Detection of Borrelia persica infection in Ornithodoros tholozani using PCR targeting rrs gene and xenodiagnosis

Relapsing fever caused by Borrelia persica, is an acute tick-borne disease which is transmitted by soft ticks of Ornithodoros tholozani to human. Value of PCR and xenodiagnosis for detection of B. persica in O. tholozani ticks was compared. Sixty-four Borrelia- free ticks were fed on infected guinea pigs and used for the experiments. For xenodiagnosis, a group of 32 ticks in subsequent blood meal were fed on sterile guinea pigs and the indication of B. persica in the animal blood was tested 5-14 days later by dark-field microscopy. For PCR, all 64 ticks were subjected to PCR against B. persica rrs gene [16S-rDNA]. Also sensitivity of PCR in terms of minimum detectable number of spirochetes as well as the effects of tick sex and post digestion was tested. PCR revealed B.persica DNA in 98.4% ticks, in which B. persica were found in 25.0% by xenodiagnosis. PCR was enough sensitive to give positive results for DNA of 1 spirochete. PCR success rates were similar for male or female ticks. Course of time did not affect the efficacy of PCR and similar results were observed for ticks of immediately fed, semior completely gravid or completely digested blood ones. Our results indicate that due to very low specificity and time consuming, xenodiagnosis is not a useful method whereas PCR method has advantages for study the Borrelia prevalence in ticks

Molecular detection of leishmania infantum in naturally infected phlebotomus perfiliewi transcaucasicus in bilesavar district, Northwestern Iran

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. 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. 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. 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

[Determination of parasite species of cutaneous leishmaniasis using nested PCR in Damghan-Iran, during 2008]

Cutaneous leishmaniases with two forms of rural and urban is the endemic diseases and as a health problem in our country. Identification of parasite species and type of disease is very important for treatment of disease as well as for planning of control program. The microscopic observations by Giemsa-stained smears is the most common laboratory test for the diagnosis of cutaneous leishmaniasis, but the determination of parasite species is impossible and utilization of other ways such as biochemical and molecular methods is required. This study was carried out to determine the parasite species caused cutaneous Leishmaniasis by Nested PCR in Damghan, Iran. This descriptive study was performed on 67 patients with dermal lesions that referred to Damghan health center laboratory in Iran during 2008. The patient's information were recorded in questionnaire. DNA of Giemsa-stained slides from patients was extracted and evaluated by specific primers of kinetoplast DNA using Nested PCR. Leishmania parasites were observed in 57 patients under light microscope. The 10 patients were infected by other dermal diseases. The PCR result showed the parasite presence in lesions of 57 patients is Leismania major. 54% of patients were male and 46% were female. 72% of the patients were lived in rural areas. 50.9% of disease was observed in over 25 years old patients. Hands were the most common region of ulcer [44.7%]. 48% of the patients had one ulcer and the other patients had two or more ulcers. High prevalence [31.6%] of disease was observed in October. This study showed that zoonotic cutaneous leishmaniasis to be prevalent in this area and Nested PCR method is a sensitive and accurate to leishmania species characterization

Utility of filter paper for preserving insects, bacteria, and host reservoir DNA for molecular testing

Appropriate methodology for storage biological materials, extraction of DNA, and proper DNA preservation is vital for studies involving genetic analysis of insects, bacteria, and reservoir hosts as well as for molecular diagnostics of pathogens carried by vectors and reservoirs. Here we tried to evaluate the utility of a simple filter paper-based for storage of insects, bacteria, rodent, and human DNAs using PCR assays. Total body or haemolymph of individual mosquitoes, sand flies or cockroaches squashed or placed on the paper respectively. Extracted DNA of five different bacteria species as well as blood specimens of human and great gerbil Rhombomys opimus was pipetted directly onto filter paper. The papers were stored in room temperature up to 12 months during 2009 until 2011. At monthly intervals, PCR was conducted using a 1-mm disk from the DNA impregnated filter paper as target DNA. PCR amplification was performed against different target genes of the organisms including the ITS2-rDNA of mosquitoes, mtDNA-COI of the sand flies and cockroaches, 16SrRNA gene of the bacteria, and the mtDNA-CytB of the vertebrates. Successful PCR amplification was observed for all of the specimens regardless of the loci, taxon, or time of storage. The PCR amplification were ranged from 462 to 1500 bp and worked well for the specified target gene/s. Time of storage did not affect the amplification up to one year. The filter paper method is a simple and economical way to store, to preserve, and to distribute DNA samples for PCR analysis

Larvicidal activity of essential oils of apiaceae plants against malaria vector, anopheles stephensi

Plant extracts and oils may act as alternatives to conventional pesticides for malaria vector control. The aim of this study was to evaluate the larvicidal activity of essential oils of three plants of Apiaceae family against Anopheles stephensi, the main malaria vector in Iran. Essential oils from Heracleum persicum, Foeniculum vulgare and Coriandrum sativum seeds were hydro distillated, then their larvicidal activity were evaluated against laboratory-reared larvae of An. stephensi according to standard method of WHO. After susceptibility test, results were analysis using Probit program. Essential oils were separated from H. persicum, F. vulgare and C. sativum plants and their larvicidal activities were tested. Result of this study showed that F. vulgare oil was the most effective against An. stephensi with LC[50] and LC[90] values of 20.10 and 44.51 ppm, respectively. All three plants essential oil can serve as a natural larvicide against An. stephensi. F. vulgare oil exhibited more larvicidal properties

Study on presence of borrelia persica in soft ticks in Western Iran

A molecular survey was conducted to investigate the presence of pathogenic Borrelia persica species causing the tick borne relapsing fever [TBRF] in Takistan district Qazvin Province, western Iran. A number of 1021 soft ticks were collected from 31 villages including previously reported infected and none-infected TBRF cases and individually examined for the presence of B. persica DNA by conventional PCR targeting the 16SrRNA. A total of 1021 soft ticks of three species of Ornithodouros tholozani [120: 11.75%]. O. lahorensis [461: 45.15%] and Argaspersicus [440: 43.1%] were collected and tested against Borrelia infection. Soft ticks were more prevalent [67%] in infected areas than none infected areas. The rate O. tholozani in infected areas was much greater [29 times] than none infected areas. Ninety seven percent of soft ticks in none infected areas were of O. tholozani. Sixteen [16.7%] ticks of tested [n=95] O, tholozani were infected with B. persica. Three [1.3%] out of 205 soft ticks of O. lahorensis were positive for Borrelia sp., and no infection was observed in A persicus. Taq/ RFLP analysis and sequence analysis of the positive PCR products showed the presence of B. persica. The RFLP analysis showed that the positive ticks of O. lahorensis were infected with unknown Borrelia species. This study showed that although there were no TBRF cases in Takisan. but still infected O. tholozani, the known vector of TBRF. presented in the region. Control measures needs to be fulfilled in Thakisan

Blood meal identification in field-captured sand flies: comparison of PCR-RFLP and ELISA assays

We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method. This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006-2007. For PCR-RFLP a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b [cyt b] gene followed by digestion of the PCR products by Hae III enzyme. The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow [54%], human [10%], dog [4%], human and cow [21%], dog and cow [14%], and human and dog [3%]. The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advantages and limitations of PCR and ELISA assays are discussed

Molecular detection of leishmania major in the vectors and reservoir hosts of cutaneous leishmaniasis in Kakaleh district, Golestan province, Iran

An epidemiological study was carried out on the vector[s] and reservoir[s] of cutaneous leishmaniasis in rural areas of Kalaleh District, Golestan Province during 2006 - 2007. Totally 4900 sand flies were collected using sticky papers and were subjected to molecular methods for detection of leishmanial parasite. Phlebotomus papatasi was the common species in outdoor and indoor resting places. Employing PCR technique showed only 1 out of 372 P. papatasi [0.3%] was positive to parasite due Leishmania major. Sixteen rodent reservoir hosts were captured by Sherman traps and identified as Rhombomys opimus. Microscopic investigation on blood smear of the animals for amastigote parasites revealed 6[37.5%] infected rodents. Infection of these animals to L. major was then confirmed by PCR against rDNA loci of the parasite. This is the first molecular report of parasite infection of both vector [P. papatas] and reservoir [R. opimus] to L. major. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and R. opimus was the most important host reservoir for maintenance of the parasite source in the area

Effect of post ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes

Identification of host blood meal in haematophagous arthropods is an important element in their rule in transmission of vector borne diseases. The effects of post ingestion and physical conditions that killed mosquitoes are stored on the success of detecting blood meal DNA of Anopheles stephensi and Culex quinquefasiatus was investigated by polymerase chain reaction [PCR] amplification at the human mitochondrial DNA cytochromeB [CytB] gene. Host DNA extracted from the blood meal up to 33 h post ingestion in both species acts as an efficient template for PCR amplification. However more DNA concentration needs for meals digested longer time. Successful PCR amplification among meals digested for 36 h dropping to a faint band. There were no differences between PCR success rate for sampled stored at +4°C or -20°C, but less successful products were observed in samples kept at 4°C for periods longer than 30 h digestion. The results of this study is important in malaria epidemiological studies to provide valuable information about the degree of contact between human hosts and mosquito vectors, impact of vectors controls such bed nets and repellents, and the transmission dynamics of human malaria and other vector-borne diseases