Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3’UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis.The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental  Leishmania . At least 21 Leishmania species that are members of the subgenera Viannia, Leishmania, and Mundinia have been reported as human pathogens. So far, no vaccine is available for human leishmaniasis (L. (Mundinia) martiniquensis (L. (Mundinia) orientalis  braziliensis DNA found in C. ignacioi, C. insignis, and C. foxi and L. (Leishmania) amazonensis DNA detected in C. filariferus and C. flavivenula in Brazil (Forcipomyia (Lasiohelea) Kieffer, with L. (Mundinia) macropodum parasites has been reported (L. (Mundinia) enriettii and L. orientalis in a laboratory colony of C. sonorensis reveals that both Leishmania species can complete their development to late-stage parasites found in the stomodeal valve, a position suitable for transmission by bite  chancei   , all memn nature .Trypanosoma spp. parasites has been detected in several wild caught Culicoides spp.   . Experim species .Leishmania and Trypanosoma), natural infections of Culicoides biting midges with monoxenous trypanosomatids have been reported. Herpetomonas ztiplika was isolated from a female C. kibunensis caught while attacking buzzard (Buteo buteo) nestlings  and stray dogs in Egypt (Herpetomonas (Nasua nasua), marmosets , bats , crab-eating foxes (Cerdocyon thous), and ocelots  in Brazil have been found infected in nature by Crithidia mellificae, a monoxenous trypanosomatid classically associated with honeybees (Cr. mellificae is detected in a nectar-feeding bat (Anoura caudifer) in Brazil  to investigate trypanosomatids in Culicoides biting midges collected from an endemic area of leishmaniasis and a mixed farm of chickens, goats, and cattle in southern Thailand, (2) to analyze blood meals of engorged midges, and (3) to characterize any trypanosomatids successfully isolated into culture. Our study provided evidence for C. peregrinus as a potential vector of L. martiniquensis and revealed a novel thermotolerant Crithidia trypanosomatid that could infect mouse cells in vitro.Observation and isolation of live The use of animals in this study was approved by the animal research ethics committee of Chulalongkorn University Animal Care and Use Protocol (CU-ACUP), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA No. 023/2564 and COA No. 011/2564).Collection of biting midges was conducted in two locations, Location 1: Tambon Tha Ruea (TR), an endemic area of leishmaniasis (8°22′42.0”N 99°58′32.0″E) and Location 2: Tambon Khuan Phang (KP), a mixed farm of chickens, goats, and cattle (8°09′35.2”N 99°56′33.8″E), Nakhon Si Thammarat province, southern Thailand. In each location, wild biting midges were collected using five Center for Disease Control and Prevention (CDC) miniature light traps (25 W bulb) with ultraviolet (UV) light for 4 nights in December 2021. Traps were operated from 6.00 pm to 10.00 pm. The midges were kept in plastic boxes covered by two layers of insect nets with moisture papers on the top and transported to the Vector Biology and Vector Borne Disease Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University.Culicoides species.Live insects were placed on ice to immobilize before investigating under a binocular stereoscopic microscope . Males were separated from females by their morphology. Species identification of females was carried out using a taxonomic key according to morphological characters, namely wing spot patterns and head features  . In eachSchneider’s Insect medium (SIM) , pH 6.8 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (hi-FBS) , 100 μg/mL penicillin–streptomycin and 250 μg/mL gentamicin  was used to culture trypanosomatids from insects. Each positive sample in PBS was transferred into a 25 ml flask containing 5 mL SIM, pH 6.8 supplemented with 10% (v/v) hi-FBS and 25 μg/ml gentamicin (SIM complete), and incubated at 26°C. An axenic culture was established by serial dilution and subpassage. Promastigotes were mixed with 7.5% (v/v) glycerol in SIM complete and stored at −80°C.Trypanosomatid samples isolated directly from insects or cultures in SIM with supplements were smeared on microscope slides and air-dried. The slides were then fixed in absolute methanol and stained with 5% (v/v) Giemsa’s stain solution . The morphological characteristics and morphometry of the trypanosomatids were examined under a light microscope  at 1,000 × magnification. Light microscopy (LM) images of 50 parasites in each stage were used for morphometry, including: cell body length, cell body width, anterior end to kinetoplast distance, anterior end to nucleus distance, and length of flagellum. The measurement results were presented as mean ± standard deviation.Culicoides species, LCO1490 primer (5 ´–GGTCAACAAATCATAAAGATATTGG−3 ´) and HCO2198 primer (5 ´–TAAACTTCAGGGTGACCAAAAAA TCA−3 ´) (COI) following the polymerase chain reaction (PCR) method described by cyt b) gene sequence using the primers cyt bb1 (5 ´–CCATCMAACATYTCADC ATGAAA−3 ´) and cyt bb2 (5 ´–GCHCCTCAGAATGAYATTTG KCCTCA−3 ´) as described by Leishmania species in gDNA samples extracted from insects and/or cultures, primers LeF (5 ´–TCCGCCCGAAAGTTCACC GATA−3 ´) and LeR (5 ´–CCAAGTCATCCATCGCGACACG−3 ´) (Leishmania HSP70-type I (HSP70-I) genes as described by Genomic DNA of biting midges and trypanosomatids was extracted using a genomic DNA purification kit  according to the manufacturer’s instructions. For molecular identification of TCA−3 ´)  were useACG−3 ´) , were usACG−3 ´)  were useFor molecular identification of trypanosomatids, primers, TRY927F (5 ´–GAAACAAGAAACACGGGAG −3 ´) and TRY927R (5 ´–CTACTGGGCAGCTTGGA−3 ´), were used to amplify approximately 927 bp of the small subunit ribosomal RNA (SSU rRNA) gene  of gDNA For trypanosomatids successfully grown in culture, primers M200 (5 ´–ATGGCTCCVVTCAARGTWGGMAT−3 ´) and M201 (5 ´–TAKCCCCACTCRTTRTCRTACCA −3 ´) for the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene  were useCOI gene of Culicoides biting midges, the cyt b gene of vertebrates, ITS1 and 3’UTR-HSP70-I genes of Leishmania parasites, and SSU rRNA and gGAPDH genes of trypanosomatids were analyzed by comparison with the GenBank database using a BLAST searchLeishmania parasites and trypanosomatids were aligned and trimmed using MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms version 10.2.6  and 3’UTR-HSP70-I (817 bp). For phylogenetic analysis of Crithidia sp., evolutionary models of GTR + I + G were chosen for SSU rRNA (720 bp) and gGAPDH (654 bp). Genetic distances were calculated using the Kimura 2-parameter (K2P) model implemented in MEGA  glutaraldehyde in 0.1 cacodylate buffer (pH 7.2) overnight at 4°C. Fixed cells were washed twice in PBS and post-fixed with 1% (w/v) osmium tetroxide in PBS for 1 h. The samples were then dehydrated in a graded series of ethanol. Samples were critical point dried in liquid CO2 and coated with gold particles in a sputter-coating apparatus. Samples were observed under a scanning electron microscope, JEOL JSM-6610LV , operated at 15 kV. Samples for TEM were prepared as for SEM, except that 2% (w/v) osmium tetroxide were used to post-fix for 2 h at room temperature. After dehydration in a graded series of ethanol, the samples were incubated overnight in an epoxy resin (PolyBed 812)/acetone solution (1:1), and then embedded in pure resin and polymerized for 48 h at 60°C. ultra-thin sections were stained with 4% (w/v) uranyl acetate and 3% (w/v) lead citrate and observed under a transmission electron microscope, JEM-2200FS , operated at 200 kV.Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the ultrastructural morphology of the cultured trypanosomatids. For SEM, live trypanosomatids were pelleted at 1,600 × 4 parasites/mL (day 0). The initial inoculum was transferred into a 25 cm3 flask containing 5 ml of the culture medium for 14 flasks. Seven flasks were incubated at 26°C and the rest were incubated at 37°C with 5% CO2. One flask at each temperature was taken out daily, and a cell scraper  was applied to remove any adherent cells from the base of the flask. Ten microliters of parasites were collected and mixed at a 1:1 ratio with formaldehyde when all parasites were alive or 0.4% trypan blue solution  when nonviable forms of parasites were present in the cultures. Live cells were counted using a Neubauer chamber. Also, the cultures were smeared on slides and stained with Giemsa’s staining solution for morphological examination by LM. Experiments were performed on three independent replicates run in duplicate.Trypanosomatids were removed from −80°C, grown in SIM complete at 26°C for 4 days, and subpassaged into new SIM complete for growth analysis. The parasites were counted using a Neubauer chamber  from an initial inoculum of 4 × 10Mus musculus)  were obtained using the method described previously  and L. martiniquensis promastigotes (from Day 5) were used to infect PEMs at a ratio of 10:1 cells to macrophages. After incubation for 3 h at 37°C and 5% CO2, the coverslips were washed with pre-warmed serum-free RPMI 1640 medium for three times, replaced with 10% hi-FBS RPMI-1640 medium, and incubated at the same conditions. Coverslips were removed from the culture plates and stained with Giemsa’s staining solution every 24 h until 96 h post-infection. Two hundred macrophages were counted to determine the infection rate and average number of intracellular parasites per macrophage. The infection index, and the intracellular parasite multiplication ratio were determined  from BALB/c  and 6 males from Tha Ruea and 724 females  and 15 males from Khuan Phang , C. mahasarakhamense , and C. oxystoma . In Khuan Phang seven species were identified, C. peregrinus , C. imicola , C. shortti , C. huffi , C. mahasarakhamense , C. palpifer , and C. oxystoma . Thus, in both locations C. peregrinus was the most abundant species. For engorged females (n = 14), all blood host identifications were of Bos indicus cattle (GenBank accession no. OR088254–OR088267).A total of 1,094 biting midges of the genus an Phang . No gravC. peregrinus by wing spot patterns . Molecular identification (see below) of the KP10 sample revealed that this midge was co-infected with L. martiniquensis and a possible novel Crithidia species  in 26 out of the 100 samples of parous females from the two locations. However, no trypanosomatids were found in any nulliparous flies. All infected flies were identified as patterns  and molepatterns . None ofpatterns . Interesus, KP10 . In this foregut  and Crit species . Trypano species . In the stigotes ,F, nectostigotes , and lepstigotes , however methods . The preC. peregrinus collected in Tha Ruea (TR2 and TR3) and two from C. peregrinus collected in Khuan Phang (KP1 and KP4)  . UnfortuLeishmania species, 100 gDNA samples extracted from the dissected insects were subjected to the PCR amplification of the ITS1 and 3’UTR-HSP70-I regions. Four samples of the dissected insects, from TR17, KP10, KP17, and KP19, were positive for Leishmania and their ITS1 amplicons were successfully sequenced. Moreover, a second molecular marker, 3’UTR-HSP70-I, was used to confirm the identification of the Leishmania species of the samples. BLAST analysis of the ITS1 and 3’UTR-HSP70-I sequences of the samples revealed that the ITS1 sequences were closest to that of L. martiniquensis, GenBank accession number MK603827.1, with 98.94% identity, whereas the 3’UTR-HSP70-I sequences were very similar (98.64 and 99.77% identity) or identical (100% identity) to that of L. martiniquensis, GenBank accession number MK607435.1, and another one was identical to L. martiniquensis, GenBank accession number MK607437.1  for ITS1  with 97.00–97.74% identity. The other five sequences were also very similar to another Crithidia sp. reported from Thailand by L. martinquensis and Crithidia, as suspected from dissection for KP10.The SSU rRNA of 24 samples were successfully amplified and sequenced allowing the identification of other trypanosomatids. BLAST analysis showed that among these, 19 sequences were similar to each other and to a Crithidia sp. Clade A , and were designated as Crithidia sp. Clade B. The availability of cultures for CLA-TR2, CLA-TR3, CLA-KP1, and CLA-KP4 enabled additional DNA extractions and analysis of their gGAPDH sequences. These gGAPDH sequences also clustered together with 98% bootstrap values (K2P 0.31–0.77%) (Cr. fasciculata (GenBank accession number AF047493.1) and Cr. brachyflagelli (GenBank accession number JF717835.1) with 94.39–94.61% identity (Cr. thermophila sequences (K2P 3.77–4.25%), like what was observed in the SSU rRNA analysis , designated here as  Clade A . The cor1–0.77%) . Amongstidentity . Howeveranalysis .C. peregrinus were found infected with Crithidia sp. Clade A organisms and four females with Crithidia sp. Clade B organisms. Successful cultures were obtained only from insects TR2 and TR3, both Clade A organisms  also co-infected with Crithidia sp. Clade A organisms. In Khuan Phang, twelve females were infected with Crithidia sp. Clade A organisms and one female with Crithidia sp. Clade B organisms. In this area the infection rate of the C. peregrinus with L. martiniquensis was 6%, with one insect (TR10) again co-infected with Crithidia sp. Clade A organisms, but also in two other insects (KP17 and KP19) that only contained Leishmania. We also successfully cultured two Crithidia sp. Clade A from two C. peregrinus (KP1 and KP4)  .Crithidia sp. Clade A organisms to be related to Cr. thermophila, an unusual thermotolerant trypanosomatid. To examine whether Clade A organisms might have similar properties further experiments were performed on strain CLA-KP1. Morphological and ultrastructural analyses of cultures in SIM complete at 26°C revealed two distinct morphotypes, choanomastigotes with rounded posterior ends and promastigotes with elongated posterior ends  with a doubling time of approximately 13.5 and 15.6 h for 26°C and 37°C, respectively. After 4 days of culture, at 26°C, the cultures entered the stationary phase and reached their peak on day 6 with a density of 1.9 × 108 cells/ml. The cultures remained in this phase to the last day of the experiments. At 37°C, CLA-KP1 reached maximum growth on day four with a density of 9.8 × 107 cells/mL, remained in stationary phase for 2 days, then the population dropped dramatically after 6 days of culture until the end of the experiment with a density of 1.0 × 106 cells/ml. The average parasite density at 26°C was significantly greater than 37°C after 5 days of culture  because after that the parasites disappeared. A decrease in the intracellular parasite multiplication ratio was also observed in L. martiniquensis after 24 h post-infection, however, the Leishmania parasites were still be found in the PEMs at the end of the experiment (96 h) (Crithidia sp. CLA-KP1 could infect and persist in the PEMs up to 48 h but could not multiply within them.The ability of CLA-KP1 to infect and multiply in PEMs of BALB/c mice was determined and compared with iquensis . Crithidiquensis –J. At 24t (96 h) . In summL. martiniquensis and L. orientalis has been experimentally demonstrated in vivo  is typical of endemic areas, where only a small proportion of collected vectors are usually found harboring parasites. A recent study focused on the residences of two recently diagnosed visceral leishmaniasis patients in Songkhla Province, Thailand, indicated a local infection rate of 21.2% in Culicoides midges, including C. peregrinus  culture medium (L. macropodum from midges of the genus Forcipomyia (Diptera: Ceratopogonidae)  . Adding gonidae)  have beecultures . Using oble ones . Thus, t vectors .Leishmania parasites, monoxenous trypanosomatids, insect-restricted parasites, are more easily isolated and cultured in the laboratory  medium at 23°C have been reported  (Cr. mellificae (36–37°C) (Crithidia sp. related to Cr. fasciculata (37°C) (Crithidia sp. CLA-KP1 was able to grow and survive at 37°C in culture for at least 7 days and the density of the cells at day 7 was still high (1.0 × 106 cells/mL), although the average parasite density at 26°C was significantly greater than 37°C after 5 days of culture. In addition, Crithidia sp. CLA-KP1 could infect and persist appear in the PEMs for up to 48 h. The results suggested that Crithidia sp. CLA-KP1 is a thermotolerant monoxenous trypanosomatid that could live in a wide range of temperature, 26–37°C.The ability to survive at human body temperature and multiply in mammalian macrophage cells is a hallmark of dixenous parasites such as  mammals . In addidiseases  or with diseases . Moreovea (34°C) , Cr. mel36–37°C) , and a Ca (37°C) . In our Crithidia sp. related to Cr. fasciculata are able to infect THP-1 and J774 cells and transform back to promastigotes in culture media but the percentage of infection is much less than that of the clinical isolate of L. major . Analysis of genome sequences of all strains of Crithidia sp. CLA will be performed to compare the evolutionary relationship between this newly isolated trypanosomatid and other related parasites. A description of the detailed molecular characterization and taxonomic assignment will be provided in the future.In conclusion, here we provide microscopic analyses together with molecular identification of naturally occurring infections of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The use of animals in this study was approved by the animal research ethics committee of Chulalongkorn University Animal Care and Use Protocol (CU-ACUP), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA No. 023/2564 and COA No. 011/2564). The study was conducted in accordance with the local legislation and institutional requirements.NJ and PS: conceptualization, methodology, and formal analysis. SK, NJ, CM, TP, RA, TY, UP, and AJ: investigation. SK, PS, and NJ: visualization. NJ, SK, TP, and CM: writing – original draft preparation. NJ and PB: writing-review and editing. NJ: funding acquisition. All authors have read and agreed to the published version of the manuscript.This work was supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) (grant number GCUGR1125651016M).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fasciola hepatica and Fasciola gigantica, is a global zoonotic helminthic disease. The livestock and human are the final hosts of the parasites. Northern Iran is an important endemic region for fascioliasis. Few studies have been conducted on the characterization of Fasciola isolates from eastern regions of the Caspian littoral of the country.Fascioliasis, caused by the liver flukes F. hepatica, F. gigantica and intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools.The aim of the present study was to identify Fasciola spp. were collected from Golestan slaughterhouse during 2019–2020. The worms were morphometrically studied using a calibrated stereomicroscope. Genomic DNA was extracted from all samples, and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was performed on internal transcribed spacer (ITS1) region using Rsa1 restriction enzyme. All the isolates were then analysed by multiplex PCR on Pepck region.Livestock livers naturally infected with Fasciola isolates were collected from the infected livers, including 94 sheep, 12 cattle and 4 goats. Morphometric analysis of 61 adult Fasciola isolates indicated that, 44 and 17 isolates belonged to F. hepatica and F. gigantica, respectively. Eighty‐one and 29 isolates belonged to F. hepatica and F. gigantica using ITS1‐RFLP, respectively. However, Pepck Multiplex PCR indicated 72 F. hepatica, 26 F. gigantica and 12 intermediate/hybrid forms. All 12 hybrid isolates were found in sheep host. Two isolates were identified as F. gigantica using morphometry and F. hepatica using both molecular methods.A total of 110 F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province.The present study confirmed the existence of both  Fasciola hepatica, Fasciola gigantica and the intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools. From a total of 110 Fasciola isolates collected from 94 sheep, 12 cattle and 4 goats, 72 isolates (65.4%) were identified as F. hepatica, 26 isolates (23.6%) as F. gigantica and 12 isolates (11.0%) as the hybrid forms. The present study confirmed the existence of both F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province. The aim of the present study was to identify  Fasciola species cause significant economic losses due to liver rot, high morbidity rates and decreased milk, meat and wool production  on the nuclear internal transcribed spacers (ITS1 and/or ITS2) , cattle (12) and goats (4) during routine veterinary inspections between 2019 and 2020, in the central abattoir of Golestan province, northern Iran .A total of 110 2.2For morphometric analysis, the worms were washed in PBS and fixed and tied between two slides. Morphology and morphometric characters of the flukes were studied using a microscope equipped with a drawing tube. The morphological parameters were recorded in an Excel spreadsheet. Morphometric analysis was performed using following criteria: body length (BL), body width (BW), body length to width ratio (BL/BW) and cephalic cone length.F. hepatica and F. gigantica, and one‐way analysis of variance was used to find any significant differences between the means of morphometric values in flukes separated from different hosts  using a pestle. Then the lysate transferred to a micro tube and incubated at 60°C for 3 h after adding 20 μL proteinase K. The lysate from each sample was subjected to DNA extraction using a conventional phenol–chloroform method with minor modifications  and ITS1‐R (5′‐TTGGCTGCGCTCTTCATCGAC‐3′)  at 80 V for 1 h. A 100‐bp DNA ladder  was run to show the size of DNA fragments in each gel. Gels were visualized and photographed on a transilluminator .Ten microliters of each ITS1‐PCR product were digested for 4 h at 37°C by Rsa1 restriction endonucleases , in a final volume of 30 μL according to the manufacturer instructions. Restriction fragments along with a 50‐bp DNA ladder  were separated by electrophoresis through 2.5% agarose gels, stained with SYBR green, and visualized by UV transilluminator.To amplify partial PEPCK gene, a multiplex PCR was employed using the following primers: Fh‐pepck‐F (5′‐GATTGCACCGTTAGGTTAGC‐3′), Fg‐pepck‐F (5′‐AAAGTTTCTATCCCGAACGAAG‐3′) and Fcmn‐pepck‐R (5′‐CGAAAATTATGGCATCAATGGG‐3′)  at 80 V for 40 min. A 100‐bp DNA ladder  was run to show the size of amplicon in each gel. DNA fragments were visualized and photographed on a transilluminator .The multiplex PCR amplification was performed in a thermocycler  under the following temperature conditions: 94°C for 5 min as initial denaturation followed by 30 cycles at 94°C for 30 s as denaturation, 59°C for 30 s as annealing, 72°C for 1 min as extension and a final extension at 72°C for 10 min. In each set of multiplex‐PCR, DDW was used for negative control. Two known 33.1Fasciola isolates, morphometric analysis was performed on 61 specimens as some of the helminths were juvenile worms without full‐grown reproductive organs. The morphometric characteristics of isolated flukes are summarized in Table F. gigantica (2.5–4.76 cm) was greater than F. hepatica (1.8–3 cm). The differences between the BL, CL, in the two species were significant (p < 0.05). Based on BL/BW and BW/CW ratios, 44 and 17 specimens were morphologically identified as F. hepatica and F. gigantica, respectively. No hybrid forms were identified by morphometric analysis. The measure of BL/BW has been considered one of the useful indices for differentiating F. hepatica from F. gigantica. The BL/BW ratios of 1.29–2.80 and 3.40–6.78 are considered for F. hepatica and F. gigantica, respectively  ratios of 2.1–5.55 and 1.5–4 and (VS/OS) ratios of 0.3–2.5 and 1–3.8 are considered for F. hepatica and F. gigantica.Of 110 3.2F. hepatica and 60, 170 and 360 bp for F. gigantica isolates  and 29 (26.4%) isolates were identified as F. hepatica and F. gigantica, respectively. Pepck‐Multiplex PCR amplified a 241 bp fragment for F. hepatica, 510 bp for F. gigantica. The hybrid forms demonstrated both fragments in gel electrophoresis  as F. gigantica, whereas 12 isolates (11.0%) were identified as hybrid forms. All 12 hybrid Fasciola specimens were isolated from sheep host. All four goat isolates were identified as F. hepatica by morphometry as well as the both molecular methods. Overall, 63 (67%), 19 (20.2%) and 12 (12.8%) of 94 sheep isolates were belonged to F. hepatica, F. gigantica and hybrid forms, respectively. Five (41.66%) and 7 (57.33%) cattle isolates belonged to F. hepatica and F. gigantica, respectively. Two sheep isolates, first identified as F. gigantica using morphometry, were characterized as F. hepatica by the both molecular methods. Moreover, a sheep isolate identified as F. hepatica using ITS1‐RFLP was characterized as F. gigantica using morphometry and Pepck multiplex‐PCR. Another sheep isolate, indicated as F. gigantica using ITS1‐RFLP, was found to be F. hepatica by using morphometry and Pepck multiplex‐PCR.In ITS1‐PCR a 680 bp fragment was visualized after gel electrophoresis Figure . ITS1‐RFs Figure . Using Is Figure . Using P4F. hepatica and F. gigantica, the main Fasciola species infecting human and animals, has been performed using morphologic and morphometric methods. However, the two species cannot be accurately discriminated by these methods. In addition, during past decade, the intermediate forms of Fasciola have been described from some Asian and African countries where both F. hepatica and F. gigantica coexist. Hybrid Fasciola flukes are believed to be originated from the interspecific hybridizations occurring throughout East, South‐east and South Asian countries. However, to our knowledge, the distribution of hybrid Fasciola flukes in the regions between Central Asia and the Middle East has not been investigated. Several previous reports attempted to identify the species of Fasciola flukes from these regions using morphological and morphometric features as well as conventional molecular markers    and F. gigantica (27.87%). ITS1‐PCR–RFLP showed that 73.6% and 26.4% of 110 young and adult Fasciola isolates belonged to F. hepatica and F. gigantica, respectively, with no hybrid form. However, Pepck‐Multiplex PCR reveled 65.5%, 23.6% and 10.9% of 110 isolates as F. hepatica, F. gigantica and Fasciola hybrid form, respectively. It confirms that morphometric and PCR–RFLP analyses cannot accurately detect Fasciola species. Previous study in Golestan province using morphological method indicated 11.84% of sheep and cattle isolates belonged to Fasciola sp.; however, this was not confirmed by molecular methods  among 110 isolates. All four goat isolates in the present study were F. hepatica, which is in concordance with previous studies in Iran  from buffalo and goat in Mazandaran province, northern Iran. Akhlaghi et al. (2018) reported 2 out of 44 sheep isolates as Fasciola sp., employing cox1 and ITS1 sequencing. Phylogenetic analysis indicated that the two Fasciola sp. isolates were located in a same clade with an intermediate form of Fasciola sp. from Japan. The present study also reports Fasciola intermediate/hybrid form in 12 sheep isolates from Golestan province using Pepck‐Multiplex PCR.The hybrid form may occur in some endemic areas where both r et al.  using IT5F. hepatica, F. gigantica and the intermediate/hybrid forms in sheep, goat and cattle in the northern province of Golestan. Findings of the present study indicate the coexistence of both species of Fasciola and a high potential of fascioliasis transmission in the livestock of the region. The study presents the first molecular evidence of intermediate/hybrid forms of Fasciola from the sheep in this province.This study confirmed the presence of Conceptualization; funding acquisition; methodology; project administration; resources; supervision; validation; writing – original draft; writing – review and editing: Mitra Sharbatkhori. Data curation; investigation; methodology; software; validation; visualization; writing – original draft; writing – review and editing: Saeid Nasibi. Formal analysis; methodology; software; writing – original draft; writing – review and editing: Mohammad Ali Mohammadi. Data curation; investigation; methodology; project administration; writing – original draft; writing – review and editing: Mojgan Aryaeipour. Investigation; methodology; validation; writing – original draft; writing – review and editing: Saber Raeghi. Conceptualization; formal analysis; methodology; project administration; validation; visualization; writing – original draft: Majid Fasihi Harandi.The authors declare no conflict of interest.Golestan University of Medical Sciences, Gorgan, Iran, Grant Number: 17305.https://publons.com/publon/10.1002/vms3.1189.The peer review history for this article is available at


Leishmania and transmitted through the bite of sand flies vector. Macrophages (MΦ), the target cells of Leishmania parasites, are phagocytes that play a crucial role in the innate immune microbial defense and are antigen-presenting cells driving the activation of the acquired immune response. Exploring parasite–host communication may be key in restraining parasite dissemination in the host. Extracellular vesicles (EVs) constitute a group of heterogenous cell-derived membranous structures, naturally produced by all cells and with immunomodulatory potential over target cells. This study examined the immunogenic potential of EVs shed by L. shawi and L. guyanensis in MΦ activation by analyzing the dynamics of major histocompatibility complex (MHC), innate immune receptors, and cytokine generation. L. shawi and L. guyanensis EVs were incorporated by MΦ and modulated innate immune receptors, indicating that EVs cargo can be recognized by MΦ sensors. Moreover, EVs induced MΦ to generate a mix of pro- and anti-inflammatory cytokines and favored the expression of MHCI molecules, suggesting that EVs antigens can be present to T cells, activating the acquired immune response of the host. Since nano-sized vesicles can be used as vehicles of immune mediators or immunomodulatory drugs, parasitic EVs can be exploited by bioengineering approaches for the development of efficient prophylactic or therapeutic tools for leishmaniasis.Leishmaniasis is a parasitic disease caused by different species of  Leishmaniasis is a neglected tropical disease, affecting mainly underdeveloped regions. It is the second disease with the highest mortality rate  and the third in disability-adjusted life years (DALYs), behind malaria and schistosomiasis . This diLeishmania co-infection [Cutaneous manifestations such as localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), mucocutaneous leishmaniasis (MCL), and post-kala-azar dermal leishmaniasis are usually characterized by inflammatory skin lesions at different levels, which can be disfiguring and very damaging of people’s quality of life . Cutaneonfection . Thus, tLeishmania infection. Cells belonging to innate immunity can sense invading organisms through receptors that recognize highly conserved molecular patterns associated with pathogens (PAMPs). These pattern recognition receptors (PRR) include the family of Toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors [The innate immune response is essential in the defense against eceptors .Leishmania promastigotes are deposited in the dermis of the host, being recognized, and phagocytosed by phagocytes. Upon contact with the pathogen, macrophages (MΦ) are activated and differentiated into professional antigen-presenting cells (APCs), capable of processing and presenting antigens to lymphocytes, establishing the communication between the innate and the adaptive immune response [+ T cells) and class II molecules (MHCII) that are present to T helper lymphocytes (CD4+ T cells). During the blood meal of the female phlebotomine vector in the vertebrates, response . Promastresponse , drivingLeishmania parasites over the host’s immune system. To avoid cytotoxic CD8+ T cell activation, the parasite selectively impairs the release of interleukin (IL)-12 and interferon-gamma (IFN-γ). Instead, the parasite directs infected MΦ to produce anti-inflammatory cytokines, such as IL-10, IL-4, and transforming growth factor beta (TGF-β), which are known for their inhibitory action on MΦ functions, promoting the parasite persistence [There is increasing evidence of the immune modulatory effect exerted by sistence ,12,13. TAn element that has been considered key in host–parasite interactions are the extracellular vesicles (EVs). These nano-sized lipid vesicles, shed by all living cells, generally transport proteins, lipids, and nucleic acids, and have the potential to immunomodulate the target host cells. EVs are classified according to their biogenesis and size. Exosomes originate in the endosomal membrane, constitute multivesicular bodies that fuse with the plasma membrane, and are then shed into the extracellular environment . AccordiPlasmodium [Trypanosoma cruzi [Schistosoma [Trypanosoma brucei brucei [Leishmania [Leishmania EVs can carry virulence factors and modulate MΦ activity to prevent parasite death by inhibiting the secretion of pro-inflammatory cytokines. The main virulence factor present in exosomes is the metalloprotease glycoprotein of 63 kDa (gp63), which can regulate transcription factors of target MΦ, such as NF-κB [Leishmania, has been growing, aiming to develop efficient prophylactic and therapeutic strategies [Several studies have already evaluated the use of exosomes in the treatment of diseases such as COVID-19 ,17,18, casmodium ,24, Trypma cruzi , Schistoistosoma , Trypanoi brucei , and Leiishmania . Accordiishmania , Leishmaas NF-κB . Howeveras NF-κB . Thus, trategies .Leishmania causing CL in the new world into murine MΦ activation. L. guyanensis causes different clinical forms varying from LCL to MCL, and L. shawi causes a less common CL clinical form. Therefore, the current study aimed to evaluate the effect of EVs shed from two species of Leishmania spp. (L. shawi and L. guyanensis) on murine MΦ cell line, EVs of Leishmania axenic promastigotes were isolated from the culture medium and morphologically characterized by Scanning Electron Microscopy (SEM), dynamic light scattering (DLS), and Electrophoretic Light Scattering (ELS). The protein composition of EVs was examined by SDS-PAGE gel and zymography and the interplay of EVs with MΦ was analyzed by multiparametric flow cytometry and fluorescence microscopy. Moreover, to examine the effect of EVs on MΦ immune activation, gene expression of cell sensors and cytokines was evaluated by real-time PCR (RT-qPCR), MHCI and MHCII surface expression was assessed by multiparametric flow cytometry, and NO and urea production was analyzed by colorimetric assays. To evaluate the effect of EVs shed by promastigotes of LC-causing ®, Nuaillé, France) supplemented with 10% inactivated fetal bovine serum , 100 U·mL−1 of penicillin and 100 μg·mL−1 of streptomycin , pH 7.2, at 37 °C in a humid atmosphere with 5% CO2. Cells were centrifuged at 300× g for 10 min and transferred to a fresh medium supplemented with 10% exosome-depleted inactivated FBS  to be used in the following assays.Macrophagic mouse cell line P388D1  previously isolated from a mouse lymphoma was maintained in RPMI 1640 culture medium (BiowestL. shawi (MHOM/BR/96/M15789) and L. guyanensis (M19663) (SNGPGCTA—certificate ref. A095CE9) promastigotes were maintained in Schneider’s Drosophila medium (Biowest®), supplemented with 10% heat-inactivated FBS, 100 U·mL−1 of penicillin, and 100 μg·mL−1 of streptomycin and were incubated at 26 °C. Parasites in the logarithmic growth phase were centrifuged at 1800× g for 10 min and further used to isolate EVs and also to infect MΦ. L. shawi and L. guyanensis kept in Schneider’s medium were centrifuged at 1800× g. A fresh medium supplemented with exo-free FBS was added, and parasites were incubated at 26 °C for 24 h. Cultures were again centrifuged at 1800× g for 10 min, and the pellet was transferred to the fresh medium and incubated at 26 °C for 72 h. After this period, the cultures were centrifuged at 1800× g for 10 min to remove the parasites, and supernatants were further centrifugated at 2000× g for 30 min to remove cellular debris. The supernatant was collected, and the exosome isolation reagent  was added at a ratio of 1:2, according to the manufacturer’s instructions, and the supernatant was incubated for 24 h at 4 °C. After incubation, the EV solution was centrifuged at 10,000× g for 1 h at 4 °C. Pellets (rich in EVs) were resuspended in 1× phosphate-buffered saline (PBS) and used immediately or stored at −80 °C for further assays. In parallel, sterile Schneider’s medium supplemented with 10% exo-free FBS followed the same protocol of EV isolation, and the obtained solution was used as a negative control of the EV isolation method (ImC). The proteins in the final EV solution were quantified in the NanoDrop 1000® spectrophotometer .Viable promastigotes of L. shawi and L. guyanensis promastigotes of the logarithmic growth phase were centrifuged at 1800× g for 10 min, and the obtained pellet was washed twice with 1× PBS. The supernatants were discarded, and the pellet was resuspended in 200 μL of 1× PBS. The parasites were subjected to three cycles of freezing and thawing followed by shaking to promote cell lysis and the release of soluble proteins. After these cycles, the lysed parasites were centrifuged at 1800× g for 10 min, and the total soluble protein of the supernatants was quantified in the NanoDrop. L. shawi and L. guyanensis soluble antigens (Ags) were stored at −80 °C until further use.L. shawi and L. guyanensis promastigotes were used, as well as viable promastigotes of both species. The topography analysis of the EVs was performed using scan electron microscopy (SEM). For this analysis, EVs isolated from ®) overnight to increase adherence and later placed in a 24-well plate. Then, parasites were left to adhere to the coverslips, followed by a fixation step with PBS 4% paraformaldehyde  for 30 min at 4 °C. For EVs, coverslips were rinsed three times with distillate water, treated with 0.5% osmium tetroxide (Sigma-Aldrich®), and washed again. A fixative solution of 1% tannic acid (Sigma-Aldrich®) was added for 30 min. EVs were fixed to coverslips with 2.5% glutaraldehyde, 0.1 M sodium cacodylate buffer, and pH 7.4 for 2 h at 4 °C, and then coverslips were washed. Afterward, both parasites- and EV-coverslips were washed and dehydrated by sequential addition of 30%, 50%, 70%, 80%, and 90% ethanol for 5 min each. Coverslips were immersed in 100% ethanol and then treated with hexamethyldisilane solvent (Sigma-Aldrich®), coated with gold-palladium, and mounted on stubs to be observed under an ultra-high resolution scanning electron microscope . Acquired images were analyzed using ImageJ software to estimate the vesicle diameter.For the promastigotes, round glass coverslips were immersed into poly-D-Lysine  in Malvern ZetaSizer equipment , at a constant temperature of 25 °C and with a detector placed at 90°. EV zeta potential (ζ), which is related to membrane charge and is an important indicator of the stability of colloidal dispersion, was evaluated using electrophoretic light scattering (ELS) at pH 7.5 in a Malvern ZetaSizer equipment . ImC was also analyzed for the diameter and zeta potential of its constituents.L. shawi and L. guyanensis EVs (50 μg of protein) were added to the gel, along with a 4× loading buffer  supplemented with 1:10 of β-mercaptoethanol. ImC was also used to disclose protein components that did not correspond to parasite EVs. For the zymography assay, L. shawi and L. guyanensis EVs (50 µg of protein) were added to the gel with a 4× loading buffer free of β-mercaptoethanol. Protein characterization of EVs was performed by acrylamide gel electrophoresis (10%) with sodium dodecyl sulfate (10% SDS-PAGE) and zymography (with 0.41% gelatin). ® Brilliant Blue G 250 (Sigma-Aldrich®) and destained with a solution of 10% acetic acid and 40% methanol to visualize the protein bands. For the zymography assay, the gel was incubated with Triton-X100 for 1 h and then incubated for 18 h with 0.5M Tris-HCl buffer (pH 7.5), 0.2 M NaCl, 0.005 M CaCl2, and 0.02% Brij35. Afterward, the gel was stained and destained following the same steps of SDS-PAGE. After the end of the run, the SDS-PAGE was stained with Coomassiewww.gelanalyzer.com).The molecular mass of the bands found in ImC and EV samples was determined by comparison with the 10–250 kDa molecular weight (MW) marker  using the GelAnalyzer 19.1 software (18(7) , which incorporates into lipid membranes increasing fluorescence, was used to EV stains.To follow the interplay of parasite EVs with MΦ, the lipophilic fluorescent cationic dye DiR’. DilC18 for 2 h at 26 °C and then were passed through columns  and centrifuged at 750× g for 3 min to remove the unincorporated dye. In parallel, 1× PBS was incubated with DilC18 and passed through the column to be used as a negative staining control and to assess the capacity of the column to retain non-bonded DilC18. On the other hand, MΦ were directly incubated with the dye to be used as a positive control. Stained EVs and the negative control were incubated with MΦ for 4 h, 24 h, and 48 h at 37 °C in a humid atmosphere with 5% CO2. At each time point, negative and positive as well as EVs-incubated MΦ were washed with 1× PBS, and samples were then analyzed by multiparametric flow cytometry and fluorescence microscopy. Samples were acquired by a flow cytometer analyzer , and the proportion of positive cells, as well as the mean fluorescence intensity (MFI), were evaluated.EVs were incubated with DilCTM with DAPI, Sigma-Aldrich®). The slides were observed under a fluorescence microscope , and images were acquired. For microscopy examination, cells were fixed with 2% paraformaldehyde for 30 min at 4 °C, and MΦ nuclei were stained with DAPI (Fluroshield6 cells·mL−1) were separately incubated at 37 °C in a humid atmosphere with 5% CO2, for 4 h, 24 h, and 48 h, with (i) viable promastigotes of L. shawi or L. guyanensis (ratio cell: promastigote 1:3), (ii) 40 μg·mL−1 of L. shawi or L. guyanensis soluble Ag, and (iii) L. shawi (LsEVS) or L. guyanensis (LgEVs) EVs at concentrations of 5, 10, 20, and 45 μg·mL−1. In parallel, (iv) resting MΦ and (v) MΦ stimulated with Phorbol-12-myristate-13-acetate  at 0.2 µg·mL−1 were also evaluated.To evaluate the effect of parasite EVs on MΦ activity, cells  was used. Resazurin is a low-fluorescent blue compound that is reduced to resorufin by metabolically active cells, resulting in highly fluorescent pink staining [To assess the viability of MΦ that were incubated with promastigotes or EVs, resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) metabolization assay , 40 µg·mL−1 soluble Ag, and promastigotes (1:3), and were incubated at 37 °C with 5% CO2 for 24 h, 48 h, and 72 h. Three controls were used: resting MΦ , MΦ stimulated by 0.2 µg·mL−1 phorbol myristate acetate (PMA), and cell death control (DC) where 2% paraformaldehyde was added to MΦ. Paraformaldehyde inactivates cell metabolism, and these cells are considered non-viable cells. Then, resazurin solution in 1× PBS (0.067 µg·mL−1) was added to all wells and incubated for 1 h at 37 °C. After incubation, absorbances were read at 595 nm, after excitation at 535 nm, in a TRIADTM 1065 fluorimeter . MΦ were added to 96-well plates at a concentration of 1 × 10®, Newark, CA, USA) and Nitrate/Nitrite colorimetric assay kit , respectively, according to the manufacturer’s instructions. The chromogenic reagent present in the urea kit reacts specifically with urea, developing a colorimetric complex that can be analyzed by spectroscopy at a wavelength of 430 nm (TRIADTM 1065 fluorimeter (DYNEX Technologies), a color intensity that is directly proportional to the concentration of urea in the sample. The nitrate/nitrite concentration was determined using a two-step process: first, it converts nitrate to nitrite using the nitrate reductase, and in the second step utilizes the Griess Reagent to convert the nitrite into the azo compound with the color purple that can be photometrically measured for absorbance at 540 nm. The final results were normalized to an RPMI-supplemented medium and presented as fold change to resting MΦ (non-stimulated cells). To evaluate the final products that result from the activation of L-arginine pathways, MΦ supernatants from all experimental conditions (as described in 2.9) were centrifuged to remove cell debris and used to quantify urea and NO production using the Urea Assay Kit (BioChainw/v) were added . Cells were acquired by a flow cytometer (CytoFlex), and MFI (median fluorescence intensity) values were analyzed and presented as fold change to resting MΦs (non-stimulated cells).Expression of MHCI and MHCII molecules on the surface of MΦ exposed to parasites and stimulated by PMA, Ag, and EVs and in resting MΦ was analyzed by multiparametric flow cytometry. After 24 h, 48 h, and 72 h of incubation, cells were harvested and washed three times with 1× PBS and mouse-monoclonal MHCI , and MHCII  antibodies diluted in 1× PBS 2% albumin ((IL-)1β, IL-4, IL-10, IL-12p40, and tumor necrosis factor (TNF)-α, the total RNA was extracted using the RNA extraction kit  following the manufacturer’s instructions. The quantity and purity of isolated RNA were evaluated in the NanoDrop. cDNA synthesis was performed using NZY first-strand cDNA synthesis kit (NzyTech), followed by real-time semi-quantitative RT-PCR gene expression analysis using primers specific for mouse MΦ , 0.15 µL of each primer (forward and reverse), 2 µL of sample cDNA, and 7.7 µL of ultra-pure water. Samples were then amplified in the BioRad thermocycler (CFX Connect BioRad). Gene amplification conditions included 39 cycles of denaturation  and annealing for 30 s. Finally, the extension was performed at 50 °C for 15 min. The housekeeping gene HPRT was used to perform a baseline of gene expression in each analyzed sample (ΔCt). Resting cells (non-stimulated macrophages) were collected at each time point and considered as the negative control and used to perform the relative quantification at each time point (∆∆Ct). The results of the relative analysis were obtained through the formula 2−∆∆Ct [To evaluate the relative gene expression of Toll-like and NOD-like innate immune receptors in MΦ exposed to parasites and stimulated by EVs, Ag, and PMA as well as the generation of pro-inflammatory and anti-inflammatory interleukin mouse MΦ . Primer a 2−∆∆Ct .t-test was used to compare the means between the two groups and analyze the differences between the experimental conditions and the controls of each method. The unidirectional ANOVA test was used to compare the mean among samples in groups in the following situations: (i) to analyze the effect of the time  on the same experimental conditions and (ii) to analyze the statistical significance of the crescent EV concentrations for a defined Leishmania species. A significance level of 5% (p < 0.05) was used as indicative of statistical significance. Data analysis was performed using the GraphPad Prism 9 software .Three independent experiences with a minimum of triplicates per experimental condition were performed. After verifying the normality of the sample by the Shapiro–Wilk test, the parametric Student’s L. shawi and L. guyanensis promastigotes appear mostly spherical, with a smooth membrane  and also the presence of vesicles with a diameter compatible with microvesicles (LsEVs—199.53 ± 21.02 and LgEVs—195.93 ± 39.05 nm)  with a variable size dispersion compatible with the presence of exosomes (30–100 nm) and microvesicles (100–2000 nm). The EV profile observed was similar between the two species of  noticed A. DLS an9.05 nm) A,B. L. shawi and L. guyanensis showed negative values, with an average of −11.78 ± 0.36 mV and−9.87 ± 0.57 mV, respectively. When compared to ImC (control), there were statistically significant differences, pointing out to the successful isolation of intact Leishmania-derived EVs (LsEVs p = 0.0002 and LgEVs p = 0.001). Since most cellular membranes are negatively charged, the zeta potential of a nanoparticle can influence its tendency to interact and permeate other cell membranes. However, nanoparticles with a zeta potential between−10 and +10 mV are considered approximately neutral [Leishmania EVs can interact with other cell membranes in a non-disruptive way. In addition, the range of zeta potential obtained for Leishmania EVs is indicative of their ability to flocculate, generating EV aggregates that may promote their interaction with cell membranes. The analysis of the zeta potential  defined as the voltage at the edge of the diffuse layer where it meets the surrounding liquid and, therefore, indicative of the presence of intact lipid membranes in suspension, revealed important differences between the EVs extracted from promastigotes and the negative control (ImC) C. The ze neutral , suggestL. shawi and L. guyanensis showed the presence of four protein fractions with molecular masses of approximately 50 kDa, 63 kDa, 70 kDa, and 80 kDa , which is considered an important virulence factor of Leishmania parasites and has also been identified in EVs of other species of Leishmania.The evaluation of the EV protein profiles from both d 80 kDa . These pLeishmania EVs interacted with phagocytic cells, DilC18-stained EVs were incubated with murine P388D1 macrophages. The observation of cells by fluorescence microscopy reveals that after 4 h of incubation L. guyanensis and L. shawi EVs were incorporated by MΦ. EV incorporation by MΦ was visible through the accumulation of stained vesicles bonded to the cells  evidence residual fluorescence (The uptake of DilCrescence B. p < 0.0001), reaching maximum values at 24 h and maintained at 48 h. Interestingly, LsEVs incubated MΦ showed higher MFI when compared with MΦ incubated with LgEVs (p = 0.0009) (MΦ incubated with stained LsEVs and LgEVs for 4 h showed a higher MFI increase when compared with unstained MΦ ( 0.0009) C. Taken together, these results indicated that MΦ fast incorporates EVs and that the density of LsEVs incorporation is more intense in comparison with LgEV. L. guyanensis and L. shawi EVs and before evaluating the effect of these nano-sized vesicles on the immunological activity of MΦ, the potential effect of EVs, promastigotes, and parasite Ags on MΦ viability were assessed. Resting MΦ was considered to represent 100% viability.After protein analyses of p < 0.001). Remarkably, Leishmania EVs did not alter MΦ viability significantly, although they interacted directly with MΦ cell membrane as previously observed . MΦ innate immune sensors can recognize parasite antigens, signaling downstream pathways that can lead to MΦ immune activation, resulting in the synthesis of immune mediators. Therefore, the gene expressions of cell membrane TLR2, TLR4, TLR9, NOD1, and NOD2 fold increase, evidencing that MΦ were able to express these innate immune pattern recognition receptors (PRRs). In contrast, parasite Ag induced a low gene expression of these sensors. However, after 72 h of incubation, L. shawi Ag caused an increase in TLR9 gene expression (p = 0.0087). After 24 h of incubation , PMA stiTLR2, TLR9, NOD1, and NOD2 genes was observed in MΦ exposed to L. shawi promastigotes or LsEVs. Interestingly, both parasites promoted increasing levels of intracellular TLR9  and cytoplasmic NOD1  at all time points, suggesting parasite internalization by MΦ.Regarding the innate immune receptors, the greatest upregulation of TLR2 gene expression was detected in MΦ stimulated with 45 µg·mL−1 of EVs at all time points . In contrast, low generation of TLR4 and NOD2 was observed in EV-stimulated MΦ during the 72 h of the study.An increase in TLR9 and NOD1 (p= 0.0385) gene expression, followed by a decrease after 48 h of stimulation. The highest concentration of LgEVs promoted the upregulation of cytoplasmic NOD2 (p = 0.0309) gene expression. Thus, these results indicated that parasite EVs could be recognized by macrophage PRRs. MΦ stimulated with LsEV45 exhibited an early increase in intracellular PRRs Leishmania species. L. shawi promastigotes and LsEVs were more efficient in modulating the cell membrane TLR2 (p = 0.0002), the endocytic TLR9 (p= 0.0152), and the cytoplasmatic NOD1 (p < 0.0001) and NOD2 (p < 0.001) when compared with L. guyanensis. Overall, at 24 h of incubation, the higher concentration of LsEVs exerted a more pronounced effect on MΦ innate immune receptors. However, differences in the MΦ’s profiles were observed among these two Leishmania host cell and also make part of the first line of defense against these parasites. Upon stimulation, MΦ can synthesize pro- and anti-inflammatory cytokines that direct the immune activation of other cells. Thus, the effect of EVs shed by L. shawi and L. guyanensis parasites on MΦ gene expression of proinflammatory cytokines  and anti-inflammatory cytokines (IL-4 and IL-10) was examined. MΦ are the IL-4, indicating that MΦ were able to generate cytokines (p < 0.0001)  .L. shawi Ag promoted a significant increase in IL-1β (p = 0.0049) and IL-12p40 (p = 0.0014), and L. guyanensis Ag caused an increase in IL-1β at 48 h (p = 0.0334) and 72 h of stimulation (p = 0.0098). At 24 h of incubation, L. shawi parasites promoted the upregulation of IL-1β (p = 0.0063) and TNF-α (p = 0.0070) gene expression. Increased IL-1β gene expression was also observed after 72 h (p = 0.0174). However, L. guyanensis promastigotes needed 72 h to induce the upregulation of IL-1β (p = 0.0082), IL-12p40 (p = 0.0016), and TNF-α (p = 0.0081). IL-4 gene expression was stimulated at all time points by both parasite species . After 48 h (p = 0.0255) and 72 h (p = 0.0328) of exposure to L. guyanensis parasites, MΦ evidenced the upregulation of IL-10 gene expression. After 72 h of stimulation, L. shawi and L. guyanensis EVs induced in MΦ an increase in IL-1β generation. The higher concentrations of LsEVs  induced IL-1β upregulation at all time points. MΦ stimulated with LgEVs also exhibited the upregulation of IL-1β after 24 h of stimulation (pLgEV20 = 0.0096 and pLgEV45 = 0.0400), and the lowest concentration of LgEVs was also able to induce a sustained increase in IL-1β gene expression . Although presenting fluctuations related to stimulation time and concentration, IL-12p40, a subunit of IL-12 and IL-23 proinflammatory cytokines, was induced early by EVs. After 24 h of stimulation, the two highest concentrations of LgEVs upregulated IL-12p40, followed by a decrease . MΦ stimulated with 20 (p = 0.0127) and 45 µg·mL−1 (p = 0.0086) of LsEVs also showed an early increase in IL-12p40 gene expression. However, the highest concentration of LsEVs promoted the upregulation of this cytokine for 48 h (p = 0.0057), followed by a significant decrease (p72h < 0.0001). In contrast, downregulation of TNF-α was found in MΦ exposed to EVs. Moreover, EVs highly induced IL-4 upregulation, which can interfere with the inflammatory immune response. MΦ stimulated with the higher concentration of LsEVs and LgEVs showed the increase of IL-4 gene expression at all time points .Increased gene expression of IL-10, a regulatory cytokine that contributes to the balance of immune response. During the first 48 h of stimulation, the highest concentration of LgEVs induced MΦ to a transitory IL-10 upregulation (p = 0.0098). Furthermore, at 72 h of stimulation, 20 µg·mL−1 of LsEVs led to a significant increase of IL-10 (p = 0.0175). The two highest concentrations of LgEVs also caused an early transitory increase in IL-10 gene expression , whereas 10 µg·mL−1 of LgEVs needed 72 h of stimulation to induce IL-10 upregulation (p = 0.0470). Parasite EVs also promote MΦ to upregulate L. shawi and L guyanensis EVs, L. shawi EVs seemed to generate higher cytokines levels . Moreover, EV concentration and stimulation time also seemed to play a crucial role in cytokines generation.Taken together, these results indicated that EVs induced MΦ to generate pro- and anti-inflammatory cytokines. Although there were no critical differences between Leishmania survival.The microbicide activity of EV-stimulated MΦ was examined by the ability of these cells to metabolize arginine, leading to pro-inflammatory MΦ (M1-MΦ) that produce NO or anti-inflammatory MΦ (M2-MΦ), which leads to urea synthesis. Thereby, M2-MΦ has an important role in tissue repair but favors parasite replication. In contrast, NO is important for the resolution of parasitic infection, as it provides an environment hostile to L. shawi and L. guyanensis promastigotes for 72 h (p = 0.0073) and 48 h (p = 0.0228) showed a peak of de novo urea production, respectively. On the other hand, MΦ incubated with L. shawi EVs , L. shawi Ag , L. guyanensis EVs , or L. guyanensis Ag  exhibited a reduction in de novo urea production when compared to promastigote infection . L. shawi Ag did not seem to influence NO production, while L. guyanensis Ag triggered low NO levels (p24h= 0.0006).The production of NO by MΦ exposed to parasites or stimulated with EVs, Ag, and PMA for 24 h, 48 h, and 72 h was analyzed. PMA-stimulated MΦ produced high levels of NO at all time points, indicating that MΦ were functional and able to produce NO. On the contrary, MΦ exposed to promastigotes of both −1 of EVs shed by both species of Leishmania showed an early induction of NO synthesis, followed by a decrease . Furthermore, NO synthesis seemed to be dependent on EV concentration, being greater at the highest concentration of EVs.MΦ stimulated with 10, 20, and 45 μg·mLL. shawi and L. guyanensis were able to activate MΦ, directing NO production. However, these cutaneous species of Leishmania seemed to exert different effects on de novo production of urea by MΦ. L. shawi EVs impaired urea production, whereas L. guyanensis EVs induced an early boost of urea.Taken together, these results indicated that in contrast with the parasite, EVs shed by MHC molecules complexed with parasite antigens can interact with T lymphocytes by presenting the antigens, which may lead to T cell immune activation. Therefore, it was analyzed the expression of MHC class I and II molecules in MΦ after stimulation with LsEVs and LgEVs to address the potential ability of MΦ to present antigens to lymphocytes. Representative flow cytometry plots and histograms for all experimental conditions are presented in L. shawi and L. guyanensis virulent promastigotes induced an accentuated reduction in the density of MHC molecules  that was not recovered during the 72 h of the study (p < 0.0001). However, MΦ stimulation with L. shawi Ag caused an early increase of the MFI MHCI molecules, followed by a progressive decrease . On the other hand, L.guyanensis Ag restrained MHCI MFI . In contrast, when stimulated by LsEVs and LgEVs, it was observed that the expansion of the MHCI molecules resulted in MFI increasing during the first 24 h of the study  (−1), driving an accentuated expansion of these MHC molecules . At 72 h of stimulation, EVs led to a moderate expansion of these molecules.As a positive control of inflammation due to its activation of the nuclear factor-κB, PMA-stimulated MΦ showed a high MFI of MHCI and low MHCII molecules at all time points, indicating that MΦ were able to present antigens. Interestingly, MΦ exposed to  0.0074) . Moreove 0.0074) . After 4 0.0074)  with theL. shawi and L. guyanensis EVs, directing the early increase in MHCI molecules and a later increase in MHCII expression, which could be involved in the antigenic presentation to T cells.Taken together, these results pointed towards the immunological activation of murine MΦ by Leishmania EVs is still limited, especially in what concerns the influence of EVs on the host’s immune response. Therefore, the current study investigated the modulation of MΦ immune response by EVs shed by two species of Leishmania that cause human disease, L. shawi and L. guyanensis. Both these species belong to the subgenus Viannia and, according to Cupolillo and collaborators (1994) [L. shawi. L. shawi was first described in the Amazon region in 1989 by Lainson and collaborators [L. shawi usually infects monkeys Cebus apella and Chiropotes satanus, sloths Choloepus didactylus and Bradypus tridactylus, as well as coatis Nasua nasua, and is rarely detected in humans. L. guyanensis constitutes a well-documented agent for MCL in humans and animals and is usually associated with the presence of ulcerative lesions that can progress to mucosal tissue destruction [Leishmania reservoirs, can lead to an increase in the number of infections for both Leishmania species. Extracellular vesicles are lipid-bilayer nano-sized vesicles shed by mammal cells and also by parasites, which can circulate in the extracellular microenvironment. Parasite EVs carry macromolecules that can be transferred to host cells, including immune cells, interfering with their normal activity. Despite there being an upsurge of new information about EVs in almost all domains of biomedical sciences in the last few years, the information available on s (1994) , are monborators , and in borators  showed ttruction . In thestruction . NeverthLeishmania parasites to secrete EVs was demonstrated in 2010 by Silverman and colleagues [L. shawi (LsEVs) and L. guyanensis (LgEVs) promastigotes can include exosomes and microvesicles. The small vesicles (30 to 100 nm) of endosomal origin are identified as exosomes. Their biogenesis inside the cell includes the formation of multivesicular bodies by the invagination of endosomal membranes and their release in the extracellular space upon fusion with the plasma membrane [Leishmania virulent promastigotes are coated by a glycocalyx that plays an important role in the initial interaction between the parasite and its host environment. Gp63, also known as major surface protease, leishmanolysin, or promastigote surface protease, is the most abundant protein covering Leishmania promastigotes and is considered a major virulence factor in Leishmania infection [Leishmania infection are a consequence of the ability of gp63 to (i) inactivate the factor C3b of the complement system by generating the C3bi factor that prevents the formation of the membrane attack complex, which leads to promastigotes lysis, (ii) degrade components of the extracellular matrix, facilitating parasite migration, and (iii) cleave intracellular substrates, which ensures intra-macrophage parasite survival and disease progression [Leishmania parasites, is also a main constituent of Leishmania shed EVs, pointing out the potentially crucial role of the vesicles in the early host–parasite communication. [L. amazonensis EVs with different gp63 cargo displayed distinctive macrophage immunomodulatory capabilities and that the high expression of gp63 was essential to sustain the CL pathology, therefore confirming gp63 as a primordial component of EVs in augmenting the cutaneous inflammatory response in Leishmania spp. infection. Other proteins can make part of EVs cargo, such as HSP83/90 (heat shock proteins) and Leishmania elongation factor 1 α (EF1α). HSP are molecules that play an important role in the immune response, promoting cytokine release by immune cells [L. infantum, L. donovani, L. major, and L. mexicana [L. shawi and L. guyanensis EVs cargo. Recent studies on L. donovani and L. braziliensis EVs have described the presence of small non-coding RNAs, particularly tRNA-derived small RNAs in parasitic EVs [The term EVs describes a heterogeneous population of membrane-enclosed vesicles that cannot replicate and are naturally released by prokaryotic and eukaryotic cells. EVs contain biologically active molecules, including proteins, nucleic acids, lipids, and carbohydrates, and participate in cell-to-cell communication by transferring their cargo content into the recipient cell . The abilleagues . Accordimembrane . On the membrane . In the nfection ,45. Somegression ,47. Inteication. . In a reication.  demonstrne cells  and actine cells , contribmexicana ,53,54,55itic EVs . HoweverLeishmania EVs could interact with other cell membranes in a non-disruptive way. Although L. shawi and L. guyanensis EVs were fast incorporated by MΦ, L. shawi EVs seemed to be faster and highly bound to MΦ. Therefore, different LsEVs and LgEVs dynamics may reveal some interesting details of parasite strategy to subvert the host’s immune response, but it can also differ according to the host, reflecting Leishmania’s host-specific adaptation. The strong EVs incorporation, which did not interfere with MΦ viability but modulated the gene expression of cytoplasmic (NOD1) and endocytic (TLR9) innate receptors, indicated that EVs (or its cargo) were internalized by MΦ. The incorporation of EVs by MΦ was previously demonstrated by Silverman and collaborators [Leishmania EVs by the recipient cells. Using different analytic methodologies, the present study demonstrated that LsEVs and LgEVs interacted with MΦ, being internalized by the cells. The zeta potential value obtained for LsEVs and LgEVs demonstrated that these nanoparticles were electrically neutral , suggestborators , but furLeishmania parasites triggers a range of intracellular signals that promote the production of immune mediators (cytokines and chemokines), which lead to the activation of the host’s immune system, influencing the type and duration of the immune response. Therefore, the findings of the current study indicated that L. shawi and L. guyanensis EVs could be recognized by these sensors. In both L. shawi and L. guyanensis infected MΦ, as well as in EV-stimulated MΦ, the transmembrane innate immune receptor TLR2 exhibited higher upregulation, pointing throughout the recognition of parasite and EVs antigens. This sensor can be activated by parasite lipophosphoglycan, which is highly expressed in the Leishmania cell membrane. According to Jafarzadeh and coworkers [Leishmania, triggering a protective immune response or leading to disease development. L. shawi and L. guyanensis EVs signalization through TLR2 promoted an early boost of NO, which pointed throughout the classical activation of MΦ that it could favor parasite elimination. A study by Polari and coworkers [L. braziliensis, the patient’s MΦ increased TLR2 and TLR4 and triggered TNF-α and IL-10. However, despite the fact that EV-stimulated MΦ evidenced TLR2 upregulation during the entire study, NO production was fast abrogated, pointing to a short duration of the activation of MΦ classical pathway. Furthermore, signalization of the TLR4 downstream pathway seemed to be crucial for the efficient expression of inducible nitric oxide synthase (iNOS) [Leishmania antigens [de novo urea production indicated that EVs could induce the activation of MΦ alternative pathway. Overall, the engagement of TLRs by parasite antigens appeared to be dependent on the infecting Leishmania species and mammal host considered in each study. In addition, diverse Leishmania species appeared to trigger different TLRs in order to control the host’s immune response. The endocytic transmembrane TLR9 was signalized by unmethylated CpG motifs of DNA [L. shawi parasites and Ag seemed to be recognized by TLR9, although without generating substantial levels of IL-12. However, L. shawi and L. guyanensis EVs were early recognized by TLR9 associated with the generation of IL-12p40. Despite the fact that there are only a few available studies characterizing nucleic acids carried out by trypanosomatid EVs, recently, Douanne and colleagues [Leishmania EVs. Thus, it is possible that L. shawi and L. guyanensis EVs carry parasite DNA that signalizes TLR9 of rodent MΦ.Signalization of PRRs by oworkers , signalioworkers  describee (iNOS) ,60 that antigens ,62,63,64s of DNA , leadinglleagues  reportedLeishmania infection are available. In the current study, NOD1 (NLRC1) seemed to be transiently signalized by L. shawi and L. guyanensis parasites, as well as by L. shawi EVs. In contrast, NOD2 (NLRC2) did not recognize EVs, but promastigotes of both species of Leishmania could be signalized through NOD2. The detailed study of PRR profiles appeared as a promising strategy to improve the efficacy of vaccination and therapies with parasite antigens as adjuvants [Leishmania parasites. IL-1β was one of the first cytokines to be produced and promoted the release of other cytokines, including IL-12, a heterodimeric cytokine that modulates the differentiation of Th1 cells. In contrast, cytokines such as IL-4 and IL-10 are responsible for stimulating the differentiation of Th2 cells and regulatory T lymphocytes, respectively, with negative consequences on the activation of MΦ microbicidal pathways. In the current study, IL-4 generation was induced by L. shawi and L. guyanensis promastigotes and by EVs, being the highest upregulation of this cytokine caused by the parasites. Recently, it was demonstrated that this cytokine could assume a pro-inflammatory role when in the presence of other cytokines, such as TNF-α, promoting parasite control [L. guyanensis infected MΦ, which could be related to infection pathogenesis since this parasite can cause mucocutaneous leishmaniasis. The highest pathogenicity of L. guyanensis could explain the discrete results obtained for LgEVs, as the parasite could more easily escape the host’s immune response and establish the infection. Interestingly, IL-10, a cytokine that regulates inflammatory immune response, was only induced by EVs. Although IL-10 is related to parasite persistence and dissemination and MΦ-M2 polarization, it is also a key cytokine for controlling the exaggeration of the inflammatory response associated with pathology present in parasitic diseases such as malaria, Chagas disease, and leishmaniasis [NOD-like receptor family (NLR) is localized in MΦ cytoplasm and activates signal transduction of the transcription factor NF-κB that induces the expression of pro-inflammatory genes, as is the case of cytokines  and NO production ,68,69. Adjuvants . Togethe control ,72. Howemaniasis ,74,75. TL. guyanensis infection, may prevent the development of mucocutaneous pathology. Furthermore, previous studies demonstrated that the co-inoculation of Leishmania-EVs in the host dermis during the phlebotomine blood meal worsened the pathology of the cutaneous lesion with increased expression of inflammatory cytokines [Leishmania EVs are an essential part of parasite biology and play essential roles in host communication and disease outcomes. The expression of MHC molecules by MΦ is very important as these molecules establish complexes with parasite antigens directing T-cell activation. Virulent L. shawi and L. guyanensis promastigotes markedly restrain MHCI, and MHCII molecules in MΦ, compromising the capacity of MΦ to present antigens. The decrease in MHCI molecules has been documented as a mechanism of immune subversion in viral infections [Leishmania infection. Nyambura and colleagues [L. donovani, showed that infected MΦ exhibited a decrease in MHCI and MHCII complex, but CD83 co-stimulatory molecules remained unchanged. The decrease in MHC class I and class II expression on infected cells has also been described in murine studies [+ T cells (T cytotoxic lymphocytes) and also to CD4+ T cells (T helper lymphocytes). In the context of leishmaniasis, CD8+ T cells have been shown to be protective, triggering a cytotoxic immune response that can destroy infected cells, controlling the infection, and preventing disease development [+ T cells may also exacerbate disease and the generation of anti-inflammatory cytokines, as well as regulatory cytokines, impairing the development, as a whole, of a predominant protective immune response. Even so, Leishmania-EVs appear to be directly involved in the balance of the host’s immune response, either activating cells to exert moderated parasite control or increasing disease severity. The findings of the current study (summarized in L. shawi and L. guyanensis carried parasite antigens and also seemed to carry parasite nucleic acids that could be recognized by surface and intracellular PRRs. These EVs are immunogenic and can direct MΦ activity, including the generation of cytokines and the expansion of MHC molecules, which can induce the activation of cytotoxic immune response in addition to the production of antimicrobial NO that can promote parasite inactivation. Despite not mimicking the exact effect of promastigotes in MΦ activation, EVs direct MΦ to generate a mix of regulatory and pro- and anti-inflammatory cytokines, which can lead to a balanced immune response, allowing parasite persistence in the host but avoiding excessive infection that, in the particular case of ytokines . Parasitytokines . Overallfections  and cancfections  but is alleagues , in a st studies . Intereselopment . HoweverL. shawi and L. guyanensis EVs may play a crucial role in modulating the host’s immune defense, promoting a balanced immune response against the parasite. Thus, since they can be used as vehicles of immune mediators or immunomodulatory drugs, EVs may be a promising target for the development of future prophylactic or therapeutic products/systems for cutaneous leishmaniasis, which is the most common clinical form of leishmaniasis among the disadvantaged human populations. By delivering parasite macromolecules,


Ascomycota, Oomycota, and Basidiomycota phyla. M451 exhibited significant antifungal activity with EC50 values from 34–145 μg/mL. The minimal fungicidal concentration against Fusarium oxysporum ranged from 4 to 512 μg/mL depending on the exposure times of 5 min to 24 h. M451 has the highest activity and significantly lower exposure times compared to different polyene, azole, and phenylpyrrole antifungals. The conidial germination assay revealed that M451 induced 99 and 97.8% inhibition against F. oxysporum within 5 min of exposure to 5,000 and 500 μg/mL, respectively. Germ tube elongation, spore production, and spore germination were also significantly inhibited by M451 at concentrations of ≥50 μg/mL. Based on the broad spectrum of antifungal effects across different plant pathogens, M451 could be a new chemical fungicide for plant disease management.Phytopathogenic fungi are the predominant causal agents of plant diseases. Available fungicides have substantial disadvantages, such as being insufficiently effective owing to intrinsic tolerance and the spread of antifungal resistance accumulating in plant tissues, posing a global threat to public health. Therefore, finding a new broad-spectrum fungicide is a challenge to protect plants. We studied the potency of a novel antimicrobial agent, M451, a 1,6-diaminohexane derivative, against different phytopathogenic fungi of the  All tested isolates were identified using classic cultivation methods and coupled with 16S ribosomal RNA sequencing for isolate identification and preserved as pure cultures in the collection of the Human Microbiology Institute  (The  States) . The fun States) .2.2.2PO4  is a phosphate salt of TGV-28  particularly N′-aminated) and the permeation enhancer 0.01% NaH2PO4 . TGV-28,2PO4 .2PO4 , and Maxim® XL .We used agriculture-purpose antifungals as positive controls: prothioconazole and tebuconazole (both azole derivatives), natamycin (a polyene derivative), fludioxonil, NaH2.3.2PO4 0.1%, or fludioxonil 0.25% plates. The control plate contained untreated PDA medium. To test the 13 fungal strains, 3 mm diameter mycelium plugs of each fungal strain were cut from the edge of a 5 day-old colony grown on stock PDA plates, transferred to the control and experimental Petri dishes, and incubated at 25°C for 120 h. The diameter of the colonies was measured, and the growth inhibition percentage was calculated.Mycelial growth inhibition was evaluated as previously described . For sub2.4.F. oxysporum spore production, 3 mm diameter mycelial plugs were taken from the edge of a five-day-old colony and transferred into the control and test tubes. The control tubes contained 10 mL dH20, 10 μL Tween 20, and 30 μL lactoglycerol as spore germination preventers. The test tubes contained the same mixture of fungicides at various concentrations. The spores were counted using a Malassez counting chamber  by transferring 0.2 mL from each tube.Spore production was determined as previously described . To test2.5.7 spores/mL) was placed in a series of microtubes, and 20 μL of M451 was added. The tubes were prepared in triplicate and incubated for 24 h at 25°C. After incubation, spore germination inhibition was observed under an AxioStar Plus light microscope  using a Malassez cell counting chamber. The percentage of non-germinated spores was calculated using Equation 1:Spore germination was analyzed using a previously described method , in whic%SNG: percentage of non-germinated spores.SG: number of germinated spores.SNG: number of non-germinated spores.2.6.Fusarium spp. growth.The minimal fungicidal concentration (MFC) of the tested products was assessed using the standard broth microdilution method, according to the recommendations of the Clinical and Laboratory Standards Institute with RPMI 1640  . The sta2.7.in vitro to determine the minimum concentration and exposure time required for the tested compounds to kill the fungi. We assessed the activity of serially diluted compounds against F. oxysporum as described by 5 colony-forming units (CFU)/mL) for each strain was transferred to 96-well microtiter plates . The plates were incubated for 120 h at 25°C. Then, 50 μL of the tested compounds at different concentrations were added for 5 min, 30 min, 1 h, 3 h, 6 h, and 24 h. Untreated probes were used as negative controls. After exposure, the fungal suspension was centrifuged at 4000 × g for 15 min and washed with deionized water. Centrifugation and washing were repeated twice. The probes were diluted in 11 mL phosphate-buffered saline, and the total CFU number was determined through serial dilution and plating on PDA. All assays included at least two replicates and were repeated in three independent experiments.We performed a time-kill test 2.8.F. oxysporum (107 spores/mL). One hundred microliters of this suspension were placed on control and experimental plates supplemented with M451 at various concentrations. The plates were incubated for 10 h at room temperature. Germ tube length was measured for 100 germinated spores at 400× magnification ) using Fiji software (The germ tube elongation was performed following a previously reported procedure . To testsoftware .2.9.F. oxysporum conidia suspension (106 conidia/mL) to a final concentration of 5,000, 500, 100, 50, 5, 1, or 0.5 μg/mL. After 1, 3, 6, and 24 h of incubation at 25°C, the number of germinated conidia was determined using the Malassez counting chamber in 10 fields with standard techniques . Data are presented as the mean ± standard deviation (SD) with three independent replicates. All experiments were performed in triplicate.All data were analyzed using GraphPad Prism version 9.3.1. Two-way analysis of variance (ANOVA) was used for multiple comparisons at a 95% confidence level , against different species from the Ascomycota, Oomycota, and Basidiomycota phyla. TGV-28 was highly effective against all strains, with the MFC 64.3–96.3 μg/mL . Surprisingly, the TGV-28 and NaH2PO4 combination increased the antifungal activity of TGV-28 against F. oxysporum by 49% (p < 0.05). Therefore, in subsequent experiments, we used a complex of TGV-28 and NaH2PO4 named M451.We tested TGV-28 and NaH.3 μg/mL . As expe3.2.in vitro mycelial growth of all Ascomycota, Oomycota, and Basidiomycota strains (50 of 34–145 μg/mL and a high correlation coefficient. The EC50 of M451 against Fusarium spp. (phylum Ascomycota) varied from 66 μg/mL to 145 μg/mL. The activity against other non-Fusarium representatives of the Ascomycota phylum was higher, with EC50 values ranging from 34 to 52 μg/mL. The EC50 values for Phytophtora infestans (phylum Oomycota) and Rhizoctonia solani (phylum Basidiomycota) were 58 and 53 g/mL, respectively.M451 exhibited significant activity against all tested plant-pathogenic fungal isolates and inhibited the  strains , with anp < 0.05 for the control, M451 0.1%, and M451 0.05%). Representative images of the mycelial growth inhibition of F. oxysporum by M451 are shown in We then compared the antifungal activity of M451 and fludioxonil against the same fungal isolates. M451 revealed dose-dependent activity: 0.1% M451 displayed 91–97% mycelial growth inhibition, and 0.05% M451 resulted in a slightly lower decrease in mycelial growth with 82–86.7% inhibition among different fungal pathogens . Notably3.3.F. oxysporum. The time-kill study against planktonic fungi demonstrated a higher antifungal activity of M451 at all tested time points. At the end of 24 h of exposure, the antifungal activity of M451 was 5–50 times higher than that of other antifungal agents. Notably, M451 was the only tested antifungal agent that showed antifungal activity from 5 min of exposure.3.4.F. oxysporum conidial germination  are a subject for future research and are outside the scope of this study. M451 exhibited significant antifungal activity against Fusarium spp. as a seed dressing for wheat seeds, which exceeded the commercially used fungicide Maxim XL against both seed- and soil-borne F. oxysporum infections  can be found at: VT, KKa, and GT conceived and supervised the research and edited and helped to draft the final manuscript. KKa and MV conducted experiments. KKa and KKr analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


H0), molecular weight (MW) distribution, antioxidant activity, angiotensin‐converting enzyme (ACE), α‐amylase, and α‐glucosidase inhibitory activities, and functional characteristics of hydrolysates were determined. Hydrolysis changed the amount of hydrophilic and hydrophobic amino acid composition and significantly increased the H0 values of hydrolysates, especially for CPH. The DPPH radical scavenging activity (RSA) was higher for CPH, SPH30:CPH70, and SPH50:CPH50 than SPH and SPH70:CPH30. Moreover, SPH, SPH70:CPH30, and SPH50:CPH50 showed lower MW than CPH, and this correlated with the higher hydrophilicity, and ABTS and hydroxyl RSA values obtained for SPH and the mixed hydrolysates with predominantly SPH. SPH70:CPH30 exhibited higher ACE, α‐glucosidase, and α‐amylase inhibitory activities among all samples due to its specific peptides with high capacity to interact with amino acid residues located at the enzyme active site and also low binding energy. At 15% degree of hydrolysis, both SPH and CPH showed enhanced solubility at pH 4.0, 7.0 and 9.0, emulsifying activity, and foaming capacity. Taken together, SPH70:CPH30 displayed strong antioxidant, antihypertensive, and antidiabetic attributes, emulsifying activity and stability indexes, and foaming capacity and foaming stability, making it a promising multifunctional ingredient for the development of functional food products.New mixed Alcalase‐hydrolysates were developed using corn gluten meal (CP) and soy protein (SP) hydrolysates, namely CPH, SPH, SPH30:CPH70, SPH70:CPH30, and SPH50:CPH50. Amino acid profile, surface hydrophobicity  and soy protein (SP) hydrolysates, namely CPH, SPH, SPH30:CPH70, SPH70:CPH30, and SPH50:CPH50. Amino acid profile, surface hydrophobicity (H0), molecular weight (MW) distribution, antioxidant activity, angiotensin‐converting enzyme (ACE) and antidiabetic activities, and functional characteristics of hydrolysates were determined. For CPH, protein or peptide bands with MW of 30–40, 50–55, 70–80, and 150–170 kDa were observed. For SPH, most of the bands at 70–200 kDa assigned to SP disappeared, and new bands were formed at 10–15, 15–20, 30, 40–50, and 50–60 kDa. SPH70:CPH30 revealed higher ACE, a‐glucosidase, and a‐amylase inhibitory activities among all samples. At 15% degree of hydrolysis, both SPH and CPH showed enhanced solubility at pH 3.0, 7.0, and 9.0, emulsifying activity, and foaming capacity. Taken together, SPH70:CPH30 with high antioxidant, antihypertensive, and antidiabetic attributes. These results have important implications for the designing of mixed protein hydrolysate as by‐product used for the development of novel antidiabetic, antihypertensive, and antioxidant additives for food industrial application. Soluble starch ACS reagent was purchased from Merck.Soy protein isolate  and CGM (~62% protein) were supplied by Shansong Industrial Chinese Co. Ltd. and a grain processing refinery, Golshahd Co. Ltd., respectively. Alcalase 2.4 L from 2.2g for 10 min and the obtained supernatants (rich in soluble peptides) were collected and adjusted to pH 7.0 using 1 M HCl, and spray‐dried (DORSA tech) through a drying air of 180°C (the exhausting temperature of 75–80°C) and an air flow of 0.3–0.4 MPa  and CGM (~62% protein) were prepared at 5% w/w. Soy protein isolate solution was heated at 90°C for 15 min to unfold the protein while the CGM solution was heated at 100°C for 30 min due to its low water solubility, thus the intense thermal treatment for denaturation  of NaOH needed to maintain the pH constant; Nb is the normality of the consumed base; Mp is the mass of protein in CP and SP; htot is the total number of peptide bonds in the protein substrates (considered 9.2 for CP and 7.75 for SP)  at three proportions, including 30:70, 50:50, and 70:30 (% w/w), which were referred to as SPH30:CPH70, SPH70:CPH30, and SPH50:CPH50, respectively. Unhydrolyzed proteins (CP and SP) were used as control in all analyses.2.5The amino acid profiles were determined using reversed‐phase high‐performance liquid chromatography , as described by Liu et al. . First, 2.6H0 using 1‐anilinonaphthalene‐8‐sulfonic (ANS) according to the method of He et al. (H0).The protein and hydrolysate samples were investigated for the e et al. . The sam2.7SDS‐polyacrylamide gel electrophoresis was used to estimate the MW profile of CP, SP, and their hydrolysates following the method of Rezvankhah et al. . Briefly2.8g for 10 min, and the supernatant was filtered through a 0.22 μm membrane filter. The injection volume of sample was 50 μl, and the column was eluted with 20 mM phosphate buffer containing 0.15 M NaCl (pH 7) at a flow rate of 0.5 ml/min. The spectra were monitored at a wavelength of 210 nm. Standard compounds of known MW including reduced glutathione (300 Da), glutathione disulfide (600 Da), cyanocobalamin (1355 Da), aprotinin (6500 Da), and cytochrome C  were used to prepare a standard curve, which was used to determine the MW.Gel permeation chromatography (GPC) was applied to determine MW distribution of the CP, SP, and hydrolysate samples following the method of Rezvankhah et al. . The Wat2.92.9.1AC), sample (AS), and blank (AB).Antioxidant activity was determined according to the method of Zheng et al. . 2,2 Dip2.9.2·+ scavenging activity of hydrolysates was evaluated according to the protocol reported by Amini Sarteshnizi et al. , and 2 ml of H2O2 (6 mM) were thoroughly mixed and kept for 10 min at room temperature. Then, 2 ml of salicylic acid (6 mM) was added and incubated for 30 min. The absorbance was then read at 510 nm (AS). Distilled water was used as the blank  and the control (instead of sample solution). Ascorbic acid (0.01 mg/ml) was used as a positive control. The RSA was calculated using equation The hydroxyl RSA was evaluated using the protocol reported by Zheng et al. . Two mil2.10AC), sample (AS), and blank (AB). Also, the IC50 value, the concentration of sample that inhibited 50% of ACE activity, was determined using sample concentrations of 0.1–2 mg/ml.The potential antihypertensive activity of CP, SP, and hydrolysates was assessed by the in vitro inhibition of angiotensin I‐converting enzyme (ACE) based on the method of Boye et al. . ACE inh2.112.11.1AC) and sample (AS). Sample concentrations of 10–500 μg/ml were used to determine the IC50 values.The inhibitory activity of CP, SP, and their hydrolysates against rat intestinal α‐glucosidase was assessed following the method of Karimi et al. . Briefly2.11.2g for 2 min. Then, 20 μl of the supernatant was mixed with 1 ml of PAHBAH and the solution was heated to 70°C for 10 min. The sample solutions were cooled, and absorbance values were read at 410 nm. The inhibitory activity was determined using the following equation:AS), blank , and control . Furthermore, IC50 values were determined as previously described. Acarbose, at its IC50 value (0.125 mg/ml), was used as a positive control.The α‐amylase inhibitory activity of CP, SP, and hydrolysates was determined using the method reported by Rahimi et al. . Briefly2.122.12.1g for 20 min. The supernatants were taken for protein determination based on the Bradford protocol  and emulsifying stability index  were assessed by the method of Rezvankhah et al. , I is the path length of the cuvette (m), θ is the oil volume fraction (0.25), C is the protein concentration in the aqueous phase (g/m3), ∆A=A0−A10, and ∆t=10min.Two emulsifying properties including emulsifying activity index  and foaming stability (FS) of the CP, SP, and hydrolysates were assessed following the procedure reported by Rezvankhah, Emam‐Djomeh, et al.  and Rezv2.13The experimental data were reported as means ± standard deviation. One‐way analysis of variance (ANOVA) was used to analyze the obtained data. The Duncan test was applied to evaluate the comparison of mean difference using the SPSS software .33.1Amino acid composition of the hydrolysates is presented in Table The variation in amino acid profiles could be related to the unfolding of the protein structures and exposure of the hydrophobic segments during enzymatic hydrolysis  has an important effect on the macromolecular structural stability, surface, and biological properties of proteins , followed by SPH70:CPH30 (33.50%), CP (19.30%), SPH (12.40%), and SP (9.30%). Ascorbic acid, however, exhibited higher DPPH RSA at a much lower concentration than the hydrolysates. CP and its hydrolysates had high content of hydrophobic amino acids, which may be related to their higher reactivity with DPPH radicals , CP 9.30%, SP50%), CP 9.30%, SPA previous study on chickpea protein hydrolysates obtained with Alcalase showed that a content of over 50% hydrophobic amino acids resulted in high DPPH RSA  > SPH70:CPH30 (77.40%) > SPH50:CPH50 (75.20%) > SPH30:CPH70 (73.40%) > CPH (70.60%) > ascorbic acid (54.20%) (as positive control) > SP (44.60%) > CP (40.50%), respectively. Taken together, the results suggest that SPH and CPH combined hydrolysate SPH70:CPH30 possessed the best antioxidant activities and, thus, have the potential to protect food or biological systems against oxidative damages. Zhang et al.  reported3.650 values of 0.5, 0.25, 0.38, 0.15, 0.23, 0.18, 0.21 mg/ml were obtained for CP, SP, CPH, SPH, SPH30:CPH70, SPH70:CPH30, and SPH50:CPH50, respectively. It was observed that hydrolysis of SP increased the ACE inhibitory activity as previously reported  led to mixed hydrolysates with higher α‐glucosidase inhibitory activity than CPH. Acarbose, a synthetic compound, at the same concentration exhibited much higher α‐glucosidase inhibitory activity than the crude hydrolysates. However, it has several side effects  of CP and SP were 12.43 and 41.82 mFor foaming properties, CP showed FC (0%) while SP had a FC of 81.25%. Hydrolysis significantly increased the FC, which reached 25% and 102.5% for CPH and SPH, respectively. The improvement of FC could be related to an increase in solubility of the hydrolysates. SPH30:CPH70, SPH70:CPH30, and SPH50:CPH50 had FC values of 32.50%, 40.62%, and 40%, respectively. Limited hydrolysis not only enhances protein solubility but also increases hydrophobic interactions, thus increasing FC .None of the authors declare a conflict of interest.This study does not involve any human or animal testing.