Prevalence of co-infection and genetic diversity of avian haemosporidian parasites in two rehabilitation facilities in Iran: implications for the conservation of captive raptors.

BACKGROUND: Various haemosporidian parasites infect raptors, especially captive hosts who may be more exposed. Diagnosis of threatening factors such as infectious diseases indirectly has a significant role in protecting endangered or threatened species that may boost the mortality or extinction resulting from declined reproduction. Few investigations have been performed in captive hosts to detect the prevalence of haemosporidian parasites and define genetic diversity in west Asia. For the first time, the current study was designed to determine the prevalence and genetic diversity of haemosporidian parasites in captive raptors by molecular methods in two rehabilitation facilities in North and North-east Iran and to define phylogenetic relationships of detected lineages circulating in raptors. RESULTS: Molecular characterization of the haemosporidian parasite was accomplished by PCR-based method and DNA sequencing in 62 captive raptors. The overall prevalence was ~ 36% with higher infection of Haemoproteus spp. than Leucocytozoon spp. Plasmodium infection was not detected in any host. Results showed that 22 individuals (of 10 species) were infected with unique lineages. Genus Haemoproteus was detected in 26.66% of examined individuals (of eight species) and Leucocytozoon was found in 10% of individuals (of four species). The molecular analysis could detect ten lineages (nine Haemoproteus spp. and one Leucocytozoon spp.) which were categorizes as new and six lineages which have been previously detected in the other investigations. CONCLUSIONS: The Bayesian phylogenetic analysis derived from obtained data in the present study and published lineages in previous investigations indicated the probable host specificity of Haemoproteus and Leucocytozoon parasites in several sub-clades at hosts' order and genus level. As monitoring the parasite loads of captive birds when admitted reduce the risk of infecting hosts in captivity at those locations, we designed this study to determine infection prevalence and genetic diversity of blood parasites in raptors examined in Iran. These results allow mapping of haemosporidian distribution and shed light on the depth of their diversity in Iran to protect species by identification of risk in rehabilitation facilities.

Developing a Vaccine to Block West Nile Virus Transmission: In Silico Studies, Molecular Characterization, Expression, and Blocking Activity of Culex pipiens mosGCTL-1.

BACKGROUND: Mosquito galactose-specific C-type lectins (mosGCTLs), such as mosGCTL-1, act as ligands to facilitate the invasion of flaviviruses like West Nile virus (WNV). WNV interacts with the mosGCTL-1 of Aedes aegypti (Culicidae) and facilitates the invasion of this virus. Nevertheless, there is no data about the role of mosGCTL-1 as a transmission-blocking vaccine candidate in Culex pipiens, the most abundant Culicinae mosquito in temperate regions. METHODS: Adult female Cx. pipiens mosquitoes were experimentally infected with a WNV infectious blood meal, and the effect of rabbit anti-rmosGCTL-1 antibodies on virus replication was evaluated. Additionally, in silico studies such as the prediction of protein structure, homology modeling, and molecular interactions were carried out. RESULTS: We showed a 30% blocking activity of Cx. pipiens mosGCTL-1 polyclonal antibodies (compared to the 10% in the control group) with a decrease in infection rates in mosquitoes at day 5 post-infection, suggesting that there may be other proteins in the midgut of Cx. pipiens that could act as cooperative-receptors for WNV. In addition, docking results revealed that WNV binds with high affinity, to the Culex mosquito lectin receptors. CONCLUSIONS: Our results do not support the idea that mosGCTL-1 of Cx. pipiens primarily interacts with WNV to promote viral infection, suggesting that other mosGCTLs may act as primary infection factors in Cx. pipiens.

Serological evidence of West Nile virus infection among birds and horses in some geographical locations of Iran.

Recent expansion of arboviruses such as West Nile (WNV), Usutu (USUV), and tick-borne encephalitis (TBEV) over their natural range of distribution needs strengthening their surveillance. As common viral vertebrate hosts, birds and horses deserve special attention with routine serological surveillance. Here, we estimated the seroprevalence of WNV, USUV and TBEV in 160 migrating/resident birds and 60 horses sampled in Mazandaran, Golestan, North Khorasan, Kordestan provinces and Golestan province of Iran respectively. ELISA results showed that of 220 collected samples, 32 samples (14.54%), including 22 birds and 10 horses, were positive. Microsphere immunoassay results showed that 16.7% (10/60) of horse blood samples collected in Golestan province were seropositive against WNV (7; 11.7%), Flavivirus (2; 3.3%) and seropositive for USUV or WNV (1; 1.7%). Furthermore, micro virus neutralization tests revealed that four of seven ELISA-positive bird blood samples were seropositive against WNV: two Egyptian vultures, and one long-legged buzzard collected in Golestan province as well as a golden eagle collected in North Khorasan province. No evidence of seropositivity with TBEV was observed in collected samples. We showed that WNV, responsible for neuroinvasive infection in vertebrates, is circulating among birds and horses in Iran, recommending a sustained surveillance of viral infections in animals, and anticipating future infections in humans.

Assessment the Changing Trend of Susceptibility to Two Insecticides among Field-Population Culex quinquefasciatus Compared with the Same Population Undergoing to Multiple Colonization.

BACKGROUND: During the past decade, rapid development of insecticide resistance have been reported among many species of mosquito vectors against four main categories of insecticides worldwide. The aim of the research was to assess the variation trend of susceptibility levels of Culex quinquefasciatus to two insecticides separately for the field population compared with subsequent generations of the same sample after multiple colonization. METHODS: Larvae and pupae of Cx. quinquefasciatus were collected from house sewages and reared to adult which blood-fed on roosters. Ten percent sucrose fed female mosquitoes aged 2-3 days were used for susceptibility tests with DDT and deltamethrin. Susceptibility levels was assessed in the adult stage of field stran Cx. quinquefasciatus against DDT 4.0% and deltamethrin 0.05% and continued up to next six generations undergoing multiple rearing at insectary condition. RESULTS: The susceptibility levels to DDT 4.0% did not change compared to the field with the lab population to six generations. Regarding deltamethrin 0.05%, no significant difference was shown between field strain (58.3%) and 3rd generation (52.7%) compared to the 6th one (33.8%). CONCLUSION: This finding may reflect the role of the kdr gene in resistance to organochlorine which has cross-resistance with pyrethroid insecticides. The results of this study clearly showed the irreversible trend of pyrethroid resistance among colonized mosquitoes. This is the first study of the resistance status of Cx. quinquefasciatus in Iran.

Detection of arboviruses in mosquitoes: Evidence of circulation of chikungunya virus in Iran.

Mosquitoes are vectors of viruses affecting animal and human health. In Iran, the prevalence of mosquito-borne viruses remains poorly investigated. Once infected, mosquito females remain infected for all their life making virus detections possible at early steps before infections are reported in vertebrate hosts. In this study, we used a recently developed high-throughput chip based on the BioMark Dynamic arrays system capable of detecting 37 arboviruses in a single experiment. A total of 1,212 mosquitoes collected in Mazandaran, North-Khorasan, and Fars provinces of Iran were analyzed. Eighteen species were identified, belonging to five genera; the most prevalent species were Anopheles maculipennis s.l. (42.41%), Culex pipiens (19.39%), An. superpictus (11.72%), and Cx. tritaeniorhynchus (10.64%). We detected chikungunya virus (CHIKV) of the Asian genotype in six mosquito pools collected in North Khorasan and Mazandaran provinces. To our knowledge, this is the first report of mosquitoes infected with CHIKV in Iran. Our high-throughput screening method can be proposed as a novel epidemiological surveillance tool to identify circulating arboviruses and to support preparedness to an epidemic in animals and humans.

New Insights Into Culturable and Unculturable Bacteria Across the Life History of Medicinal Maggots Lucilia sericata (Meigen) (Diptera: Calliphoridae).

Because of the nutritional ecology of dung- and carrion-feeding, bacteria are the integral part of Lucilia sericata life cycle. Nevertheless, the disinfected larvae of the blowfly are applied to treat human chronic wounds in a biosurgery named maggot debridement therapy (MDT). To realize the effects of location/diet on the gut bacteria, to infer the role of bacteria in the blowfly ecology plus in the MDT process, and to disclose bacteria circulating horizontally in and vertically between generations, bacterial communities associated with L. sericata specimens from various sources were investigated using culture-based and culture-independent methods. In total, 265 bacteria, including 20 families, 28 genera, and 40 species, were identified in many sources of the L. sericata. Culture-dependent method identified a number of 144 bacterial isolates, including 21 species, in flies reared in an insectary; specimens were collected from the field, and third-instar larvae retrieved from chronic wounds of patients. Metagenetic approach exposed the occurrences of 121 operational taxonomic units comprising of 32 bacterial species from immature and adult stages of L. sericata. Gammaproteobacteria was distinguished as the dominant class of bacteria by both methods. Bacteria came into the life cycle of L. sericata over the foods and transovarially infected eggs. Enterococcus faecalis, Myroides phaeus, Proteus species, Providencia vermicola, and Serratia marcescens were exchanged among individuals via transstadial transmission. Factors, including diets, feeding status, identification tool, gut compartment, and life stage, governed the bacteria species. Herein, we reemphasized that L. sericata is thoroughly connected to the bacteria both in numerous gut compartments and in different life stages. Among all, transstadially transmitted bacteria are underlined, indicating the lack of antagonistic effect of the larval excretions/secretions on these resident bacteria. While the culture-dependent method generated useful data on the viable aerobic gut bacteria, metagenomic method enabled us to identify bacteria directly from the tissues without any need for cultivation and to facilitate the identification of anaerobic and unculturable bacteria. These findings are planned to pave the way for further research to determine the role of each bacterial species/strain in the insect ecology, as well as in antimicrobial, antibiofilm, anti-inflammatory, and wound healing activities.

High Transmission Potential of West Nile Virus Lineage 1 for Cx. pipiens s.l. of Iran.

: Vector competence is an important parameter in evaluating whether a species plays a role in transmission of an arbovirus. Although the protocols are similar, interpretation of results is unique given the specific interactions that exist between a mosquito population and a viral genotype. Here, we assessed the infection (IR), dissemination (DR), and transmission (TR) rates of Cx. pipiens s.l., collected from Iran, for West Nile virus (WNV) lineage 1a. We showed that Cx. pipiens s.l. mosquitoes in Iran were susceptible to WNV with IR up to 89.7%, 93.6%, and 83.9% at 7, 14, and 21 days post-infection (dpi) respectively. In addition, DR and TR reached respectively 92.3% and 75.0% at 21 dpi, and the number of viral particles delivered with saliva reached up to 1.33 × 105 particles. Therefore, an unexpected high risk of WNV dissemination in the region where Cx. pipiens s.l. mosquitoes are well established should be considered carefully and surveillance measures implemented accordingly.

Dynamics of prevalence and distribution pattern of avian Plasmodium species and its vectors in diverse zoogeographical areas - A review.

Avian Plasmodium is of special interest to health care scientists and veterinarians due to the potency of causing avian malaria in non-adapted birds and their evolutionary phylogenetic relationship with human malaria species. This article aimed to provide a comprehensive list of the common avian Plasmodium parasites in the birds and mosquitoes, to specify the common Plasmodium species and lineages in the selected regions of West of Asia, East of Europe, and North of Africa/Middle East, and to determine the contribution of generalist and host-specific Plasmodium species and lineages. The final list of published infected birds includes 146 species, among which Passer domesticus was the most prevalent in the studied areas. The species of Acrocephalus arundinaceus and Sylvia atricapilla were reported as common infected hosts in the examined regions of three continents. The highest numbers of common species of infected birds between continent pairs were from Asia and Europe, and no common record was found from Europe and Africa. The species of Milvus migrans and Upupa epops were recorded as common species from Asia and Africa. The lineage of GRW11 and species of P. relictum were the most prevalent parasites among all the infection records in birds. The most prevalent genus of vectors of avian malaria belonged to Culex and species of Cx. pipiens. The lineage SGS1 with the highest number of occurrence has been found in various vectors comprising Cx. pipiens, Cx. modestus, Cx. theileri, Cx. sasai, Cx. perexiguus, Lutzia vorax, and Culicoides alazanicus. A total of 31 Plasmodium species and 59 Plasmodium lineages were recorded from these regions. SGS1, GRW04, and GRW11, and P. relictum and P. vaughani are specified as common generalist avian malaria parasites from these three geographic areas. The presence of avian Plasmodium parasites in distant geographic areas and various hosts may be explained by the movement of the infected birds through the migration routes. Although most recorded lineages were from Asia, investigating the distribution of lineages in some of the countries has not been done. Thus, the most important outcome of this review is the determination of the distribution pattern of parasite and vector species that shed light on gaps requiring further studies on the monitoring of avian Plasmodium and common vectors extension. This task could be achieved through scientific field and laboratory networking, performing active surveillance and designing regional/continental control programs of birds' malaria and other zoonotic diseases.

Identification, molecular characterization and expression of aminopeptidase N-1 (APN-1) from Anopheles stephensi in SF9 cell line as a candidate molecule for developing a vaccine that interrupt malaria transmission.

BACKGROUND: According to the World Health Organization reports, billions of people around the world are at risk for malaria disease and it is important to consider the preventive strategies for protecting the people that are living in high risk areas. One of the main reasons of disease survival is diversity of vectors and parasites in different malaria regions that have their specific features, behaviour and biology. Therefore, specific regional strategies are necessary for successful control of malaria. One of the tools that needs to be developed for elimination and prevention of reintroduction of malaria is a vaccine that interrupt malaria transmission (VIMTs). VIMT is a broad concept that should be adjusted to the biological characteristics of the disease in each region. One type of VIMT is a vector-based vaccine that affects the sexual stage of Plasmodium life cycle. According to recent studies, the aminopeptidase N-1 of Anopheles gambiae (AgAPN-1) is as a potent vector-based VIMT with considerable inhibition activity against the sexual stage of Plasmodium parasite. METHODS: Systems for rapid amplification of cDNA ends (3'-RACE) and genome walking methods were used for sequence determination of apn-1 gene from Anopheles stephensi and distinct bioinformatics software were used for structural analysis. AsAPN-1 was expressed in Spodoptera frugiperda (Sf9) insect cell line using the baculovirus expression system. Recombinant AsAPN-1 was purified under the hybrid condition and its biological activity was assayed. RESULTS: Asapn-1 gene and its coded protein from An. stephensi were characterized for the first time in this study. Subsequently, the structural features and immunological properties of its coded protein were evaluated by in silico approaches. Enzymatic activity of the recombinant AsAPN-1, which was expressed in Sf9 insect cell line, was equal to 6 unit/µl. CONCLUSIONS: Results of this study revealed that AsAPN-1 is very similar to its counterpart in An. gambiae. In silico evaluation and fundamental data which are necessary for its evaluation as a VIMT-based vaccine in the next steps were acquired in this study and those could be useful for research groups that study on malaria vaccine for countries that An. stephensi is the main malaria vector there.

Molecular characterization of RNase III protein of Asaia sp. for developing a robust RNAi-based paratransgensis tool to affect the sexual life-cycle of Plasmodium or Anopheles fitness.

BACKGROUND: According to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specific features for this goal, could be considered as effective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required information for creating an RNase III mutant Asaia bacterium. METHODS: Asaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties. RESULTS: In this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specific substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined. CONCLUSIONS: In this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

Target Site Insensitivity Detection in Deltamethrin Resistant Culex pipiens Complex in Iran.

BACKGROUND: Some mosquito species which belong to the Culex. pipiens complex are primary vectors for West Nile virus, Sindbis, Dirofilaria immitis, and many arboviruses. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene of Cx. pipiens that is inherited, is one of the important threats for the efficacy of pyrethroids insecticides. Knockdown resistance (kdr) mutation, L1014F, is a well-defined mechanism of resistance to pyrethroids and DDT in many insect species. The aim of study was to determine the mechanisms of Insecticide resistance in this species. METHODS: Specimens of Cx. pipiens, the major vector of West Nile virus, were obtained in Tehran, Iran by collecting larvae from polluted wastewater in Qarchak of Tehran. In 2016 Insecticide susceptibility tests were performed according to WHO methods with deltamethrin 0.05%. We focused on determination of this point mutation in the VGSC gene of Cx. pipiens by Real-time PCR. RESULTS: Our results revealed high levels of resistance to deltamethrin 0.05%. The lethal times i.e. LT50 and LT90 for deltamethrin were 2.1530 and 8.5117 h respectively. The result of Real-time PCR confirmed the presence of resistant genotype in all the members of tested population. This study is the first report on kdr genotyping of Cx. pipiens from Tehran and our results on the VGSC gene in position L1014F confirmed the TTA to TTT nucleotide change. CONCLUSION: This finding will provide a clue for management of insecticide resistance in mosquito which are vectors of arboviruses and decision for replacement of novel approach for vector control.

Correction: Molecular detection and genetic diversity of avian haemosporidian parasites in Iran.

[This corrects the article DOI: 10.1371/journal.pone.0206638.].

Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing.

BACKGROUND: Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia Sericata (L. sericata) collagenase (MMP-1) has been expressed in insect cells. Collagenase is an enzyme that is widely used in clinical therapy and industry. It has been indicated that collagenase is expressed and secreted in salivary glands of L. sericata while using for maggot debridement therapy. OBJECTIVES: In the present study we decided to produce the recombinant form of collagenase enzyme in Spodoptera frugiperda (SF9) insect cells using the baculovirus expression system (Bac-to-Bac). MATERIALS AND METHODS: cloned the coding sequences (residues 494-1705) of L. sericata collagenase into the pFastBacHTA as donor plasmid. After transposition in the bacmid of DH10Bac host, the bacmid was transfected into the Sf9 cell line, then the expressed recombinant collagenase (MMP-1) was purified using the Ni-NTA agarose. RESULTS: The recombinant protein was verified by Western blotting. Furthermore, the biological activity of purified protein was measured in the presence of its specific substrate and its inhibitor, which was 67 IU.mL-1 based on our results, it was revealed that the characterized gene in our previous study codes L. sericata collagenesa enzyme. CONCLUSION: Considering to the broad applications of collagenase in medical sciences, for the first time, we cloned the L. sericata collagenase (MMP-1) gene into the insect cell line to establish a method for the expression and purification of L. sericata collagenase (MMP-1). The result help for preparing and designing a safe and versatile recombinant drug in future.

Co-occurrence of pederin-producing and Wolbachia endobacteria in Paederus fuscipes Curtis, 1840 (Coleoptera: Staphilinidae) and its evolutionary consequences.

The dual occurrence of Pseudomonas-like and Wolbachia endobacteria has not been investigated in the Pederus beetles yet. We investigated pederin-producing bacteria (PPB) infection in Paederus fuscipes specimens from the southern margins of the Caspian Sea by designed genus-specific (OprF) and species-specific (16S rRNA) primers. Wolbachia infection was studied through a nested-PCR assay of Wolbachia surface protein (wsp) gene. Of the 125 analyzed beetles, 42 females (82.35%) and 15 males (20.27%) were positive to PPB infection; this is the first study reporting male P. fuscipes infection to PPB. Wolbachia infection was found in 45 female (88.23%) and 50 male (67.57%) analyzed beetles. Surprisingly, a number of 36 females (70.59%) and 13 males (17.57%) were found to be infected with both PPB and Wolbachia endosymbionts. In general, population infection rates to PPB and Wolbachia were determined to be 45.6% and 76%, respectively. The infection rates of female beetles to PPB and PPB-Wolbachia were significantly higher than males. In Paederus species, only female beetles shelter PPB and the discovery of this bacterium in adult males may reflect their cannibalistic behavior on the contaminated stages. Phylogenetic analysis showed that the sequences of OprF gene were unique among Pseudomonas spp.; however, sequences of 16S rRNA gene were related to the PPB of Pederus species. The co-occurrence and random distribution of these endobacteria may imply putative tripartite interactions among PPB, Wolbachia, and Paederus. In order to elucidate these possible tripartite interactions, further studies are required even at gender level.

Molecular detection and genetic diversity of avian haemosporidian parasites in Iran.

BACKGROUND: The mobility of birds across or between continents exposes them to numerous vectors that have the potential to transmit pathogens and spread them into new regions. A combination of rich species diversity of birds along with the small amount of molecular studies in Iran makes observing the blood parasite distribution in wild avian populations indispensable for further estimation and administration of blood parasites. METHODOLOGY/PRINCIPAL FINDINGS: In order to evaluate the infection rate and molecular context of avian blood parasites, bird samples were collected (passerine = 316 and non-passerine = 14) in eight provinces of northern Iran between June to September 2015 and 2016. All bird samples were examined for haematoprotozoan infections by morphological screening using light microscope and mtDNA cytb gene amplification. A total of 115 birds were positive for blood parasites by molecular approach (34.84% overall infection). The infection rate of Haemoproteus, Plasmodium, and Leucocytozoon were 33.03%, 1.21%, and 0.6%, respectively. Sequences analysis has detected 43 lineages in Iranian birds' hosts. Lineages were attributed to three genera Haemoproteus (n = 37), Plasmodium (n = 4), and Leucocytozoon (n = 2), of which 23 lineages fully matched previously recorded sequences in GenBank and MalAvi data reciprocities. Five lineages of ACDUM1, ACDUM2, PARUS1, PYERY01, and SISKIN1 were detected in multiple hosts' species from dissimilar families. In Bayesian tree, all sequences were clustered in three main monophyletic clades as Haemoproteus, Plasmodium, and Leucocytozoon genera. CONCLUSIONS/SIGNIFICANCE: As the first study outlining the molecular detection of hematozoa of passerines from Iran, the current study has recorded 20 new lineages for three genera of Haemoproteus, Plasmodium, and Leucocytozoon. Additional investigations into these taxa in the avifauna for the other parts of Iran may provide extra information on blood parasites, hosts relationships and distribution patterns.

Speculation on the possibility for introducing Anopheles stephensi as a species complex: preliminary evidence based on odorant binding protein 1 intron I sequence.

BACKGROUND: Anopheles stephensi is considered an important malaria vector in Iran, Asia, and recently in the Horn of Africa. Recently, Ansteobp1 intron I sequence has been introduced a new molecular marker for identification of its biological forms including, mysorensis, intermediate and type, using insectary colony specimens. METHODS: In the current study, new marker ability in molecular identification of biological forms has been evaluated with An. stephensi specimens collected from Iran and Afghanistan malarious provinces. Following DNA extraction and PCR amplification, sequence analysis and constructed phylogenetic tree revealed that type and intermediate forms are distributed in Iran. RESULTS: The specimens collected from Afghanistan identified as intermediate and mysorensis forms. Therefore, intermediate form is sympatric species in both countries. Based on the results of Ansteobp1 intron I sequences, An. stephensi could be suggested as new Anopheles complex species including An. stephensi sibling A (type form), An. stephensi sibling B (intermediate form) and An. stephensi sibling C (mysorensis form). This is the first report on the presence of An. stephensi biological forms in Afghanistan. CONCLUSIONS: Iran is going to eliminate malaria transmission from the country, precise species identification, especially in complex species will be helpful in the prevention of malaria resurgence in the country, mainly because of common fauna of Anopheles species and through border malaria and population movement within Afghanistan, Pakistan, and Iran.

The Role of Molecular Techniques on Malaria Control and Elimination Programs in Iran: A Review Article.

BACKGROUND: The aim of this review was to describe the application of molecular methods in epidemiological aspects of malaria vectors, parasites, and human hosts in Iran and their critical role in malaria control and elimination programs. METHODS: Medline, EMBASE, Web of Science, Scopus, and Google Scholar databases were searched systematically for original published papers on PCR, the molecular identification of malaria vectors, the molecular epidemiology of malaria, insecticide resistance, and drug-resistant parasites, in Iran. In total, 51 studies on molecular entomology and 36 studies on molecular parasitology of malaria and three on human host were selected. RESULTS: Molecular methods are essential for improving the detection of malaria infection and monitoring antimalarial drugs and insecticide resistance in malaria elimination settings such as Iran. CONCLUSION: The application of molecular methods may be of particular interest for malaria control/elimination programs, for monitoring progress towards malaria elimination, and for optimal orientation of program activities.

Occurrence of Haemoproteus spp. (Haemosporida: Haemoproteidae) in New Host Records of Passerine Birds from the East of Iran.

BACKGROUND: Avian haemosporidians are able to parasitize numerous bird species all over the world. The extensive range of blood parasites infection rate is between 50% and 100% or less percentage. Haemoparasites with major effects on physiology, ecology, health, population dynamics, sexual selection and production success of avian hosts may promote species extinction. METHODS: To evaluate haemosporidians infection rate in Iranian birds, 136 individuals were examined by microscopic observation of stained blood smears under light microscope. These samples belonged to 10 different families of Songbirds from the east of Iran from April to August 2014-2016. RESULTS: Fifty-one passerine birds were detected as harboring Haemoproteus spp. Furthermore, we recorded Haemoproteus spp. infection of Granativora bruniceps, Oenanthe pleschanka for the first time in the world and eight more species for Iran. CONCLUSION: Age and sampling localities do not influence the infection rate of Haemoproteus spp. from the eastern provinces of Iran. The relative high infection of avian haematozoa revealed this region might provide suitable sites for future studies on these parasites and the relationship with their hosts and vectors.

Isolation and identification of Asaia sp. in Anopheles spp. mosquitoes collected from Iranian malaria settings: steps toward applying paratransgenic tools against malaria.

BACKGROUND: In recent years, the genus Asaia (Rhodospirillales: Acetobacteraceae) has been isolated from different Anopheles species and presented as a promising tool to combat malaria. This bacterium has unique features such as presence in different organs of mosquitoes (midgut, salivary glands and reproductive organs) of female and male mosquitoes and vertical and horizontal transmission. These specifications lead to the possibility of introducing Asaia as a robust candidate for malaria vector control via paratransgenesis technology. Several studies have been performed on the microbiota of Anopheles mosquitoes (Diptera: Culicidae) in Iran and the Middle East to find a suitable candidate for controlling the malaria based on paratransgenesis approaches. The present study is the first report of isolation, biochemical and molecular characterization of the genus Asaia within five different Anopheles species which originated from different zoogeographical zones in the south, east, and north of Iran. METHODS: Mosquitoes originated from field-collected and laboratory-reared colonies of five Anopheles spp. Adult mosquitoes were anesthetized; their midguts were isolated by dissection, followed by grinding the midgut contents which were then cultured in enrichment broth media and later in CaCO3 agar plates separately. Morphological, biochemical and physiological characterization were carried out after the appearance of colonies. For molecular confirmation, selected colonies were cultured, their DNAs were extracted and PCR was performed on the 16S ribosomal RNA gene using specific newly designed primers. RESULTS: Morphological, biochemical, physiological and molecular results indicated that all isolates are members of the genus Asaia. CONCLUSIONS: Contrary to previous opinions, our findings show that Asaia bacteria are present in both insectary-reared colonies and field-collected mosquitoes and can be isolated by simple and specific methods. Furthermore, with respect to the fact that we isolated Asaia within the different Anopheles specimens from distinct climatic and zoogeographical regions, it is promising and may be concluded that species of this genus can tolerate the complicated environmental conditions of the vector-borne diseases endemic regions. Therefore, it can be considered as a promising target in paratransgenesis and vector control programs. However, we suggest that introducing the new technologies such as next generation sequencing and robust in silico approaches may pave the way to find a unique biomarker for rapid and reliable differentiation of the Asaia species.

Mosquito-borne viral diseases and potential transmission blocking vaccine candidates.

Mosquito-borne viral diseases (MBVDs) have a complex biological cycle involving vectors and vertebrate hosts. These viruses are responsible for many deadly diseases worldwide. Although MBVDs threaten mostly developing countries, there is growing evidence indicating that they are also of concern in western countries where local transmission of arboviruses such as West Nile, Zika, Chikungunya and Dengue viruses have been recently reported. The rapid rise in human infections caused by these viruses is attributed to rapid climate change and travel facilities. Usually, the only way to control these diseases relies on the control of vectors in the absence of licensed vaccines and specific treatments. However, the overuse of insecticides has led to the emergence of insecticide resistance in vector populations, posing significant challenges for their control. An alternative method for reducing MBVDs can be the use of Transmission Blocking Vaccines (TBVs) that limits viral infection at the mosquito vector stage. Some successes have been obtained confirming the potential application of TBVs against viruses; however, this approach remains at the developmental stage and still needs improvements. The present review aims to give an update on MBVDs and to discuss the application as well as usage of potential TBVs for the control of mosquito-borne viral infections.