Ticks analysis for molecular detection and phylogenetic evaluation of stray dogs infecting protozoa from Alborz, Iran.

Ticks play an important role in the transmission of parasitic diseases, especially pathogenic protozoa in canine hosts, and it is very important to determine the role and extent of their infection with these pathogens in order to determine important control strategies. This study assessed the molecular prevalence of three protozoan pathogens including Hepatozoon canis, Leishmania spp. and Babesia spp., in ticks using PCR. A total 300 stray dogs were investigated and 691 ticks (171 male, 377 female and 143 nymph) were detected directly from 45 infested dogs. Species, stage of growth, and gender were determined for each tick. DNA extracted from 224 ticks (26 male, 165 female and 33 nymph). The molecular presence of three protozoan pathogens including Hepatozoon spp. (18S rRNA gene), Leishmania infantum (kinetoplastid minicircle DNA) and Babesia spp. (ssrRNA gene) were investigated using PCR method. One species of ticks, Rhipicephalus sanguineus was identified. Two of the target pathogens, Hepatozoon spp. (7/83; 8.43 %) and Babesia spp. (1/83; 1.2 %), were detected by PCR method. Sequence analysis of the ssrRNA gene of detected Babesia spp. showed a close relationship to the deposited strains of Babesia vulpis in the gene bank. To the best of our knowledge, this is the first study to undertake a phylogenetic analysis of H. canis and Babesia spp. in stray dogs in Alborz province, Iran and the first report about molecular detection of Babesia vulpis from tick infesting dogs in Iran. According to the above results, it seems necessary to implement tick control programs in dogs.

Detection, Identification, and Characterization of Eimeria spp. from Commercial Chicken Farms in Different Parts of Iran by Morphometrical and Molecular Techniques.

PURPOSE: Coccidiosis of domestic chicken is an important disease caused by any of seven species of Eimeria which, by developing within the epithelial cells of the intestine, cause lesions therein. We carried out a study on poultry farms located in various regions of Iran to determine the incidence and spread of Eimeria species by employing a single PCR test. METHODS: A total of 64 fully confirmed clinically intestinal tracts were collected from different parts of Iran. From these 64 intestinal tracts, 82 samples were prepared from the different sites involved in the digestive tract. In morphological assessment, 23 samples could not be isolated and its information was not evaluated. RESULTS: Using morphological methods, the following seven species of Eimeria were identified: E. acervulina (15/59; 25.42%), E. tenella (30/59; 50.84%), E. maxima (12/59; 20.33%), E. praecox (1/59; 1.69%), E. necatrix (2/59; 3.38%), E. mitis (5/59; 8.47%), and E. mivati (2/59; 3.38%). Mixed infections were found in eight (13.55%) samples. In molecular assessment, 31 samples could not be isolated and its information was not evaluated. Totally, the following five species were identified using molecular methods: E. acervulina (35/51; 68.62%), E. tenella (33/51; 64.70%), E. maxima (6/51; 11.76%), E. brunetti (5/51; 9.80%), and E. necatrix (2/51; 3.92%). Mixed infections were found in 23 (45.09%) samples. CONCLUSIONS: The present study is an update on the situation of poultry coccidiosis in Iran and provides the first data on the molecular detection, identification, and characterization of Eimeria spp. in the poultry population of this country and confirmed the presence of different species of this parasite in this area. According to the results, E. acervulina and E. tenella, as the main disease-causing species, should be considered in control programs such as treatment and vaccination strategies.

Molecular detection and phylogenic characterization of Neospora caninum in naturally infected sheep in Alborz and Qazvin provinces, the north of the central region of Iran.

Neospora caninum is a protozoan coccidian parasite that can act as a cause of abortion in sheep. The aim of this study was to investigate the presence of this parasitic agent and its role in causing abortion in sheep of Iran. Between June 2019 and February 2022, 100 samples [brain (n = 39), placenta (n = 8), embryonic membrane (n = 7), cotyledon (n = 7), umbilical cord (n = 2), homogenate mixture of tissues (heart, liver, spleen and digestive track) (n = 37)] that were collected following the necropsies of 39 aborted ovine fetuses from different parts of the Alborz and Qazvin provinces, the north of the central region of Iran were employed for DNA extraction. Nc-5 was selected as the target gene sequence for amplification of DNA by using four pairs of primers in two semi-nested PCR. Samples considered positive for the presence of the NC-5 gene were examined to further confirm the presence of the ITS1 gene. Sequence of NC-5 gene was detected from the 27 tissue samples of 23 aborted ovine fetuses. The ITS1 gene sequence was detected in all of the 27 tissue samples that were positive for the NC-5 gene analysis. Brain tissue was the most studied tissue, and the highest number of positive cases was observed in this tissue. The present study updated the situation of ovine neosporosis in the central region of Iran and confirmed the presence of the N. caninum among sheep flocks' abortion.

Impact of Iran's Forest Nationalization Law on Forest Cover Changes over Six Decades: A Case Study of a Zagros Sparse Coppice Oak Forest.

Forest nationalization policies in developing countries have often led to a reduction in local forest ownership rights and short- or long-term exploitative behaviors of stakeholders. The purpose of this research is to quantify the effect of Iran's Forest Nationalization Law (FNL) in a part of Zagros Forest over a 68-year time period (1955-2022) using 1955 historical aerial photos, 1968 Corona spy satellite photography, and classification of multi-temporal Landsat satellite images. A past classification change detection technique was used to identify the extent and the pattern of land use changes in time. For this purpose, six periods were defined, to cover the time before and after the implementation of FNL. A 0.27% deforestation trend was identified over the period after the FNL. Dense and open forested area has decreased from 7175.62 ha and 68,927.46 ha in 1955 to 5664.26 ha and 59,223.38 ha in 2022. The FNL brought decisive changes in the legal and forest management systems at the state level, mainly by giving their ownership to the state. Accordingly, the FNL and the related conservation plans have not fully succeeded in protecting, rehabilitating, recovering, and developing the sparse Zagros Forest ecosystems, as their most important goals.

Modeling wildfire risk in western Iran based on the integration of AHP and GIS.

This study aimed at delineating the wildfire risk zones in a fire-prone region located in a rarely addressed area of western Iran (Paveh city) by assessing the potential of factors such as NDVI, topographic factors (elevation, slope, and aspect), land cover, and evaporation in explaining the fire occurrence probability. Analytic hierarchy process (AHP) and geographical information system (GIS) methods were used synergistically to integrate the mentioned factors into analysis, following an informed categorization of each factor based on the information on previous fire occurrence. In the AHP process, elevation and evaporation data were considered to be the most critical factors. It was found that the predicted wildfire risk areas were in agreement with past fire events by the use of the methodology proposed by this study. Accordingly, the study's final wildfire risk map indicated that approximately 64.7% of the study area is located in the high- and very high-risk zones. Land-use planners and decision-makers may use the developed map to setup and implement fire prevention strategies and enhance or develop the fire-surveillance logistics and infrastructure, including but not limited to the positions of fire watchtowers, fire lines, and fire sensors, with the aim to minimize potential fire impacts.

Morphological detection and molecular characterization of Hepatozoon spp. from venomous terrestrial snakes in Iran.

Hepatozoon spp. are blood protozoans that can infect snakes. Infections with these parasites are found in more than 200 snake species and are considered to be the most frequent hemogregarines in snakes. In this study, a total of 73 dead snakes in captivity conditions, belonging to six different species, which were collected between June 2018 to October 2020 from different parts of Iran, were examined for the presence of blood parasites with microscopy and molecular methods. DNA was extracted from the removed heart, and PCR was done with two pairs of primers targeting the 18S rRNA gene. By microscopy, Hepatozoon spp. were detected in the heart blood of 29 out of the 73 (39.72%) snakes. From these 29 positive samples, eight were found to be positive using the PCR method. This study aimed to report for the first time the molecular characteristics of Hepatozoon spp. infecting venomous terrestrial snakes in Iran.

Synthesis of Tellurium Oxide (TeO2) Nanorods and Nanoflakes and Evaluation of Its Efficacy Against Leishmania major In Vitro and In Vivo.

PURPOSE: Today, the use of natural products and nanostructures has increased. Given the reports on beneficial effects of various organotellurane compounds on types of visceral leishmaniasis, we decided to investigate the effect of TeO2 NPs on Leishmania major (L. major). Tellurium can cause cell apoptosis in cancer cells without activating the caspase-pathway. METHODS: TeO2 NPs at first synthesized and the structure was checked by XRD, SEM and EDS tests. The cytotoxic effect of TeO2 NPs against L. major promastigotes, amastigotes and macrophages was assessed by MTT test or counting. The possible apoptosis of L. major by TeO2 NPs was evaluated by flow cytometry test. For in vivo assay, the lesions of infected BALB/c mice with L. major promastigotes were treated with TeO2 NPs, then the lesion size and survival rate were evaluated. RESULTS: The synthesis of TeO2 with tetragonal structure was confirmed by XRD. The combination of nanorods and nanoflakes and the presence of Te were proven by SEM and EDS, respectively. According the effects of nanoparticle on promastigotes and amastigotes, the IC50 values of TeO2 after 72 h of incubation were 15.13 and 52.22 µg/ml, respectively. TeO2 NPs induced apoptosis in about 41% of promastigotes. The ulcer greatly healed and survival rate was higher in treated mice compared to those in control group. CONCLUSION: Based on the data, favorable anti-leishmanial properties were observed by using TeO2 NPs. TeO2 NPs have cytotoxic impacts on L. major promastigotes and amastigotes in vitro and in vivo and may be regarded as a therapy option.

Investigating in Vivo and in Vitro Effects of Ethanolic and Aqueous Extracts of Myrtle (Myrtus communis) on Leishmania major.

BACKGROUND: The extract of myrtle plant contains polyphenolic compounds that show antibacterial, antiviral, and anti-parasitic properties. We aimed to investigate the therapeutic effect of aqueous and ethanolic myrtle extract against leishmaniasis caused by L. major in vivo and in vitro conditions. METHODS: This study was carried out in Tarbiat Modares University, Tehran, Iran in 2018. Aqueous and ethanolic extract of myrtle plant at 6.25 to 400 mg/ml concentrations were tested on Leishmania major promastigotes, non-infected macrophages, and macrophages infected with amastigotes in vitro using counting, MTT and flow cytometry techniques. Then, BALB/c mice were treated with ethanolic, aqueous and a mixture of both extracts of myrtle plant. The treatment was carried out for four weeks. Then, the effectiveness of the herbal medicine was assessed by measuring wounds diameters, mice weights and their mortality rate on weekly basis. RESULTS: The IC50 values of aqueous and ethanolic extracts for promastigotes were 7.86 and 11.66 µg/mL respectively. The IC50 values of the aqueous and ethanolic extracts for amastigotes were 12.5 and 47.2 µg/mL respectively. Flow cytometry indicates 62.88% and 60.16% apoptosis induced by ethanolic and aqueous extract of myrtle plant respectively. The lowest parasitic load was seen in the group treated with ethanolic extract. CONCLUSION: The lesion sizes for treated groups with extracts were similar to those treated with glucantime. Oral administration instead of injection is another advantage of myrtle plant over glucantime, which makes the herb easy and more practical.

Design, Construction and Immunogenicity Assessment of pEGFP-N1-KMP11-GP96 (Fusion) as a DNA Vaccine Candidate against Leishmania major Infection in BALB/c Mice.

BACKGROUND: KMP-11 (Kinetoplastid membrane protein-Π) exists in all species of kinetoplastid family. It is fully conserved and the protein produced by this gene can induce a very high cellular immune response. We aimed to design a suitable construction for a Leishmania major DNA vaccine and evaluate the protective efficacy of it as a candidate for DNA vaccine against cutaneous leishmaniasis in BALB/c mice. METHODS: This experimental study was conducted in Tehran City, Iran, between April 20, 2015 and May 30, 2016. KMP-11 gene of L. major (MRHO/IR/75/ER, Iranian strain) and NT-GP96 of Xenopus GP96 DNA from a pBluescript-GP96 plasmid were amplified by PCR and the purified PCR products were cloned into the pJET1.2/blunt plasmid vector, then, subcloned into pEGFP-N1 plasmid as an expression vector. Finally, the KMP-11 gene was fused with GP96 and afterward the combination cloned in pEGFP-N1. All the cloned genes confirmed by enzyme digestions. Then, four groups of mice were immunized with PBS, pEGFP-N1, pEGFP-N1-KMP, and pEGFP-N1-fusion. Four weeks after immunization, all animals were challenged with L. major virulent promastigotes. RESULTS: The constructed fusion potentially showed an ability to elicit Th1 responses that led to cutaneous lesion healing. Interestingly, the group received KMP11-GP96 -GFP showed the highest ratio of IFN- γ /IL-4 and IgG2a/IgG1 compare to other groups. No side effect was observed after using the fusion in the mice. CONCLUSION: The constructed fusion could well stimulate both the cellular and humoral immune systems that led to cutaneous lesion healing in mice.

Evaluating the preventive and curative effects of Toxocara canis larva in Freund's complete adjuvant-induced arthritis.

Helminthic infection and the parallel host immune reactions are the results of a protracted dynamic co-interaction between the host and worms. An assessment of the effect of Toxocara canis infection on arthritis in rats stimulated by Freund's complete adjuvant (FCA) was the main purpose of the investigation. An arthritis model was established by the administration of 0.1 mL FCA in the palmar surface. Cytokine assessment, evaluating oedema and the use of a rheumatoid arthritis (RA) score provided evidence of the protective effects of T canis against adjuvant-induced arthritis (AIA). The cytokines TGF-ß, IFN-É£, IL-10 and IL-17 were measured to assess the anti-inflammatory effect of T canis infection. Besides, arthritis swelling findings were evaluated in rat paws. The data showed that T canis infection significantly modulated the immune response by alleviating inflammatory cytokines and increasing TGF-ß as an anti-inflammatory cytokine. Evaluations of arthritis swelling showed low severity and faster recuperation. These findings suggest that the products derived from T canis eggs might be a potential therapeutic candidate to treat autoimmune diseases like the arthritis.

LB broth-lyophilized Rabbit serum (LLR) as a new and suitable culture medium for cultivation of promastigotes of Leishmania major.

Fetal calf serum is the major part and the most expensive ingredient of the Leishmania culture media. Here, the efficacy of the LB broth-lyophilized Rabbit serum medium (LLR) was evaluated in cultivation of Leishmaniamajor. Conventional Luria-Bertani (LB) broth medium was prepared and autoclaved for 15 min at 121 °C and then lyophilized Rabbit serum was added at the 1, 2.5, 5 and 10 % final concentrations. The efficacy of medium was evaluated by assessing the growth ability and replication pattern of the promastigotes of L. major. According to our finding, the LLR medium with 5-10 % lyophilized Rabbit serum supported the growth of the parasites and can be used for cultivation of Leishmanian parasites with acceptable In vivo infectivity for research purpose. The ability of the parasites to survive and proliferating in the presence of lyophilized Rabbit serum indicating that this serum is a good nutritional source. This study opens a new way to make low-cost medium that could be used in cultivation of Leishmanian parasites.

Molecular detection of Toxoplasma gondii in snakes.

Toxoplasma gondii, an obligate intracellular protozoan parasite, is responsible for one of the most common zoonotic parasitic diseases in almost all warm-blooded vertebrates worldwide, and it is estimated that about one-third of the world human population is chronically infected with this parasite. Little is known about the circulation of T. gondii in snakes and this study for the first time aimed to evaluate the infection rates of snakes by this parasite by PCR methods. The brain of 68 Snakes, that were collected between May 2012 and September 2015 and died after the hold in captivity, under which they were kept for taking poisons, were examined for the presence of this parasite. DNA was extracted and Nested-PCR method was carried out with two of pairs of primers to detect the 344 bp fragment of T. gondii GRA6 gene. Five positive nested-PCR products were directly sequenced in the forward and reverse directions by Sequetech Company (Mountain View, CA). T. gondii GRA6 gene were detected from 55 (80.88%) of 68 snakes brains. Sequencing of the GRA6 gene revealed 98-100% of similarity with T. gondii sequences deposited in GenBank. To our knowledge, this is the first study of molecular detection of T. gondii in snakes and our findings show a higher frequency of this organism among them.

Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum.

BACKGROUND: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice. METHODS: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge. RESULTS: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected. CONCLUSION: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development.

A description of parasites from Iranian snakes.

Little is known of the parasitic fauna of terrestrial snakes in Iran. This study aimed to evaluate the parasitic infection rates of snakes in Iran. A total of 87 snakes belonging to eight different species, that were collected between May 2012 and September 2012 and died after the hold in captivity, under which they were kept for taking poisons, were examined for the presence of gastrointestinal and blood parasites. According to our study 12 different genera of endoparasites in 64 (73.56%) of 87 examined snakes were determined. Forty one snakes (47.12%) had gastrointestinal parasites. In prepared blood smears, it was found that in 23 (26.43%) of 87 examined snakes there are at least one hemoparasite. To our knowledge, these are the first data on the internal parasitic fauna of Iranian terrestrial snakes and our findings show a higher prevalence of these organisms among them.

Effects of sheep and mouse urine on the growth pattern of Leishmania major promastigotes.

The protozoan parasites of the genus Leishmania are the causative agents of different clinical diseases. Fetal calf serum (FCS) is the main part and the most expensive ingredient of the Leishmania culture media. Here, the efficacies of different concentrations (1%, 2.5%, 5%, and 10%) of the filtered and autoclaved sheep and mouse urine were evaluated as a growth stimulator in Leishmania culture procedure. The results indicated that culture media enriched with the filtered sheep and mouse urine supported the growth of the parasites and can be used for cultivation of Leishmania parasites. In conclusion, this study has demonstrated an alternative low-cost medium that could be used in cultivation process of Leishmania major promastigotes.

Sheep blood-LB agar base medium (SLM) as a simple and suitable medium for the cultivation of Leishmania major promastigotes.

Several methods of cultivation of Leishmania parasites are available, and among them, biphasic media like Nicolle-Novy-MacNeal medium are considered to be superior for the initial isolation of the parasites and their in vivo infectivity. In the present work, the efficacy of sheep blood-LB agar base medium (SLM) was evaluated for the cultivation of Leishmania major promastigotes. The SLM supports the proliferation of the parasites and can be used for routine isolation and cultivation of Leishmania parasites with acceptable in vivo infectivity for research purposes.

Intestinal parasitic infections among inhabitants of Karaj City, Tehran province, Iran in 2006-2008.

Karaj is an area with large influx of refugee people in Iran. To increase knowledge about parasitic infections, we carried out this research during 2006-2008. We recorded the stool examination results and some of their personal characteristics. A total of 13,915 human stools were examined, and 649 (4.7%) were positive for intestinal parasites. Among them, 13 (0.09%) had worm and 636 (4.6%) had protozoan infections. Maximum infections belonged to Giardia intestinalis, and 534 (3.8%) samples had this infection. Other parasitic infections included Entamoeba coli (0.39%), Entamoeba histolytica (0.021%), Blastocystis hominis (0.08%), Trichomonas hominis (0.1%), Iodamoeba butschlii (0.06%), Chilomastix mesnili (0.007%), Endolimax nana (0.05%), Enterobius spp. eggs (0.028%), Taenia proglottids (0.028%), and Strongyloides stercoralis larvae (0.03%). The maximum numbers of referred people to laboratories were in July and the maximum percentage of infections was in August. There is a point that all 5 Strongyloides stercoralis infections were pertained to 2008. With attention to the rate of parasitic infections (4.7%), it seems that we should take additional educational information to wide spectrum of people living in this city.