Identification of Blastocystis spp. in Urban Rodents of Different Districts in Southwestern Iran: Subtype Distribution and Possible Zoonotic Potential.

PURPOSE: Rodents are one of the most abundant and diverse species of mammals and have recently been identified as carriers of numerous human pathogens. The current study was conducted to assess the prevalence, subtype (STs) distribution, and zoonotic potential of Blastocystis spp. in various species of rodents in Shiraz, southwestern Iran. METHODS: For this aim, a total of 120 fresh fecal samples were collected from Mus musculus (n = 40), Rattus norvegicus (n = 40), and Rattus rattus (n = 40) in various municipality districts of Shiraz (6 out of 10 districts) between February and November 2020. Upon detecting parasites using light microscopy, a DNA fragment of the Blastocystis SSU rDNA gene was amplified using conventional PCR. RESULTS: By employing direct wet mount examination, 8 out of 120 fecal samples (6.7%; 2 from house mice, 3 from black rats, and 3 from brown rats) tested positive. Similarly, 5% (2/40) of house mice, 7.5% (3/40) of black rats, and 7.5% (3/40) of brown rats tested positive using the molecular method. Phylogenetic analysis revealed that the Blastocystis infecting different rodent species in Shiraz belonged to two potentially zoonotic STs (ST1 and ST4). Accordingly, rodents should not be overlooked as potential reservoirs of zoonotic Blastocystis infections. Different sampled urban districts and their statistical association with reported prevalence rates were analyzed separately. CONCLUSION:  Overall, the issue of the frequency and ST distribution of Blastocystis in urban rodents of Iran is still open to question and for a proper understanding, wider and more comprehensive studies are needed.

Use of Recombinant CP2 and CP23 Antigens of Cryptosporidium parvum for Serodiagnosis of Human Cryptosporidiosis

Background: Cryptosporidium parvum is an important coccidian parasite infecting many mammals, including human. This parasite can manifest as chronic severe diarrhea in immunocompromised individuals, especially those with AIDS. The present study reports the recombinant production of recombinant (r)P2 and rP23 antigens of C. parvum as antigens for detecting human cryptosporidiosis using indirect ELISA tests. Methods: The coding sequences of rP2 and rP23 proteins were codon-optimized, commercially synthesized and sub-cloned in the pET28a expression vector. The expressed proteins were purified by Ni-NTA column chromatography and confirmed by Western blotting. The efficacy of rP2/rP23 proteins for serodiagnosis was evaluated by positive (n = 20) and negative (n = 20) human sera, confirmed by the Ziehl-Neelsen staining as the gold standard test. Results: In ELISA test, the sera from C. parvum-infected patients reacted strongly to rP2/rP23. The sensitivity and specificity related to the diagnostic potential of rP2/rP23 in the ELISA assay were 100%. Conclusion: Our results showed that combination of rP23 and rP2 antigens in ELISA significantly increases the performance of C. parvum serodiagnosis in human cryptosporidiosis.

The level of interleukin-17, 23, and gamma interferon in cutaneous leishmaniasis patients before and after intra lesion treatment.

Leishmaniasis is a zoonotic vector-borne disease that is endemic in tropical and sub-tropical districts. The immune system response is one of the most important factors that has affected parasitic treatment. In this research, the production of IL-17 (Interleukin 17), IL-23 (Interleukin 23), and IFN-ɤ (Interferon-gamma) in peripheral blood mononuclear cells (PBMCs) isolated from patients with cutaneous leishmaniasis caused by L. major before and after treatment were compared to evaluate their roles in the recovery process. For this experimental study, we recruited 23 patients in Iran. Ten milliliters of peripheral blood samples were collected before and after one month of treatment, and PBMCs were isolated. Production of IL-17, IL-23, and IFN-ɤ was assessed by sandwich ELISA technique. The production of IFN-ɤ and IL-17 in patients (before treatment sensitive leishmaniasis and resistance leishmaniasis) was more than the healthy controls (P < 0.05). Moreover, both of the cytokines productions in sensitive leishmaniasis cases were more than the resistance leishmaniasis patients. In this study, we observed lower levels of IL-23 in patients compared to healthy controls. And among the patients, IL-23 production was lower in sensitive leishmaniasis cases (P < 0.05). Conclusion: It appears that the production of IFN-ɤ is necessary for the treatment of leishmaniasis, but further studies are required to address the role of IL-17 and IL-23 in this disease.

Serosurvey of parvovirus B19 and cytomegalovirus infections among female university students in Shiraz, Southern Iran.

Infection with parvovirus B19 and cytomegalovirus (CMV) during pregnancy might lead to fetal infection, resulting in congenital abnormalities. This study aimed to investigate the frequency of IgM and IgG antibodies against parvovirus B19 and CMV in female university students in Shiraz, in Fars province, Southern Iran. In this cross-sectional study, 370 female university students were included. Blood samples were collected from each participant and tested for anti-parvovirus B19 and CMV IgG and IgM antibodies, using commercial ELISA kits. The mean age of the participants was 24 (±7)years. Out of 370 participants, 327 (88.4%) and 9 (2.4%) were positive for IgG and IgM antibodies against CMV. Moreover, 211 (57.0%) and 4 (1.1%) of the participants were respectively positive for IgG and IgM antibodies against parvovirus B19. The difference in CMV or parvovirus B19 seropositivity between different age groups was not statistically significant (P>0.05). The findings of our study showed that more than 50% of the female university students are seropositive to CMV and parvovirus B19 infections. It highlights the importance of health education and also the laboratory screening of females at childbearing age to reduce the risk of congenital infections resulting from these viral infections.

Identification of immunoreactive proteins in secretions of Leishmania infantum promastigotes: an immunoproteomic approach.

BACKGROUND: In the Mediterranean region, Leishmania infantum is the main cause of visceral leishmaniasis. Dogs with canine visceral leishmaniasis are an important reservoir of visceral leishmaniasis. Control of canine visceral leishmaniasis could disrupt transmission of visceral leishmaniasis to humans. The secreted antigens of Leishmania promastigotes are potential stimuli of the host immune system. Proteomic techniques facilitate the identification of new protein markers. AIMS: This study aimed to identify immunoreactive proteins in the secretions of L. infantum promastigotes which could be possible targets for the diagnosis and treatment of canine visceral leishmaniasis and the development of vaccines against the disease. METHODS: Secretions of L. infantum promastigotes were obtained from the cultivation of 6 × 109 promastigotes in serum- free RPMI-1640 medium during a period of 72 h. After deionization and lyophilization, two-dimensional gel electrophoresis was used for protein separation followed by Western blotting. Thirteen common and repeatable immunoreactive spots were analysed by mass spectrometry. RESULTS: Nine proteins were identified by spectrometry. Most of these proteins were involved in metabolism pathways, survival and pathogenicity of Leishmania parasites. Phospholipase C, immune inhibitor A, chitin-binding protein and a single peptide match to chain A crystal structure of selenomethionine were observed in the secretions of L. infantum promastigotes. CONCLUSIONS: The proteins identified in metabolism pathways, survival and pathogenicity of Leishmania parasites are possible targets that could be used for the diagnosis and treatment of canine visceral leishmaniasis and the development of vaccines against the disease in the future.

An immunoproteomic approach to identifying immunoreactive proteins in Leishmania infantum amastigotes using sera of dogs infected with canine visceral leishmaniasis.

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani and Leishmania infantum. The infected dogs with canine visceral leishmaniasis (CVL) are important reservoirs for VL in humans, so the diagnosis, treatment and vaccination of the infected dogs will ultimately decrease the rate of human VL. Proteomics and immunoproteomics techniques have facilitated the introduction of novel drug, vaccine and diagnostic targets. Our immunoproteomic study was conducted to identify new immunoreactive proteins in amastigote form of L. infantum. The strain of L. infantum (MCAN/IR/07/Moheb-gh) was obtained from CVL-infected dogs. J774 macrophage cells were infected with the L. infantum promastigotes. The infected macrophages were ruptured, and pure amastigotes were extracted from the macrophages. After protein extraction, two-dimensional gel electrophoresis was employed for protein separation followed by Western blotting. Western blotting was performed, using symptomatic and asymptomatic sera of the infected dogs with CVL. Thirteen repeatable immunoreactive spots were identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Some, including prohibitin, ornithine aminotransferase, annexin A4, and apolipoprotein A-I, have been critically involved in metabolic pathways, survival, and pathogenicity of Leishmania parasites. Further investigations are required to confirm our identified immunoreactive proteins as a biomarker for CVL.

Immunoreactivity Analysis of Toxoplasma gondii Recombinant Antigen rSAG3 in Sera from Immunized BALB/c Mice and Toxoplasmosis Patients.

BACKGROUND: The coccidian protozoa Toxoplasma gondii is an obligate intracellular parasite of humans and other warm-blooded animals. Diagnosis of toxoplasmosis is of considerable medical importance for human, especially pregnant women and immunocompromised individuals. The apply of an Escherichia coli recombinant antigen(s) would be significantly useful in developing standardization of the diagnostic tests and reducing their costs. In this study, immunoreactivity of recombinant SAG3 against sera from immunized mice and human anti-T. gondii IgG positive patients was evaluated by western-blotting and enzyme immunoassay (EIA) in Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences in 2013. METHODS: Three inbreed BALB/c female mice were obtained. Two mice were injected with rSAG3 and one was remained untreated, as control. Sera from immunized mice and also pooled sera from IgG positive toxoplasmosis cases were evaluated with western-blotting. IgG antibody responses to recombinant SAG3 was measured by indirect ELISA against the negative control group. RESULTS: The rSAG3 protein reacted with sera of immunized mice and sera from patients with anti-Toxoplasma IgG antibodies in western-blot analysis. The result of ELISA showed that, there was marked differences in the absorbance values between the recombinant SAG3 immunized mice and control group. CONCLUSION: The rSAG3 showed IgG reactivity with sera from immunized mice and anti-Toxoplasma IgG patients.

Faunal distribution of fleas and their blood-feeding preferences using enzyme-linked immunosorbent assays from farm animals and human shelters in a new rural region of southern Iran.

Blood sucking insects, such as fleas, are responsible for the transmission of many infectious disease-causing agents which impose an intolerable burden on the health of people living particularly in endemic parts of the world. Fleas (Insecta: Siphonaptera) are found in many parts of the world including Iran. Both adult male and female fleas are obligatory ectoparasites. They are one of the main public health concerns as a result of their nuisance or the potential to act as vectors of a number of medically-important pathogens. The current study was conducted to examine the geographical distribution and fauna of fleas and their anthropophagic index in part of Fars province, southern Iran. This study was the first to be done in Iran. A total of 20 villages were randomly selected. From October 2011 to May 2012, adult fleas were collected by direct hand catch from human to animal shelters. Overall 848 fleas, most of which were blood-fed, were captured from the floor or the body of farm animal hosts (cattle, sheep, goat and hens). Only two different genera of fleas were identified, the main species (99.76 %) was human flea, Pulex irritans. The village of Shamsabad was the most heavily infested area. P. irritans had an anthropophagic index of 15 % using indirect enzyme-linked immunosorbent assays (ELISA). It could be concluded that P. irritans is widely distributed in this area. Based on their blood feeding activity, fleas thus posed a serious health threat to residents and their economically important livestock in this part of Iran.

Evaluation of recombinant SAG1, SAG2, and SAG3 antigens for serodiagnosis of toxoplasmosis.

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.

Genetic Variability of Antigen B2 of Human, Sheep, Goats, Camel and Cattle Isolates of Echinococcus granulosus in Iran.

BACKGROUND: Antigen B (AgB) is frequently used for immuno-diagnosis of human cystic echinococcosis (CE). Echinococcus granulosus AgBs show a high degree of genetic variability in different hosts or in different CE endemic areas. The present study aimed to evaluate the genetic polymorphisms of encoding antigen B2 gene (AgB2) among different Iranian isolates of E. granulosus. METHODS: A total of 50 CE isolates were collected from human, sheep, cattle, goat and camels, 10 isolates from each intermediate host of E. granulosus. Total genomic DNA from either protoscolices or germinal layer was extracted from each cyst and PCR-RFLP followed by DNA sequencing was used to evaluate sequence variation and polymorphism of AgB2 in the isolates. RESULTS: After the PCR amplification, using AgB2 primers, an almost 400 bp band was amplified in all of the isolates. The PCR products were digested with Alu1 restriction endonuclease. After restriction enzyme digestion with Alu1' sheep and human isolates gave a similar pattern of RFLP with the gene size of approximately 140 and 240bp and camel and goat isolates gave a similar pattern, but different from sheep and human, with the gene size of approximately 150 and 250bp. Sequence analysis showed the most genetic similarity of AgB2 between human and sheep isolates. CONCLUSION: Findings of this study revealed the differences in the sequences of AgB2 within and between the Iranian isolates of E. granulosus. These differences may affect the performance of any diagnostic test which uses AgB.