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.

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.

Molecular and Morphological Data Confirmed First Record of Abbreviata kazakhstanica Markov and Paraskiv, 1956 (Spirurida: Physalopteridea) in Iran.

BACKGROUND: The genus Abbreviata (Spirurida: Physalopteridea) currently contains 47 species. Physalopteridae nematodes infect a large number of vertebrates, including mammals, birds, reptiles and amphibians. The current study is a report of the first morphological and molecular identification of A. kazakhstanica (Spirurida: Physalopteridea) in Pseudopus apodus in Iran. METHODS: Eleven road-killed P. apodus, were collected from, Iran during 2016-2018. The nematodes were isolated from stomach. After morphological study, the genomic DNA of the parasites was extracted using CTAB method. The DNA was used for PCR amplification of cytochrome c oxidase subunit I (cox1). The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. RESULTS: After detailed microscopic examination, the A. kazakhstanica was identified. The cox1 sequences confirmed the species of helminth. The new sequences of A. kazakhstanica were submitted to GenBank under the accession number MK578751-2. CONCLUSION: Regarding the limited data on parasitological status of Iranian reptiles, more specific and comprehensive investigations are needed to identify the parasitic fauna.

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.

Assessment of DNA vaccine encoding Toxoplasma gondii microneme complete gene and IL-12 as adjuvant in BALB/c mice.

OBJECTIVES: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in humans and animals. Micronemes (MICs) are effective candidates for DNA vaccine. MATERIALS AND METHODS: In this study, we evaluated the immune response of BALB/c mice against MIC3 gene of Toxoplasma gondii and interleukin 12 (IL-12) as DNA vaccine. The MIC3 gene was cloned into the PTZ57R/T vector before sub-cloning in pcDNA3. Recombinant pc-MIC3 was transformed into Escherichia coli (TOP10 strain). The pc-MIC3 plasmid was then transfected into Chinese Hamster Ovary (CHO) cells, and the expression of the MIC3 gene was evaluated by SDS-PAGE and Western blotting. Sixty female BALB/c mice were divided into 6 groups. Each group received 3 intramuscular immunizations on days 0, 21st and 42nd using one of the following stimulants: phosphate-buffered saline, pcDNA3, pCAGGS-IL12, pc-MIC3 (100 µg), pc-MIC3 (50 µg), or combined pCAGGS-IL12 (50 µg) and pc-MIC3 (50 µg). The enzyme-linked immunosorbent assays was applied to evaluate interferon gamma (IFN-γ) and IL-4 cytokines excretion of lymphocytes stimulated with tachyzoites lysate antigen, as well as the total levels of immunoglobulin G (IgG), IgG2a and IgG1 in immunized mice sera. RESULTS: Our results showed that mice challenged with pc-MIC3 (100 µg) had the highest longevity and quantity of immunoglobulin. Moreover, the highest expression level of IFN-γ was found in mice injected with combined pcMIC3 and pCAGGS-IL12 (P<0.05). CONCLUSION: The MIC3 gene can be an efficient DNA vaccine candidate against toxoplasmosis. While, the single-gene vaccine can confer partial protection to mice against toxoplasmosis, the multigene vaccine can significantly enhance immune responses.

Molecular assessment of Neospora caninum and Toxoplasma gondii in hooded crows (Corvus cornix) in Tehran, Iran.

Neospora caninum and Toxoplasma gondii are two closely related protozoan parasites that have been detected from various species of bird hosts. However, little is known about the prevalence of N. caninum and T. gondii in crows. Hence, we examined the molecular frequency of N. caninum and T. gondii in the brain samples of hooded crows (Corvus cornix) that collected from different public parks of Tehran, Iran by nested-PCR method. We used the primers targeting the Nc5 and GRA6 genes for detection of N. caninum and T. gondii, respectively. From a total of 55 brain samples, 5 (9.9%) and 9 (16.36%) samples were positive for N. caninum and T. gondii, respectively. Sequencing of a N. caninum isolate revealed 95%-100% identity with the deposited N. caninum in GenBank. Genotyping of T. gondii isolates by PCR-RFLP analysis of the GRA6 gene revealed type III genotype in 8 isolates. The results of this study indicate that hooded crows may have a putative role in transmission of N. caninum and T. gondii to canines and felines definitive hosts, respectively.

Comparative Assessment of Induced Immune Responses Following Intramuscular Immunization with Fusion and Cocktail of LeIF, LACK and TSA Genes Against Cutaneous Leishmaniasis in BALB/c Mice.

In the present study, we evaluated induced immune responses following DNA vaccine containing cocktail or fusion of LeIF, LACK and TSA genes or each gene alone. Mice were injected with 100 µg of each plasmid containing the gene of insert, plasmid DNA alone as the first control group or phosphate buffer saline as the second control group. Then, cellular and humoral responses, lesion size were measured for all groups. All vaccinated mice induced Th1 immune responses against Leishmania characterized by higher IFN-γ and IgG2a levels compared with control groups (p < 0.05). In addition, IFN-γ levels increased in groups immunized with fusion and cocktail vaccines in comparison with LACK (p < 0.001) and LeIF (p < 0.01) groups after challenge. In addition, fusion and cocktail groups produced higher IgG2a values than groups vaccinated with a gene alone (p < 0.05). Lesion progression delayed for all immunized groups compared with control groups from 5th week post-infection (p < 0.05). Mean lesion size decreased in immunized mice with fusion DNA than three groups vaccinated with one gene alone (p < 0.05). While, lesion size decreased significantly in cocktail recipient group than LeIF recipient group (p < 0.05). There was no difference in lesion size between fusion and cocktail groups. Overall, immunized mice with cocktail and fusion vaccines showed stronger Th1 response by production of higher IFN-γ and IgG2a and showed smaller mean lesion size. Therefore, use of multiple antigens can improve induced immune responses by DNA vaccination.