Leishmania tropica Infection in Golden Jackals and Red Foxes, Israel

During a survey of wild canids, internal transcribed spacer 1 real-time PCR and high-resolution melt analysis identified Leishmania tropica in samples from jackals and foxes. Infection was most prevalent in ear and spleen samples. Jackals and foxes may play a role in the spread of zoonotic L. tropica.


The Study
Wild golden jackals, red foxes, and gray wolves were trapped at 57 different locations in Israel as part of a survey for oral rabies vaccination conducted by the Israeli Nature and Parks Authority and the Veterinary Services. DNA was extracted from ear pinna, snout, blood, and spleen by using the guanidine thiocyanate technique (11); in some animals, samples were not available from all sites. DNA from all tissues was tested for Leishmania spp. infection by internal transcribed spacer 1 (ITS1) real-time PCR and high-resolution melt analysis (ITS1-HRM) PCR (12). A 265-288-bp fragment, depending on the Leishmania species, within the ITS1 region of the leishmanial rRNA was amplifi ed as previously described (12). All samples were tested in duplicates and results were compared with those from HRM analysis of positive controls for each assay. These were L. infantum (MCAN/IL/2002/Skoshi), L. tropica (MHOM/IL/2005/ LRC-L1239), and L. major (MHOM/TM/1973/5ASKH). Negative controls included samples from jackals born and reared at a zoo in central Israel, as well as from foxes and wolves from areas in which leishmaniasis is not endemic, that were tested by PCR and found negative. All positive PCR products were purifi ed by using ExoSAP-IT (USB, Cleveland, OH, USA) and sequenced at the Center for Genomic Technologies, Hebrew University of Jerusalem. Sequences obtained were compared for similarity to sequences in GenBank by using the BLAST program (www.ncbi. nlm.nih.gov/BLAST). Positive samples also were verifi ed by kDNA PCR as described (13).
Sequences were analyzed by using MEGA version 3.0 (www.megasoftware.net). A phylogenetic tree was constructed by using the neighbor-joining method in agreement with maximum-parsimony and maximum-evolution algorithms and by using the Kimura 2-parameter model with uniform rates for transitions and transversions. Bootstrap replicates were performed to estimate the node reliability, and values were obtained from 1,000 randomly selected samples of the aligned sequence data. Sequences were compared with the following Leishmania sequences deposited in GenBank: L. tropica FJ595949 and FJ595950 from central Israel and IARA/IL/02/LRC-L910 and ISER/IL/02/ LRC-L909 from northern Israel; L. infantum (MHOM/ TN/1980/IPT1) and L. major (MHOM/TM/1973/5ASKH) were used as outgroups.
We examined 208 samples from 113 wild canids by ITS1-HRM PCR: 152 samples from 77 golden jackals, 44 from 25 red foxes, and 12 samples from 11 wolves. None of the animals had clinical signs attributed to leishmaniasis. Seven animals tested positive for L. tropica, and 1 was positive for L. infantum. The overall Leishmania infection rate for jackals was 7.8% (6/77) and for foxes 8% (2/25). All wolves were negative. Fourteen tissue samples (ear, snout, spleen, and blood) tested positive. Five (63%) of the 8 animals positive for Leishmania spp. had >2 infected tissues (Table). Ears were positive for 6 of 8 infected animals and spleen for 4 of 8 animals. The snout sample was positive for another animal for which blood also was positive. Four (15%) of 26 spleens collected were positive.
The ITS1-HRM PCR DNA product size was 265 bp for L. infantum and 273 bp for L. tropica. Sequencing verifi ed the species specifi c results. All samples positive by ITS1-HRM PCR were also positive by kDNA PCR and produced a 120-bp kDNA product.
Thirteen sequences from positive DNA products obtained by ITS1-HRM PCR were identifi ed as belonging to L. tropica, showing the closest similarity (98%-99%) to L. tropica sequences deposited in GenBank (Table). Only 1 sequence was amplifi ed by using DNA extracted from the blood of a jackal for which other tissues were not available. This sequence was closest (99.5% identity over 222 bp) to L. infantum (Table). DNA sequences from all the positive tissues belonging to an individual animal were aligned, and consensus sequences representing each animal were creat-ed. These consensus sequences were deposited in GenBank under accession nos. GU591390-GU591397 and included in the phylogenetic tree (Figure). By using 3 algorithms, sequences obtained from 7 wild canids clustered with L. tropica isolated from hyraxes in central Israel (FJ595949 and FJ595950), and the sample amplifi ed from the jackal blood clustered with L. infantum (MHOM/TN/1980/IPT1).

Conclusions
We report L. tropica infections in jackals and foxes from Israel. Sequence analysis (using 3 algorithms) of ITS1 fragments showed perfect correlation with L. tropica isolates from hyraxes in central Israel. The fi nding of L. tropica positivity in >1 tissue sample from infected asymptomatic animals implies that wild canid species could be natural hosts for this parasite. Furthermore, the relatively high percentage of infected spleens indicates that this parasite can visceralize in foxes and jackals. Unlike hyraxes, which generally stay close to their burrows in caves or boulders, jackals and foxes travel long distances, potentially transmitting L. tropica from 1 area to another, provided that competent sandfl y vectors are found. The home range of golden jackals in Israel is adapted to the food resources available. For golden jackals, it was 6.6 km 2 near settlements and 21.2 km 2 in sparsely inhabited settings (14). Foxes may foray 5.3 km and less frequently roam 7.8 km-15 km (15). Wild canids may transmit L. tropica from an area with an infected population of hyraxes to a remote naive hyrax population or be responsible for infecting humans because they tend to live peridomestically and frequently rely on human waste. The involvement of wild canids in the sylvatic life cycle of L. tropica could be crucial to understanding disease emergence in Israel, Jordan, and the Palestinian Authority (2). Populations of jackals and foxes, which were nearly eliminated in Israel during 1950-1980, have recovered and grown in parallel with the local outbreaks of L. tropica in humans (10). Further study is required to discern the potential epidemiologic role of wild canids in spreading and transmitting infection.