Evaluation of a new fusion antigen, cd loop and HAP2-GCS1 domain (cd-HAP) of Plasmodium falciparum Generative Cell Specific 1 antigen formulated with various adjuvants, as a transmission blocking vaccine.

BACKGROUND: Malaria is a major global health challenge, and for the elimination and eradication of this disease, transmission-blocking vaccines (TBVs) are a priority. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising TBV candidate, is essential for gamete fertilization. The HAP2-GCS1 domain of this antigen as well as its cd loop could induce antibodies that partially inhibit transmission of P. falciparum. METHODS: In the current study, a new synthetic fusion antigen containing cd loop and HAP2-GCS1 domain (cd-HAP) of PfGCS1 was evaluated as a transmission blocking vaccine candidate. Initially, the profile of naturally acquired IgG antibodies to the cd-HAP antigen was analysed in Iranian individuals infected with P. falciparum, to confirm that this new fusion protein has the appropriate structure containing common epitopes with the native form of PfGCS1. Then, the immunogenicity of cd-HAP was evaluated in BALB/c mice, using different adjuvant systems such as CpG, MPL, QS-21, and a combination of them (CMQ). Furthermore, the blocking efficacy of polyclonal antibodies induced against these formulations was also assessed by oocyst intensity and infection prevalence in the Standard Membrane Feeding Assay (SMFA). RESULTS: The naturally acquired antibodies (dominantly IgG1 and IgG3 subclasses) induced in P. falciparum-infected individuals could recognize the cd-HAP antigen which implies that the new fusion protein has a proper conformation that mimics the native structure of PfGCS1. Concerning the immunogenicity of cd-HAP antigen, the highest IgG levels and titers, by a Th1-type immune profile, and elevated antibody avidity were induced in mice immunized with the cd-HAP antigen formulated with a combination of adjuvants (P < 0.0001). Additionally, cytokine profiling of the immunized mice displayed that a high level of IFN-γ response, a Th1-type immune response, was produced by splenocytes from immunized mice that received cd-HAP antigen in combination with CMQ adjuvants (P < 0.0001). This formulation of cd-HAP antigen with CMQ adjuvants could reduce oocyst intensity and infection prevalence by 82%, evidenced by the SMFA and hold significant implications for future malaria vaccine development. CONCLUSION: Altogether, the results showed that cd-HAP antigen formulated with a combination of the adjuvants (CMQ), could be a promising formulation to develop a PfGCS1-based transmission-blocking vaccine.

Optimization of the heterologous expression and purification of Plasmodium falciparum generative cell specific 1 in Escherichia coli.

Generative cell specific 1 (GCS1) or Hapless2 (Hap2) is a main transmission-blocking vaccine (TBV) candidate against malaria. Experience has shown that this protein is difficult to express in heterologous hosts. In a study, Plasmodium falciparum GCS1 (PfGCS1) could be expressed in fusion with Glutathione S Transferase (GST). Since the large fusions could influence the immunogenicity of the recombinant antigens, in the current study, we tried to express PfGCS1 protein without large fusion tags with an appropriate yield and purity in E. coli. To this end, pfgcs1 gene was codon-optimized and cloned in pET23a plasmid. The expression was evaluated in different E. coli hosts [E. coli BL21(DE3), E. coli BL21(DE3) pLysS, E. coli Rosetta(DE3), and E. coli Rosettagami(DE3)] and media cultures. In addition, the effect of post-induction times, inducer concentration, temperature, and supplementation of glucose and ethanol to culture media were evaluated. The obtained results revealed that rPfGCS1 protein was expressed in all examined E. coli hosts and media cultures with different yields, with the best yield in E. coli BL21(DE3), and E. coli Rosetta(DE3) hosts in TB medium, 16 h post-induction. The expression of rPfGCS1 was confirmed by western blotting using anti-His antibodies. Expression in low temperature at 20 °C and addition of glucose and ethanol to TB media could improve the expression of rPfGCS1. We could express and purify rPfGCS1 without a large fusion protein with an appropriate yield and purity in E. coli Rosetta(DE3). We will evaluate this antigen as TBV candidate against P. falciparum transmission in the future.

Molecular characteristics of odorant-binding protein 1 in Anopheles maculipennis.

BACKGROUND: Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding proteins (OBPs), which influence the chemosensory system and behavioural responses, is essential to understanding the population structure and developing effective control measures against this vector. The present study was designed to identify and analyse the obp1 gene in An. maculipennis. METHODS: Adults of An. maculipennis sensu stricto were collected in Zanjan Province, northwest of Iran, and gDNAs of female mosquitoes were extracted. Fragments of An. maculipennis obp1 (Amacobp1) gene were amplified using degenerate and specific primers, and some of amplicons were selected for sequencing. RESULTS: Analysis of amplified products identified that the sequence of Amacobp1 gene was 1341 bp long. This gene contains three exons (5', internal, and 3'of 160, 256, and 18 bp, respectively) and encodes 144 amino acids. The sizes of introns I and II in deduced gene are 268 and 358 nucleotides, respectively. The amino acid sequence in the C-terminal of AmacOBP1 is similar to that of major malaria vector Anopheles species. However, its N-terminal has a specific signal peptide with 19 amino acids. This peptide is conserved in different studied populations, and its sequence of amino acids shows the most variation among anopheline species. CONCLUSIONS: Degenerate primers in this study are suggested for studying obp1 gene in Anopheles species. Amacobp1 gene is proposed as a molecular marker for the detection of intraspecific ecotypes and diagnosis of different species within Maculipennis Group. Moreover, the N-terminal of AmacOBP1 peptide is recommended as a molecular marker to identify the Amacobp1 expression patterns in different chemosensory organs for assessing the molecular mechanisms and developing novel behavioural disturbance agents to control An. maculipennis.

Measuring of IgG2c isotype instead of IgG2a in immunized C57BL/6 mice with Plasmodium vivax TRAP as a subunit vaccine candidate in order to correct interpretation of Th1 versus Th2 immune response.

Evaluation of the murine isotype antibodies is essential in subunit vaccine development because inbred mouse strains with diverse genetic backgrounds respond different to recombinant proteins. In this regard, the main goal of this study was to measuring and comparing the profile of IgG isotype responses in C57BL/6 mice. For this purpose, the extracellular region of plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) gene was expressed in Escherichia coli Rosetta (DE3)-pET23a. Then, the recombinant PvTRAP alone or emulsified with Freund's complete adjuvant were applied for immunization of the C57BL/6 mice. The role of antibodies and cellular immune responses induced by recombinant PvTRAP were evaluated. The results showed the level of anti-rPvTRAP IgG2c was significantly higher than IgG2a in the groups that received rPvTRAP alone (mean OD490 = 0.798 ± 0.12 and 0.39 ± 0.1, respectively) and emulsified with CFA/IFA (mean OD490 = 1.48 ± 0.07 and 0.605 ± 0.13, respectively; P < 0.05, independent sample t-test). Additionally, the immunized mice with rPvTRAP and rPvTRAP + CFA/IFA had an intermediate-avidity IgG2a antibody but high-avidity IgG2c antibody as well as the mean of serum antibody titers results exhibited that in both rPvTRAP and rPvTRAP + CFA/IFA mouse groups, IgG2a end-point titer (1:3200 and 1:25,600, respectively) was noteworthy lower than IgG2c (1:25,600 and 1:102,400, respectively). Moreover, the results revealed the eliciting significant levels of IFN-γ (P < 0.05, independent sample t-test) and no detectable level of IL-4 in the mouse groups received rPvTRAP alone and emulsified with CFA/IFA as compared to the mouse control groups. In general, our results showed that for correctly interpreting of Th1 immune responses in C57BL/6 mouse strain it is critical to measure IgG2c instead of IgG2a along with IFN-γ.

Correction: Untangling population structure and genetic diversity of reticulocyte binding protein 2b (PvRBP2b) erythrocytic stage vaccine candidate in worldwide Plasmodium vivax isolates.

[This corrects the article DOI: 10.1371/journal.pone.0266067.].

Genotyping Plasmodium vivax isolates infecting Anopheles stephensi, an Asian main malaria vector.

Malaria is a serious vector-borne infectious disease in Iran and Anopheles stephensi has long been suspected as a main malaria vector. However, opinions about its vectorial capacity in supporting Plasmodium species and strains are not clear. This study investigates the susceptibility of an Asian main malaria vector, An. stephensi, to Plasmodium vivax isolates, collected from tropical region of Iran. P. vivax gametocytes which used in ex vivo assay to An. stephensi infection were genotyped by using PCR-RFLP and sequencing. A 650-bp fragment was amplified from patients, followed by RFLP analysis using Alu I restriction enzyme determined the presence of P. vivax VK210 variants. Sequence analysis also showed 100% similarity with the previously reported VK210 sequences of haplotype B from Iran. This is the first study that reports An. stephensi mysorensis is susceptible to P. vivax VK210 haplotype, VK210B. This finding help in useful in better understanding the composition and interaction of Anopheles-Plasmodium and its implication in targeting the main malaria vector for control and elimination programs in Eastern Mediterranean region.

Analysis of Fcgamma receptor IIa (cd32) gene polymorphism and anti-malarial IgG subclass antibodies to asexual blood-stage antigen of Plasmodium falciparum in an unstable malaria endemic area of Iran.

One of the main host genetic factors involved in inflammation, immune responses and pathogenesis of malaria is FcγRIIa (cd32) gene. A single point mutation at position 131 replace an arginine (R) with a histidine (H) that can affect the affinity of the receptor for human IgG subclasses. This investigation was designed to explore the polymorphisms at FcγRIIa gene in association with both anti-malarial total IgG antibody and IgG subclass profiles to C-terminal region of Plasmodium falciparum merozoite surface protein 1 (PfMSP-1(19)). In this study, 166 infected patients with P. falciparum who are living in a malaria endemic area of Iran were studied using PCR-RFLP and ELISA methods. The results showed that the frequency of FcγRIIa-R/R131, -R/H131 and -H/H131 genotypes was 9.6%, 42.8% and 47.6%, respectively. Level of total IgG to recombinant PfMSP-1(19) antigen showed that there was no difference among the FcγRIIa-R/R131, -R/H131 and -H/H131 groups. With regards to the IgG subclasses, the anti-malarial IgG1 antibodies predominated. Also, there was a significant difference between the frequency of positive responders for anti-PfMSP-1(19) IgG and IgG1 antibodies in P. falciparum-infected individuals with FcγRIIa-R/R131, -R/H131 or -H/H131 genotypes (P<0.05, X(2) test). Regarding to IgG2-PfMSP-1(19) antibody, 27.27% (FcγRIIa-R/R131), 25.71% (FcγRIIa-R/H131) and 22.2% (FcγRIIa-H/H131) of IgG responders showed positive antibody response. Taken together, this study is the first report that exhibits the high frequency of both FcγRIIa-H131H genotypes and H131 allele in the Baluchi ethnic group, which was similar to the Fulani ethnic group. The present results provide additional data to understand the role of FcγRIIa-131 genotypes in the pathogenesis of malaria.

Isolation and identification of microbiota of Culex quinquefasciatus for their application as paratransgenic tools in vector control.

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 sequencing, 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, Aeromonas, Arthrobacter, Asaia, Enterobacter, Gluconobacter, Klebsiella, Lysinibacillus, Micrococcus, Psuedomonas and Serratia. 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 populations. This data can be utilized to interfere with the transmission of pathogens and design new strategies for the control of mosquito-borne diseases.

Untangling population structure and genetic diversity of reticulocyte binding protein 2b (PvRBP2b) erythrocytic stage vaccine candidate in worldwide Plasmodium vivax isolates.

BACKGROUNDS: Plasmodium vivax is the predominant Plasmodium species distributed extensively in the Americas and Asia-Pacific areas. Encoded protein by Plasmodium vivax Reticulocyte Binding Proteins (PvRBPs) family member are of critical prominence to parasite invasion and have been considered the significant targets in development of malaria vaccine for the blood stage. As high genetic polymorphism of parasites may impede the effectiveness of vaccine development, more research to unraveling genetic polymorphism of pvrbp2b from various geographical regions seems indispensable to map the exact pattern of field isolates. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to determine the sequences of Iranian pvrbp2b (nt: 502-1896) gene and then, to ascertain polymorphism of pvrbp2b gene, recombination, the level of genetic distances, evaluation of natural selection, and the prediction of B-cell epitopes of Iranian and global P. vivax isolates. Pvrbp2b partial gene was amplified and sequenced from 60 Iranian P. vivax isolates. Iranian pvrbp2b sequences as well as 95 published sequences from five countries were used to evaluate the genetic diversity and neutral evolution signature in worldwide scale. A total of 38 SNPs were identified among 60 Iranian pvrbp2b sequences (32 non-synonymous and 6 synonymous mutations), and 32 amino acid substitutions were observed in 29 positions as compared to Sal-1 sequence. Worldwide sequence analysis showed that 44 amino acid changes had occurred in 37 positions of which seven polymorphic sites had trimorphic mutations while the rest was dimorphic. The overall nucleotide diversity for Iranian isolates was 0.00431 ± 0.00091 while the level of nucleotide diversity was ranged from 0.00337 ± 0.00076 (Peru) to 0.00452 ± 0.00092 (Thailand) in global scale. CONCLUSIONS/SIGNIFICANCE: Of amino acid substitutions, 12 replacements were located in the B-cell epitopes in which nine polymorphic sites were positioned in N-terminal and three polymorphic sites in predicted B-cell epitopes of C-terminal, signifying both variable and conserved epitopes for vaccine designing. Using the achieved outcome of the current investigation interrogate questions to the selection of conserved regions of pvrbp2b and understanding polymorphism and immune system pressure to pave a way for developing a vaccine based on PvRBP2b candidate antigen.

Molecular Detection of Plasmodium Infection among Anophelinae Mosquitoes and Differentiation of Biological Forms of Anopheles Stephensi Collected from Malarious Areas of Afghanistan and Iran.

Background: Updated information on the vectorial capacity of vectors is required in each malarious areas as well in Iran and its neighboring countries such as Afghanistan. The aims of this study were to investigate the potential infection of about 800 specimens collected from malarious areas of Afghanistan and Iran, and to differentiate biological forms of Anopheles stephensi. Method: Two molecular markers, 18S RNA gene subunit and AsteObp1 intron I, were used respectively for investigation Plasmodium infection and identifying the biological forms of An. stephensi. Results: Plasmodium infection was detected in 4 pools of Afghanistan specimens, including An. stephensi, collected from Nangarhar. Individually examination showed infection in 5 An. stephensi (infection rate: 1.25), to P. falciparum (2), P. vivax (2) and a mix infection. Out of five infected specimens, three were intermediate forms and two were mysorensis. No infection was found in specimens collected from Iran (Chabahar County), probably due to the active malaria control program in south-east of Iran. Conclusion: The key role of An. stephensi, as a known Asian malaria vector, was re-emphasized in Afghanistan by the results achieved here. The fauna of vectors and the pattern of biological forms of An. stephensi are similar in both countries that urge regional investigations to provide evidence-based and applied data for decision-maker in malaria control.

Plasmodium vivax: prevalence of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax clinical isolates from Pakistan.

The main aim of the present study was to investigate the frequency of SNPs-haplotypes of dhfr and dhps genes associated to sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax clinical isolates circulating in a malaria endemic area, Pakistan. All 164 collected isolates were analyzed for SNPs-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of pvdhfr and 383 and 553 of pvdhps genes using PCR-RFLP methods. All examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 15.2% and 53.6% of isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B (79.3%) was the most prevalent variant. The combination of pvdhfr and pvdhps haplotypes demonstrated nine distinct haplotypes. The three most prevalent haplotypes were I(13)P(33)F(57)S(58)T(61)S(117)I(173)/A(383)A(553) (43.9%), I(13)P(33)F(57)S(58)T(61)N(117)I(173)/A(383)A(553) (33.6%) and I(13)P(33)F(57)R(58)T(61)N(117)I(173)/A(383)A(553) (12.2%). The presence of mutant haplotypes is worrying and indicates the emergence of drug tolerant/resistant P. vivax isolates in Pakistan in near future.

Relationship between Wolbachia infection in Culex quinquefasciatus and its resistance to insecticide.

Many studies have been done on the various factors affecting resistance to insecticides. The relationship between Wolbachia bacteria and resistance to insecticides is one of the factors that has attracted a lot of attentions. Wolbachia are obligatory intracellular endosymbionts that naturally occur in a wide range of arthropods and nematodes, including the mosquito Culex quinquefasciatus. Initially, the presence of bacteria was proved by molecular assays. Then the resistance level of this species was evaluated in adults against DDT 4.0% and deltamethrin 0.05% using the standard WHO guideline. After elimination of Wolbachia by tetracycline and its proof by molecular assays, the susceptibility tests were conducted again on uninfected line. Finally, the two lines were compared in terms of responding to insecticides. The findings indicated that there is no significant correlation between susceptibility of two lines in response to DDT 4.0% while they represented a significant correlation for deltamethrin (P =0.00). We propose that Wolbachia bacteria increase the susceptibility to deltamethrin but they show neutral effect on DDT susceptibility in Cx. quinquefasciatus. However, more studies on other vectors and insecticides still need to be done.

Haemoproteosis and avian malaria in Columbidae and Corvidae from Iran.

Avian malaria (Plasmodium) and related genera (Haemoproteus and Leucocytozoon) are diverse and widespread parasites. Despite the extent of knowledge on avian haemosporidian parasites, information about domestic and wild bird's blood parasites is overall insufficient in Iran. Prevalence of the haemosporidian parasites' and phylogenetic relationship of lineages are studied by using molecular and morphological results of 152 examined hosts belonging to 17 species. Molecular analysis for haemosporidian detections demonstrated overall prevalence 22.36%. Inspected hosts mostly belonging to Common Pigeons (Columba livia) parasitized by Haemoproteus spp., and Hooded Crows (Corvus cornix) and Carrion Crow (C. corone) were identified as hosting Plasmodium spp. Detected lineages COLIV03, COQUI05, LINN01, ROFI04 and SGS01 are identified as new reports from Iran. We detected no evidence of Leucocytozoon lineages, while the high prevalence of H. columbae was found in Common Pigeons. Such investigation on avian blood parasites contributes to providing new information on the prevalence, epidemiology and geographical distribution of haemosporidian parasites circulating in domestic, pets and wild birds.

Molecular Monitoring of Knockdown Resistance in Head Louse (Phthiraptera: Pediculidae) Populations in Iran.

Knockdown resistance (kdr) is a common mechanism of insecticide resistance in head lice to the conventionally used pyrethroid pediculosis and can be the result of various amino acid substitutions within the voltage-sensitive sodium channel (VSSC). In this study, 54 sequences from varied specimens were investigated to monitor well-known resistance mutations and probable new mutations. The Pediculus humanus capitis de Geer specimens were collected from 13 provinces in Iran. The specimens were stored in 70% ethanol until DNA extraction and PCR amplification of ~900-bp fragment of VSSC. The sequences were analyzed using different bioinformatics software for the detection of well-known kdr substitutions and additional mutations potentially associated with kdr resistance in head lice. There were six new and an old (haplotype I) kdr haplotypes within the Iranian head louse population. K794E, F815I, and N818D amino acid substitutions were reported for the first time. The P813H mutation was the most prevalent amino acid substitution in eight provinces. Among 53 sequences, 26 (49%) were homozygous susceptible, and 27 (51%) were heterozygotes. Thus, 51% of the head lice collected in Iran harbored only the P813H allele. The exact test for the Hardy-Weinberg (H-W) equilibrium showed that genotype frequencies differed significantly from the expectation in East-Azerbaijan and Tehran provinces. Moreover, these populations had an inbreeding coefficient (Fis) <0, indicating the excess of heterozygotes. This observation suggests that the populations of head lice from Iran are currently under active selective pressure. For the rest of the populations, H-W equilibrium and the expectations were significantly in harmony. The results of the current study highlight molecular techniques in the accurate detection of resistance genotypes before their establishment within the head louse population. Accurate detection of resistant genotypes seems to be helpful in decision-making on lice control programs and resistance monitoring and management.

Cloning, expression and transmission-blocking activity of anti-PvWARP, malaria vaccine candidate, in Anopheles stephensi mysorensis.

BACKGROUND: Notwithstanding progress in recent years, a safe, an effective and affordable malaria vaccine is not available yet. Ookinete-secreted protein, Plasmodium vivax von Willebrand factor A domain-related protein (PvWARP), is a candidate for malaria transmission-blocking vaccines (TBVs). METHODS: The PvWARP was expressed in Escherichia coli BL21 using the pET-23a vector and was purified using Ni-NTA affinity chromatography from a soluble fraction. Polyclonal antibody was raised against rPvWARP and transmission blocking activity was carried out in an Anopheles stephensi-P. vivax model. RESULTS: Expression of full length of PvWARP (minus signal peptide) expression showed a 35-kDa protein. The purified protein was recognized by mouse polyclonal antibody directed against rPvWARP. Sera from the animals displayed significantly a blocking activity in the membrane feeding assay of An. stephensi mysorensis. CONCLUSIONS: This is the first report on P. vivax WARP expression in E. coli that provides an essential base for development of the malaria TBV against P. vivax. This may greatly assist in malaria elimination, especially in the oriental corner of WHO Eastern Mediterranean Regional Office (WHO/EMRO) including Afghanistan, Iran and Pakistan.

Detection of mixed Plasmodium falciparum & P. vivax infections by nested-PCR in Pakistan, Iran & Afghanistan.

BACKGROUND & OBJECTIVES: Species identification and information on transmission pattern of malaria parasite in any malaria endemic area is key to success for a malaria control programme. In this investigation, malaria diagnosis using molecular method was used to assess the transmission pattern of malaria parasite in three malaria endemic regions: Afghanistan, Iran and Pakistan. METHODS: Blood samples were collected from the patients presenting with vivax malaria from Afghanistan (n=108), Iran (n=200) and Pakistan (n=199). Malaria parasite detection was made by the gold standard (microscopy) and also nested-PCR assay, using 18S small sub-unit ribosomal RNA (ssrRNA) gene. RESULTS: Based on microscopy method, the level of mixed infection was zero to 2.5 per cent; however, nested-PCR assay detected 6.5, 22 and 23.5 per cent mixed infections in samples collected from Afghanistan, Iran and Pakistan, respectively. The present results showed that the co-infection of P. vivax with P. falciparum was frequent in malaria endemic regions of Iran and Pakistan. INTERPRETATION & CONCLUSION: The present data suggest the need for improving microscopy diagnosis method and the clinician should also have careful clinical observation, along with the reports on Giemsa- stained thick blood films, particularly in summer time when P. vivax is predominant. Also sharing information on transmission pattern of mixed infection among these countries may help in designing better control strategies for malaria.

Population genetic structure analysis of thrombospondin-related adhesive protein (TRAP) as a vaccine candidate antigen in worldwide Plasmodium falciparum isolates.

Antigenic diversity is a major concern in malaria vaccine development that requires to be considered in developing a malaria vaccine. Plasmodium falciparum thrombospondin-related adhesive protein (PfTRAP) is a leading malaria vaccine candidate antigen. In the current study, we investigated the level of genetic diversity and natural selection of pftrap sequences in P. falciparum isolates from Iran (n = 47). The gene diversity of Iranian pftrap sequences was also compared to available global pftrap sequences deposited in the GenBank or PlasmoDB databases (n = 220). Comparison of Iranian PfTRAP sequences with T9/96 reference sequence showed the presence of 35 amino acid changes in 32 positions and a limited variation in repeat sequences, leading to 13 distinct haplotypes. The overall nucleotide diversity (π) for the ectodomain of Iranian pftrap sequences was 0.00444 ± 0.00043, with the highest diversity in Domain IV. Alignment comparison of global PfTRAP sequences with T9/96 reference sequence indicated 96 amino acid replacements as well as extensive variable repeat sequences (9-23 repeats), which led to 192 haplotypes. Among the global isolates, the lowest nucleotide diversity was detected in French Guianan (0.00428 ± 0.00163) and Iranian (0.00444 ± 0.00043) pftrap sequences, and the most variation was observed in domains II and IV in all populations. The dN-dS value displayed the evidence of positive selection due to recombination and immune system pressure. The Fst analysis revealed a gene flow between African populations; however, genetic differentiation observed between Iranian and other populations probably was due to gene flow barriers. Both conserved and variable epitopes were predicted in B- and T-cell epitopes of PfTRAP antigen. The obtained results from this study could be helpful for developing a PfTRAP-based malaria vaccine.

Detection of haemosporidian parasites in wild and domestic birds in northern and central provinces of Iran: Introduction of new lineages and hosts.

Haemosporidian parasites characterize multi-host and multi-parasite structures which are prevalent among wild bird populations. Here, determination of host records, estimation of the prevalence and diversity of haemosporidian lineages were performed in wild and domestic birds in 11 provinces in Iran. To our knowledge, for the first time in this region, molecular characterization of haemosporidians in migratory water birds, raptors, and domestic birds was carried out: blood or tissue samples were collected from 246 birds belonging to 36 species, 12 families, and 11 orders. The prevalence of Plasmodium, Haemoproteus, and Leucocytozoon were documented as 1.21%, 3.65%, and 0.4%, respectively. Of 36 birds' species inspected in this investigation, 13 individuals of 9 species were parasitized by blood parasites. To our knowledge, five lineages including hANACRE03, hAYTFER01, hAYTFER02, hAQUCYR01, and hSTAL06 were found as un-described lineages, while six known lineages of hLK03, pLK05, lTUSW04, pSW5, hMILANS02, and hHAECOL1 were recorded in hosts within novel geographical regions. Such results are required to fill the gaps in understanding the geographical distribution patterns of wildlife related vector-borne parasites in migratory birds as potential carriers, raptors with high vulnerability, and domestic birds as pet or with economic value.

Molecular characterization of antifolates resistance-associated genes, (dhfr and dhps) in Plasmodium vivax isolates from the Middle East.

BACKGROUND: In Iran, co-infections of Plasmodium vivax and Plasmodium falciparum are common and P. vivax infections are often exposed to sulphadoxine-pyrimethamine (SP). In the present study, the frequency distribution of mutations associated to SP resistance was investigated in pvdhfr and pvdhps genes from field isolates. METHODS: Clinical isolates of P. vivax were collected in two different malaria endemic regions in northern and south-eastern Iran, between 2001 and 2006. All 189 collected isolates were analysed for SNP/haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of pvdhps genes using nested PCR-RFLP methods RESULTS: All 189 examined isolates were found to carry wild-type amino acids at positions 13, 33, 61 and 173, while 57L and 58R and 117N mutations in pure form was detected among 1.1%, 17.5% and 26% examined samples, respectively, with no polymorphisms in different loci of dhps genes. Based on size polymorphism of pvdhfr genes at repeat region, among northern isolates, the frequency distribution for type A and B were 2.2% and 97.8% respectively. However, in southern samples the prevalence of type A, B and C were 7%, 89.5% and 7.7%, respectively. Mixed genotype infections (type B and C) were detected in only 4.2% (6/143) of southern, but in none of the northern isolates. The combination of pvdhfr and pvdhps haplotypes among all 189 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were I13P33F57S58T61S117I173/A383A553 (65.6%) and I13P33F57S58T61N117I173 (16.4%). Two other alleles with one point mutation I13P33F57R58T61S117I173/A383A553 and two mutations I13P33F57R58T61N117I173/A383A553 accounted for 7.4% and 9.5% of the total isolates. CONCLUSION: The present molecular data provide important information for making decisions on population based drug use in Iran. In addition, since October 2005, with more availability of SP as first-line treatment, P. vivax isolates are more exposed to SP and the selection or spread of resistant pvdhfr and pvdhps alleles might increase in the near future in this region.

Analysis of von Willebrand factor A domain-related protein (WARP) polymorphism in temperate and tropical Plasmodium vivax field isolates.

BACKGROUND: The identification of key molecules is crucial for designing transmission-blocking vaccines (TBVs), among those ookinete micronemal proteins are candidate as a general class of malaria transmission-blocking targets. Here, the sequence analysis of an extra-cellular malaria protein expressed in ookinetes, named von Willebrand factor A domain-related protein (WARP), is reported in 91 Plasmodium vivax isolates circulating in different regions of Iran. METHODS: Clinical isolates were collected from north temperate and southern tropical regions in Iran. Primers have been designed based on P. vivax sequence (ctg_6991) which amplified a fragment of about 1044 bp with no size variation. Direct sequencing of PCR products was used to determine polymorphism and further bioinformatics analysis in P. vivax sexual stage antigen, pvwarp. RESULTS: Amplified pvwarp gene showed 886 bp in size, with no intron. BLAST analysis showed a similarity of 98-100% to P. vivax Sal-I strain; however, Iranian isolates had 2 bp mismatches in 247 and 531 positions that were non-synonymous substitution [T (ACT) to A (GCT) and R (AGA) to S (AGT)] in comparison with the Sal-I sequence. CONCLUSION: This study presents the first large-scale survey on pvwarp polymorphism in the world, which provides baseline data for developing WARP-based TBV against both temperate and tropical P. vivax isolates.