الفهرس 
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Abstract
Background:
Leishmaniasis is one of the eight priority targets of the WHO and represents a major health problem in many parts of the world especially Mediterranean and Middle East countries. In Egypt, the number of cases is probably consistently under-reported; about 15-25 suffers CL and none suffer VL since 1995 according to ministry of health annual report. The wide distribution of CL was observed throughout the last decade in Northern and Southern Sinai. L. major was the culprit parasite investigated from cases, sandflies, and reservoirs. P. papatasi was the potent vector throughout all endemic foci. Screening of endemic foci is of necessity to predict the risk that disease can cause.
In December, 2006, doctors from the local hospital in El Barth village, Rafah, Northern Sinai, Egypt, clinically diagnosed ten cases of CL. On the basis of this information, preliminary field investigations ”observations” including sandflies, rodents and Leishmania parasite were studied in El Barth village to understand the transmission cycle in the region and for the implementation of strategic vector control measures and disease prevention tactics of cutaneous leishmaniasis in this recent focus of the disease throughout North Sinai, Egypt.
Two approaches were involved in the current study; Field approach and experimental one. Here is the summary to the results obtained in this study;
A) Field approach:
1. Sandfly identification and parasite isolation:
A total of 2049 sandflies were identified to the species level. P. papatasi accounted for 81.6% (1138♀♀, 536 ♂♂) of the identified flies and the remaining flies, 18.4%, (169♀♀, 206 ♂♂) were P. sergenti. Based on microscopy, seven P. papatasi had Leishmania-like flagellates in their midgut, of which four produced viable cultures identified as L. major.
2. Sandfly activity rhythm:
Both sandfly species, P. papatasi and P. sergenti, exhibited nocturnal activity and peaked after midnight (24:00 – 2:00 a.m.). The weighted average of caught sandflies is plotted against the collection time. A gradual increase in the numbers of captured sandflies was recognized starting from 18:00 till the peak at midnight then a gradual decrease took place till dawn 4:00 – 6:00. Just after sunrise, (during the month of May sun rises a few minutes past 6:00) sandflies were absent from sticky paper traps recovered.
3. Rodent Species:
Eighty-one individuals from 5 rodent species were collected: Gerbillus pyramidum floweri (n = 29), G. andersoni (n = 26), Meriones sacramanti (n = 18), M. crassus (n= 7) and Mus musculus (n = 1). Of 25 rodents with cutaneous lesions, 19 (all of genus Gerbillus) produced positive amastigote smear tests and 14 (11 G. pyramidum floweri and 3 G. andersoni) produced viable parasite cultures. Cultures from 2 G. pyramidum floweri were identified as L. tropica (samples 6 and 7) and the remaining rodent-derived cultures harbored L. major.
4. Leishmania species identity.
Twenty-four out of 28 Sinai isolates were positive for Leishmania DNA using the LEIS primer-probe set, however, only 19 of these were also positive for L. major DNA. The remaining 5 isolates (samples 4--8) were positive for Leishmania DNA but negative to L. major DNA confirming the presence of Leishmania parasites (Table 7). ITS1 DNA sequences were obtained for samples number 2 (accession FJ460456), 4 (FJ460457), 5 (FJ460459) and 6 (FJ460458). Sample 2 was confirmed as L. major; the sequence is identical to several L. major sequences deposited in GenBank including isolates from Iran, Kenya, Jericho and Sinai. Of the remaining 3 samples, samples 4 and 6 were identical and differed from sample 5 at 3 of 322 nucleotides. These samples had highest overall BLAST similarity to a L. tropica strain from Sudan (97% identity, MHOM/SU/60/OD).
A maximum parsimony gene tree constructed with these sequences and homologues from GenBank shows a tight clustering of L. major sequences, including sample 2, and a looser, but definitive clustering of samples 4-6 with other L. tropica sequences. Phylogenetic analysis yielded a single best tree when using maximum parsimony, 500 bootstraps, and completes deletion of gaps; the 348 nucleotide alignment had 33 parsimony informative sites. The Sinai L. tropica sequences ally most closely to an Iranian isolate (MHOM/IR/02/Mash_10), principally due to shared similarity at the 3’ end of the fragment. RFLP analysis supports these results. Samples 2 and 3 and the L. major reference DNA produced fragments characteristic of L. major while samples 4--8 and the L. tropica reference DNA had the pattern typical for L. tropica when RFLPs procedure was applied.
5. Visceralization of L. tropica in the naturally infected Gerbillus pyramidum floweri.
Visceral infection was detected in only a single rodent out of 19 (5.26%). The spleen of infected rodent was large in size, dark in color, soft, and fragile versus the normal one.
B) Experimental approach:
1. Susceptibility of different rodent species to Leishmania tropica:
The susceptibility of different rodent species to L. tropica were arranged in ascending pattern from BALB/C, golden hamster (Mesocricetus auratus), G. pyramidum, G. andersoni, M. sacramanti, Mus musculus and M. crassus. BALB/C and golden hamster (experimental animals) were more susceptible to L. tropica strain MHOM/EG/06/RTC66 as compared to the wild rodents depending on the time required for the first appearance of lesion(s). M. crassus, M. musculus appeared to be more tolerant to L. tropica infection, since, longer periods of 71.5 ± 7.6 and 70.2 ± 7.9, respectively were required for the first appearance of the lesion.
2. Experimental infection of P. papatasi and P. sergenti with L. major:
L. major promastigotes began to appear in the anterior and posterior midgut of both species. On the 3rd day, midgut infection rates were 73.3%, 50% for P. papatasi and P. sergenti, respectively. On 5th – 7th days post-infection, infection rates were heavy (93.3-100%) in P. papatasi, on the other hand were lower in P. sergenti (30–60%). By the 5th day; L. major migrated anteriorly in only P. papatasi to colonize in anterior midgut, and stomodeal valve. In P. sergenti, only a single female maintained the parasite stomodeal valve by the 5th day. By the 5th day, among the positive females, stomodeal valve was colonized by promastigotes in 85.7% P. papatasi, and 16.7% P. sergenti of infected flies. The latter finding reflects the vector-parasite interaction in case of P. papatasi and L. major.
3. Experimental infection of P. papatasi and P. sergenti with L. tropica:
Susceptibility of P. sergenti to L. tropica originally isolated from the study area was high (66.7- 93.3%). In contrast, P. papatasi infection rates were low (26.7-53.3%). Transformation of amastigote form was observed within the first 24 hours post-infection. The promastigotes migrate to stomodeal valve on 1, 3 days post-infection in P. sergenti and P. papatasi, respectively. By the 5th day, the pharynx was colonized by promastigotes in some positive females; 50 % and 16.7 % for P. sergenti and P. papatasi, respectively. On the 7th day, the infection rate in proboscis of P. sergenti was high (78.6% of positive females) in comparison to that of P. papatasi (37.5 % of positive females). Throughout this time, the promastigotes intensity is heavy in P. sergenti and low in P. papatasi although their forward migration to proboscis of both species.
4. Experimental transmission of L. major and L. tropica to BALB/C mice by the bite of P. papatasi and P. sergenti.
Four out of nine mice used in experiment developed characteristic Leishmania lesion/s; only a single BALB/c mouse out of three exposed to the bites of P. papatasi/ L. major and the three BALB/c mice exposed to the bites of P. sergenti/ L. tropica. None of rodents subjected to the bites of P. papatasi infected with L. tropica developed any characteristic lesion/s.
A foot lesion was observed on the footpad of BALB/C mouse after 38 days from feeding of P. papatasi infected with L. major whereas a mean of 18±2 days were required for lesion appearance after the bites of P. sergenti infected with L. tropica.