Borrelia theileri infections in Rhipicephalus annulatus ticks from the north of Iran.

Ticks serve as vectors and reservoirs of various Borrelia species, potentially causing diseases in humans and animals. Mazandaran, a fertile green land in northern Iran, provides ample grazing grounds for livestock and harbors at least 26 hard tick species. This study investigated Borrelia infection in hard ticks from forest areas in this region and compared their genetic identity with the species data in the GenBank database. A total of 2,049 ticks were collected manually from mammalian hosts or using dragging and flagging methods. These ticks were then grouped into 190 pools and 41 individuals based on host, species, developmental stage, and gender. A real-time PCR (qPCR) detected Borrelia DNA in 26 pools from female, male, and nymph of Rhipicephalus annulatus (n = 17) and Ixodes ricinus (n = 9) ticks and one individual female Haemaphysalis punctata tick. The generated partial flaB and glpQ sequences from qPCR-positive Rh. annulatus ticks exhibited the highest identities of 98.1-100% and 98.2% with Borrelia theileri and closely related undefined isolates. Additionally, in phylogenetic analysis, these sequences clustered within well-supported clades with B. theileri and the closely related undefined isolates from various geographic regions, confirming the presence of B. theileri in the north of Iran. Divergence in B. theileri flaB and glpQ sequences across various geographical areas suggests potential subspeciation driven by adaptations to different tick species. This divergence in our flaB sequences implies the possible introduction of B. theileri-infected ticks from different geographical origins into Iran.

First Paleoparasitological Report on the Animal Feces of Bronze Age Excavated from Shahr-e Sukhteh, Iran.

Shahr-e Sukhteh (meaning burnt city in Persian) in Iran is an archeological site dated back to around 3,200-1,800 BC. It is located in Sistan and Baluchistan Province of Iran and known as the junction of Bronze Age trade routes crossing the Iranian plateau. It was appointed as current study area for paleoparasitological investigations. Excavations at this site have revealed various archeological materials since 1967. In the present study, sheep and carnivore coprolites excavated from this site were analyzed by means of rehydration technique using TSP solution for finding helminth eggs. Dicrocoelium dendriticum, Capillaria sp., and Taenia sp. eggs were identified, while some other objects similar to Anoplocephalidae and Toxocara spp. eggs were also retrieved from the samples but their measured parameters did not match those of these species. The present paper illustrates the first paleoparasitological findings of Bronze Age in eastern Iran supporting the economic activities, peopling, and communication as well as the appropriate condition for zoonotic helminthiasis life cycle in Shahr-e Sukhteh archeological site.

Detection and molecular identification of leishmania RNA virus (LRV) in Iranian Leishmania species.

Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

Eimeria leuckarti in equid coprolites from the Sassanid Era (2nd-6th century CE) excavated in Chehrabad Salt Mine archaeological site, Iran.

OBJECTIVE: This study reports coccidian oocysts in an equid coprolite dated to the Sassanid Empire (2nd-6th century CE) recovered in Chehrabad Salt Mine archaeological site, Iran. METHODS: Between 2015 and 2017, an archaeoparasitological investigation led to the discovery of an equid coprolite in the Chehrabad Salt Mine archeological site, (Douzlakh), western Iran. Samples were rehydrated using trisodium phosphate solution and were examined by light microscopy. RESULTS: Seven oocysts of Eimeria leuckarti (Flesch, 1883) were identified; they were in various stages of sporulation. CONCLUSION: This is the first report of ancient coccidian oocysts from equids. The importance of this observation is discussed, and current knowledge of eimeriid oocysts at archaeological sites is reviewed. SIGNIFICANCE: The observations of E. leuckarti increases current knowledge of parasite biodiversity in ancient Iran when it rested along the Silk Road, a network of trade routes connecting the East and West that was central to economic, cultural, political, and religious interactions between these regions, and to livestock movement that could contribute to the transmission of the parasites from/to other regions. LIMITATIONS: The contextual information about animal species present in and around the Salt Mine during its working periods, including Achaemenid dynasty (6th to 4th century BCE) and Sassanid era (2nd to 6th century CE), is very limited and does not allow secure conclusions regarding the host origin of the coprolites. SUGGESTIONS FOR FURTHER RESEARCH: Application of molecular biology tools to identify the correct host origin of the coprolites and to detect more parasite species is advocated.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Molecular investigation of Coxiella burnetii and Francisella tularensis infection in ticks in northern, western, and northwestern Iran.

Tularemia and Q fever are endemic diseases in Iran; however, little information is available on the prevalence of the causative agents, Coxiella burnetii and Francisella tularensis, in Iranian ticks. This study investigated C. burnetii and F. tularensis among hard ticks in this country. We collected ticks from livestock and other mammals in Guilan, Mazandaran, Golestan (northern Iran), Kurdistan (western Iran), and West Azerbaijan (northwestern Iran) provinces. Genomic DNA from collected ticks was extracted and screened for C. burnetii and F. tularensis using Real-time PCR. A total of 4,197 ticks (belonging to 12 different species) were collected, and Ixodes ricinus (46.4%), Rhipicephalus turanicus (25%), and Rhipicephalus sanguineus sensu lato (19.1%) were the most collected species. Of 708 pooled tick samples, 11.3% and 7.20% were positive for C. burnetii and F. tularensis, respectively. The genus of Rhipicephalus had the highest (18.3%) C. burnetii infection among the collected tick pools (P<0.001). Furthermore, the most positive pools for F. tularensis belonged to Haemaphysalis spp. (44.4%). Kurdistan had the most significant percentage of C. burnetii-infected ticks (92.5%), and there was a meaningful relationship between the provinces and the infection (P< 0.001). The ticks from Golestan exhibited the highest F. tularensis infection rate (10. 9%), and the infection showed no significant relationship with the provinces (P = 0.19). Ticks collected from grasslands had a higher Coxiella burnetii infection rate than those collected from animals (39.4% vs. 7.9%; p<0.01). However, ticks collected from animal surfaces had a slightly higher rate of Francisella tularensis infection than those collected from grasslands (7.6% vs. 3.9%; p = 0.24). Here, we demonstrated the presence of both pathogens in the north (Guilan, Mazandaran, and Golestan provinces), the west (Kurdistan province), and the northwest (West Azerbaijan province) of Iran. The public health system should pay particular attention to tick bites in veterinary medicine and humans.

Fasciola Infection Unexpectedly Found During Cholecystectomy: Review on How to Avoid Increasing Surgery Interventions in Non-human Endemic Areas.

PURPOSE: Fascioliasis is caused by Fasciola hepatica of almost worldwide distribution and F. gigantica in wide regions of Asia and Africa. Their adult stage develops in the biliary canals and gallbladder. Infection follows an initial, 3-4 month long invasive, migratory or acute phase, and a several year-long biliary, chronic or obstructive phase. METHODS: The unexpected finding of a fasciolid inside the gallbladder during a cholecystectomy for obstructive lithiasis suspicion in a patient is reported from an area of Iran where human infection had been never reported before and studies on fascioliasis in livestock are absent. RESULTS: The fluke obtained was phenotypically classified as F. hepatica by morphometry and genotypically as F. gigantica by mtDNA cox1 fragment sequencing, although with F. hepatica scattered mutations in species-differing nucleotide positions. The clinical, radiological, and biological signs observed at the acute and chronic phases often lead to some misdiagnosis. Serological methods may be useful in cases of negative coprology. Diagnostic techniques with insufficient resolution leading to unnecessary invasive interventions are analyzed. The way to avoid unnecessary surgery is described, including analyses to be made, diagnostic tools to be used, and aspects to be considered. CONCLUSION: Reaching a correct diagnosis in the confusing presentations avoids procedure delays and unnecessary surgery. A correct drug treatment may be sufficient. Except in extreme pathological presentations, lesions decrease in number and size and finally disappear or calcify after a successful treatment. Finally, the need to increase awareness of physicians about fascioliasis is highlighted, mainly in non-human endemic areas.

Metagenomics Characterization of Ixodes ricinus Intestinal Microbiota as Major Vector of Tick-Borne Diseases in Domestic Animals.

Background: Understanding the microbiota of disease vectors can help for developing new strategies to prevent the transmission of vector pathogens. Ixodes ricinus is one of the most notorious tick vectors with increasing importance in Iran and other parts of the world while there is limited data on its microbiota. This study aimed to use metagenomics for identifying the I. ricinus tick's microbiota of Iran. Methods: A total of 39 adult ticks were collected from Mazandaran (21 females), Gilan (17 females), and Golestan (1 male). Five tick pools prepared from 39 adults of I. ricinus were subjected to metagenomics analysis. The data were analyzed by targeting the V6 region of the 16S rRNA gene by Illumina 4000 Hiseq sequencing. Results: Among hundreds of intestinal microbiota identified by metagenomics, various pathogenic microorganisms distributed in 30 genera and species including those responsible for tick-borne diseases resided in the genera Coxiella, Rickettsia, and Burkholderia were found. Conclusion: Our results indicated that metagenomics identifies bacteria genera and species which cannot be easily recognized by routine methods. The presence of such pathogenic bacteria indicates the importance of possible zoonotic diseases in this region which could affect public health. These results further substantiate the importance of advanced metagenomics analyses to identify neglected tick-borne pathogens which enable researchers to provide efficient mapping roads for the management of emerging and re-emerging infectious diseases.

Phylogenetic analysis of the spirochete Borrelia microti, a potential agent of relapsing fever in Iran.

We report a role for Borrelia microti as a cause of relapsing fever in Iran supported by robust epidemiological evidence. The molecular identity of this spirochete and its relation with other relapsing fever borreliae have, until now, been poorly delineated. We analyzed an isolate of B. microti, obtained from Ornithodoros erraticus ticks, by sequencing four loci (16S rRNA, flaB, glpQ, intragenic spacer [IGS]) and comparing these sequences with those of other relapsing fever borreliae. Phylogenetic analysis using concatenated sequences of 16S rRNA, flaB, and glpQ grouped B. microti alongside three members of the African group, B. duttonii, B. recurrentis, and B. crocidurae, which are distinct from B. persica, the most prevalent established cause of tick-borne relapsing fever in Iran. The similarity values for 10 concatenated sequences totaling 2,437 nucleotides ranged from 92.11% to 99.84%, with the highest homologies being between B. duttonii and B. microti and between B. duttonii and B. recurrentis. Furthermore, the more discriminatory IGS sequence analysis corroborated the close similarity (97.76% to 99.56%) between B. microti and B. duttonii. These findings raise the possibility that both species may indeed be the same and further dispel the one-species, one-vector theory that has been the basis for classification of relapsing fever Borrelia for the last 100 years.

Molecular surveillance for Rickettsia spp. and Bartonella spp. in ticks from Northern Iran.

Tick-borne zoonotic diseases pose a threat to public health; hence, identifying the pathogenic agents associated with them is critical. The prevalence of Bartonella and Rickettsia in Iran is unknown. This study aimed to detect Rickettsia spp. and Bartonella species in ticks in northeast Iran and conduct phylogenetic analysis on these bacteria. Ticks from the sample bank in the Research Center for Emerging and Re-emerging Diseases were included in this study. The ticks were collected in 2017 and 2018 from domestic animals (sheep, goats, cows, camels, horses, dogs, and donkeys) and rodents in Golestan, Mazandaran, and Guilan provinces. Molecular methods were used to examine the DNA extracted from these samples to detect Rickettsia spp. and Bartonella species. The study examined a total of 3999 ticks. Ixodes ricinus (46.4%), Rhipicephalus turanicus (26.3%), and Rhipicephalus sanguineus (17.1%) were the most prevalent species. Among 638 DNA pools, real-time-PCR detected Rickettsia spp. in 161 (25.2%), mostly belonging to Rh. sanguineus (48.9%) and Rh. turanicus (41.9%). Golestan Province had the highest number of positive pools (29.7%). No positive samples for Bartonella were detected in a 638 pooled samples. Eight distinct Rickettsia species were detected in 65 sequenced samples, the majority of which were R. massiliae (n = 32, 49.2%) and R. sibirica (n = 20, 30.8%). Other species included R. rhipicephali (n = 3), R. aeschlimannii (n = 5), R. helvetica (n = 5), R. asiatica (n = 4), R. monacensis (n = 6), and R. raoultii (n = 1). The research findings may provide helpful information about tick-borne Rickettsiae in Iran and help to clarify the role of these arthropods in maintaining these agents. Rickettsia species were found to be circulating in three Northern provinces; thus, it is recommended that this disease be considered in the differential diagnosis of febrile diseases caused by tick bites and febrile diseases with skin rashes such as Crimean-Congo hemorrhagic fever (CCHF).

Dermatoparasitoses in Referral Patients to the Laboratory.

Background: Dermatoparasitic infestations due to the mites Demodex spp. and Sarcoptes scabie are prevalent dermatological disorders worldwide. Methods: Referral patients from the Departments of Dermatology, Infectious Diseases, and from the psychologists, in some cases, to the laboratory of Medical Helminthology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran were examined and documented for demodicosis and scabies from March 2009 to December 2020. All patients' data were collected and then analyzed statistically by SDATA version 14, using the Chi-square test. Results: Out of 494-suspected patients suffering from dermal disorders, 99 patients (20.04%) and 20 cases (4.04%) were found infested with demodicosis and scabies, respectively. Most demodicosis cases belonged to the 46-60 year age group while the infestation rate of scabies was higher in the age group under 5 years (P=<0.0001). Demodicosis was seen more prevalent in women than men, and scabies were higher in men (P =0.15). The cases of demodicosis in fall and scabies in winter and spring were more frequent. Demodicosis picked up in 2015 and 2017 (P=0.03), while the prevalent year for scabies was in 2016 (P=0.77). Both current ectoparasites declined dramatically by Covid-19 pandemic. Conclusion: Demodicosis and scabies have been found correlated with age, and no statistical association was seen between the gender and seasonal factors. Besides, the obvious decline of demodicosis and scabies infestation rates during the Covid-19 outbreak can mention that social distance and hygiene standards have negative effects on dermatoparasites transmission.

Development of an Indirect Fluorescent Antibody (IFA) Assay for the Detection of Leishmania RNA Virus 2 (LRV2) in Leishmania Parasites.

Background: Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Methods: Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. Results: LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Conclusion: Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features.

Identification of Intestinal Fungal Microflora and Bacterial Pathogens in the Collected Adult Ixodes ricinus from the Northern Provinces of Iran.

Background: Ticks are vectors of many pathogens that involve various important diseases in humans and animals, they have several diverse hosts consequently can retain a diverse group of indigenous microbes, from bacteria to fungi. Little is known about the prevalence and diversity of tick microflora colonizing the midgut and their effects on ticks and their interaction. This information is important for development of vector control strategies. Methods: This study was carried out in northern Iran during autumn 2019. Ticks, Ixodes ricinus caught alive on the bodies of domestic animals in the fall. The tick homogenate was prepared. The identification of fungal isolates was carried out according to a combination of macro and microscopic morphology and molecular sequencing. Pathogenic bacteria of the family Borreliaceae, Francisella tularensis, Borrelia burgdorferi and Coxiella burnetii were tested by real-time PCR. Results: A total of 133 mature I. ricinus ticks were collected from domestic animals, including 71.5% cattle and 28.5% sheep. The tick frequency rates were 87.21% for Mazandaran, 8.28% for Golestan and 4.51% for Gilan Provinces. Total prevalence of fungal tick contamination was 53.4% (75/133) of which Trichoderma harzianum (57%) was the most prevalent species followed by Aspergillus spp. (42%), Mortierella alpine (19%) and Penicillium polonicum (14%). All tick samples were negative for three pathogenic bacteria including Francisella tularensis, Coxiella burnetii, and Borrelia burgdorferi by real-time PCR analysis. Conclusion: These results show a first picture of the microbial diversity of ticks and highlight the importance of microbiota and their role in host-pathogen interaction.

Genomic palaeoparasitology traced the occurrence of Taenia asiatica in ancient Iran (Sassanid Empire, 2th cent. CE-6th cent. CE).

Palaeoparasitology investigates parasitological infections in animals and humans of past distance by examining biological remains. Palaeofaeces (or coprolites) are biological remains that provide valuable information on the disease, diet, and population movements in ancient times. Today, advances in detecting ancient DNA have cast light on dark corners that microscopy could never reach. The archaeological site of the Chehrabad salt mine of Achaemenid (550-330 BC) and Sassanid (third-seventh century AD) provides remains of various biotic and abiotic samples, including animal coprolites, for multidisciplinary studies. In the present work, we investigated coprolites for helminth eggs and larvae by microscopy and traced their biological agents' DNA by Next Generation Sequencing. Our results revealed various helminths, including Taenia asiatica, the species introduced in the 1990s. Implementing advanced modern molecular techniques like NGS gives a paramount view of pathogenic agents in space and time.

Borrelia duttonii-like spirochetes parasitize Meriones persicus in East Azerbaijan Province of Iran.

In Iran, Borrelia persica and Borrelia microti/microti-like borreliae have been established as causative agents of tickborne relapsing fever (TBRF). However, the epidemiology of two previously described species, Borrelia balthazardi and Borrelia latyschewii (latychevi), has remained elusive for many years. We investigated Borrelia infection in various rodents and small mammals in the TBRF endemic East Azerbaijan Province, northwestern Iran, where B. perisca and B. balthazardi might coexist. Among trapped animals (n=210), a 16S real-time PCR detected Borrelia DNA in 11 Meriones persicus. Multilocus sequence analysis (MLSA) using six different loci, including four coding regions (flaB, glpQ, groEL, p66) and two non-coding (rrs, IGS) followed by phylogeny revealed considerable sequence identity between the borreliae detected, B. microti, and East African Borrelia duttonii, and Borrelia recurrentis. Our results indicate that B. microti and microti-like borreliae, including the specimens previously characterized in the south of Iran and the present study, are different ecotypes of B. duttonii, i.e., exhibiting a single species/entity or descendants of a recent common ancestor. Our findings also suggest that the species we had long coined as B. balthazardi and the microti-like borreliae detected herein might be the same.

Serological Detection of Trichinellosis among Suspected Wild Boar Meat Consumers in North and Northeast of Iran.

BACKGROUND: Trichinellosis is a foodborne zoonosis disease worldwide. Humans acquire infection by ingesting raw or uncooked animal flesh containing viable Trichinella larvae. The most common reservoirs of this helminth are pigs and wild boars. In northern Iran, hunting and consuming wild boars meat by some communities, including ethnic Armenians, may expose them to trichinellosis. Here, we investigated anti-Trichinella IgG antibodies in high-risk individuals in northeastern Iran. METHODS: From Mar to Aug 2020, we collected 189 blood samples from individuals with a history of wild boar meat consumption and examined the sera for anti-Trichinella IgG antibodies using a commercial ELISA kit (NovaTec Immunodiagnostica GmbH, Germany). Sera from 30 individuals with no history of eating wild boar meat was used to determine the range of actual negative values and possible cross-reactivity with other similar antigens. RESULTS: Of the 189 participants, 5 (2.6%) had anti-Trichinella IgG antibodies (OD, 1.176 ±0.154). None of the 30 negative controls became positive (OD, 0.198 ± 0.044). The age, gender, occupation, and education showed no significant association with Trichinella seropositivity rate (P>0.05). All five seropositive cases were among 112 individuals (4.46% seropositivity) that resided in the western part of the study area, stretching from Behshar to Gorgan. CONCLUSION: Eating wild boar meat might expose individuals to trichinellosis in the north and northeast of Iran. Further studies with more individuals from different parts of the country and confirmation of the ELISA by additional tests like Western blot will give a more in-depth insight into human trichinellosis epidemiology in Iran.

Molecular variation and distribution of Anopheles fluviatilis (Diptera: Culicidae) complex in Iran.

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.

Development of A Loop-Mediated Isothermal Amplification (LAMP) Assay for Detection of Relapsing Fever Borreliae.

BACKGROUND: This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of tick-borne relapsing fever in resource-limited areas. METHODS: A set of six primers were designed based on the conserved regions of the Glycerophosphodiester phosphodiesterase (glpQ) gene of Borrelia species. For sensitivity assay, serial dilutions of a recombinant plasmid containing a 219bp sequence of the glpQ were prepared and used as the template DNA. The LAMP reactions containing the six primers and the reagents required for amplification were incubated at 60-65 °C for 60min in a Loopamp real-time turbidimeter. For the specificity test, DNA from 14 other bacteria were included in the assays, and double-distilled water was used as the negative control. Also, DNA from dried blood spots (DBSs) of spirochetemic mice, and blood samples from relapsing fever-suspected patients were examined by the LAMP along a Borrelia-specific nested PCR that targets the rrs-rrl-IGS region. RESULTS: The LAMP detected as low as 90glpQ copies in reactions. The primers reacted with DNA from DBS of spirochetemic mice showing spirochete concentrations of ≤ one per a 1000X microscopic field. In clinical samples, the LAMP assay showed a higher sensitivity compared to nested-PCR. The LAMP specificity was 100%, as the primers did not react with other bacteria DNA. CONCLUSION: The high sensitivity and specificity of the test, along with the simplicity of the DNA extraction procedure, make the LAMP a reliable and adaptable tool for the diagnosis of tick-borne relapsing fever in rural endemic areas.

Infection of hard ticks in the Caspian Sea littoral of Iran with Lyme borreliosis and relapsing fever borreliae.

The Caspian Sea littoral of Iran is home to various hard tick species, including Ixodes ricinus, the notorious vector of Lyme borreliosis (LB) in Eurasia. Here, in this area, we examined I. ricinus and other hard ticks, along with common rodents and small mammals for LB and relapsing fever (RF) borreliae infection. Ticks were collected from various mammalian hosts, including sheep, goats, cattle, camels, horses, dogs, donkeys, rodents, and hedgehogs. Rodents and small mammals were live-captured from different habitats. A real-time PCR for 16S rRNA sequence revealed borrelial DNA in 71 out of 501 (≈14 %) specimens belonging to I. ricinus and Rhipicephalus ticks. None of the rodents and small mammals showed borrelial infection in the viscera. PCR amplification and sequencing of a 600-bp sequence of the flaB identified Borrelia bavariensis, Borrelia garinii, Borrelia afzelii, and Borrelia valaisiana, and the RF Borrelia, B. miyamotoi in I. ricinus ticks. The RF-like Borrelia in Rhipicephalus ticks shared the highest identity (98.97 %) with an isolate infecting Haemaphysalis megaspinosa ticks in Japan. Our phylogeny and BLAST analysis suggest the range extension of the European I. ricinus-associated borreliae into the north of Iran.