Emergent spotted fever group Rickettsiae infections among hard ticks in Islamic Republic of Iran.

Background: Tick-borne rickettsioses have become a health concern worldwide following the increasing incidence in recent decades. However, there is limited information about these diseases in Islamic Republic of Iran. Aim: This cross-sectional study was conducted to estimate the Rickettsia infection among ixodid ticks collected from cattle, sheep and goats in Islamic Republic of Iran. Methods: The DNA of ixodid ticks collected from cattle, sheep and goats in 54 villages of Zanjan Province, Islamic Republic of Iran, were collected and analysed using a spectrophotometer. Rickettsial-positive samples were screened by targeting the htrA gene and fragments of gltA gene were analysed. The variables were analysed using descriptive statistics and the χ2 test was used to compare the variables. Results: A total of 528 ticks were tested. Overall, Rickettsia infection rate was 6.44%. Nine of the 12 tick species were infected. Rickettsial positive rates in Hyalomma marginatum and Dermacentor marginatus were 21.33% and 12.77%, respectively. R. aeschlimannii, the predominant rickettsia, was detected only in Hy. marginatum. R. raoultii, R. sibirica and R. slovaca comprised about half of the positive ticks and were recovered from more than one tick species. Conclusion: Considering the discovery of infected ticks in the Islamic Republic of Iran, there is a need to establish a tick control programme in the country, paying attention to populations at high-risk.

A comprehensive survey of permethrin resistance in human head louse populations from northwest Iran: ex vivo and molecular monitoring of knockdown resistance alleles.

BACKGROUND: Head louse infestation is an important public health problem, and expanding resistance to permethrin is a major challenge to its control. The mapping and detection of pyrethroid resistance are essential to the development of appropriate treatments and ensure the effectiveness of current measures. The aim of this study was to present the phenotypic and genotypic basis of permethrin resistance and identify knockdown resistance (kdr) mutations in head louse populations in northwestern Iran. METHODS: Adult head lice were collected from 1059 infested girls in Ardebil, East Azerbaijan, West Azerbaijan and Zanjan Provinces, northwestern Iran. The toxicity of permethrin and the possible synergistic effect of piperonyl butoxide (PBO) on this toxicity were assessed using bioassays. Fragments of voltage-sensitive sodium channels (vssc) and cytochrome b (cytb) genes were amplified and analyzed for the detection of knockdown resistance (kdr) mutations and mitochondrial groups. Moreover, genotypes of the two hot spot regions of the vssc gene were determined by melting curve analysis of amplicons. RESULTS: A total of 1450 adult head lice were collected during 2016-2021. Live lice were exposed to a dose of 1% permethrin for 12 h, and the median lethal time (LT50) and time to achieve 90% mortality (LT90) were determined to be 6 and 14.8 h, respectively. Topical application of 2 and 16 ng permethrin per louse resulted in 25% and 42.11% mortality, respectively. Pre-exposure of samples to 3% piperonyl butoxide had no synergistic effect on the effects of permethrin. Analysis of the 774-bp vssc gene fragment showed the presence of the M815I, T917I and L920F mutations, wild-type and T917I mutation, in 91.6%, 4.2% and 4.2% of samples, respectively. Investigation of the mitochondrial cytb gene demonstrated the predominance of clade B. The frequency of domain II segment 4 (S4)-S5 kdr genotypes in mitochondrial groups was identical, and heterozygotes were present in 93.5% of samples. A significant difference was detected in the frequency of domain IIS1-S3 kdr genotypes, and the frequency of resistant alleles and heterozygotes was higher in clade B than in clade A. CONCLUSIONS: The presence of kdr mutations in the vssc gene and the non-synergist effect of PBO indicate that pyrethroid target site insensitivity is the main resistance mechanism. This phenomenon and the high frequency of resistant alleles necessitate that new pediculosis management programs be developed. Further studies need to be conducted to identify all factors contributing this resistance and to develop alternative pediculicides.

Molecular Survey of Mitochondrial Genes in Different Populations of the Black Fat-Tailed Scorpion, Androctonus crassicauda.

Background: Androctonus crassicauda is the most medically relevant scorpion and understanding its genetic forms is essential for improvement of anti-venom sera, and risk management of scorpionism. Present study was designed to identify the variations of mitochondrial genes in different populations of A. crassicauda. Methods: Adults of A. crassicauda were collected from Zanjan Province during 2016-2017. Genomic DNA of samples was extracted and fragments of mitochondrial 16S, COI and ND1 genes were amplified and some of the amplicons were sequenced. Haplotype of samples were identified by multiple alignment of sequences, then phylogenetic trees of haplotypes were constructed. Results: Fragments of 352bp, 618bp and 680bp were amplified from 16S, COI and ND1 genes respectively. Nucleotide sequence in COI fragments was conserved, however, five haplotypes with some specific polymorphic sites were detected in 16S and ND1 fragments. Haplotype I was dominant and found in all areas. Other haplotypes were rare and limited to specific regions. Analysis of the phylogenetic trees inferred from 16S and COI genes, confirmed a strong positive correlation between geographic and genetic distance. Conclusion: Mitochondrial COI, 16S and ND1 genes were detected suitable for identifying the population structure. Five genotypes were found using 16S and ND1 genes. To prepare and improve the anti-venoms quality, additional studies are necessary to identify the toxin electrophoretic profile and geographical/ecological niche models of these genotypes in future.

Morphometric Indices and Venom Protein Profile in Different Populations of Androctonus crassicauda.

Background: Androctonus crassicauda is the most medically relevant animal and understanding its morphological characteristics is essential in the production of antiscorpion sera. Methods: Adults of A. crassicauda were collected from different areas of Zanjan Province and the morphometric parameters and the cuticular fluorescence patterns of samples were studied. The crude venom of samples was extracted by electric stimulation, and their biochemical properties were analyzed by the SDS-PAGE method. Results: Values of the morphometric parameters depended on sex and altitude of the area. Except for values of the pectinal organ, these parameters in females were higher than in males. No significant difference was in the number, shape, and intensity of cuticular fluorescence patterns. The body length of males in high and lowlands was 72.53±1.53 and 77.33±2.70mm, respectively. Females' body lengths in that area were 81.66±2.19 and 86.55±2.33mm, respectively. Analysis of toxin proteins showed two isotypes that the 12, 13, 15, 16, 18, and 19kDa proteins were in all areas. However, the 41 and 74kDa proteins, and 46 and 63kDa proteins were detected in low and highlands, respectively. Conclusion: Black fat-tailed scorpion has a considerable dominancy and developing preventive programs and providing treatment facilities in studied areas are necessary. Values of the morphological parameters and venom electrophoresis patterns depended on the geographical location. Therefore, pool crude toxin is suggested for the production of effective antivenoms. Moreover, additional field complementary works in the geographic information system based niche modeling and mass fingerprinting of scorpion venoms are suggested for screening effective isotypes.

High levels of pyrethroid resistance and super-kdr mutations in the populations of tropical bed bug, Cimex hemipterus, in Iran.

BACKGROUND: The tropical bed bug, Cimex hemipterus, is an important ectoparasite causing various health problems. This species is mainly confined to tropical regions; however, insecticide resistance, global warming, and globalization have changed its distribution map. Molecular information on pyrethroid resistance, which is essential for the development of control programs, is unknown for C. hemipterus in expanded areas. The present study was designed to determine the permethrin resistance status, characterize the pyrethroid receptor sites in voltage-gated sodium channel (vgsc) gene, and identify the resistance-related mutations in the populations of tropical bed bug in Iran. METHODS: Live bed bugs were collected, and adults of C. hemipterus were selected for bioassay and molecular surveys. Bioassay was performed by tarsal contact with permethrin 0.75% in mixed-sex of samples. Conventional and quantitative TaqMan and SYBR Green real-time PCR assays were conducted to characterize the vgsc gene and genotypes of studied populations. RESULTS: In the bioassay tests, the mortality rates were in the range of 30.7-38.7% and 56.2-77.4% in 24 and 48 h, respectively. The knockdown rates of studied populations were in the range of 32.2-46.6% and 61.5-83.8% in the first and second days, respectively. The KT50 and KT90 values in the Cimex lectularius Kh1 strain were presented as 5.39 and 15.55 h, respectively. These values in the selected populations of C. hemipterus varied from 27.9 to 29.5 and from 82.8 to 104.4 h, respectively. Knockdown time ratios (KR50 and KR90) in these populations varied from 5.17 to 6.17-fold compared with those of the C. lectularius Kh1 strain. Fragments of vgsc gene with 355 bp and 812 bp were amplified. Analysis of sequences revealed the A468T substitution, kdr-associated D953G, and super-kdr M918I and L1014F mutations in all populations. CONCLUSIONS: The specific/sensitive, safe, and rapid diagnostic assays developed in this study are recommended for detection of kdr/super-kdr mutations and frequency of mutant alleles. The presence of super-kdr mutations and high resistance to permethrin in all the populations necessitate the reconsideration of control approaches against C. hemipterus.

Diversity of mitochondrial genes and predominance of Clade B in different head lice populations in the northwest of Iran.

BACKGROUND: The head louse, Pediculus humanus capitis, is the most important ectoparasite causing many health problems. Several linkages are presented for this parasite, each representing a particular geographical distribution, prevalence rate, vector competence, susceptibility to pediculicides, and infestation rate. Determining the genetic nature of these linkages is necessary to identify the population structure and also to develop and monitor control programmes against head lice. This study was designed to analyse cox1 and cytb genes and determine the mitochondrial clades in head lice populations in the northwest of Iran. METHODS: Adult head lice were collected from infested females of Ardabil, East and West Azerbaijan, and Zanjan Provinces from 2016 to 2018. Partial fragments of the mitochondrial genes cox1 and cytb were amplified by PCR and some of the amplicons were sequenced. All confirmed sequences were analysed, and the frequency of each mitochondrial clade was determined in the studied areas. RESULTS: A total of 6410 females were clinically examined, and 897 adult head lice were collected from 562 infested cases. Genomic DNA was extracted from 417 samples, and fragments of cox1 and cytb genes were amplified in 348 individuals. Analysis of the 116 sequences showed the 632-bp and 495-bp fragments for cox1 and cytb genes, respectively. The nucleotide and haplotype diversities of cytb and cox1 genes were 0.02261 and 0.589 and 0.01443 and 0.424, respectively. Sequence analysis indicated 6 haplotypes clustered in two clades, A and B. The relative prevalence of clade B was 73% for cytb and 82% for cox1 gene. Haplotypes of clade B were found in all the studied areas, while those of clade A were observed only in rural and suburban areas. CONCLUSIONS: To our knowledge, this is the first study investigated deeply the field populations of Pediculus and documented two clades in the Middle East. The considerable prevalence of pediculosis in the studied areas requires authorities' attention to establish effective control and preventive measures. Given the role of cytb in monitoring population groups, application of this marker is suggested for future epigenetic studies to evaluate the factors affecting the abundance of these clades.

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.

First report of target site insensitivity to pyrethroids in human flea, Pulex irritans (Siphonaptera: Pulicidae).

The human flea, Pulex irritans, is the most important ectoparasite of humans. Intensive use of pyrethroids for its control has led to insecticide resistance. Monitoring pyrethroid resistance and its underlying mechanisms is essential for flea control. The aims of this study were to identify the susceptibility status of human flea to permethrin and to detect the presence of knockdown resistance (kdr) mutation and its frequency in populations of P. irritans. Adults of P. irritans were collected from Zanjan Province, northwest of Iran, during 2013-2017. Different populations of this flea were exposed to permethrin 0.75% for one and 8 h and then the mortality rate, as well as KD50 and KD95 times were calculated. Total RNA and gDNA of samples were extracted, and the fragments of cDNA encoding the partial voltage-gated sodium channel (VGSC) peptides were amplified using degenerated primers. Specific PCR and TaqMan real-time assays were conducted to characterize the vgsc gene and to detect the presence of mutation and genotyping of the populations. Mortality rates were in the range from 32% to 67% for one-hour and 73% to 90% for eight-hour exposure to permethrin 0.75%. KD50 and KD95 times varied in a range from 46 to 241 and 177 to 899 min, respectively. Sequencing of 70 amplified fragments of gDNA resulted in a 578-bp product. These fragments contained two introns (92 and 63 bp) and three exons (141, 189, and 92 bp) encoding 138 amino acids that encompassed IIS4-IIS6 and the partial linker between domains II and III of VGSC. All the studied populations showed L1014F mutation, substitution of CTT for TTT at the 1014 allele. The result of TaqMan assay for 624 samples showed 96.6% homogenous and 3.36% heterozygous mutant. The development of permethrin resistance and the presence of the L1014F mutation at high frequency in flea populations indicate that pyrethroids are likely ineffective in controlling human flea. Therefore, novel alternative control methods are needed to combat this human ectoparasite.

Molecular survey of ITS1 spacer and Rickettsia infection in human flea, Pulex irritans.

The human flea is an important ectoparasite causing serious public health problems worldwide. Planning and monitoring the control programs against this vector require the knowledge of population structure and vector competence. This study was carried out to identify molecular structure of internal transcribed spacer 1 (ITS1) of ribosomal gene and its capability in the survey of Pulex irritans populations as well as to investigate Rickettsia infection in these populations. Flea samples were collected via human baits from animal farms in two districts of Zanjan Province, northwest of Iran. The ITS1 region and the partial Rickettsia gltA gene were amplified from the samples of human flea, and 30 amplicons were sequenced. The 1136 collected fleas consisted of 1079 (94.98%) P. irritans, 36 (3.17%) Ctenocephalides canis, and 21 (1.85%) Ctenocephalides felis. Molecular investigation of 182 human fleas detected the infection of Rickettsia sp. in 4.9%. The ITS1 region covered 957 bp and contained three tandem units of 98-99 bp, starting at positions 145, 245, and 331. Multiple alignments of ITS1 sequences showed single-nucleotide polymorphism at position 798, which caused the substitution of cytosine for adenine in the novel haplotype. High frequency of P. irritans and its Rickettsia infection requires the application of vector control measures, and full characterization of Rickettsia sp. and its potential to cause disease in humans. Regarding the consistency of ITS1 region and its ability to differentiate insect communities, further investigations are recommended to identify the role of selective factors in maintenance of this spacer.

Repellency Effect of Essential Oils of some Native Plants and Synthetic Repellents against Human Flea, Pulex irritans (Siphonaptera: Pulicidae).

BACKGROUND: Fleas are important vectors of human and animal disease, and control measures for protection against their bites and flea-borne diseases are necessary. METHODS: The essential oils (EOs) of four native medicinal plants, Ziziphora tenuiore, Myrtus communis, Achillea wilhelmsii and Mentha piperita were isolated by hydrodistillation technique and analyzed by GC-MC. The repellent activity of EOs and synthetic compounds, DEET and permethrin, were assayed on human subjects against field collected fleas. The effective doses of 50% and 90% of EOs and synthetic compounds were estimated by probit analysis of dose and response regression line. RESULTS: Analysis of EOs revealed about 19 major components. All oils were found to be more repellent (ED50 range= 208-955µg cm-2) than DEET and permethrin (ED50 range= 27-182 × 103µg cm-2). Thyme and myrtle oils showed high repellent activities and among the total detected terpenes, thymol (36.26%) and α-pinene (32.5%) were the major components of those oils respectively. CONCLUSION: Low repellent potency of DEET and permethrin against fleas might be related to flea olfactory system and further molecular and electrophysiological studies are required to conceive new ideas for the discovery and development of the next generation of repellents. Based on high repellent activity of thyme and myrtle essential oils against Pulex irritans further studies should be staged to develop their appropriate effective formulations. Likewise, field trials should be carried out to evaluate the operational feasibility and dermal toxicity over a long period.

First Record of Urogenital Myiasis Induced by Megaselia scalaris (Diptera: Phoridae) from Iran.

Myiasis is the infestation of organs and tissues of human and animals with fly larvae. This article reports an 18 year-old man with urogenital myiasis, the passing of live Megaselia scalaris larvae in the urine, from Zanjan City, northwest of Iran. We discourse the importance of diagnosis and management of urogenital myiasis in medicine.