Assessment of tissue levels of miR-146a and proinflammatory cytokines in experimental cerebral toxoplasmosis following atovaquone and clindamycin treatment: An in vivo study.

BACKGROUND: Despite recent advances for treating cerebral toxoplasmosis (CT), monitoring the parasite burden and treatment response is still challenging. miRNAs are small non-coding RNAs with regulatory functions that can be used in diagnosis and treatment monitoring. We investigated the changes in miR-146a, BAG-1 gene, IL-6, and IL-10 tissue levels in the brain of BALB/c mice with chronic CT caused by the PRU strain of T. gondii following anti-parasitic and antibiotic treatment. METHOD: Fifty-three 6-to 8-week-old BALB/c mice were infected using intraperitoneal inoculation of cerebral cysts of T. gondii PRU strain and then divided into five groups as follows: group 1 included mice treated with 100 mg/kg/d Atovaquone (AT), group 2 included mice treated with 400 mg/kg/d clindamycin (CL), group 3 included mice treated with combination therapy (AT + CL), group 4 included infected untreated mice as a positive control (PC), and; group 5 included uninfected untreated mice as negative control (NC). After the completion of the treatment course, tissue level of mir-146a, miR-155, BAG-1 gene, IL-6, and IL-10 was investigated with real-time polymerase chain reaction. The IL-6/IL-10 ratio was calculated as an indicator of immune response. Moreover, brain cyst numbers were counted on autopsy samples. RESULTS: miR-146a, IL-6, IL-10, and BAG-1 genes were expressed in PC, but not in the NC group; miR-146a, IL-6, IL-10, and BAG-1 gene expression were significantly lower in AT, CL, and AT + CL compared with PC. MiR-146a and BAG-1 levels in AT and CL were not different statistically, however, they both had lower levels compared to AT + CL (P < 0.01). There was no difference in the expression of IL-6 and IL-10 between treatment groups. BAG-1 expression was significantly lower in AT, than in CL and AT + CL (P < 0.0089 and < 0.002, respectively). The PC group showed a higher ratio of IL-6/IL-10, although this increase was not statistically significant. It is noteworthy that the treatment with AT reduced this ratio; in the inter-group comparison, this ratio showed a decrease in the AT and AT + CL compared to the PC. The number of brain tissue cysts was significantly lower in AT, CL, and AT + CL, than in PC (p < 0.0001). AT had significantly lower brain cysts than CL and AT + CL (P < 0.0001). CONCLUSION: It seems that the factors studied in the current research (microRNA and cytokines) are a suitable index for evaluating the response to antiparasitic and antibiotic treatment. However, more studies should be conducted in the future to confirm our findings.

Occurrence and genetic characterization of Echinococcus granulosus sensu lato from domestic animals in Central Iran.

BACKGROUND: The species complex of Echinococcus granulosus sensu lato (s.l.) causes cystic echinococcosis distributed worldwide. There is no genotype information from hydatid cysts in the intermediate hosts in Central Iran. Therefore, in this study, we analyzed the hydatid cysts in livestock slaughtered in an abattoir in this region. Six hundred fifty-seven hydatid cysts were isolated from 97 animals, including sheep, cattle, camels, and goats slaughtered in Yazd abattoir from September 2018 to January 2020. The demographic data was collected as well as cyst location, fertility, and viability. Out of 657 samples, 164 samples were genotyped. Then, phylogenetic analysis was performed using MEGAX. Statistical analyses were done using SPSS version 16.0 by chi-square with a significant difference of less than 0.05. RESULTS: Out of 164 samples, the G1-G3 complex genotype had the most frequency in samples, with 135 cases recognized. The G6/G7 was observed in 19 isolates and G5 was reported in nine samples. One sample was detected as Taenia hydatigena. CONCLUSIONS: This study showed that G1-G3 and G6/G7 genotypes were presented in all animals, but G5 was reported only in cattle, goats, and camels. It is the first molecular identification of cystic echinococcosis in Central Iran. Hence, reporting G5 in livestock in this area should be considered due to transmission to humans.

Protective immunity induced with a DNA vaccine encoding B- and T-cells multi-epitope SAG1, ROP16, MIC4, GRA12, M2AP, and multi-epitope ROP8 against acute and chronic toxoplasmosis in BALB/c mice.

BACKGROUND: T. gondii infection is characterized by a high global prevalence. Nearly, 16-40% of people have been infected by T. gondii. Although T. gondii often causes subclinical infection, it may cause severe complications in newborns with congenital infection and immunocompromised individuals. Constant attempts of scientists have made valuable findings in the development of T. gondii candidate vaccines. However, an effective vaccine has not been successfully developed yet. In this study, multi-epitope SAG1, MIC4, ROP16, M2AP, GRA12, and multi-epitope ROP8 were injected into BALB/c mice intramuscularly, as cocktailed plasmids or as single-gene plasmids to assess the immune response against chronic and acute Toxoplasma infection. METHODS: BALB/c mice were immunized on days 0, 21, and 42. The immune responses of both vaccinated and control groups were evaluated using cytokine and antibody measurements, lymphocyte proliferation assay, survival time, and average number of cysts in each brain. RESULTS: The results indicated that DNA vaccination using multi-epitope ROP8 and multi-epitope SAG1, ROP16, MIC4, GRA12, M2AP could elicit both cellular and humoral immune responses, and enhanced the survival time in BALB/c mice. Also, the administration of multi-epitope ROP8 plus multi-epitope SAG1, ROP16, MIC4, GRA12, M2AP could enhance the concentrations of IgG antibody, elicit a mixed IgG1/IgG2a reaction with the predominance of the IgG2a, increase the release of IFN-γ cytokine, prolonge the survival time, and reduce the brain cysts. CONCLUSIONS: Here, we report that vaccination using cocktailed plasmids could induce better protective immunity compared to single plasmid for acute and chronic T. gondii infection.

Spontaneous abortion among Toxoplasma gondii IgG seropositive women: Molecular detection, genotype identification, and serological assessment with conventional ELISA and avidity ELISA.

OBJECTIVES: It has been generally believed that women who exposed to Toxoplasma gondii before pregnancy and have anti-T. gondii IgG antibody are immunized and their newborns will be protected from congenital infection. This study is aimed to investigate the role of T. gondii infection in spontaneous abortion through serological and molecular methods in southern Iran. STUDY DESIGN: Blood samples were taken from 50 spontaneously aborted mothers and anti-T. gondii antibodies were assessed using conventional enzyme-linked immunosorbent assay (ELISA) and avidity ELISA methods. The placenta and blood samples of aborted women were used for detection of the parasite's DNA by polymerase chain reaction (PCR) method targeting the RE gene. The parasite genotypes were determined by PCR-restriction fragment length polymorphism (RFLP) method using SAG3 and GRA6 genes. RESULTS: IgG antibody was detected in 28% (14/50) of mothers, but all samples were negative for IgM antibody. In the avidity ELISA test, 26% (13/50) of the samples had a high avidity index, suggesting chronic infection, while a low avidity index was detected in one case (2%), which suggests acute infection. The parasite's DNA was detected in 18% (9/50) and 14% (7/50) of blood and placenta samples, respectively. All DNA positive samples were IgG positive. All isolates were belonged to the T. gondii type III genotype. CONCLUSION: The results suggest that T. gondii seropositive women are not protected from congenital transmission. However, the results should be interpreted cautiously until further studies will be confirmed these results.

Multi-epitope vaccine expressed in Leishmania tarentolae confers protective immunity to Toxoplasma gondii in BALB/c mice.

Current study deals with a novel multi-epitope vaccine designed in silico and its confirmation experiments for potential efficacy in BALB/c mice. Major histocompatibility complex (MHC)-binding and B-cell binding epitopes of five Toxoplasma antigens (SAG1, ROP16, GRA12, MIC4 and M2AP) were predicted. Selected epitopes were fused together using SAPGTP linker, and antigenicity, allergenicity, physico-chemical features, secondary and tertiary structures and validations were all performed via bioinformatics servers. Then, vaccine construct was cloned into pLEXSY-neo 2.1 vector. After Leishmania tarentolae transfection, live recombinant and wild parasites were subcutaneously injected into 6-8 week female BALB/c mice and immune responses were measured. Results showed that the peptide possessed 282 amino acid residues with average molecular weight of 28.06 kDa. About 90% of the peptide residues were incorporated in favored and allowed regions of the Ramachandran plot. Vaccinated mice showed remarkably elevated levels of specific antibodies (P < 0.05) with predominance of IgG2a production. Also, a Th1 immune response with production of IFN-γ and relatively increased survival rate against intraperitoneal challenge with RH strain was demonstrated in immunized mice than control groups (P < 0.05). Also, very low levels of IL-4 were demonstrated, which showed statistically significant association with controls (P < 0.05). The findings clarified that multi-epitope vaccine expressed in Leishmania tarentolae induced significant immune responses against acute toxoplasmosis.

Structural predication and antigenic analysis of ROP16 protein utilizing immunoinformatics methods in order to identification of a vaccine against Toxoplasma gondii: An in silico approach.

Toxoplasmosis, caused by Toxoplasma gondii, is a common parasitic disease, affecting almost one-third of the world's population. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the production of appropriate vaccines against this pathogen is an important goal to avoid toxoplasmosis. considering the role of rhoptry antigens like ROP16 in virulence and satisfactory immunogenicity, they can be used as promising vaccine candidates against T. gondii. In the present study, an in silico approach was used to analyze various aspects of the ROP16 protein, including physicochemical characteristics, the potential epitopes of B and T-cells, the secondary and tertiary structure, the subcellular localization, the transmembrane domain, and other important features of this protein using several bioinformatics tools to design a proper vaccine against T. gondii. The results showed that ROP16 protein includes 93 potential post-translational modification sites. The secondary structure of the ROP16 protein comprises 34.23% alpha-helix, 54.46% random coil, and 11.32% extended strand. Moreover, several potential B- and T-cell epitopes were identified for ROP16. Based on the results of Ramachandran plot, 84.64% of the amino acid residues were located in the favored, 10.34% in allowed, and 5.02% in outlier regions. Furthermore, the results of the antigenicity and allergenicity assessment noted that this protein was immunogenic and non-allergenic. Our findings suggested that structural and functional predictions applied to ROP16 protein using in silico tools can reduce the failure risk of the laboratory studies. This research provided an important basis for further studies and also developed an effective vaccine against acute and chronic toxoplasmosis by various strategies. Further studies are needed on the development of vaccines in vivo using ROP16 alone or in combination with other antigens in the future.

Computational probing of Toxoplasma gondii major surface antigen 1 (SAG1) for enhanced vaccine design against toxoplasmosis.

The SAG1 is a tachyzoite-specific protein critical for Toxoplasma gondii (T. gondii) adhesion to surface receptors of the host cells. In this study we've comprehensively excavated the sequence of SAG1 using online bioinformatics servers toward better vaccine design against toxoplasmosis. Web-based tools were used to assess the physico-chemical properties, post-translational modifications (PTMs), transmembrane domains, subcellular localization, secondary and 3D structures, as well as B-cell, Cytotoxic T cells (CTL) and major histocompatibility complex (MHC) epitopes. The 336 amino acid sequence possessed a molecular weight of 34829.02 D, aliphatic index of 80.15 and GRAVY score of 0.129. There was 47 PTM sites without any transmembrane domains. Also, the SAG1 protein was appointed to be immunogen and non-allergen. The secondary structure comprised 62.5% random coil, 26.79% extended strand and 10.71% alpha helix. Ramachandran plot of the refined model demonstrated 94.4% residues in the favored region, 4.8% in allowed region and 0.8% in outlier region. Additionally, various potential B-cell (linear and conformational), CTL and HTL epitopes were predicted for T. gondii SAG1. This in silico investigation would be a premise for appropriate immunization strategies against toxoplasmosis. More studies are anticipated to be done empirically using SAG1 immunoprotective epitopes combined with other antigenic compounds.

An unlabelled probe-based real time PCR and modified semi-nested PCR as molecular tools for analysis of chloroquine resistant Plasmodium vivax isolates from Afghanistan.

BACKGROUND: Plasmodium vivax resistance to chloroquine (CQ) has been reported from many endemic regions in the world. Plasmodium vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are possible markers for CQ-resistance in P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. The present work aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax. METHODS: Plasmodium vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. A new asymmetric qPCR and melting analysis assay based on unlabelled probe developed for scanning of K10 insertion in pvcrt-o gene. RESULTS: The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces. Of the 36 samples evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province, respectively, possessed K10 insertion, confirmed by either sequencing and unlabelled probes. CONCLUSION: Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance in P. vivax populations in Afghanistan. Furthermore, unlabelled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

Characterization of extracellular proteases of Acanthamoeba genotype T4 isolated from different sources in Iran.

The members of the genus Acanthamoeba are ubiquitous, free-living amoebae found in various environments. The amoebae can cause severe complications in both, immunocompetent and immunocompromised individuals. The aim of this study was to characterize extracellular proteases of Acanthamoeba isolates from different sources belonging to genotype T4 as well as the determination of the pathogenicity potential to correlate pathogenicity with protease activity and protease banding pattern. A total of 19 isolates (11 clinical and 8 environmental) were cultured axenically, then the pathogenicity of the isolates was assessed by osmo- and thermo- tolerance tests. An applied colorimetric method using azocasein as a substrate was used for the extracellular protease activity assay. Protease characterization was carried out by zymography analysis with and without protease inhibitors. Assessment of the pathogenicity potential using physical parameters revealed that 2 (25%), 2 (25%), and 4 (50%) of the environmental isolates were potential pathogens, weak potential pathogens, and non-pathogens, respectively. According to our results, this protease activity assay can be a powerful tool for differentiating pathogenic and non-pathogenic strains of Acanthamoeba.

Molecular detection of Neospora caninum in house sparrows (Passer domesticus) in Iran.

Neospora caninum is an intracellular protozoan parasite with a wide range of intermediate bird hosts. There is little information describing the prevalence and genetic characterization of N. caninum in bird hosts worldwide and in Iran. In this study, a total of 217 brain samples of house sparrow (Passer domesticus) were examined for N. caninum presence by nested polymerase chain reaction targeting the Nc-5 gene. N. caninum DNA was detected in 3.68% (8/217) of sparrows. Sequencing of the Nc5 genomic DNA revealed 97-99% of similarity with N. caninum sequences deposited in Genbank. To our knowledge, this study is the first molecular evidence of N. caninum DNA in bird hosts in Iran. The results of this study highlight the role of the house sparrow (Passer domesticus) in maintaining and spreading N. caninum infection to canines in the feral and domestic environment.

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR-RFLP in Iran.

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral, parasitic sexually transmitted infection in the world. At present, little is known regarding the degree of strain variability of T. vaginalis. A classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, drug resistance, pathogenesis and transmission of T. vaginalis. Eight different types of actin genes have been identified by PCR-RFLP in T. vaginalis; the purpose of this study is to determine the genotypes of this parasite in Karaj city, Iran. Forty-five clinical T. vaginalis isolates from vaginal secretions and urine sediment were collected from Karaj city from 2012 through 2014. DNA was extracted and the actin gene was amplified by nested-PCR; all samples were positive. To determine the genetic differences, sequencing on seven samples was conducted. Then, all PCR products were digested with HindII, MseI, and RsaI restriction enzymes. Of 45 isolates, 23 samples (51.1%) were of actin genotype G, 11 samples (24.4%) of genotype E, six samples (13.3%) of genotype H, three samples (6.6%) of genotype I, and two samples (4.4%) were mixed genotypes of G and E. Genetic diversity of T. vaginalis isolates is notable. The actin genotype G may be the dominant genotype in Karaj city, Iran.

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.

Biochemical Properties and Immunogenic Epitopes of Echinococcus granulosus Glutathione S-Transferase as a Vaccine Target: In-Silico Study.

Background: The current in silico study was done to determine the primary biochemical features and immunogenic epitopes of Echinococcus granulosus glutathione S-transferase protein as a potential vaccine candidate. Methods: Several web tools were employed to predict physico-chemical properties, antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, subcellular localization, signal peptide, transmembrane domain, secondary and tertiary structure followed by refinement and validations. In addition, B-cell epitopes were predicted and were screened using various web servers, while MHC-binding and CTL epitopes were predicted using IEDB and NetCTL servers, respectively. Results: The protein had 219 residues with a molecular weight of 25.55 kDa and alkaline isoelectric pH (7.5). This protein was stable, thermo-tolerant (aliphatic index: 78.04) and hydrophilic (GRAVY: -0.440). The predicted antigenicity scores were low and the protein was nonallergenic in nature. There were no transmembrane domain and signal peptide in the sequence. Moreover, several B-cell, MHC-binding and CTL epitopes were found in the EgGST protein, which could be further used in multi-epitope vaccines. Conclusion: Further studies are needed on the development of vaccines in vivo using EgGST alone or in combination with other antigens in the future.

In Silico Analysis of the ROP29 Protein as a Vaccine Candidate Against Toxoplasma gondii.

The progression of Toxoplasma gondii (T. gondii) invasion is aided by rhoptry proteins (ROPs), which are also crucial for the parasite's survival in host cells. In this study, in silico analysis was performed to examine the various aspects of the ROP29 protein, such as physicochemical properties, potential T- and B-cell epitopes, and other significant features. The research revealed that there were 55 possible sites for posttranslational modification in the ROP29 protein. The secondary structure of the ROP29 protein consists of a random coil, an alpha-helix, and an extended strand, which account for 49.69%, 36.81%, and 13.50%, respectively. Moreover, a number of putative T- and B-cell epitopes for ROP29 were found. The Ramachandran plot showed that 88.91% (crude model) and 97.54% (refine model) of the amino acid residues were located in the favored regions. Also, the testing of this protein's antigenicity and allergenicity showed that it was nonallergenic and immunogenic. Our results suggested that employing in silico tools to apply structural and functional predictions to the ROP29 protein can lower the likelihood that laboratory investigations will fail. This research served as a crucial foundation for further research. More research is required in the future in suitable animal model employing ROP29 alone or in combination with other antigens.

Antimalarial Effects of Nano Chloroquine Loaded Curcumin In vivo.

BACKGROUND: Malaria is still the deadliest parasitic disease caused by Plasmodium spp. Due to drug resistance and their unpleasant side effects, of conventional researchers are enormously seeking to achieve antimalarial drugs with more curative effective, less toxic and cost-affordable drugs using more advanced technology such as nanodrugs. PURPOSE: The present study aimed to examine the antimalarial effects of a novel synthesized nonochloroquine-loaded curcumin relying on dendrimer G2 in susceptible mice. METHODS: Antimalarial activity and toxicity of the nanocomposite were examined on BALB/C mice with microscopy, checking RBCs morphology and related enzymatic activity rate. RESULTS: The maximum inhibitory effect of the nanocomposite was seen at 10 mg/kg, killing 98% of P. berghei compared to sole chloroquine, whereas ED50 was reported at 5.5 mg/kg. The safety of the synthesized nanocomposite was confirmed with biochemical tests with no detrimental effects on mice. The sustainability and longevity of the nanodrug increased significantly with the NDC-CQ assay compared to the control groups. CONCLUSION: The study showed that nonochloroquine-loaded curcumin had a promising inhibitory effect on P. berghei growth in infected mice compared to standard drugs. However, further studies and clinical trials with large samples are recommended to study different aspects of using nanodrug.

Association Between Wolbachia Infection and Susceptibility to Deltamethrin Insecticide in Phlebotomus papatasi (Diptera: Psychodidae), the Main Vector of Zoonotic Cutaneous Leishmaniasis.

Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis. Wolbachia is a symbiotic alphaproteobacteria of arthropods that can be involved in susceptibility or resistance. This study aimed to investigate the relationship between Wolbachia and Deltamethrin susceptibility/resistance in Ph. papatasi. Deltamethrin filter papers (0.00002%) were used to test sand fly field collected from southern Iran. After the test, PCR amplification of the Wolbachia surface protein gene (wsp) was used to measure Wolbachia infection rate in the killed, surviving, and control groups. Result: The rates of infection by Wolbachia strain (wPap, super group A) differed between killed (susceptible) and surviving (resistant) Ph. papatasi specimens. The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) in resistant individuals with the same genetic background. This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. In addition, the results showed that Deltamethrin can act as a PCR inhibitor during detection of Wolbachia in Ph. papatasi. Conclusion: Results of this study show that Wolbachia is associated with Deltamethrin susceptibility level in Ph. papatasi. Also, as Deltamethrin has been identified as a PCR inhibitor, great care must be taken in interpreting Wolbachia infection status in infected populations. The results of this study may provide information for a better understanding of the host-symbiont relationship, as well as application of host symbiosis in pest management.

Anti-parasitic activity of a chimeric peptide Cecropin A (2-8)-Melittin (6-9) (CM11) against tachyzoites of Toxoplasma gondii and the BALB/c mouse model of acute toxoplasmosis.

Toxoplasmosis is a zoonotic disease that infects most animals, including humans. Pyrimethamine/sulfadiazine is the standard treatment for toxoplasmosis. Although this treatment has been successful, it is often associated with side effects that cannot be tolerated. Therefore, various compounds have been proposed as alternative treatments for toxoplasmosis. Antimicrobial peptides (AMPs) act on various pathogens, from viruses to protozoa. The purpose of the present study was to evaluate the effects of CM11 on in vitro and in vivo Toxoplasma gondii infection. For in vitro experiments, VERO cells were treated with different concentrations of CM11 (1-128 µg/ml) compared to sulfadiazine (SDZ) (0.78-100 µg/ml). MTT and lactate dehydrogenase (LDH) assays evaluated the cell viability and plasma membrane integrity. Then, the inhibitory concentration (IC50) values were determined for treating tachyzoites of T. gondii before or on cells previously infected. Annexin V-FITC/propidium iodide (PI) staining was used to distinguish viable and apoptotic cells. The effect of CM11, SDZ, and a combination of CM11 and SDZ was evaluated in the BALB/c mouse model of acute toxoplasmosis. CM11 was effective on tachyzoites of T. gondii and had a time and dose-dependent manner. The results of the MTT assay showed that the CC50 values of CM11 and SDZ were estimated at 17.4 µg/ml and 62.3 µg/ml after 24-h, respectively. The inhibitory concentration (IC50) of CM11 and SDZ on infected cells was estimated at 1.9 µg/ml and 1.4 µg/ml after 24-h, respectively. The highest rate of apoptosis (early and late) in high concentrations of SDZ and CM11 was determined for tachyzoites (2.13 % and 13.88 %), non-infected VERO cells (6.1 % and 19.76 %), and infected VERO cells (7.45 % and 29.9 %), respectively. Treating infected mice with CM11 and a combination of CM11 and SDZ had increased survival time. Based on the mentioned results, it can be concluded that CM11 has a beneficial effect on tachyzoites of T. gondii in vitro. The result of the mouse model suggests that CM11, either alone or in combination with other chemotherapeutic agents, could be a potential therapeutic for toxoplasmosis. Hence, antimicrobial peptides could be applied as promising anti-toxoplasma agents for treating toxoplasmosis.

Genotyping of Acanthamoeba Isolated from Hospital Environments and Thermal Water of Recreational Baths in Markazi Province, Iran.

Background: Due to the opportunism character of Acanthamoeba, the presence of this parasite in the thermal water of recreational baths and hospital environments can be a risk to the health of staff, patients and others. The aim of this study was to investigate the distribution of potentially pathogenic Acanthamoeba genotypes isolated from the hospital environment and the thermal water of recreational baths in Markazi Province, central Iran. Methods: Overall, 180 samples including thermal water from recreational baths in Mahallat City and dust, soil and water from different hospitals of Arak, Farahan and Komijan cities, central Iran were collected. The presence of Acanthamoeba was investigated using microscopic examination and molecular methods. The PCR and sequencing was performed based on a specific 18S fragment of ribosomal DNA. Results: Based on the microscopic survey, totally 134 positive samples were detected including 35% in thermal water samples and 44.7% in hospital samples. In molecular analysis, 53.5% of the samples were identified as Acanthamoeba and 46.7% as Protacanthamoeba bohemica. The genotypes were detected as T4 (33.3%), T2 (10%), T11 (6.7%), and T5 (3.3%). Conclusion: The T4 was the most common genotype found in hospitals sampling sites while the T2 genotype and P. bohemica were detected in thermal water sampling sites.

Molecular Characterization of Liver Fluke Isolated from Sheep, Goat and Cattle in Sulaymaniyah, Iraq.

Background: We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP). Methods: The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using Hpy188III and Dra II restriction enzymes, besides DNA sequencing. Results: The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed Fasciola hepatica, F. gigantica, and F. intermediate, where 28 of them displayed F. hepatica (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the F. gigantica (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to F. intermediate (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to F. hepatica, F. gigantica and F. intermediate. Conclusion: All three main species are present in the study area and F. hepatica predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.

Berberine improves inhibitory avoidance memory impairment of Toxoplasma gondii-infected rat model of ketamine-induced schizophrenia.

BACKGROUND: Memory impairment caused by Toxoplasma gondii infection has been documented. Berberine (BRB) is well known for its enhancing effects on memory and has shown promising results. However, the impact of BRB on T. gondii infection and schizophrenia-induced consolidation and reconsolidation memory impairment is still unclear. Here; we examined the effect of BRB on the inhibitory avoidance (IA) memory consolidation and reconsolidation impairment induced by T. gondii infection, and ketamine (Ket) as a pharmacological model of schizophrenia. Also; the brain-derived neurotrophic factor (BDNF) levels in the medial prefrontal cortex (mPFC) and hippocampus were analyzed. METHODS: Rats were infected with T. gondii RH strain or received Ket (30 mg/kg/day) intraperitoneally (i.p) for at least five consecutive days (as the model of schizophrenia). Then followed by oral administration with BRB (25 mg/kg/day) for five days. Finally, the IA memory retention test was examined 48 post-conditioning, and BDNF was measured. RESULTS: Results indicated IA memory impairment in T. gondii-infected animals since lower step-through latency (STL) was observed than in control animals. We found significant (P = 0.01, P = 0.001) elevations in STL and a significant decrease (P = 0.001) in total time spent in the dark area following BRB administration in infected and Ket-treated rats, indicating improvement (increased STL) in consolidation and reconsolidation memory. Moreover, BDNF levels were reduced (P = 0.01) in the hippocampus and mPFC regions of both T. gondii- infected and Ket-induced groups, which remarkably enhanced after BRB treatment. Furthermore; we found that BRB administration notably increased the mPFC BDNF levels in mPFC (P < 0.01) and hippocampus (P = 0.001) in the Ket-treated and rats infected with T. gondii. CONCLUSION: Taken together; BRB may be a valuable preclinical treatment for improving memory impairment through BDNF expression in PFC and hippocampus, therefore; BRB is suggested for memory disturbances induced by T. gondii infection.