Effects of morphine on the biomass and development rate of Chrysomya albiceps (Diptera: Calliphoridae), a forensically important species.

The aim of this study was to determine the effects of morphine on the biomass and development rate of Chrysomya albiceps (Diptera: Calliphoridae). C. albiceps, a well-known forensically important species which is among the first wave of faunal succession on human cadavers, which makes it a valuable source of information for the estimation of postmortem interval (PMI). Antemortem exposure to substances such as drugs and toxins may have an effect on the biomass and/or on the development rate of insects that feed on carcass, which may directly affect PMI estimation. In this study, three rabbits were administered 12.5, 25 or 50 mg/ml of morphine sulfate via ear perfusion over a period of 3 hours, and a fourth rabbit, which did not receive morphine, was used as a control. The rabbits were sacrificed using chloroform 30 minutes after morphine administration. The tissues were analyzed for the presence of morphine using HPLC-UV. Morphine was detected in all tissues of rabbits that received morphine, except in the bile and spleen of the rabbit which received 12.5 mg/ml dose of morphine. The presence of morphine in rabbit tissues retarded larval development rate, but accelerated the puparial development rate. The rate of development of C. albiceps larvae that fed on rabbits which received 25 and 50 mg/ml dosages of morphine was 9 days each. However, the rate of larval development was similar in the 12.5 mg/ml morphine group and the control; 6 days. Results of this study show that an underestimation of the postmortem interval of 72 h based on larval development and an overestimation of 24 to 48 h based on puparial development is possible if the presence of morphine in tissues is not considered. Moreover, the decreased larval development rate caused an increase larval length and weight compared with the control group. In this study, we found a strong correlation between the concentration of morphine administered and concentrations in rabbit tissues. In the estimation of PMI, it is recommended that effects of drugs such as morphine on the development of carcass colonizers be considered.

Detection of Leishmania major DNA within wild caught Phlebotomus papatasi and species composition of sand flies in endemic focus of cutaneous leishmaniasis, in western Iran.

Cutaneous leishmaniasis is one of the most important public health problem in many developing countries. The present study was conducted to determine the vector(s), the parasite and the species composition of sand flies in the Dehloran County during May-November 2012. Sand flies were collected by sticky traps and mounted in Puri's medium for species identification. Polymerase chain reaction (PCR) techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism (RFLP) were used for identification of DNA of Leishmania parasites in infected sand flies. A total of 82443 specimens comprising 15 species of sand flies (5 Phlebotomus and 10 Sergentomyia) were collected and identified. The species of Phlebotomus papatasi was dominant in outdoor and indoor resting places. Among the 280 specimens of female P. papatasi tested by PCR of kDNA, ITS1-rDNA genes of the parasite followed by RFLP, only 5 of them (1.8 %) were positive to Leishmania major parasites. This is the first molecular detection of leishmania infection of P. papatasi to L. major in this region. The results indicated that, P. papatasi was only species found infected by L. major and the principal vector of disease agent to human.

First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in South-eastern Iran.

OBJECTIVE: To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis. METHODS: Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer (ITS1) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Leishmania species in sand flies specimens. RESULTS: Three out of 115 females of Phlebotomus sergenti (P. sergenti) (2.6%) were positive to Leishmania tropica (L. tropica). CONCLUSIONS: This is the first report on P. sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HRM analysis. This method is rapid, sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.

Natural infection of wild caught Phlebotomus tobbi to Leishmania infantum in East Azerbaijan province, northwestern Iran.

BACKGROUND & OBJECTIVES: Zoonotic visceral leishmaniasis is caused by Leishmania infantum, which is transmitted to humans by bites of phlebotomine sandflies and is one of the most important public health problems in Iran. To detect and identify the Leishmania parasites and their corresponding vector(s), an investigation was carried out in Azarshahr County, a new and important focus of the disease in East Azerbaijan province in northwestern Iran during late April to late October 2010. METHODS: Sandflies were sampled using sticky papers (A4 white paper soaked in castor oil) from inside and outside of the houses and animal shelters, close to the vegetation and crevices. The head and three last abdomen segments of the specimens were removed and mounted in Puri's medium for species identification. The rest of body was subjected to molecular methods for detection of leishmanial parasites. RESULTS: Among 400 female sandflies tested by polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP), only 2 out of 8 Phlebotomus tobbi were positive to L. infantum parasites. CONCLUSION: The results indicated that, P. tobbi was the only species found infected by L.infantum and the principal vector of the disease agent to human.

Molecular Detection of Leishmania infantum in Naturally Infected Phlebotomus perfiliewi transcaucasicus in Bilesavar District, Northwestern Iran.

BACKGROUND: Visceral leishmaniasis is caused by Leishmania infantum, transmitted to humans by bites of phlebotomine sand flies and is one of the most important public health problems in Iran. To identify the vector(s), an investigation was carried out in Bilesavar District, one of the important foci of the disease in Ardebil Province in northwestern Iran, during July-September 2008. METHODS: Using sticky papers, 2,110 sand flies were collected from indoors (bedroom, guestroom, toilet and stable) and outdoors (wall cracks, crevices and animal burrows) and identified morphologically. Species-specific amplification of promastigotes revealed specific PCR products of L. infantum DNA. RESULTS: SIX SAND FLY SPECIES WERE FOUND IN THE DISTRICT, INCLUDING: Phlebotomus perfiliewi transcaucasicus, P. papatasi, P. tobbi, P. sergenti, Sergentomyia dentata and S. sintoni. Phlebotomus perfiliewi transcaucasicus was the dominant species of the genus Phlebotomus (62.8%). Of 270 female dissected P. perfiliewi transcuacasicus, 4 (1.5%) were found naturally infected with promastigotes. CONCLUSION: Based on natural infections of P. perfiliewi transcaucasicus with L. infantum and the fact that it was the only species found infected with L. infantum, it seems, this sand fly could be the principal vector of visceral leishmaniasis in the region.

Phlebotomus perfiliewi transcaucasicus, a vector of Leishmania infantum in northwestern Iran.

Visceral leishmaniasis is caused by Leishmania infantum, which is transmitted to humans by bites of phlebotomine sand flies and is one of the most important public health problems in Iran. To identify the vector(s), an investigation was carried out in Germi district, an important focus of the disease in Ardebil province in northwestern Iran, during July-September 2004 and 2005. Using sticky papers, CDC light traps and aspirators, 3,560 sand flies were collected and identified to species. Host bloodmeal preference and Leishmania infections in female specimens were evaluated using enzyme-linked immunosorbent assay (ELISA) for the former and microscopic examination followed by polymerase chain reaction (PCR) assay using species-specific kinetoplast minicircle primers for the latter. Nine sand fly species are present in the district, including Phlebotomus kandelakii Shchurenkova, Phlebotomus perfiliewi transcaucasicus Perfil'ev, Phlebotomus major Annandale, Phlebotomus balcanicus Theodor, Phlebotomus halepensis Theodor, Phlebotomus brevis Theodor & Meshghali, Phlebotomus papatasi Scopoli, Sergentomyia dentata Sinton, and Sergentomyia sintoni Pringle, with P. p. transcaucasicus being the most prevalent representative of the genus Phlebotomus at 45%. The anthropophilic index for P. p. transcaucasicus was 36.3%, indicating a strong preference for humans. Of 905 female P. p. transcuacasicus dissected, 10 (1.1%) were found naturally infected with promastigotes. Species-specific amplification of promastigotes eluted from Giemsa-stained slides revealed specific PCR products of L. infantum DNA. Based on its high anthropophily and natural infections with L. infantum, and the fact that it was the only species found infected with L. infantum, we conclude that P. p. transcaucasicus is the principal vector of L. infantum in northwestern Iran.

Identification of Sand flies of the Subgenus Larroussius based on Molecular and Morphological Characters in North Western Iran.

BACKGROUND: THE ADULT FEMALE SAND FLIES (DIPTERA: Psychodidae) of the subgenus Larroussius are important vectors of Leishmania infantum (Kinetoplastida: Tripanosomatidae) in Meshkinshahr district, Northwest of Iran. Four Phlebotomus (Larroussius) species are present in this area, i.e. Phlebotomus (Larroussius) kandelakii, P. (La.) major, P. (La.) perfiliewi and P. (La.) tobbi. The objective of the present study was to identify and distinguish the females of P. perfiliewi, P. major and P. tobbi, in this district. METHODS: Adult sand flies were collected with sticky papers, CDC light traps, and aspirator in 2006. Individual sand flies of this four species from thirty different locations were characterized morphologically and by comparative DNA sequences analyses of a fragment of mitochondrial gene Cytochrome b (Cyt b) and nuclear gene Elongation Factor 1-alpha (EF-1α). PCR amplification was carried out for all three species P. major, P. perfiliewi and P. tobbi in the subgenus Larroussius. RESULTS: Phylogenetic analyses of P. major populations in this study displayed two different populations and genetic diversity. Spermathecal segment number, pharyngeal armature and other morphological characters of these three species were examined and found to present consistent interspecific differences. CONCLUSION: According to our findings, the phylogeny of Cyt b and EF-1α haplotypes confirms the relationships between P. major, P. tobbi and P. perfiliewi as already defined by their morphological similarities.

Molecular data on vectors and reservoir hosts of zoonotic cutaneous leishmaniasis in central Iran.

Due to the increasing number of positive cases of cutaneous leishmaniasis with occurrence of new foci, a study was carried out to investigate on vectors and reservoirs of the disease in the Shahrood district, central Iran during 2005-2006. Sandflies and rodents were collected using sticky papers and Sherman live traps respectively More than 1700 sandflies were collected and identified, mainly Phlebotomus papatasi species. RAPD-PCR analysis of sandflies showed that 10% of P. papatasi and 4.2% of P. caucasicus were naturally infected with Leishmania major. Two species of rodents, potential reservoirs, Rhombomys opimus (92.5%) and Nesokia indica (7.5%) were trapped in the district. Microscopy identification from rodents confirmed that 91.9% of the Rhombomys opimus were positive to amastigotes. Species identification of isolated parasites revealed Leishmania major DNA in the infected Rhombomys using RAPD-PCR technique. This epidemiological data highlight the importance of the disease in the region and could help people involved in control programs.

Vectors and reservoirs of cutaneous leishmaniasis in Marvdasht district, southern Islamic Republic of Iran.

An epidemiological study was made of vectors and reservoirs of cutaneous leishmaniasis in rural regions of Marvdasht, Fars province, southern Islamic Republic of Iran during 2003-04. Using live traps, 126 rodents were collected: 75.4% were Meriones libycus, 14.3% Cricetulus migratorius and 10.3% Microtus arvalis. Eight out of 95 Meriones libycus (8.4%) were found to be infected with Leishmania major, identified by nested-PCR; none of the other rodents were positive. Female sandflies were collected from indoor locations: 75% were Phlebotomus papatasi and only 2.7% were found naturally infected with L. major. This is the first report of P. papatasi as a proven vector of zoonotic cutaneous leishmaniasis in this area.

PCR based on identification of vectors of zoonotic cutaneous leishmaniasis in Shahrood District, central of Iran.

A study was made in rural region of Shahrood city, Semnan province in the central of Iran during 2005 to investigate of vectors of zoonotic cutaneous leishmaniasis. Sticky traps and an aspirator were used for collecting of sandflies. Three species of Phlebotomus papatasi, P. caucasicus and Sergentomyia sintoni were collected and identified and the first species was dominant (53%). Nested PCR method were employed for identifying of isolated parasites of dissected female of sandflies. Among the dissected sand flies 3 out of 24 (12.5%) Phlebotomus papatasi and 2 out of 48 (4.2%) Phlebotomus caucasicus were found naturally infected with promastigotes. Species-specific amplification ofGiemsa staind promastigote slides revealed specific PCR production of Leishmania major DNA in the infected P. papatasi and P. caucasicus sand flies. Having high prevalence and infection rate provide enough evidence to incriminate of P. papatasi as the main and proven vector of cutaneous leishmaniasis to human in the region and the species of P. caucasicus play the second role for maintenance of disease between rodents.

Vectors and reservoirs of cutaneous leishmaniasis in Marvdasht district, southern Islamic Republic of Iran

An epidemiological study was made of vectors and reservoirs of cutaneous leishmaniasis in rural regions of Marvdasht, Fars province, southern Islamic Republic of Iran during 2003-04. Using live traps, 126 rodents were collected: 75.4% were Meriones libycus, 14.3% Cricetulus migratorius and 10.3% Microtus arvalis. Eight out of 95 Meriones libycus [8.4%] were found to be infected with Leishmania major, identified by nested-PCR; none of the other rodents were positive. Female sandflies were collected from indoor locations: 75% were Phlebotomus papatasi and only 2.7% were found naturally infected with L. major. This is the first report of P. papatasi as a proven vector of zoonotic cutaneous leishmaniasis in this area

Meriones libycus is the main reservoir of zoonotic cutaneous leishmaniasis in south Islamic Republic of Iran.

A study was made in rural regions around Neiriz city, Fars province in the south of the Islamic Republic of Iran during 2002-03 to further investigate a new focus of zoonotic cutaneous leishmaniasis. Meriones libycus was the dominant rodent (100% of 65 rodents collected) and 4.6% were naturally infected with the amastigote form of Leishmania spp. Promasigotes were identified as L. major. This confirms M. libycus as the main reservoir host of zoonotic cutaneous leishmaniasis in southern parts of the country.

Phlebotomus (Paraphlebotomus) alexandri: a probable vector of Leishmania infantum in Iran.

The incidence of human visceral leishmaniasis (VL) caused by Leishmania infantum, which is endemic in several parts of Iran, has recently increased in the Nourabad-Mamassani district of Fars province, in the south of the country. Between 2003 and 2005, 12,688 sandflies were caught in this focus, using CDC miniature light traps, sticky traps and aspirators, in an attempt to identify the main vector of L. infantum. When 120 of the parous, female Phlebotomus (Paraphlebotomus) alexandri caught were individually checked for Leishmania infection, in an assay based on PCR and kinetoplast minicircle primers (LINR4 and LIN17), five (4.2%) of the flies were found to be infected with L. infantum. Thirty-nine (32.5%) of the 120 blood-fed Ph. alexandri checked in an ELISA were found positive for human blood. Since Ph. alexandri was the third-most common species caught (representing over 17% of the total catch), was clearly anthropophilic, and was not infrequently infected with L. infantum, it is probably an important vector in the Nourabad-Mamassani focus. Although other sandfly species have been found naturally infected with promastigotes in Iran, Ph. alexandri is the first that has been proven to harbour L. infantum in the country.

Meriones libycus is the main reservoir of zoonotic cutaneous leishmaniasis in south Islamic Republic of Iran

A study was made in rural regions around Neiriz city, Fars province in the south of the Islamic Republic of Iran during 2002-03 to further investigate a new focus of zoonotic cutaneous leishmaniasis. Meriones libycus was the dominant rodent [100% of 65 rodents collected] and 4.6% were naturally infected with the amastigote form of Leishmania spp. Promasigotes were identified as L. major. This confirms M. libycus as the main reservoir host of zoonotic cutaneous leishmaniasis in southern parts of the country