J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 97 http://jad.tums.ac.ir Published Online: June 30, 2022 Original Article Identification of Intestinal Fungal Microflora and Bacterial Pathogens in the Collected Adult Ixodes ricinus from the Northern Provinces of Iran Manijeh Yousefi-Behzadi1,2, Neda Moazzezy3, Mahdi Rohani1,4, Saied Reza Naddaf5, Ehsan Mostafavi1,2, Ali Mohamadi1, Masoomeh Shams-Ghahfarokhi6, Nasrin Pashootan7, *Mehdi Razzaghi-Abyaneh7 1Department of Epidemiology and Biostatistics, Research Centre for Emerging and Reemerging Infectious Diseases, Pasteur Institute of Iran, Tehran, Iran 2National Reference Laboratory of Plague, Tularemia and Q Fever, Research Centre for Emerging and Reemerging Infectious Diseases, Pasteur Institute of Iran, Akanlu, Kabudar-Ahang, Hamadan, Iran 3Molecular Biology Department, Pasteur Institute of Iran, Tehran, Iran 4Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran 5Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran 6Department of Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 7Department of Mycology, Pasteur Institute of Iran, Tehran, Iran *Corresponding author: Dr Mehdi Razzaghi-Abyaneh, E-mail: mrab442@pasteur.ac.ir (Received 03 Oct 2020; accepted 20 Apr 2022) Abstract 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 Borre- lia burgdorferi by real-time PCR analysis. Conclusion: These results show a first picture of the microbial diversity of ticks and highlight the importance of micro- biota and their role in host-pathogen interaction. Keywords: Microflora; Ixodes ricinus; Fungal species; Mycoflora; Microbiome Introduction Arthropods are vectors of many pathogens that involve various important diseases in hu- mans and animals (1). These organisms are he- matophagous which feed on blood and most of these arthropods are blood eaters and during engorgement transmit or acquire microorganisms (2) on the other hand, arthropods have several diverse hosts consequently can retain a diverse group of microbes indigenous, from bacteria to fungi (3, 4). A single tick can carry different pathogens, co-infections are common and can make diagnosis and treatment difficult (4). Ix- odidae ticks are important in the transmission of a variety of zoonotic microorganisms (viruses, Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://mail.yahoo.com/neo/b/compose?to=mrab442@pasteur.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 98 http://jad.tums.ac.ir Published Online: June 30, 2022 bacteria and protozoan) (5). Ticks are not only carriers of pathogens, but also a diverse group of commensal and symbi- otic microorganisms as bacteria, viruses and fun- gi are also present in ticks whose biology and their effects on ticks and their interaction re- main largely unexplored or very often neglect- ed (6, 7). Tick-borne diseases seem to be a real challenge threatening public and economic health. Some of them can cause physical and cognitive damage which can be very painful (8). Lyme disease is the most common tick- borne pathogen in the temperate woodlands of North America, Europe and Asia and is caused by some members of Borrelia burgdorferi. The past 20 years the number of reported cases has tripled in the United States and has also in- creased in parts of Europe (9). The first endemic case of lyme borreliosis was reported in 1997 in Iran proving the existence of the spirochete B. burgdorferi which had not previously been found in ticks in this region (10). In 2001 a 9-year- old boy was admitted to Children's Medical Center with a final diagnosis of Lyme disease (https://acta.tums.ac.ir/index.php/acta/article/ view/3187). In 2020, Naddaf et al. reported for the first time the existence of the infection of I. ricinus ticks in the littoral of the Caspian Sea by the spirochetes Lyme borreliosis (11). The tularemia caused by Francisella tu- larensis, zoonosis, is characterized by high mor- bidity and mortality rates in more than 190 dif- ferent mammal species, including humans (12). In Iran, positive serological tests were first re- ported in 1973, in wildlife and domestic live- stock in the northwestern and southeastern parts of the country. The first human case was re- ported in 1980 in the southwest of Iran, and recent studies conducted among at-risk popu- lations in the western, southeastern, and south- western parts of Iran. The presence of F. tu- larensis in ticks was confirmed in the prov- ince of Kurdistan (western Iran) and the pos- sible role of ticks in the transmission of the pathogen to livestock and humans by bites has been demonstrated in this region (13). Q fever is a zoonosis with a worldwide dis- tribution is caused by Coxiella burnetii, many species of mammals, birds, and ticks are res- ervoirs of C. burnetii in nature. Coxiella bur- netii infection is most often latent in animals (14). According to a study published in 2019 in the case of infective endocarditis (IE) pa- tients hospitalized in Rajai Cardiovascular Med- ical and Research Center from August 2015 to September 2017, a high prevalence of Q fever was revealed (15). Coxiella burnetii DNA or its antibodies have frequently been detected in ruminants. Since these animals can transmit the infection to humans, Q fever can be a poten- tial health problem in Iran (16). Anaplasmosis is a zoonotic disease, de- scribed in various domestic animals and hu- mans and the bacterium in question is Anaplas- ma phagcytophilum transmitted by ticks with a worldwide distribution (17). In Iran, in 2014 in the province of Mazandaran, we have the first investigation of tick-borne Anaplasma infec- tions in domestic animals and humans (18). Fun- gal microbiota comprises an important part of total microbiota of vertebrates and non-verte- brates (19). The determination of the tick fun- gal microflora (microbiota) and the interactions between its symbiotic microorganisms in the context of pathogen transmission will likely reveal new perspectives and spawn new para- digms for tick-borne diseases (20). The pre- sent study for the first time aimed to determine the diversity of the fungal microflora that col- onize in the middle intestine of mature I. rici- nus ticks in the three provinces of northern Iran. Materials and Methods Study area and population This study was conducted in northern Iran consists of the southern border of the Caspian Sea and the Alborz mountains with an area of 58,167Km2 in three provinces of Gilan (37.280 N, 49.59o E) Mazandaran (36.2262° N, 52.5319° E) and Golestan (37.2898° N, 55.1376° E). The forest cover of northern Iran has a total of http://jad.tums.ac.ir/ https://acta.tums.ac.ir/index.php/acta/article/view/3187 https://acta.tums.ac.ir/index.php/acta/article/view/3187 J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 99 http://jad.tums.ac.ir Published Online: June 30, 2022 3,400,000 hectares of forest on the northern slopes of the Alborz Mountains and the coastal provinces of the Caspian Sea (Fig. 1). General characteristics of the climate are too much rain in all seasons, especially in autumn and win- ter, relatively high humidity in all seasons and low temperature difference during the day due to the presence of moisture. Domestic animals such as cattle, sheep, and dog due to the large number of forests, can easily move between the forest and the herd. Sampling Sampling of ticks on domestic animals (cat- tle, sheep) was carried out in autumn 2019 (from 6 to 11 November) during the period of multi- plication of ticks and was carried out at vari- ous sites in the provinces of northern Iran in- cluding Mazandaran, Gilan and Golestan (Ta- ble 1), (Fig. 1). The sample size was estimat- ed at 100 ticks but given the number of ticks collected at this time of the year to increase the accuracy of the study, all 133 ticks col- lected were entered in this study and collected according to their animal host in 14 sampling sites (Table 1). Ticks caught alive on the bodies of domes- tic animals living in barn using forceps and ster- ile disposable material, were then collected ac- cording to their host. The ectoparasites were identified directly under a stereomicroscope without mounting. All ectoparasites have been identified at the species level using available taxonomic morphological key of ticks (21). Ticks identified as mature I. ricinus were kept for this study. A number of 133 I. ricinus ticks were isolated from two domestic animals, cat- tle and sheep from a total of 14 sampling sites (Table 1). Preparation of ticks’ homogenate The whole ticks were placed in sterile tubes containing 70% ethyl alcohol for 3min then rinsed with sterile water three times to avoid transportation of microorganisms from the out- side environment and from the surface of the ticks. Homogenization of the samples (ticks) is done with a QIAGEN Tissue Lyser II ho- mogenization device, the samples were placed in sterile microtubes containing metal willow and 300μl of RTL RNA buffer extraction kits (QIAGEN) were added to the microbial tubes. According to the manufacturer's instructions, the QIAGEN Tissue Lyser II microtubes were homogenized at 30 Hertz (HZ) for 3min to ob- tain a uniform suspension. The topical solution was removed and transferred to a new sterile microtube to continue the extraction process. One part was used fresh for fungus culture and the rest for DNA extraction and the molecular process. Mycological Identification Culture based identification Ticks’ homogenate was diluted with Phos- phate Buffer Saline (PBS) to give a final vol- ume of 100µl and diluted solutions these were spread on to Sabouraud Dextrose Agar (Pep- tone 1%, Glucose 2%, Agar-agar 1.5%; Merck, Germany) and Potato Dextrose Agar (Potato infusion 20%, Dextrose 2%, Agar 2%) plates and incubated for 2 weeks at 25 °C. The plates were periodically checked for fungal colonies. Identification of fungal isolates was performed according to a combination of macro and mi- croscopic morphology. Molecular identification DNA extraction DNA was extracted and purified from fungal colonies using the following method, briefly, 10–20mm3 of the fresh colonies grown on Sabouraud glucose agar (Difco, Detroit, MI, USA) were added to 1.5ml tubes that con- tained 300µl of lysis buffer (200M Tris-HCl, pH 7.5; 25mM ethylenediaminetetraacetic acid (EDTA); 0.5% w/v sodium dodecyl sulfate; and 250mM NaCl) and crushed with a conical grind- er (Micro Multi Mixer; IEDA Co. Ltd., Tokyo, Japan) for 1min. The samples were incubated in a boiling water bath for 10min, mixed with 150µl of 3.0M sodium acetate, kept at −20 °C http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 100 http://jad.tums.ac.ir Published Online: June 30, 2022 for 10min, and centrifuged at 12,000rpm for 10min. The supernatant was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and once more with chloroform. The DNA in supernatant was precipitated with 250µl iso- propanol, washed with 300µl of 70% ethanol, air dried, and rehydrated in 50µl ultrapure wa- ter, and stored at −20 °C until use. Polymerase Chain Reaction (PCR) and Se- quencing For the polymerase chain reaction with the specific ITS region primers, ITS1 and ITS4 (Ta- ble 2). Each mixture contained 2.5µl of 10× re- action buffer, 0.5µM of universal fungal pri- mers forward ITS1 and reverse ITS4, 400µM of deoxynucleoside triphosphate, 1.25U of Taq DNA polymerase (Takara, Japan), 1µl of DNA extracted and enough ultrapure water to reach a final reaction volume of 25µl. The PCRs were programmed for preheating at 96 °C for 6min followed by 35 cycles at 94 °C for 1min, 56 °C for 1min, and 72 °C for 45s, and a final ex- tension step at 72 °C for 10min. Five micro- liters of the PCR products were electrophoresed onto1.5% agarose gel in Tris-Acetate-EDTA (TAE) buffer (Tris 40mM, acetic acid 20mM, EDTA 1mM), stained with 0.5µg/ml of ethidi- um bromide, and observed and photographed under ultraviolet irradiation. The PCR products of the ITS region were purified using a QI quick purification kit (Qiagen, Valencia, CA, USA), sequenced in both directions using the same primers. Molecular detection of bacterial pathogens The whole genomic DNA extracted from ticks were screened for members of the family Borreliaceae, F. tularensis and C. burnetii by targeting 16SrRNA, ISFTu2 and IS1111 genes respectively, using primers and probes listed in Table 2. The final 20μl reactions contained 10μl master mix 2× (Amplicon, Denmark), 500 nM of each primer, 200nM probe, and 4μl (50 nM) of the template DNA. Amplifications were performed in a Rotor-Gene 6000 instrument (Corbett Life Science, Sydney, Australia) for an initial denaturation at 95 °C for 10min, fol- lowed by 45 cycles of denaturation at 95 °C for 15sec, and annealing at 60 °C for 60sec. DNA of B. borgdorferi senso stricto (Ampli- run® Borrelia DNA control), the DNA of F. tularensis subsp holarctica NCTC 10857 and plasmid contain IS1111 from C. burnetii were included as positive controls in all the assays. PCR with no sample DNA was considered as negative control. Results Ticks collected A total of 133 mature I. ricinus ticks were collected from domestic animals including 71.5 % cattle and 28.5% sheep. The tick frequency was 87.21% for Mazandaran, 8.28% for Go- lestan and 4.51% for Gilan (Table 1), (Fig. 2). Detection of fungal species The isolated extract of tick midgut was cul- tured on Sabouraud dextrose agar (E-Merck, Germany) medium to isolate the possible fun- gal species. The morphological and then mo- lecular identification have proven the fungal species include 57% Trichoderma harzianum (MT804339, MT803548, MT809136), 42% Aspergillus spp., 14% Penicillium polonicum (MT809131) and 19% Mortierella alpine (MT803487). Among the 14 sampling sites, ticks from seven sites were contaminated with fungal species from four distinct genera (Fig. 3). Detection of Bacterial pathogens Real-time PCR for three bacterial pathogens including F. tularensis, C. burnetii, B. burgdor- feri was negative for all 133 ticks obtained from 14 sampling sites (Table 1). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 101 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 1. Details of location and host of fungal species (Trichoderma harzianum, Mortierella alpine, Aspergillus sp., Penicillium polonicum) were found in Ixodes ricinus from the northern provinces of Iran, 2019 Province City Distract N E Host No. of sampled ticks (contaminated ticks) Fungal spp. Mazandaran Mahmoud-abad Boondeh 36°34'16.0" 52°14'16.0" Cattle 7 (5) T. harzianum Mazandaran Chamestan Joorband 36°26'42.2" 52°07'37.9" Cattle 6 NF Mazandaran Chamestan Joorband 36°26'45.2" 52°07'26.4" Sheep 2 NF Mazandaran Chamestan Joorband 36°26'39.2" 52°07'32.1" Cattle 6 NF Mazandaran Amol Razageh 36°19'50.0" 52°21'42.0" Sheep 6 NF Mazandaran Amol Esku Mahalleh 36°24'19.7" 52°18'30.8" Cattle 7 NF Mazandaran Chamestan Joorband 36°26'15.5" 52°07'15.3" Cattle 14 (6) (14) T. harzianum M. alpine Mazandaran Amol Komdarreh 36°23'55.2" 52°25'51.7" Cattle 20 NF Mazandaran Savadkuh Chay Baq 36°20'30.4" 52°52'05.9" Sheep 11 (9) (5) T. harzianum, Aspergillus sp. Mazandaran Tonekabon Goli Jan 36°49'04.1" 50°48'45.8" Cattle 16 (10) (5) Aspergillus sp., P. polonicum Mazandaran Nowshahr Musa Abad 36°37'40.2" 51°30'39.0" Sheep 11 (10) (8) T. harzianum, Aspergillus sp. Mazandaran Fereydunkenar Boneh Kenar 36°39'24.0" 52°30'22.3" Cattle 10 (9) (8) T. harzianum, Aspergillus sp. Golestan Kordkuy Valaghuz 36°45'59.2" 54°07'10.2" Cattle 11 NF Gilan Rudsar Ahmad Abad 37°10'13.5" 50°16'57.3" Cattle 6 (4) (5) T. harzianum P. polonicum NF: Not Found Fig. 1. Map of tick sampling areas in three northern provinces of Iran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 102 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 2. Primers and probes used for molecular detection of three pathogens Francisella tularensis, Coxiella burnetii, Borrelia burgdorferi and fungal species Target (gene) Primer/probe Sequence (5’ 3’) Product (bp) ISFtu2 (F. tularensis) ISFtu2Fw ACAAGAAGTCATGCTTGATTCAAC 144 ISFtu2Rv GGATTACCTAAAGCATCAGTCATAGC Probe FAM-ATAGCAAGAGCACATGCTTGTGCTACGG- TAMRA 16S rRNA (Borrelia) 6BOR16SFw GGTCAAGACTGACGCTGAGTCA 135 6BOR16SRv GGCGGCCACTTAACACGTTAG Probe FAM-TCTACGCTGTAAACGATGCACACTTGGTG –BHQ-1 IS1111 (C. burnetii) tmQ-koorts4-Fw AAAACGGATAAAAAGAGTCTGTGGTT 70 tmQ-koorts4-Rv CCACACAAGCGCGATTCAT tmQ-koorts4-probe FAM-AAAGCACTCATTGAGCGCCGCG-TAMRA Fungal ITS ITS1 TCCGTAGGTGAACTGCGG 600-750 ITS4 TCCTCCGCTTATTGATATGGC Fig. 2. The frequency (left panel) and prevalence (right panel) of fungal contamination of the Ixodes ricinus ticks col- lected in three Northern provinces in Iran, 2019 Fig. 3. Distribution of fungal genera and species in contaminated Ixodes ricinus ticks in Northern provinces of Iran, 2019 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 103 http://jad.tums.ac.ir Published Online: June 30, 2022 Discussion To our knowledge this project was the first attempt to determine the fungal community that inhabits the middle intestine of ticks in the three provinces of northern Iran and contamination with three bacterial pathogens that transmitted to human by ticks, using culture and molecu- lar methods. Ixodes ricinus ticks cover a wide geograph- ic region in the EU (22), I. ricinus is an indig- enous hard tick species having a wide geo- graphical distribution (23), Portugal to Russia and from North Africa to Scandinavia. This wide geographical distribution entails that this tick species could have a role in transmission of tick-borne disease in wide geographic areas (24). A survey of ticks was carried out in four dif- ferent geographic areas of Iran, where the ma- jority of domestic ruminants in Iran exist (18, 19) showed Ixodidae ticks were found through- out the year. The highest number of adult Ix- odidae ticks was generally found from April to August, and Ixodes ticks were present in the Caspian region and in the south and west of country (24-27). The number of ticks obtained is very im- portant in Mazandaran which agrees with the previous studies on the abundance of this tick according to favorable climatic conditions in the Northern provinces of Iran (28, 29). Ixodes ricinus is a three-host tick: larvae, nymphs and adults feed on different hosts where larvae and nymphs prefer small to medium-sized animals and adults tend to feed on large ani- mals (30). In this study we have chosen adult ticks because of their size, it is especially the adult females fed or in the process of being engorged with blood which are the most de- tectable, because they are much larger than during the other stages of development and the amount of blood they eat on large animals such as cattle and sheep. We were expecting to have a much more varied number of fungi species. As this study is the first done by this approach, we have not the element of comparison but in a study car- ried out on the midgut of sand flies they found six fungi species of which two fungi species are common with our study (Penicillium and Aspergillus) (31). Another most observed spe- cies in this study was Actinomycetes that is a shared bacterial species with fungi and among 14 sample sites in 83.4%, this species of bac- teria was present. The fungal species isolated in this study are among the saprophytes which can be pathogenic in insects, plants and humans (32). Studies have shown that fungi can have an entomopathogenic effect under different envi- ronmental conditions (33). The predominant pathogenic genera isolated from soil in the United States for winter tick larvae (Derma- centor albipictus) being Aspergillus spp, Beau- veria bassiana, Mortierella spp, Mucor spp, Paecilomyces spp, Penicillium spp. and Tricho- derma spp. (34). On I. ricinus ticks, the most important tick species in Europe, susceptibil- ity to entomopathogenic fungi shows particu- larly high potential efficacy and the predomi- nant species of isolated entomopathogenic fun- gi were hyphomycetes, Paecilomyces farino- sus and Verticillium lecanii, Beauveria bassi- ana, B. brongniartii, P. fumosoroseus and V. aranearum (35). The secondary metabolites of fungi may be involved in their entomopatho- genic effect, Aspergillus flavus is effective on insect Heliozella stenella by secreting aflatox- in, caused histological changes in even at very low doses. Metarhizium anisopliae secretes Dis- truxine (a) Distruxine (b) when injected into wax and silk moth. Beauvaria bassiana (White mus- curdine fungus) secret Beauvaricine is the pep- tide depsi (36). The susceptibility to entomopath- ogenic fungi against two of the most important tick species in Europe: I. ricinus and Derma- centor reticulatus shows the potential efficacy particularly greater in I. ricinus (37). The def- inition of the tick's microbiome and interac- tions between the tick and its symbiotic bacte- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 104 http://jad.tums.ac.ir Published Online: June 30, 2022 ria in the context of pathogen transmission like- ly reveal new knowledge for controlling tick- borne diseases (38). In this study we have tried to answer this question even though it is very early because in the studies already show mi- crobiome of I. ricinus ticks evaluated over the last 10 years in Austria reveals a number of bacteria were affiliated with the genera Rick- ettsia, Bartonella and Borrelia (39) which are known to be pathogenic and transmitted by ticks indicating that the tick microbiome is mainly composed of gram-negative bacteria of the phylum Proteobacteria and intracellular bac- terial endosymbionts which regulate the repro- ductive capacity and vectoral competence of ticks, also maintain the integrity of the epithe- lial barrier of the tick gut (6). Although I. rici- nus is of great importance to public health, its microbial communities remain largely unex- plored to this day. A pool of adults of I. ricinus collected from two distinct geographic regions of northern It- aly showed a total of 108 genera belonging to all bacterial phyla and pathogenic bacteria, such as Borrelia, Rickettsia and Candidatus neoehr- lichia (40). The microbiome currently focuses mainly on its eubacterial members, but the mi- crobiome is also made up of viruses and eukar- yotic microbes such as protozoa, nematodes, and fungi, and their interactions within and be- tween realms could further modulate the hu- man health (41). On the other hand, ecological analysis revealed that the composition of bac- terial communities depending on the geograph- ic regions and the life stages of the ticks. This finding suggests that the environmental con- text (abiotic and biotic factors) and host selec- tion behaviors affect their microbiome (40). Entomopathogenic fungi are known to in- fect different tick species and their effective- ness is very strain specific. Numerous studies show the important role of pathogenic fungi in the control of insects, including Ixodes (35, 37, 42–44). Although entomopathogenic fun- gi have been widely used for agricultural and forestry pest control, little effort has been made to assess the applicability of the biological con- trol potentials of entomopathogenic fungi against ticks, vectors of human and animal diseases. it can also be said that the susceptibility of ticks to a particular fungus depends largely on the genera and species of ticks; species of fungus, strain, concentration of conidia;, temperature, humidity and geographical area always trying to understand the biology of the fungus, which is necessary to better understand its proper use in field conditions (40). In the case of Fusarium stem rot caused by Fusarium graminearum strongly affecting the productivity of corn crops by modifying the plant microbiome by promot- ing the colonization of roots by Trichoderma harzianum in the corn rhizosphere, first, we in- crease plant growth and further protect the en- vironment from harmful agrochemical effects by replacing it with a biological control agent (45). Conclusion Results of the present study revealed that different human and animal pathogenic fungal genera are among microbial flora of I. ricinus ticks in Northern provinces of Iran. These re- sults represent only a first image of the mi- crobial diversity of the tick. Further studies are needed to determine the roles these genera play in ticks and their effects on human health. These studies could bring us closer to the discovery of the causative agent and interaction between them to know the system of transmission of these agents even save them by ticks. Acknowledgments Authors wish to thank the personal of My- cology department of the Pasteur Institute of Iran for their kind help in culturing tick sam- ples. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 97–107 M Yousefi-Behzadi et al.: Identification of … 105 http://jad.tums.ac.ir Published Online: June 30, 2022 Conflict of interest Authors declare that there is no conflict of interest. Ethical considerations There was no human and animal study that needs ethical concerns. References 1. Irwin PJ, Jefferies R (2004) Arthropod-trans- mitted diseases of companion animals in Southeast Asia. Trends Parasitol. 20(1): 27–34. 2. Krenn HW, Aspöck H (2012) Form, func- tion and evolution of the mouthparts of blood-feeding Arthropoda. Arthropod Struct Dev. 41(2): 101–118. 3. 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