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Background and Objectives: Although the study on the bacteria residing in the mid-gut, salivary gland, and reproductive organs of insect vectors have drawn appeal to the host-pathogen interactions, we know comparatively less about microbiota that naturally exist in different mosquito organs within Iran. Materials and Methods: In the current investigation, PCR assay by using 16S rRNA gene amplification and DNA sequenc- ing, in addition to the traditional culture-based approach utilized for the detection of cultivable bacterial assemblages in mid-gut and reproductive tracts of Culex quinquefasciatus. Results: The identified bacteria isolated from different tissues of 45 individuals were consisted of Achromobacter, Aero- monas, Arthrobacter, Asaia, Enterobacter, Gluconobacter, Klebsiella, Lysinibacillus, Micrococcus, Psuedomonas and Serra- tia. The results showed that Proteobacteria was the most prevalent phylum in both genders' mid-gut and reproductive tracts, and Asaia was the most common bacteria that originated in adult females and males’ tissues. Conclusion: These outcomes recommend that the discovered microbiome may span through Cx. quinquefasciatus popula- tions. This data can be utilized to interfere with the transmission of pathogens and design new strategies for the control of mosquito-borne diseases.

Background The spread of Plasmodium vivax strains resistant to chloroquine (CQ) has posed a challenge to control strategies aimed at eliminating malaria. Molecular analysis of candidate resistance markers is very important for monitoring the P. vivax resistance to CQ in different endemic regions. In the present study, the multidrug resistance 1 (pvmdr1) gene, a possible marker for CQ resistance in P. vivax, was evaluated by molecular methods. Methods A simple PCR–RFLP method was developed for mutation analysis in pvmdr1 gene. A number of 120 blood spots were obtained from patients with P. vivax mono-infection in 2021. All of the samples were collected from Pakistani patients who travelled to Iran. Results None of the samples had any mutation at codon 976 of pvmdr1, while the 1076 mutation was detected in 96.2% of the examined isolates. Only two pvmdr1 haplotypes were identified, including the single mutant (Y976/1076L) as the most prevalent haplotype (with 96.2% frequency) and the wild type (Y976/F1076; with 3.8% frequency). Conclusions In this study, the major CQ resistance-mediating mutation and multiple mutant haplotypes of the pvmdr1 gene was not detected. However, continuous monitoring of drug resistance markers and close supervision of the efficacy of CQ is essential to detect the potential emergence of CQ-resistant P. vivax isolates in Iran. This data is important for performing future epidemiological surveillance to monitor CQ resistance in this endemic area and the bordering regions.

CRISPR-mediated integration could be used to develop the recombinant CHO (rCHO) cells by knock-in into the hotspot loci. However, low HDR efficiency besides the complex donor design is the main barrier for achieving so. The recently introduced MMEJ-mediated CRISPR system (CRIS-PITCh) uses a donor with short homology arms, being linearized in the cells via two sgRNAs. In this paper, a new approach to improve CRIS-PITCh knock-in efficiency by employing small molecules was investigated. Two small molecules, B02, a Rad51 inhibitor, and Nocodazole, a G2/M cell cycle synchronizer, were used to target the S100A hotspot site using a bxb1 recombinase comprised landing pad in CHO-K1 cells. Following transfection, the CHO-K1 cells were treated with the optimum concentration of one or combination of small molecules, being determined by the cell viability or flow cytometric cell cycle assay. Stable cell lines were generated and the single-cell clones were achieved by the clonal selection procedure. The finding showed that B02 improved the PITCh-mediated integration approximately twofold. In the case of Nocodazole treatment, the improvement was even more significant, up to 2.4-fold. However, the combinatorial effects of both molecules were not substantial. Moreover, according to the copy number and out-out PCR analyses, 5 and 6 of 20 clonal cells exhibited mono-allelic integration in Nocodazole and B02 groups, respectively. The results of the present study as the first attempt to enhance the CHO platform generation by exploiting two small molecules in the CRIS-PITCh system could be used in future researches to establish rCHO clones.

Background: Myiasis is the infestation of flies' larvae in living or dead tissues of the human body and animals. Ophthalmomyiasis is divided into internal and external types and thelarvae penetrate eyes in the internal type. This study aimed to examine larval morphology and molecular identification of flies causing ophthalmomyiasis and determine its incidence rate in referred patients to an ophthalmology clinic in Shiraz during 2019. Materials and Methods: During one year, all larvae were isolated from patients’ eyes. These larvae were identified using the morphological method according to the 1965 diagnostic key of Zumpt. Molecular confirmation was performed using a pair of specific primers for the Cytochrome Oxidase I (COI) gene in the next step. The expected amplicons were sequenced, and their results were aligned and analyzed using the nucleotide BLAST. Results: Overall, 224 fly larvae were isolated from 36 patients. According to the morphological analysis, all larvae were Oestrus ovis. Molecular analysis confirmed morphological results. Patients presenting acute conjunctivitis symptoms had a mean age of 34 ± 2 years, and there were four women (11%) and 32 men (89%). The highest incidence were recorded in the autumn season (55.8%). Morphological results of flies were confirmed by amplifying the expected size of the CO1 gene through conventional PCR. Conclusion: The cases of ophthalmomyiasis are higher than those mentioned in the published articles and this might be due to the absence of a regular monitoring program to care for the ophthalmomyiasis cases in the public health system. Therefore, due to the importance and potential incidence of this disease in Fars province, establishing a disease care program is necessary for the health surveillance system.

Cystic Echinococcosis (CE) is a medically important disease that is caused by the metacestodes of Echinococcus granulosus. Human hydatid is considered an endemic disease in specific regions of Iran. The goal of the present study was to determine the genetic diversity of E. granulosus from the paraffin-embedded human tissue samples which were collected from the endemic regions of Iran. Fifty-five formalin-fixed and paraffin-embedded hydatid cysts (FFPE) of humans, which had been removed surgically, were obtained from the South Khorasan and Sistan and Baluchistan provinces. These regions are related to the East and Southeast regions of Iran, respectively. The cox1 and nad1 genes from mitochondria were amplified from the extracted DNA and sequenced. The sequences were edited using the BioEdit software. Furthermore, phylogenetic and genetic diversity analyses were performed. Sequencing of the cox1 and nad1 genes from the 44 CE samples was done successfully. Genetic analysis revealed that 38 (86.3%) and 6 (13.6%) of the isolates were G1- and G6-genotypes, respectively. In general, eight and six haplotypes were identified by cox1 and nad1 genes analysis, respectively. For G1 strains, the haplotype diversity index was higher for the cox1 gene (0.6 ± 0.07) in comparison with the nad1 gene (0.4 ± 0.09). The findings of the present study showed that the sheep strain (G1) and the less important camel strain (G6) play the main roles in the transmission cycle of CE in the East and Southeast regions of Iran. Therefore, these results could be useful for managing the hydatid disease control programs in the studied and other similar areas.

Plasmodium falciparum is the parasite responsible for the disease malaria. In vitro cultivation of mature gametocytes of P. falciparum plays a central role in evaluating and developing the transmission-blocking drugs and sexual stage vaccines. These types of preventive molecules are crucial for controlling malaria in the future. Among different Plasmodium species that are involved in human malaria, only P. falciparum is cultivable. Therefore, an efficient method is required for in vitro culture of P. falciparum producing mature and infective gametocytes. This chapter describes a reliable and efficient protocol for the production of adult and infective gametocytes that is suitable for small- and large-scale culture.

The main objectives of developing vaccines to prevent malaria transmission are malaria control and preventing the reemergence of the disease in endemic regions. Molecular and in silico characterization of a candidate molecule is the first step in the vaccine design process. Determining the sequence and amplification of full-length cDNA copies from the mRNA transcripts is often challenging. The methods in this chapter provide a protocol for the rapid amplification of cDNA ends (RACE) and genome walking. Carboxypeptidase B2 enzyme from A. stephensi (CPBAs-2) was selected as the target molecule and the steps in its characterization and in silico analysis are explained in this chapter.

Malaria is one of the most important infectious diseases in the world, especially in the developing countries of the tropics. There are many difficulties in malaria research, including in vitro culture of the targeted parasites, in vitro production of mature and infectious gametocytes, and animal models. A rodent malaria parasite model is a promising solution to alleviate the stated problems. These parasites are very similar in physiology, life cycle, and structure to human malaria parasites and could help us understand the biology of human malaria. Creating the genetically modified parasites and specific animal mouse models for distinct human illnesses increases the utility of this approach and pre-evaluation strategy. Among the four rodent malaria parasites, P. berghei is the well-studied parasite.

Traditional and modern approaches have been applied to combat the malaria disease. Malaria eradication is a priority in several developing countries. Transmission-blocking vaccines are one of the suggested solutions for malaria eradication. Therefore, there is a demand for introducing the new targets and evaluation methods. Standard membrane feeding assay is the base of the evaluation process of transmission-blocking candidate molecules. Hence, this process is explained in this chapter in detail.

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Larval therapy with Lucilia sericata is a promising strategy in wound healing. Axon guidance molecules play vital roles during the development of the nervous system and also regulate the capacity of neuronal restoration in wound healing. Netrin-1, one of the proteins that larvae secrete, plays a useful role in cell migration and nerve tissue regeneration. The UNC-5 receptor combines with a netrin-1 signal and transmits the signal from one side of the membrane to the other side, initiating a change in cell activity. In the current study, we identified the full length of the UNC-5 receptor mRNA in L. sericata using different sets of primers, including exon junction and specific region primers. The coding sequence (CDS) of the UNC-5 receptor was sequenced and identified to include 633 base-pair nucleic acids, and BLAST analysis on its nucleotide sequence revealed 96% identity with the Lucilia cuprina netrin-1 UNC-5 receptor. The protein residue included 210 amino acids (aa) and coded for a protein with 24 kD weight. This gene lacked the signal peptide. Furthermore, the UPA domain is conserved in UNC-5. It lied at the interval of 26–131 aa. We identified the CDS of netrin-1 UNC-5 receptor in L. sericata. It could be applied to research activities implementing a new essential component design in wound healing.

Introduction Proteus mirabilis is a biofilm-forming agent that quickly settles on the urinary catheters and causing catheter-associated urinary tract infections. Thus, the spread of multidrug-resistant P. mirabilis isolates, with the ability to form a biofilm that carries integron, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated colistin resistance genes (mcr), represents a severe threat to managing nosocomial infectious diseases. This study is aimed at surveying the prevalence of ESBL, integrase, and mcr genes of P. mirabilis, isolated from the catheter, to assess the differences in their antimicrobial susceptibility and clonal dissemination. Method Microtiter plate assay was adopted to measure biofilm formation. The antimicrobial susceptibility was assessed by the disk diffusion method. Antimicrobial resistance genes (intI1, intI2, intI3, blaTEM, blaCTX-M, blaSHV, mcr1, and mcr2) were detected by PCR. All of the isolates were characterized by repetitive sequence-based PCR. Result From 385 collected catheters in patients admitted to the intensive care unit (ICU), 40 P. mirabilis were isolated. All of the isolates could form a biofilm. Proteus spp. had intrinsic resistance to tetracycline (95%) and nitrofurantoin (92.5%), which explains the high resistance prevalence. The most widely resistant antibiotic was trimethoprim-sulfamethoxazole (75%). Thirty-three (82.5%) isolates were classified as multidrug resistance (MDR). The prevalence of intI1 and intI2 genes was 60% and 25%, respectively. In 6 (15%) isolates, both genes were detected. The most frequent ESBL gene detected in all of the isolates was blaTEM. Also, no detection for mcr1 and mcr2 antibiotic resistance genes was reported. Rep-PCR identified 39(GTG)5 types (G1–G39) of 40 isolates that 38 isolates had unique patterns. Conclusion In this study, 82.5% of isolates were MDR with high antibiotic resistance to trimethoprim-sulfamethoxazole. The intI1 and blaTEM were the most prevalent genes in the integrase and ESBL gene family. High diversity was seen in the isolates with Rep-PCR. The increasing rate of MDR isolates with a high prevalence of resistance genes could be alarming and demonstrate the need for hygienic procedures to prevent the increased antibiotic resistance rate in the future.

Plasmodium vivax is the most widespread malaria species parasitizing humans outside Africa, with approximately 100 million cases reported per year. Most human cases of P. vivax are asymptomatic with low parasitemia, making active case detection-based elimination programme challenging and less effective. Despite the widespread distribution of P. vivax, no effective vaccines are currently available. Transmission blocking vaccines have recently emerged as potential vaccine candidates to reduce transmission rates to below the essential levels required for the maintenance of the parasite life cycle. Here, we demonstrated that P. vivax was the predominant species found in a malaria-endemic area, although P. vivax/P. falciparum co-infections were also common. Through genomic sequence analysis and neighbor-joining algorithms, we demonstrated limited genetic heterogeneity in the P. vivax transmission-blocking vaccine candidate Pvs48/45 among clinical isolates of P. vivax. Restricted genetic polymorphism occurred at both nucleotide and amino acid levels. The most frequent mutation was A � G at nucleotide position 77 (46.7), whereas the least frequent was C � T at nucleotide position 1230 (3.3). The occurrence of single nucleotide polymorphisms (SNPs) distribution at 6/8 positions (75) led to changes in amino acid sequences in the Pvs48/45 loci, whereas 2/8 (25) of SNPs resulted in no amino acid sequence variations. Consistently, the nucleotide diversity in the Pvs48/45 locus among the P. vivax population studied was extremely low (I� = 0.000525). Changes in amino acid sequences in the Pvs48/45 protein did not result in substantial conformational modifications in the tertiary structures of these proteins. Unveiling the population genetic structure and genetic heterogeneity of vaccine target antigens are necessary for rational design of transmission-blocking antibody vaccines and to monitor the vaccine efficacy in clinical trials in endemic areas for malaria. © 2021 Elsevier B.V.

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.

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.

Additional file 4. Predicted peptides which are presented by MHC-II from AgAPN-1.

Additional file 2. Antigenic peptides of AgAPN-1.

Additional file 1. Antigenic peptides of AsAPN-1.

Additional file 3. Predicted peptides which are presented by MHC-II from AsAPN-1.

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.

The current study was performed to determine Hirudinea species diversity collected from rice fields of Guilan province from June 2012 to June 2020. This study reports the most recent information on leech species (Annelida, Clitellata) in this region. Morphological characters and DNA barcode analysis were used to identify the collected samples. According to the acquired results, there were two leech families in this zone (Glossiphoniidae and Hirundinidae). Based on morphological criteria 5 species of leech including Hirudo orientalis, Hirudo medicinalis, Hirudo verbana, Helobdella stagnalis and Placobdella costata belonging to 3 genera were identified. For the first time, Hirudo verbana which predominantly bred in leech farms and used as medicinal leech from southeastern Europe and Turkey, was reported in our study. The knowledge on leech fauna in Iran is incomplete. Therefore, more comparative studies based on molecular and morphological techniques should be conducted on different regions. This study was performed to identify the distribution of Hirudinida from Bandar Anzali's wetland of Guilan province, Iran using genetic sequence assessment.

Malaria is still a serious disease, killing hundreds of thousands of people every year. Previous studies on the Aminopeptidase N1 (APN1) protein from Anopheles gambiae (the main malaria vector in Africa) revealed that it is a receptor for sexual stage of Plasmodium falciparum in midgut of this mosquito and can be inhibited by polyclonal antibodies, producing against the N-terminal of this protein. Despite its importance this gene and its protein have not yet been characterized in Anopheles stephensi (the main malaria vector in Eastern Mediterranean Regions and Indochina). In this paper, total cDNA of APN1 from Anopheles stephensi (APN1As) was identified by using Rapid Amplification of cDNA Ends (RACE), real-time PCR was applied to evaluate amount of mRNA level after blood-feeding, and several bioinformatics tools were used for more characterization of identified sequences. Result showed that this protein has highly similarity that of aminopeptidase N1 from Anopheles gambiae , especially in N-terminal part of this protein. Also, expression level of this gene significantly increases after blood-feeding. Therefore, as this gene and its protein have high similarity that APN1Ag, if biological tests confirm, it could be a candidate for transmission blocking vaccines in malaria endemic countries.

Non-invasive therapeutic methods have recently been used in medical sciences. Enzymes have shown high activity at very low concentrations in laboratories and pharmaceutical, enabling them to play crucial roles in different biological phenomena related to living organism, especially human medicine. Recently, using the therapeutic methods based on non-invasive approaches has been emphasized in medical society. Researchers have focused on producing medicines and tools reducing invasive procedures in medical. Collagenases are proteins which catalyze chemical processes and break the peptide bonds in collagen. Collagen may be generated more than the required amount or produced in unsuitable sites or may not degrade after a certain time. In such cases, using an injectable collagenase or its ointment can be helpful in collagen degradation. In both in vitro and in vivo tests, it has been revealed that collagenases have several therapeutic properties in wound healing, burns, nipple pain and some diseases including intervertebral disc herniation, keloid, cellulite, lipoma among others. This review describes the therapeutic application of collagenase in medical sciences and the process for its production using novel methods, paving the way for more effective and safe applications of collagenases.

BackgroundThe recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi revealed only one gene encoding the leucine-rich repeat immune factor APL1, while in Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in A. stephensi have not yet been specifically examined.MethodsThe APL1 locus was manually sequenced to confirm the computationally predicted single APL1 gene in A. stephensi, and APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in A. stephensi and A. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium, and expression analysis.ResultsAPL1 is present as a single ancestral gene in most Anopheles including A. stephensi, but has expanded to three paralogs in an African lineage that includes only the Gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in A. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted A. stephensi is rescued by antibiotic treatment, suggesting that bacteria are the cause of mortality, and that the unique APL1 gene is essential for host survival. Successful Plasmodium development in A. stephensi depends upon APL1 activity for protection from high host mortality, probably caused by exposure to enteric bacteria when parasites cross the midgut epithelial barrier. In contrast, silencing of all three APL1 paralogs in A. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single APL1 gene is regulated by both the Imd and Toll immune pathways, while control by the two pathways is subdivided to different paralogs in the expanded APL1 locus.ConclusionsAPL1 underwent neofunctionalization with both loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the Gambiae complex in Africa.

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.

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, and Quillaja saponaria Molina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity of Escherichia coli-expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in female Anopheles stephensi mosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development in A. stephensi. However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) in A. stephensi. It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria.

Background Plasmodium falciparum parasite is the most deadly species of human malaria, and the development of an effective vaccine that prevents P. falciparum infection and transmission is a key target for malarial elimination and eradication programmes. P. falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate. A comparative study was performed to characterize the immune responses in BALB/c mouse immunized with Escherichia coli-expressed recombinant PfCelTOS (rPfCelTOS) in toll-like receptor (TLR)-based adjuvants, CpG and Poly I:C alone or in combination (CpG + Poly I:C), followed by the assessment of transmission-reducing activity (TRA) of anti-rPfCelTOS antibodies obtained from different vaccine groups in Anopheles stephensi. Methods The aim of the current work was achieved by head-to-head comparison of the vaccine groups using conventional and avidity enzyme-linked immunosorbent assay (ELISA), immunofluorescence test (IFAT), and standard membrane feeding assay (SMFA). Results Comparing to rPfCelTOS alone, administration of rPfCelTOS with two distinct TLR-based adjuvants in vaccine mouse groups showed a significant increase in responses (antibody level, IgG subclass analysis, avidity, and Th1 cytokines) and was able to induce reasonable transmission-reducing activity. Also, comparable functional activity of anti-rPfCelTOS antibodies was found in group that received antigen in either CpG or Poly I:C (69.9%/20% and 73.5%/24.4%, respectively, reductions in intensity/prevalence). However, the vaccine group receiving rPfCelTOS in combination with CpG + Poly I:C showed a significant induction in antibody titers and inhibitory antibodies in oocysts development (78.3%/19.6% reductions in intensity/prevalence) in An. stephensi. Conclusions A key finding in this investigation is that rPfCelTOS administered alone in BALB/c mouse is poorly immunogenic, with relatively low IgG level, avidity, inhibitory antibodies, and mixed Th1/Th2 responses. However, immunological characteristic (IgG level, cytophilic IgG2a and IgG2b, avidity, and Th1 cytokines) and TRA of anti-rPfCelTOS significantly enhanced in the presence of co-administration of TLR-based adjuvants, confirming that targeting TLRs would be an effective means for the enhancement of inducing TRA against rPfCelTOS. Electronic supplementary material The online version of this article (10.1186/s12936-019-2773-3) contains supplementary material, which is available to authorized users.

Malaria is a vector-borne infectious disease that is considered a priority of the World Health Organization due to its enormous impacts on global health. Plasmodium spp. (Haemosporida: Plasmodiidae), Anopheles spp. (Diptera: Culicidae), and a suitable host are the key elements for malaria transmission. To disrupt the parasitic life cycle of malaria or prevent its transmission, these three key elements should be targeted by effective control strategies. Development of vaccines that interrupt malaria transmission is one of the solutions that has been recommended to the countries that aim to eliminate malaria. With respect to the important role of Anopheles stephensi in malaria transmission and involvement of Anopheles carboxypeptidase B1 in sexual parasite development, we characterized the second member of cpb gene family (cpbAs2) of An. Stephensi to provide some basic information and evaluate significance of cpbAs2's role in complementing sexual plasmodium development role of cpbAs1. The cpbAs2 mRNA sequence was characterized by 3' and 5' RACE and the structural features of its coded protein were studied by in silico modeling. The coding sequence and gene structure of cpbAs2 were determined empirically and compared with the in silico predictions from the An. stephensi genome sequencing project. Furthermore, homology modeling revealed that its structure is very similar to the structurally important domains of procarboxypeptidase B2 in humans. This study provides basic molecular and structural information about another member of the cpb gene family of An. stephensi. The reported results are informative and necessary for evaluation of the role of this gene in sexual parasite development by future studies.

Dirofilaria immitis is endemic in the southern parts of Iran where there is a favourable environment for the development of Anopheles stephensi. The aim of the present study was to evaluate the susceptibility of A. stephensi as D. immitis vector under laboratory conditions. Anticoagulated dog blood containing 1,500 microfilariae ml–1 was artificially administered to 140 mosquitoes of the test group (T group), while 94 mosquitoes were left as a control (C group). Blood-fed mosquitoes of the T group were dissected for morphological and molecular analyses at pre-set time points to observe the developmental stage of D. immitis. The results showed that the average number of the microfilariae ingested per female in T group was 9.6. At the end of the study, 16 mosquitoes developed infective thirdstage larvae of D. immitis, and 49 mosquitoes survived at end of incubation period, giving an estimated infection rate of 32.6% and the vector efficiency index of 17%. The infection rate and vector efficiency index suggested that A. stephensi could be considered as a potential vector of D. immitis., Нематоды Dirofilaria immitis обычны в южных провинциях Ирана, где складываются походящие условия для развития комаров Anopheles stephensi. Проведено исследование способности A. stephensi служить вектором-переносчиком для нематод D. immitis в лабораторных условиях. Кровь собак с добавлением антикоагулянтов, содержащие 1,500 микрофилярий в 1 мл была использвоана для заражения голодавших самок комаров. Всего 140 комаров было использовано для эксперимента (Т группа) и 94 особи комаров служили контролем (C группа). Комаров из Т группы вскрывали через определенные промежутки времени для морфологического и молекулярного выявления развивающихся стадий D. immitis. Результаты показали, что в среднем каждая самка комара заглатывала 9.6 микрофилярий. В конце эксперимента было установлено, что 49 самок пережили условия данного опыта (32.6%) и у 16 самок комаров развились инвазионные личинки 3-й стадии D. immitis. Таким образом, индекс эффективности переноса составил 17%. Сделан вывод о способности A. stephensi служить вектором для D. immitis.

We report a case of urinary myiasis occurring in a 60-yr-old Iranian male patient with urinary tract problems and a history of travel to Thailand who was referred to Shafagh Medical Laboratory in Tehran (Iran). Larvae excreted in the patient's urine were confirmed by morphological identification key and DNA barcoding to belong to the species Megaselia scalaris Loew, which is known as the scuttle fly. Based on the patient's history, he was infected with M. scalaris in Thailand. To the best of our knowledge, this is the first report of urinary myiasis caused by M. scalaris in Thailand.

Malaria, AIDS and tuberculosis are considered as the important infectious diseases in the world. Malaria has a close relationship with the climate and life style of the human. Therefore, malaria is known as the disease of the poor countries. With regards to its importance, WHO invited scientists with different specialties for programming the second global malaria eradication program? The recommendation of the vaccine consultative group was development of the vaccines that interrupt malaria transmission (VIMTs). VIMTs are composed of different type of vaccines which vector based TBVs are one of them. Vector based TBVs have advantages in compare other type VIMTs such as impact on the parasite life cycle and mosquito fitness simultaneously. Therefore, targeting the vector and parasite antigens simultaneously can be considered as a robust tool to reach the aim of global malaria eradication program.

A given amino acid sequence can be encoded by a huge number of different nucleic acid sequences. These sequences, however, prove not to be equally useful. The choice of sequence can significantly impact the expression of an encoded protein. As regards the importance of protein-coding sequence and promising industrial and medicinal applications of Clostridium histolyticum collagenase, this study examined the codon optimization of the Col H gene so as to enhance collagenase expression in Escherichia coli (E. coli). The coding region of mature Col H gene was optimized according to the codon usage of E. coli using Gene Designer software (DNA 2.0). The results revealed that relative frequency of codon usage in Col H gene was adapted to the most preferred triplets in E. coli in such a way that codon usage bias in E. coli was enhanced after codon optimization. Similarly, the higher level of collagenase expression was more likely the result of substituting rare codons with optimal codons. As has been reported elsewhere, the findings from this study suggest that codon optimization provides a theoretical improvement in Col H gene expression in E. coli. In spite of that, experimental research is needed to confirm the improvement.

Anopheles (Cellia) stephensi Liston 1901 is known as an Asian malaria vector. Three biological forms, namely "mysorensis", "intermediate", and "type" have been earlier reported in this species. Nevertheless, the present morphological and molecular information is insufficient to diagnose these forms. During this investigation, An. stephensi biological forms were morphologically identified and sequenced for odorant-binding protein 1 (Obp1) gene. Also, intron I sequences were used to construct phylogenetic trees. Despite nucleotide sequence variation in exon of AsteObp1, nearly 100% identity was observed at the amino acid level among the three biological forms. In order to overcome difficulties in using egg morphology characters, intron I sequences of An. stephensi Obp1 opens new molecular way to the identification of the main Asian malaria vector biological forms. However, multidisciplinary studies are needed to establish the taxonomic status of An. stephensi.

Malaria is one of the most important infectious diseases in the world, and it has many economic and social impacts on populations, especially in poor countries. Transmission-blocking vaccines (TBVs) are valuable tools for malaria eradication. A study on Anopheles gambiae revealed that polyclonal antibodies to carboxypeptidase B1 of A. gambiae can block sexual parasite development in the mosquito midgut. Hence, it was introduced as a TBV target in regions where A. gambiae is the main malaria vector. However, in Iran and neighboring countries as far as China, the main malaria vector is Anopheles stephensi . Also, the genome of this organism has not been sequenced yet. Therefore, in this study, carboxypeptidase B1 of A. stephensi was characterized by genomic and proteomic approaches. Furthermore, its expression pattern after ingestion of Plasmodium falciparum gametocytes and the effect of anti-CPBAs1 antibodies on sexual parasite development were evaluated. Our results revealed that the cpbAs1 expression level was increased after ingestion of the mature gametocytes of P. falciparum and that anti-CPBAs1 directed antibodies could significantly reduce the mosquito infection rate in the test group compared with the control group. Therefore, according to our findings and with respect to the high similarity of carboxypeptidase enzymes between the two main malaria vectors in Africa ( A. gambiae ) and Asia ( A. stephensi ) and the presence of other sympatric vectors, CPBAs1 could be introduced as a TBV candidate in regions where A. stephensi is the main malaria vector, and this will broaden the scope for the potential wider application of CPBAs1 antigen homologs/orthologs.

The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The gram-negative and gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries.

Additional file 3: Table S1. Primer sequences.

Additional file 3: Table S1. Primer sequences.

Background and Objectives: HIV-1 and HCV infections are life threatening problems in patients who receive blood products. Serological methods have proven useful in detecting these infections, but there are setbacks that make it challenging to detect these infectious agents. By the advent of Nucleic Acid Testing (NAT) methods, especially in multiplex format, more precise detection is possible. Materials and Methods: We have developed a multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV. Primers were designed for highly conserved region of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex RT-PCR were performed. Results: Analytical sensitivity was considered to be 100 and 200 copies/ml for HIV-1 and HCV, respectively. High concentration of one virus had no significant effect on the detection of the other one with low concentration. By analysis of 40 samples, clinical sensitivity of the assay was determined to be 97.5%. Using different viral and human genome samples, the specificity of the assay was evaluated to be 100%. Conclusions: The aim of this study was to develop a reliable, rapid and cost effective method to detect HIV-1 and HCV simultaneously. Results showed that this simple and rapid method is perfectly capable of detecting two viruses in clinical samples.