Molecular Detection of Dipylidium caninum in Isolated Fleas of Dogs in Meshkinshahr County, Northwest of Iran.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as humans, livestock, dog, cat, rabbit, squirrels, rats, and mice. The desire for blood feeding from warm-blooded animals has led to becoming an intermediate host for some tapeworms like Dipylidium. The aim of this study was to detect D. caninum larval infection in fleas of dogs living in Mesh-kinshahr County, northwest of Iran. Methods: Fleas were collected from 42 dogs using brushing the hair in Meshkinshahr for one year (2014-2015). After the morphological study, fleas were preserved in 90% ethanol for molecular identification. After DNA extraction, the 28S ribosomal RNA gene (∼670 bp) of D. caninum was amplified using specific primers. Finally, the PCR products were sequenced. Results: Overs, 974 fleas were collected from the dogs. In the morphological study, three species Ctenocephalides canis, Ct. felis, and Pulex irritans were identified. PCR and sequence analysis results showed that 4 isolates Ct. Canis were infected with D. caninum. Also, no positive specimens were isolated from the other two species. Conclusion: Ct. canis is reported as the most important species of fleas in transferring D. caninum in that region.

Molecular Identification of Leishmania infantum kDNA in Naturally Infected Dogs and Their Fleas in an Endemic Focus of Canine Visceral Leishmaniasis in Iran.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. The existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mechanism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. The aim of this study was to assess Leishmania infantum natural infection of dog fleas in northwest Iran in 2018. Methods: A total of 20 infested domestic dogs (Canis lupus familiaris) were randomly selected from 5 villages. Fleas were collected using brushing against dog hairs and fine forceps. Then, they were morphologically identified and preserved in ethanol for molecular assay. The kinetoplast DNA of the parasite was used for detection of Leishmania infantum using a semi-nested polymerase chain reaction (PCR) assay. Results: The human flea, Pulex irritans, and the cat flea, Ctenocephalides felis were identified on 40% and 35% of dogs, respectively. The results of PCR indicated that L. infantum was found in the Ctenocephalides canis (75%) and C. felis (66.7%) collected from infected dogs. No leishmanial infection was observed in P. irritans. Conclusion: It is concluded that fleas could be infected by Leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined.

Pulex irritans on Dogs and Cats: Morphological and Molecular Approach.

Background: The painful bite of Pulex irritans; causes wound on the host body and is a vector for Bartonella bacteria species, which can cause trench fever, Rickettsia species, Rocky Mountain spotted fever and Mediterranean spotted fever. We conducted a study to find out the hosts, abundance, as well as the molecular characteristics of this flea species in Meshkin-Shahr County, Ardabil Province, northwest of Iran. Methods: After collecting fleas from different reservoir hosts and transferring them to the laboratory, identification was done using different morphological characters as well as the internal transcribed spacer1 (ITS1) molecular marker. Results: This morphological study indicated that from the 1053 fleas, which were collected from 162 different animals, including cats and dogs, 74 specimens belonged to human flea, Pulex irritans. In addition, in molecular analysis showed a high sequence similarity (99.5%) with the P. irritans counterparts from Spain country and Zanjan of Iran available in GenBank. Conclusion: Pulex irritans species is an obligatory hematophagous ectoparasite of human and animals. Therefore, considering the relatively high frequency of this species on the body of cats and dogs, it is suggested to conduct more studies on its distribution and the possibility of being a vector of pathogens among these animals and human. The results of these studies will be used to compile and review the control programs of this vector.

Molecular Identification and Phylogenetic Classification of Leishmania spp. Isolated from Human Cutaneous Leishmaniasis in Iran: A Cross-sectional Study.

BACKGROUND: In Iran, both forms of cutaneous (CL) and visceral leishmaniasis (VL) have been reported; so the accurate species identification of the parasite(s) and the analysis of genetic diversity are necessary. METHODS: The smears were collected from lesions samples of 654 patients with CL, who attended local health centers in 12 provinces of Iran during 2013-2015. The smears were checked for the presence of amastigotes by light microscopy. DNA of 648 Leishmania isolates, amplified by targeting a partial sequence of ITS (18S rRNA-ITS1-5.8S rRNA-ITS2) gene. Twenty-five of all the amplicons were sequenced and analyzed with restriction fragment length polymorphism (RFLP) using the Taq1 enzyme. RESULTS: All the smears were positive microscopically. The PCR-RFLP analysis revealed that 176 (27%) CL patients were infected with L. tropica and, 478 (73%) with L. major. The dominant species in all over Iran is L. major. The sequencing results of all CL patients and RFLP analysis confirmed each other. Based on our phylogenetic tree, 25 ITS DNA sequences were grouped into two clusters representing L. major and L. tropica species. Phylogenetic tree derived from the ITS sequences supports a clear divergence between L. major from the other species. CONCLUSION: Discrimination of Iranian Leishmania isolates using ITS gene gives us this opportunity to detect, identify, and construct the phylogenetic relationship of Iranian isolates.

Molecular Identification and Intra-species Variations among Leishmania infantum Isolated from Human and Canine Visceral Leishmaniasis in Iran.

BACKGROUND: In Iran, both forms of cutaneous (CL) and visceral leishmaniasis (VL) have been re-ported; so the accurate species identification of the parasite(s) and the analysis of genetic diversity are necessary. METHODS: The investigation was conducted from 2014 to 2015 in the northwest and south of Iran, where VL is endemic (7 provinces). Blood samples of patients and infected dogs were collected and sera separated for serologic examinations (DAT, rK39). Spleen or bone marrow samples from infected dogs were also collected to confirm the infection. DNAs of 70 samples amplified by targeting a partial sequence of ITS (18S rRNA-ITS1-5.8S rRNA-ITS2) gene. All the amplicons were sequenced and analyzed with restriction fragment length polymorphism (RFLP) using the TaqI enzyme. RESULTS: The cause of all 70 VL cases, were L. infantum, so, the dominant specie is L. infantum. The sequencing results of all VL cases and RFLP analysis corroborate each other. Discrimination of Iranian Leishmania isolates using ITS gene gives us this opportunity to detect, identify and construct the phylogenetic relationship of Iranian isolates. In addition, detection and differentiation of Leishmania spp. DNA was confirmed by amplification of variable area of the minicircle kDNA (conserved sequence blocks (CSB)). CONCLUSION: Low divergence and high likelihood were seen among L. infantum isolates of human and dogs from Iran with a very slight divergence was seen between isolates from northwest and south of Iran, thus grouped in a unique clad. No correlation was observed between intraspecies divergence and geographic distribution of the isolates.

Transmission of Leishmania infantum by Rhipicephalus sanguineus (Acari: Ixodidae) in Dogs.

BACKGROUND: Rhipicephalus sanguineus is the most widely distributed tick in the world, which is partly due to its biological flexibility and the global distribution of its major host, the domestic dog. In Mediterranean region it could be principal reservoir host for Leishmania infantum, usually transmitted by the phlebotomine sand flies. In this study, we evaluated the vector potential of R. sanguineus in transmitting L. infantum to uninfected dogs. METHODS: During 2014, five dogs with clinical manifestations of canine visceral leishmaniasis (CVL), high anti-Leishmania antibody titers and tick infestation, were selected from CVL endemic areas (Tehran and Alborz provinces). At least, twenty live ticks were removed from each infected dog. After morphological identification, the ticks were divided into two groups; ticks belonging to the first group were dissected for parasitological examinations and semi-nested PCR assay, and those of the second group were selected for the transmission of CVL caused by L. infantum to uninfected dogs. Following tick infestation, all uninfected dogs were kept for 9 months and examined monthly for clinical and serological tests. RESULTS: Nearly, 67% of ticks were infected by L. infantum using the semi-nested PCR. All other parasitological tests of ticks were negative. Clinical examinations and serological tests of the investigated dogs revealed negative results. Nested-PCR test results performed on splenic biopsy samples of dogs were also negative. CONCLUSION: L. infantum-positive R. sanguineus ticks were unable to transfer L. infantum from infected dogs to healthy ones. The detection of L. infantum DNA in ticks collected from naturally infected dogs by semi-nested PCR does not prove their vectorial competence.

The PCR-RFLP-Based Detection and Identification of the Leishmania Species Causing Human Cutaneous Leishmaniasis in the Khorasan-Razavi Province, Northeast of Iran.

BACKGROUND: Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (ACL), and Leishmania major, which causes zoonotic cutaneous leishmaniasis (ZCL), are endemic in Iran. METHODS: Cross-sectional study was designed to identify Leishmania species in cutaneous leishmaniasis patients who referred to Mashhad Health Centers from 2013 to 2014 using ITS-PCR-RFLP technique. First, physical examinations were performed in all suspected patients and CL cases were confirmed with microscopical examinations. A questionnaire was prepared and completed for each confirmed patient and DNA from each lesion smear was extracted, separately. The ribosomal internal transcribed spacer was amplified with appropriate primers and PCR products were digested by enzyme Taq1 restrict enzyme. RESULTS: From all patients, 51 cases (54.3%) were men and 43 of them (45.7%) were women. The most frequent age group was 20-29 years old (27.2%). Hands, face and feet were the most common sites for appearance of skin lesions. All of the 94 cases (100%) tested found to be positive by ITS-PCR-RFLP. Overall, Leishmania species were identified in all of the 94 lesion smears which 33 (35%) of them were L. major and 61 (65%) of the remained isolates were identified L. tropica. CONCLUSION: Characterization of Leishmania isolates collected from different parts of Khorasan-Razavi Province showed that L. tropica is predominant agents of CL, especially in large and medium sized cities such as Mashhad and Shandiz. Moreover, this study revealed that ITS-PCR-RFLP based on our designed primers is a suitable method for species characterization.

Detection of Cryptosporidium and Giardia (oo)cysts by IFA, PCR and LAMP in surface water from Rasht, Iran.

BACKGROUND: Cryptosporidium and Giardia in water supplies is acknowledged as a public health problem. In the present study, we applied immunofluorescence assay (IFA), PCR and loop-mediated isothermal amplification (LAMP) for the detection of the two protozoa. METHODS: Over a period of 12 months, surface water samples were collected from two rivers in the north of Iran, and filtrated by 142 mm membrane filters. At each sampling point 10 L water were used for IFT and the10 L were analysed using molecular methods. RESULTS: In 15/40 samples, (oo)cysts were detected by one of the IFA, PCR or LAMP methods. Five samples that were Cryptosporidium-negative by IFA were positive by LAMP. A total of 10 out of 13 samples that were Giardia-positive by IFA were also positive by PCR. IFA revealed high levels of Giardia, with 1-1800 cysts and 1-16 Cryptosporidium oocysts detected per 10 L. CONCLUSION: The study reveals that the investigated water supplies were contaminated by Cryptosporidium and Giardia. The LAMP assay has advantages for detection and screening of these protozoa at relatively low concentration in water samples. The three assays applied are complimentary but no single one will give the true prevalence of these parasites in surface water samples. However, each method has its own advantages and disadvantages dependent of the aim and the study design; a combination of detection methods should be applied to discover whether water is, or is not, contaminated with (oo)cysts. This is the first report on the occurrence of (oo)cysts in Iranian surface waters to compare the results of parasite detection obtained with the different methods.