Molecular Characterization and Phylogenetic Analysis of Theileria spp. and Babesia spp. Isolated from Various Ticks in Southeastern and Northwestern Regions of Iran.

INTRODUCTION: Piroplasms are hemoprotozoa comprising heterogeneous tick-borne parasites, which are differentiated into three genera, namely Babesia, Theileria, and Cytauxzoon. The aim of this study was to determine the prevalence, molecular identification, and phylogenetic relationship of both Theileria spp. and Babesia spp. in tick species isolated from different domestic animals from two different geographical locations of Iran. MATERIALS AND METHODS: A total of 930 ticks collected from goats, sheep, and cattle were examined for the presence of Theileria spp. and Babesia spp. using PCR targeting 18S rRNA gene followed by sequencing. Sequence analysis was performed based on the data published in the GenBank on Theileria spp. and Babesia spp. isolates using bioinformatic tools, such as the standard nucleotide BLAST. RESULTS: A 390 or 430 base pair fragment of 18S rRNA gene of Theileria and Babesia species was successfully amplified in 17.2% of the examined ticks (16of 93). Genome of Theileria or Babesia species was detected in 4 ticks collected in Heris, including 3 Dermacentor marginatus and 1 Rhipicephalus sanguineus, and also in 12 ticks collected in Chabahar, including 10 R. sanguineus and 2 D. marginatus. Partial analysis of 18S rRNA gene sequence of the four D. marginatus, collected from goats and sheep in Heris, showed that they were infected with Theileria spp. that were 95-97% identical to Iranian Theileria ovis present in the GenBank database (GenBank acc. no. KP019206.1). While the five R. sanguineus, collected from sheep and goats in Chabahar, were infected with Babesia spp. that were 91-97% identical to Iranian Babesia ovis present in the GenBank database (GenBank acc. no. AY362829.1: KT587794.1). CONCLUSION: The prevalence of Babesia and Theileria is different in southeastern and northwestern parts of Iran, with higher prevalence of babesiosis in the southeastern region and that of theileriosis in the northwestern region. Sequence analysis of 18S rRNA gene revealed that T. ovis and B. ovis are genetically polymorphic in these regions.

Molecular Characterization and Phylogenetic Analysis of Anaplasma spp. and Ehrlichia spp. Isolated from Various Ticks in Southeastern and Northwestern Regions of Iran.

INTRODUCTION: Anaplasma/Ehrlichia species are tick-transmitted pathogens that cause infections in humans and numerous domestic and wild animal species. There is no information available on the molecular characteristics and phylogenetic position of Anaplasma/Ehrlichia spp. isolated from tick species from different geographic locations in Iran. The aim of this study was to determine the prevalence, molecular characteristics, and phylogenetic relationship of both Anaplasma spp. and Ehrlichia spp. in tick species isolated from different domestic animals from two different geographical locations of Iran. METHODS: A total of 930 ticks were collected from 93 cattle, 250 sheep, and 587 goats inhabiting the study areas. The collected ticks were then investigated for the presence of Anaplasma/Ehrlichia spp. using nested PCR based on the 16S rRNA gene, followed by sequencing. Sequence analysis was done based on the data published in the GenBank on Anaplasma/Ehrlichia spp. isolates using bioinformatic tools such as the standard nucleotide BLAST. RESULTS: Genome of Anaplasma or Ehrlichia spp. was detected in 14 ticks collected in Heris, including 5 Dermacentor marginatus, 1 Haemaphysalis erinacei, 3 Hyalomma anatolicum, and 4 Rhipicephalus sanguineus, also in 29 ticks collected in Chabahar, including 14 R. sanguineus, 8 D. marginatus, 3 Hyalomma Anatolicum, and 4 Hyalomma dromedarii. Partial analysis of the 16S rRNA gene sequence of positive samples collected from goats and sheep showed that they were infected with Anaplasma/Ehrlichia spp. that were 94-98% identical to ovine Anaplasma and 91-96% identical to Neoehrlichia and Ehrlichia spp. CONCLUSION: The various ticks identified in this study suggest the possible emergence of tick-borne diseases in animals and humans in these regions. R. sanguineus and D. marginatus seem to be predominant vectors responsible for anaplasmosis in these regions. Partial sequence analysis of the 16S rRNA gene showed that A. ovis is genetically polymorphic in these regions. Furthermore, an association between the genetic heterogeneity of this microorganism and the geographical regions of Anaplasma strains was found. This study also showed that those ticks that were collected from the same geographical origin were infected with closely related strains of Anaplasma.

Discrimination of Paederus fuscipes and Paederus littoralis by mtDNA-COI PCR-RFLP.

BACKGROUND: Linear dermatitis is endemic in Iran where most cases occur in the Caspian Sea coast and Fars province. The disease is caused by beetles of the genus Paederus which are active from early spring to beginning of autumn although its incidence rises from May to August. The classic taxonomy of Paederus spp. is based on the male genitalia that is very complex and needs expertise. In this study, we report a DNA-based method to discriminate Paederus fuscipes and Paederus littoralis (=syn: P. lenkoranus, P. ilsae). METHODS: Type specimens were collected from north and south of Iran. Molecular typing of the species was performed using restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified fragments of mtDNA-COI. RESULTS: Sequence analyses of the data obtained in this study showed significant DNA polymorphisms. There were 89 substitutions between COI sequences of the two species. The mtDNA-COI fragment comprises several useful species-specific restriction sites comprising HaeIII that could result in distinctively different species-specific PCR-RFLP profiles. The HaeIII enzyme cuts the 872 bp PCR amplicon of P. littoralis into 737 and 100 bp and two small nonvisible bands whereas it does not cut P. fuscipes amplicon into fragments. CONCLUSION: This study demonstrates that molecular typing is useful method and allows one to differentiate between two species and is recommended for discrimination of other Paederus species, which morphologically are indistinguishable or very difficult to be distinguished.

Molecular Detection of Leishmania major and L. turanica in Phlebotomus papatasi and First Natural Infection of P. salehi to L. major in North-East of Iran.

BACKGROUND: Leishmaniasis is an important public health disease in many developing countries as well in Iran. The main objective of this study was to investigate on leishmania infection of wild caught sand flies in an endemic focus of disease in Esfarayen district, north east of Iran. METHODS: Sand flies were collected by sticky papers and mounted in a drop of Puri's medium for species identification. Polymerase chain reaction techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism were used for identification of DNA of Leishmania parasites within infected sand flies. RESULTS: Among the collected female sand flies, two species of Phlebotomus papatasi and Phlebotomus salehi were found naturally infected with Leishmania major. Furthermore, mixed infection of Leishmania turanica and L. major was observed in one specimen of P. papatasi. Sequence analysis revealed two parasite ITS1 haplotypes including three L. major with accession numbers: KJ425408, KJ425407, KM056403 and one L. turanica. (KJ425406). The haplotype of L. major was identical (100%) to several L. major sequences deposited in GenBank, including isolates from Iran, (Gen Bank accession nos.AY573187, KC505421, KJ194178) and Uzbekistan (Accession no.FN677357). CONCLUSION: To our knowledge, this is the first detection of L. major within wild caught P. salehi in northeast of Iran.

Application of Flumethrin Pour-On on Reservoir Dogs and Its Efficacy against Sand Flies in Endemic Focus of Visceral Leishmaniasis, Meshkinshahr, Iran.

BACKGROUND: Visceral leishmaniasis (VL) is one of the most important parasitic zoonotic diseases in the world. Domestic dogs are the main domestic reservoirs of VL in endemic foci of Iran. Various methods, including vaccination, treatment of dogs, detection and removal of infected dogs have different results around the world. General policy on control of canine visceral leishmaniasis is protection of them from sand fly bites. The aim of this study was evaluation of pour-on application of flumethrin on dogs against blood-feeding and mortality of field-caught sand flies. METHODS: Once every 20 days from May untill September 2013, the treated and control dogs were exposed with field caught sandflies for 2 hours under bed net traps. After the exposure time, both alive and dead sand flies were transferred in netted cups to the laboratory. The mortality rate of them was assessed after 24 hours. The blood-fed or unfed conditions were determined 2 hours after exposure to the dogs under stereomicroscope. RESULTS: The blood feeding index was varied from 12.0 to 25.0 % and 53.0 to 58.0 % for treated and control dogs respectively (P< 0.0001). The blood feeding inhibition was 75.0-87.0 % and 41.0-46.0 % for the control and treated dogs (P< 0.0001), respectively.The total mortality rate was 94.0-100 % and 19.0-58.0 % respectively for the treated and control groups (P< 0.001). CONCLUSTION: Application of pour-on flumethrin on dogs caused 90-100 % mortality until 2.5 month and inhibited the blood-feeding of sand flies.

ABO blood groups of residents and the ABO host choice of malaria vectors in southern Iran.

Recent epidemiological evidences revealed the higher prevalence of 'O' blood group in the residents of malaria-endemic areas. Also some data indicated preference of mosquitoes to 'O' group. The aim of this study was to determine ABO group ratio in the residents as well as ABO group preference of Anopheles in two malaria endemic areas in south of Iran. Agglutination method was used for ABO typing of residents. Field blood fed Anopheles specimens were tested against vertebrate DNA using mtDNA-cytB PCR-RFLP and then the human fed specimens were tested for ABO groups using multiplex allele-specific PCR. A total of 409 human blood samples were identified, of which 150(36.7%) were 'O' group followed by 113(27.6%), 109(26.7%), and 37(9.0%) of A, B, and AB groups respectively. Analyzing of 95 blood fed mosquitoes revealed that only four Anopheles stephensi had fed human blood with A(1), B(1), and AB(2) groups. Result of this study revealed high prevalence of O group in south of Iran. To our knowledge, it is the first ABO molecular typing of blood meal in mosquitoes; however, due to low number of human blood fed specimens, ABO host choice of the mosquitoes remains unknown. This study revealed that ABO blood preference of malaria vectors and other arthropod vectors deserves future research.