High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia.

BACKGROUND: Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia. METHODS: Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers. RESULTS: The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples. CONCLUSIONS: The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.

Atelerix algirus, the North African Hedgehog: Suitable Wild Host for Infected Ticks and Fleas and Reservoir of Vector-Borne Pathogens in Tunisia.

Small wild mammals are an important element in the emergence and transmission of vector-borne pathogens (VBPs). Among these species, hedgehogs have been found to be a reservoir of VBPs and host of arthropod vectors. Surveillance of VBPs in wildlife and their arthropods are crucial in a one health context. We conducted an exploratory study to screen Atelerix algirus hedgehogs and their infesting ticks and fleas for VBPs using a high throughput microfluidic real-time PCR system. Tested biopsies from hedgehogs were found to be naturally infected by Theileria youngi, Hepatozoon sp., Ehrlichia ewingii, Coxiella burnetii, and Candidatus Ehrlichia shimanensis. Similarly, Haemaphysalis erinacei and Rhipicephalus sanguineus tick species were infected by Ehrlichia ewingii, Rickettsia spp., Rickettsia massiliae, Borrelia sp., Coxiella burnetii, Rickettsia lusitaniae and Anaplasma sp. Archaeopsylla erinacei fleas were infected by Rickettsia asembonensis, Coxiella burnetii, and Rickettsia massiliae. Co-infections by two and three pathogens were detected in hedgehogs and infesting ticks and fleas. The microfluidic real-time PCR system enabled us not only to detect new and unexpected pathogens, but also to identify co-infections in hedgehogs, ticks, and fleas. We suggest that hedgehogs may play a reservoir role for VBPs in Tunisia and contribute to maintaining enzootic pathogen cycles via arthropod vectors.

Presence of Sergentomyia (Parrotomyia) lewisi (Diptera: Psychodidae) in Tunisia.

In Tunisia, 17 phlebotomine sand fly species are reported, belonging to genera Phlebotomus and Sergentomyia. However, subsequent to faunal studies and outbreaks of leishmaniasis in different regions of the country, unrecognized sand fly species could exist. Indeed, we report in this study the presence of Sergentomyia (Parrotomyia) lewisiParrot 1948. A brief collection of phlebotomine sand flies was undertaken in August 2016 in Khbina locality, in Sidi Bouzid governorate situated in Central Tunisia, which constitutes an old focus of Zoonotic Cutaneous Leishmaniasis. Sand flies were collected, using CDC light traps that were placed overnight, in different biotopes (inside habitations, outdoors, and within animal shelters). Specimens were collected and morphologically identified. Measurements were taken with an ocular micrometer. Two female specimens of the species Se. (Parrotomyia) lewisi were collected. One specimen is here described and measured. A comparison of its taxonomic characters to the holotype from Sudan and a specimen from Algeria is also presented. This species was until now only reported from Sudan, Ethiopia, Algeria, and Morocco. It is here described for the first time in Tunisia, which raises the Tunisian sand fly fauna to seven subgenera and 18 species.

Paraechinus aethiopicus (Ehrenberg 1832) and Atelerix algirus (Lereboullet 1842) hedgehogs: Possible reservoirs of endemic leishmaniases in Tunisia.

Rodents and dogs are the confirmed leishmaniases reservoir hosts in Tunisia. Recently, we described hedgehog Leishmania (L.) major and L. infantum infection in an L. infantum endemic area in the North-West. In order to assess if the observation could extend to other endemic areas and to highlight the potential role of hedgehogs as reservoir host, we aimed here at investigating their Leishmania infection in different foci in Tunisia located along a North-South transect, during and outside different transmission seasons. Based on morphological criteria, 2 hedgehogs' species, Atelerix algirus and Paraechinus aethiopicus were identified. Cytologic analysis showed presence of amastigotes in 9/22 samples corresponding to 4 Atelerix algirus specimens. Also, by combining 3 PCR tests targeting repeated DNA fragments using 13A/13B, Lei70R/Lei70L and nested T2/B4-L1/L4 specific primers, all hedgehogs (N = 12) showed a Leishmania infection. The infection rates were very high on spleen (91.66%), kidney (91.66%), blood (90.90%), liver (83.33%) and eye swabs (100%). Parasites were also detected in peritoneum. Three hedgehogs were found infected with L. infantum and the only Paraechinus aethiopicus specimen with L. major. A mixed L. major and L. infantum infection was identified in 8 animals, while the last one also had an L. tropica infection. Interestingly, 2 animals had skin lesions infected with L. major while all others appeared asymptomatic. There was a correlation between infected status and epidemiological profiles of the localities. Sequences and phylogeny indicated micro-heterogeneity and lack of correlation with sampling, season, or localities. We confirmed natural infection of Atelerix algirus and originally of Paraechinus aethiopicus in Tunisia. High rate of asymptomatic infection, parasitemia, proximity to transmission cycles, epidemiological patterns of infection together with hedgehogs' abundance, lifespan and lifestyle corroborate the hypothesis they constitute reservoir hosts.

Molecular Characterization of Leishmania Parasites in Giemsa-Stained Slides from Cases of Human Cutaneous and Visceral Leishmaniasis, Eastern Algeria.

BACKGROUND: In Algeria, visceral leishmaniasis (VL) is due to Leishmania (L.) infantum, while three cutaneous forms (CL) are caused by Leishmania major, Leishmania tropica and Leishmania infantum. In this study, the use of Giemsa-stained slides was evaluated with two PCR techniques, in Eastern Algeria. MATERIALS AND METHODS: A total of 136 samples corresponding to 100 CL smears (skin scrapings) and 36 VL slides (bone marrow aspirates) collected from 2008 to 2014 were tested. Upon DNA extraction, two PCRs were used to amplify the ribosomal Internal Transcribed Spacer 1 (ITS1) and mini-exon genes. Amplified products were digested (PCR-RFLP) and profiles analyzed for Leishmania species identification. A statistical analysis was also performed. RESULTS: ITS1-PCR was found significantly more sensitive than mini-exon-PCR (77.95% positives vs. 67.65%; p = 0.001). Comparison of PCR positivity showed statistically significant differences between old and recently prepared slides suggesting a better use of recent slides in PCR analyses. For species identification, PCR-restriction fragment length polymorphism (RFLP) results of ITS1 and mini-exon were concordant. L. infantum was identified from VL cases and L. infantum, L. major, and L. tropica from CL ones. According to geographical origin, L. infantum was found in North-Eastern provinces, while L. major was distributed from the North to the Center-East of Algeria. Interestingly, two L. tropica samples were identified in Annaba, located far North-East Algeria. CONCLUSION: Distribution of leishmaniasis in Eastern parts of Algeria, besides finding of L. tropica in the far North, is in this study described for the first time using molecular tools, thus confirming the usefulness of slides for PCR identification of Leishmania parasites in retrospective epidemiological investigations.

Natural infection of Algerian hedgehog, Atelerix algirus (Lereboullet 1842) with Leishmania parasites in Tunisia.

In Tunisia, Leishmania parasites are responsible of visceral leishmaniasis, caused by Leishmania infantum species while three cutaneous disease forms are documented: chronic cutaneous leishmaniasis due to Leishmania killicki, sporadic cutaneous form (SCL) caused by L. infantum and the predominant zoonotic cutaneous leishmanaisis (ZCL) due to Leishmania major. ZCL reservoirs are rodents of the Psammomys and Meriones genera, while for SCL the dog is supposed to be a reservoir. Ctenodactylus gundii is involved in the transmission of L. killicki. However, other mammals could constitute potential reservoir hosts in Tunisia and other North African countries. In order to explore the role of hedgehogs as potential reservoirs of leishmaniasis, specimens (N=6) were captured during July-November period in 2011-2013 in an SCL endemic area in El Kef region, North-Western Tunisia. Using morphological characteristics, all specimens were described and measured. Biopsies from liver, heart, kidney and spleen of each animal were used to extract genomic DNA, which was further used in PCR assays to assess the presence of Leishmania parasites. Different PCRs targeting kinetoplast minicircles, ITS1, mini-exon genes and a repetitive Leishmania- specific sequence, were applied. To further identify Leishmania species involved, RFLP analysis of amplified fragments was performed with appropriate restriction enzymes. Using morphological characters, animals were identified as North African hedgehogs, also called Algerian hedgehogs, that belong to the Erinaceidae family, genus Atelerix Pomel 1848, and species algirus (Lereboullet, 1842). PCR results showed in total that all specimens were Leishmania infected, with different organs incriminated, mainly liver and spleen. Results were confirmed by direct sequencing of amplified fragments. Species identification showed that all specimens were infected with L. major, three of which were additionally co-infected with L. infantum. The present study demonstrates, for the first time in Tunisia, natural infection of hedgehog animals (Atelerix algirus) by the Leishmania parasites species L. major and L. infantum. L. major is also detected for the first time in wild animals captured in the North Western part of the country; likewise for the co-infection of these animals by the 2 Leishmania species. This mammal could play a potential reservoir role in epidemiology of SCL or ZCL and could contribute to emergence or extension of ZCL in the studied region.

Screening and characterization of RAPD markers in viscerotropic Leishmania parasites.

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5' end transversions, and presence of inter- and intra- taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.

Evaluation of a gp63-PCR based assay as a molecular diagnosis tool in canine leishmaniasis in Tunisia.

A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non-endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8-95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5-71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.

Natural infection of Phlebotomus (Larroussius) langeroni (Diptera: Psychodidae) with Leishmania infantum in Tunisia.

Phlebotomine sand flies were captured from an active transmission focus of sporadic cutaneous leishmaniasis, caused by Leishmania infantum, in El Kef region, northern Tunisia. Both Phlebotomus perniciosus and P. langeroni were found. Phlebotomus langeroni females showed a statistically significant intradomiciliary dominance (P<0.01 for the 2003 and 2004 seasons) when compared to animal shelters. During the 2003 season, dissection of collected female specimens showed the presence of flagellates within the digestive tracts of two P. perniciosus among 1086 observed, but none in 232 P. langeroni. Amplification of kinetoplast minicircles of Leishmania parasites was applied to DNA samples extracted from 298 frozen females including 249 P. perniciosus, 36 P. langeroni, 5 P. longicuspis and 8 P. perfiliewi and revealed by radioactive probe hybridization. Two P. langeroni females showed a signal of the size expected for L. infantum (800bp) indicating infection with these parasites. However, this PCR-hybridization method failed to identify any positive P. perniciosus females in pools of specimens. These results show for the first time the natural infection of P. langeroni with L. infantum in Tunisia, and support the existence of different L. infantum transmission cycles in Tunisia, with a potential role for P. langeroni as a vector.

Occurrence of Leishmania infantum cutaneous leishmaniasis in central Tunisia.

Cutaneous leishmaniasis (CL) due to Leishmania infantum occurs sporadically in Tunisia where its distribution is confined to the northern parts of the country. However, during the past decade there have been occasional repeated reports of cases from areas in central Tunisia, known to be free of CL. Epidemiological, clinical and parasitological data regarding these patients were collected and analysed. Data were very suggestive of the sporadic form of CL due to L. infantum. The parasites contained within the lesions of some of the patients were characterised by two different previously described PCR assays, each having different resolutive powers. The first assay, which amplified complete kDNA minicircles, showed a fragment size characteristic of the L. donovani complex; whilst the second consisted of a PCR-RFLP analysis targeting the gp63 coding sequences that confirmed assignment of the parasites to L. infantum species while illustrating its differences from the reference isolate. These findings confirm the aetiology of CL in the concerned areas in central Tunisia and suggest that L. infantum CL might be more prevalent and widespread than previously thought, or possibly emerging in these areas.