Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

PURPOSE: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. METHODS: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. RESULTS: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. CONCLUSION: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

Evaluation of a novel Echinococcus granulosus recombinant fusion B-EpC1 antigen for the diagnosis of human cystic echinococcosis using indirect ELISA in comparison with a commercial diagnostic ELISA kit.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus sensu lato (s.l.). A large proportion of the patients are asymptomatic at the early and late stages of the disease. CE diagnosis is mainly based on imaging techniques. Laboratory diagnosis including antibody-antigen (recombinant or fusion recombinant) can be used for the diagnosis and follow up of CE and alveolar echinococcosis (AE), but need optimization and standardization. This study aimed to evaluate the efficacy of a recombinant B-EpC1 (rB-EpC1) fusion antigen comprising B1, B2, B4, and EpC1 antigens of E. granulosus using indirect ELISA in comparison with a commercial ELISA kit for the serodiagnosis of CE. The recombinant protein was expressed in the expression host, E. coli BL21, and purified. This recombinant antigen was then evaluated by indirect ELISA and compared to the commercial CE diagnostic kit (Vircell, Spain). The study samples included 124 human sera consisting of 62 sera of patients with CE, and 62 sera of individuals without clinical evidences of CE and specific anti-CE antibodies in routine indirect ELISA. The diagnostic sensitivity and specificity of the indirect rB-EpC1-ELISA test for detection of specific anti-hydatid cyst antibodies in human CE were 95.2% and 96.8%, respectively. Also, the diagnostic sensitivity and specificity of the commercial ELISA test were 96.8% in this study. Initial evaluation of the recombinant fusion antigen (B-EpC1) was promising for the detection of CE by ELISA in clinical settings. Standardization and evaluation of recombinant fusion protein require further studies.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

Proteomic characterization of hydatid cyst fluid: two-dimensional electrophoresis (2-DE) setup through optimizing protein extraction.

OBJECTIVE: Proper characterization of hydatid cyst fluid (HCF) is useful for diagnostic and follow up purposes of cystic echinococcosis/hydatidosis, which is an important zoonotic disease. In this regard, proteomics methods are very helpful. The present study was conducted to compare three protein extraction methods for HCF collected from sheep liver hydatid cysts including, trichloracetic acid (TCA)/Acetone precipitation, TCA/Acetone along with dialysis, and combination of 2-D Clean-up Kit and dialysis followed by two-dimensional electrophoresis (2-DE), to achieve better resolution in the proteomic characterization of HCF proteins. RESULTS: The 2-DE of TCA/Acetone products showed a lot of smears in the background of gels; TCA/Acetone with dialysis showed greatly reduced smears while the 2-D Clean-up Kit together with dialysis showed sharp spots and least smears. Three-dimensional images of separated spots created by Progenesis SameSpots software showed the best result was achieved by 2-D Clean-up Kit and dialysis.

Molecular identification of Leishmania species isolated from patients with cutaneous leishmaniosis in gonbad Kavoos, northeastern of Iran using hSP70 and ITS-based PCR-RFlP.

Cutaneous leishmaniosis (CL) is mainly caused by Leishmania major (rural-type) and Leishmania tropica (urban-type). CL is a major health problem in many regions of the world, and it is associated with health complications and economic loss. The identification and differentiation of Leishmania species are critical because the prevention and control methods, as well as management and therapeutic strategies, are different for each type of CL. The present study aimed to identify the parasite species responsible for CL in the study area using ITS1 and HSP70- based PCR-RFLP methods. A total of 147 stained slides were prepared from samples collected from CL patients, and these slides were positive for amastigotes of Leishmania species on microscopic examination. Forty-three Giemsastained slides with 2+ to 4+ grades were selected for molecular studies for the identification of the Leishmania species. DNA was extracted from the selected slides for the molecular studies. The amplification of HSP70 and ITS1 genes was performed by the PCR method. The PCR products were digested with the HaeIII restriction enzyme, and banding patterns of all samples were compared with reference strains. Overall, patterns of all the samples were found to correspond to the reference strains of L. major based on RFLP-PCR targeting HSP70 and ITS1 genes of the parasite, demonstrating the dominance of L. major as the causative agent of zoonotic cutaneous leishmaniosis (zCL) in the study area. This area is endemic for zoonotic CL, and further studies are required to determine the reservoir and natural infection of sand flies in this county.

Western Blot Analysis of Leishmania infantum Antigens in Sera of Patients with Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is endemic in the northwest and south of Iran. Untreated cases of VL could cause death. The aim of the present study was to evaluate the diagnostic performance of western blotting to detect a specific immunodominant proteins pattern for Leishmania infantum infection using human sera infected with VL. METHODS: We studied a panel of 122 cryopreserved human serum samples from the leishmaniasis Research Laboratory, Tehran University of Medical Sciences, Tehran, Iran from 2010 to 2017.Serum samples were collected from visceral (Group I, n: 43) and cutaneous leishmaniasis (CL) (Group II, n: 8) patients, healthy individuals from endemic (Group III, n: 13) and non-endemic (Group IV, n: 16) areas for VL, and patients with other infectious diseases (Group V, n: 42). Total antigens were prepared from the Iranian strain of L. infantum promastigote form. RESULTS: In western blotting method, 34 protein bands of 14 to 163 kDa were recognized using the sera of VL patients. The polypeptide fractions with the highest frequency including 29, 51, and 62 kDa fractions were detected using 81.4%, 79%, and 81.4% of the sera, respectively. These bands were not detected using the sera of the negative control. Moreover, 19-23, 27, 31-35, 143-163, and 109 kDa fractions were detected specifically using the sera of the patients with VL. CONCLUSION: This technique could be a primary step for further exploration of VL immunodominant antigens for cloning (or any technique) further investigations for future planning.

Spironucleus muris and Eperythrozoon coccoides in Rodents from Northwestern Iran: Rare Infections.

BACKGROUND: Rodents perform a crucial role in dispersal of zoonosis causes globally. We aimed to investigation about infection levels of parasitic agents in rodents' population in Meshkinshahr areas, northwest of Iran from Apr to Sep 2014. METHODS: Two hundred four rodents were trapped and anaesthetized. A sample of blood was collected via cardiopuncture from each one. Thin and thick blood smears were prepared and stained with Giemsa. All stained smear were examined under light microscopy with high magnification by two expert microscopists. Every suspected unicellular observed were measured microscopically and compared with key references to diagnose. RESULTS: Captured rodents were identified as three genera including Meriones persicus, Mus musculus, Cricetulus migraturius. Protozoa identified in this study were included of Spironucleus muris and Eperythrozoon coccoides, these parasites were observed in blood smear of 0.98% of rodents. S. muris and E. coccoides were seen in M. musculus and C. migraturius, respectively. CONCLUSION: The present study increases awareness about Eperythrozoonosis in rodents and its potential transmission to domestic animals and even to human in rural districts in Iran. Moreover, the attack of Spironucleus on the mucus of colon and its systemic risk was confirmed.

Ferredoxin Gene Mutation in Iranian Trichomonas vaginalis Isolates.

BACKGROUND: Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO) to its active form (CPR). Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied. METHODS: Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations. RESULTS: In four isolates (8.69%) point mutation at nucleotide position -239 (the translation start codon) of the ferredoxin gene were detected in which adenosine were converted to thymine. CONCLUSION: Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.