799321 and the
261
other strains used in the study is marked in bold.
262 263
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11
1
1
3
Figure 2. TN (Tunisia) DZ (Algeria) MA (Moroco) FR (France) SU (Soviet Union) ES (Spain) PT (Portugal) IT (Italy) UK (United Kingdom)
Canis lupus familiaris Homo sapiens Herpestes ichneumon Lycalopex vetulus
MH799321 (Herpestes ichneumon) (2012)
45 39
JQ609530 (1998)(a) JQ609526 (1970)(b) AF103740 (1988) KX098512 AF169131 (1988)(c) JQ609527 (1998)(d) AJ275332 (1997) AF184044 (1980) JQ609524 (1980)
86
KX098510 AF169133 (1985) AF169140 AF169139 KX098513 KX098511 93
AF103735 (1978) KX098514 100
AF167716 (1992) AF190476 (1984) AF190475 (1991)
0.020 (a) Identical
to sequences to sequence (c) Identical to sequences (d) Identical to sequence (b) Identical
JQ609531 (1998) and JQ609533 (1997) JQ609528 (2000) KX098509 and AF103741 (1978) JQ609529 (2003)
Highlights •
Leishmania sp. occurrence in wild carnivores (n=132) from Portugal was assessed by PCR
•
Egyptian mongoose, red fox, stone marten, common genet and European badger were tested
•
Five Egyptian mongoose were confirmed Leishmania positive by kDNA-PCR
•
The 402-nt long sequence of mongoose kDNA amplicon displayed high similarity (>97,51%) with L. infantum from dogs and humans
•
Phylogenetic analysis by Maximum Likelihood clustered mongoose strain with L. infantum from Portugal
•
L. infantum strains circulating in sylvatic animals are genetically related with urban strains
Conflict of Interest Statement On behalf of all authors, the corresponding author states that there is no conflict of interest.