Iranian Journal of Parasitology

Comparative Analysis of Nanos and Ago Genes Expression in the Germinative Cells Isolated from Germinal Layer and the Neck Region of Echinococcus granulosus

Sun, 09 Jun 2024 14:39:21 +0430

Background: We aimed to evaluate the differential expression of nanos and ago genes in the protoscoleces, germinal layer, the neck, and the sucker regions of adult Echinococcus granulosus.

Methods: The study was conducted in 2018 at the Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, Kerman, Iran. In the present study E. granulosus protoscoleces were cultured in a di-phasic medium to obtain strobilated worms. The strobilated worms were harvested and using a sterile razor blade, the neck region was separated. In the molecular study the neck sections were compared with the tissues derived from the suckers from the same worm. The primers were specifically designed for RT-qPCR on nanos and ago. The germinative cells were isolated from the cyst germinal layer and cultured in DMEM for further molecular studies. The Immunohistochemical profile was designed to explore the nature of nanos protein in the strobilated worms. Differences between and within groups were statistically assessed relative to the protoscoleces.

Results: An increasing nanos gene expressions were found in sucker, neck, cells and germinal layer in comparison to the protoscoleces. The expression of ago gene was decreased in sucker, cell and germinal layer, and increased in the neck region in comparison to the protoscoleces. The results showed that both genes were expressed in all developmental stages of E. granulosus.

Conclusion: nanos and ago genes were differentially expressed at different developmental stages of E. granulosus and may contribute to differentiation of the parasite.

Phylogeography, Genetic Diversity and Population Structure of Echinococcus granulosus Sensu Stricto Inferred by Mitochondrial DNA Markers between Southeast of Iran and Pakistan

Sun, 09 Jun 2024 15:05:35 +0430

Background: Current study was designed to provide a better insight into the circulating genotypes, genetic diversity, and population structure of Echinococcus spp. between southeast of Iran and Pakistan.
Methods: From Jun 2020 to Dec 2020, 46 hydatid cysts were taken from human (n: 6), camel (n: 10), goat (n: 10), cattle (n: 10) and sheep (n: 10) in various cities of Sistan and Baluchestan Province of Iran, located at the neighborhood of Pakistan. DNA samples were extracted, amplified, and subjected to sequence analysis of cox1 and nad1 genes. Results: The phylogeny inferred by the Maximum Likelihood algorithm indicated that G1 genotype (n: 19), G3 genotype (n: 14) and G6 genotype (n: 13) assigned into their specific clades. The diversity indices showed a moderate (nad1: Hd: 0.485) to high hap- lotype diversity (cox1: Hd: 0.867) of E. granulosus s.s. (G1/G3) and low nucleotide diver- sity. The negative value of Tajima’s D and Fu’s Fs test displayed deviation from neutrali- ty indicating a recent population expansion. A parsimonious network of the haplotypes of cox1 displayed star-like features in the overall population containing IR9/PAK1/G1, IR2/PAK2/G3 and IR18/G6 as the most common haplotypes. A pairwise fixation index (Fst) indicated that E. granulosus s.s. populations are genetically moderate differen- tiated between southeast of Iran and Pakistan. The extension of haplotypes PAK18/G1 (sheep) and PAK26/G1 (cattle) toward Iranian haplogroup revealed that there is dawn of Echinococcus flow due to a transfer of alleles between mentioned populations through transport of livestock or their domestication.
Conclusion: The current findings strengthen our knowledge concerning the evolution- ary paradigms of E. granulosus in southeastern borders of Iran and is effective in control- ling of hydatidosis.

Designing a Multiple-Epitope Vaccine Candidate against Leishmania major and Leishmania infantum for Monocyte-Derived Exosome Preparation

Sun, 09 Jun 2024 15:25:32 +0430

Background: Leishmania is a vector-borne protozoon, which causes visceral, cutaneous and mucocutaneous leishmaniosis in human and animals. Monocyte-derived exosome vaccines can be used as prophylaxis and immunotherapy strategies. The aim of this study was to design a multiple-epitope candidate vaccine using leishmaniolysin (GP63) and rK39 proteins against Leishmania major and L. infantum for monocyte-derived exosome preparation.

Methods: This study was carried out in Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, 2023–2024. Effective immunodominant epitopes were selected from two antigenic proteins of GP63 and rK39 using various immunoinformatics and bioinformatics approaches. Vibrio cholerae β-subunit was used as an adjuvant to stimulate immune responses. Then, appropriate linkers were selected for the fusion of epitopes. The 3D model of candidate vaccine was predicted and validated.

Results: This designed candidate vaccine could effectively be used as a prophylaxis strategy against leishmaniosis.

Conclusion: A candidate vaccine was designed using bioinformatic and immunoinformatic studies with virtual acceptable quality; however, effectiveness of this vaccine should be verified through further in-vitro and in-vivo studies.

The Prevalence of Cryptosporidium spp. and Giardia duodenalis in Marmota himalayana (Rodentia: Sciuridae) in the Qinghai Tibetan Plateau area, China

Sun, 09 Jun 2024 19:07:04 +0430

Background: Cryptosporidium and Giardia are well-known important intestinal zoonotic pathogens that can infect various hosts and cause diarrhoeal diseases. We aimed to determine the epidemiological prevalence and molecular characterization of Cryptosporidium and Giardia species in Himalayan marmot (Marmota himalayana, class Marmota) in the Qinghai Tibetan Plateau Area of Qinghai Province, Northwest China.

Methods: Overall, 243 Himalayan marmot fecal samples were collected in 2017 and in 2019 and a two-step nested PCR technique was performed to amplify the fragments of the SSU rRNA gene of Cryptosporidium and 18S ribosomal RNA gene of Giardia. Molecular characterization of Cryptosporidium was performed with the primary primers N-DIAGF2 and N-DIAGR2, the secondary primers CPB-DIAGF and CPB-DIAGR. Similarly, molecular characterization of Giardia was used the first primers Gia2029 and Gia2150c, the secondary primers RH11 and RH4. The positive PCR products were sequenced and the sequences were processed by Clustal Omega and BLAST. Phylogenetic analysis was achieved by NJ method in MEGA.

Results: The infection rate of Cryptosporidium spp. and G. duodenalis was 4.9% (12/243) and 0.8% (2/243) in M. himalayana, respectively. Cryptosporidium spp. are characterized as novel genotypes Cryptosporidium marmot genotype I (n=3) and Cryptosporidium marmot genotype II (n=9); G. duodenalis assemblage A (n=2) was found in M. himalayana.

Conclusion: This is the first report of Cryptosporidium spp. and G. duodenalis infections in M. himalayana in Qinghai of Northwest China. The results indicate the existence of Cryptosporidium species and G. duodenalis infections that may have a potential public health significance.

Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing

Sun, 09 Jun 2024 19:16:30 +0430

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multilocus sequence typing (MLST).

Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes.

Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite.

Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.

Isolation and Molecular Identification of Vahlkampfiidae and Vermamoeba Vermiformis from Fresh Vegetables: A Neglected Source of Infections

Marziye Fatemi, Maryam Niyyati, Seyed Ahmad Karamati, Hamed Mirjalali — Sun, 09 Jun 2024 19:21:30 +0430

Background: Naegleria spp., Tetramitus spp., and Vermamoeba vermiformis are potential pathogenic free-living amoebae (FLA) causing diseases such as keratitis, meningoencephalitis, and lung infections. We aimed to investigate the presence of Vahlkampfiidae and V. vermiformis in raw vegetables commonly consumed in Iran.

Methods: Totally, 70 samples of vegetables samples including watercress (22), leeks (12), parsley (10), basil (13) and mint (13) were collected from municipal markets of Tehran, the capital of Iran during June to October 2021. After washing vegetables, samples were cultivated onto 2% non-nutrient agar (NNA) medium. After morphological confirmations, DNA was extracted and identical fragments of the FLA were amplified and sequenced.

Results: Out of 70 cultured samples, 11 samples (15.71 %) were morphologically positive, of which four and seven were V. vermiformis and Vahlkampfiidae isolates, respectively. According to the PCR/sequencing results two, one, one, and one strains belonged to N. australiensis, N. americana, Vahlkampfia sp., V. inornata, and T. aberdonicus, respectively. All Vermamoeba genus were characterized as V. vermiformis.

Conclusion: The results of current study revealed the contamination of fresh raw vegetables with Vahlkampfiidae and V. vermiformis. In addition, to our knowledge this is the first report of T. aberdonicus in raw vegetables. Our findings highlight the public health importance of vegetables in transmission of FLA, as well as the potential role of FLA in transmission of potential pathogenic microorganisms via consuming of fresh raw vegetables.

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania

Sun, 09 Jun 2024 19:28:45 +0430

Background: Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L.

Methods: we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo).

Results: The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter.

Conclusion: Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.

Efficacy of a Combined Herbal Product Containing Indigofera argentea, Pistacia atlantica and Salvia hispanica in Zoonotic Cutaneous Leishmaniasis Patients Treated along Glucantime ®

Sun, 09 Jun 2024 19:33:00 +0430

Background: Leishmaniasis is highly prevalent worldwide, and while conventional medicine offers numerous treatment methods for cutaneous leishmaniasis, Iranian traditional medicine suggests various remedies. We aimed to evaluate the efficacy of an herbal combination containing Indigofera argentea leaves, Pistacia atlantica resin, and Salvia hispanica seeds in patients with zoonotic cutaneous leishmaniasis.

Methods: This study was conducted at the Leishmaniasis Department of Chabahar Health Center in southeastern Iran in 2021. It was a double-blinded randomized clinical trial involving 68 patients enrolled after clinical diagnosis, examination of smear samples using Giemsa staining, and PCR confirmation. The volunteers were randomly divided into intervention and control groups. Both groups received 'Glucantime ® as the primary medication weekly until complete healing or up to 12 weeks. Glucantime ® was administered intralesionally at a dosage of 0.1 cc on the wound's margin, with repeat injections at 1 cm intervals along the wound edge when necessary. In addition to the main drug, the intervention group received the herbal product topically twice daily until wound healing or for up to 4 weeks, while the control group received a placebo in the same manner.

Results: 82.35% of patients in the intervention group and 20.58% in the control group achieved complete healing within four weeks. There was a significant difference between the two groups (P < 0.001).

Conclusion: The herbal product demonstrated effectiveness in treating patients with zoonotic cutaneous leishmaniasis.

Association between Toxocara Spp. Exposure and Schizophrenia: A Case-Control Study

Sun, 09 Jun 2024 19:40:33 +0430

Background: Toxocariasis is a zoonotic disease caused by the larvae of Toxocara canis or Toxocara cati. Patients with schizophrenia may be at higher risk of infection, possibly due to their cognitive and personal self-care impairments. We aimed to assess the association between Toxocara spp. exposure and schizophrenia.

Methods: This case-control study was conducted on 109 patients with schizophrenia admitted to Ibn Sina Hospital, Shiraz, Iran, and 104 age- and gender-matched healthy controls from May to September 2021. A questionnaire was obtained and serum samples were tested for IgG antibodies to Toxocara excretory/secretory (TES) antigens using an enzyme-linked immunosorbent assay (ELISA).

Results: Anti-Toxocara IgG was detected in 12 schizophrenic patients and 10 control subjects, giving respective seroprevalences of 11.0% (95% confidence interval [95% CI]=5.8–18.4%) and 9.6% (95% CI=4.7–17.0%). Univariate logistic analyses estimated an odds ratio (OR) of 1.16 (95% CI=0.44–3.16); however, it was not statistically significant (P=0.915). Individuals with a history of eating unwashed vegetables or fruits (23.1%, 95% CI=9.0–43.6, crude odds ratio [COR]=3.21, 95% CI=1.13–9.13) and rural residency (19.5%, 95% CI=8.8–34.9, COR=2.74, 95% CI=1.06–7.05) had significantly higher rates of seropositivity using the univariate logistic analyses. After multivariate logistic analyses, the differences were not statistically significant.

Conclusion: The toxocariasis seroprevalence among schizophrenic and healthy participants was not significantly different (11% vs. 9.6%). Since the disease severity, onset, and cognitive sequelae are not the same among schizophrenic patients, clinically matched studies with larger samples are required to address the current inconsistency between the studies.

Cytotoxic and Immunomodulatory Activity of Curcumin and Chitosan on Experimental Toxoplasmosis

Sun, 09 Jun 2024 19:40:59 +0430

Background: Toxoplasma gondii is a pathogenic parasite with worldwide distribution. We investigated curcumin and chitosan in combination on the viability of T. gondii tachyzoites in silico, in vitro and in vivo.

Methods: A 3D model was employed in Urmia University of Medical Sciences, Urmia, Iran in 2021 to study the interaction between curcumin and dihydrofolate reductase (DHFR). Ramachandran root-mean-square deviation and VERIFY3D validated the model. Cytotoxicity of curcumin and chitosan was evaluated by MTT viability assay. BALB/c mice infected with 10⁴Toxoplasma organisms were treated with curcumin, chitosan, and the combination of curcumin+chitosan. Serum levels of inducible NO synthetase (iNOs), interferon gamma (IFN-γ), interleukin (IL)-5, glutamate oxaloacetic transaminases (SGOT), and glutamic pyruvate transaminase (SGPT) were determined.

Result: Curcumin-DHFR and curcumin-DHPS (dihydropteroate synthase) interactions and calculated enzyme energy indicated an excellent affinity for curcumin with DHFR, but not DHPS. MTT results of concurrent treatments demonstrated IC₅₀rates of 0.1, 0.05, and 0.01 mg/ml at 24, 48, and 72h, respectively. IFN-γ, IL-5 and iNOs levels in curcumin+chitosan treated mice were 1.71, 0.51, and 1.51 IU/L, while those of SGOT and SGPT were 76 and 84 IU/L, respectively.

Conclusion: The combination of curcumin and chitosan increased survival time of infected mice by seven days. Curcumin and chitosan in combination regulated the immune system and reduced liver damage, potentially forming the basis of a new treatment for toxoplasmosis.

Evaluation of Nanonanoliposomal Curcumin on Cutaneous Leishmaniasis Skin Lesions Caused by Leishmania major in BALB/c Mice

Sun, 09 Jun 2024 19:51:10 +0430

Background: Curcumin is an extract of rhizome turmeric (diferuloylmethane), with antioxidant, anti-inflammatory, antimicrobial, and anti-parasitic properties, which making it a potential candidate for the treatment of leishmaniasis. The aim of the presented study was to evaluate curcumin as possible candidate for treatment of cutaneous leishmaniasis.

Methods: We investigated the physicochemical properties and anti-leishmanial effects of nanoliposomal curcumin (40, 80, and 120 μM) in Leishmania major (MRHO/IR/75/ER) infected BALB/c mice at the faculty of Veterinary Medicinem University of Tehran, Iran. For this aim, L. major promastigotes (MHROM/IR/75/ER) at stationary phase (2×10⁶) were inoculated sub-cutaneously into the upper area of the tail in BALB/c mice (six groups, n= 10 per group). For evaluation of nanoliposomal curcumin, the zeta potential, particle size and stability of nanoliposomal curcumin was determined. Furthermore, the anti-leishmanial effects of nanoliposomal curcumin formulation on the lesion sizes was determined and the parasite burden in the leishmania induced lesion was performed using semi quantitative PCR.

Results: Treatment of L. major infected BALB/c mice with nanoliposomal curcumin led to a reduction in the kinetic of the skin lesion size development. The semi quantitative PCR analysis of DNA extracted from the lesions showed reduction of parasite burden. The most effective treatment could be found in 80 μM nanoliposomal curcumin. Treatment with Glucantime, as a positive control, also showed a nearly similar effect compared to the effect of 80 μM nanoliposomal curcumin.

Conclusion: Nanoliposomal curcumin could be considered as a potential drug against cutaneous leishmaniasis caused by L. major in susceptible animal models.

Investigation of Mitochondrial Cytb Gene Region of Both Echinococcus granulosus Eggs from Dogs and Cystic Echinococcosis Isolates Obtained from Sheep and Cattle by Molecular Methods

Sun, 09 Jun 2024 19:51:26 +0430

Background: We aimed to determine the common Echinococcus granulosus genotypes in Ağrı, Türkiye and to obtain information on the transmission of this parasite.

Methods: Cystic echinococcosis samples from 100 slaughtered cattle and 100 slaughtered sheep and faecal samples from 200 stray dogs were included in 2021. Collected cyst fluid samples and faces were examined microscopically. DNA was isolated from the germinal membrane of the cysts and from the parasite eggs in the stool samples. The mitochondrial cytb gene region of the parasite was amplified by PCR. Genotypes were determined using the Basic Local Alignment Search Tool (BLAST) after sequence analysis of PCR amplicons.

Results: The highest percentage of cysts was found in the lungs of sheep and the liver of cattle. In addition, 75% of sheep cysts and 25.6% of cattle cysts were fertile. Taenia spp./Echinococcus spp. eggs were found in 6% of the faeces of 200 dogs examined microscopically. E. granulosus eggs were detected in 4 out of 50 stool samples analysed by PCR. All samples analysed by sequence analysis were identified as E. granulosus s.s. G1 genotype. Sequence comparison revealed revealed one or more-point mutations in different regions of the five samples.

Conclusion: E. granulosus s.s. G1 genotype, known as sheep strain, is common in the Ağrı, Türkiye. The controlled slaughter of livestock, especially sheep, and the avoidance of feeding hydatid cyst organs to dogs, together with public education, were necessary to prevent the spread of the disease.

Diagnosis and Treatment of Fasciola Infection with Endoscopic Retrograde Cholangiopancreatography (ERCP) in Ardabil, Northwest of Iran: Two Case Reports

Shabnam Asfaram, Behnam Sajedi, Behnam Mohammadi-Ghalehbin — Sun, 09 Jun 2024 20:01:27 +0430

Here we report biliary fascioliasis in two women 27 and 54 years old from Ardabil Province, Iran who presented with nausea, anorexia, weight loss, and pain between shoulder blades. Endoscopic ultrasound showed distal common bile duct strictures (CBD) and a lesion in its proximal. Endoscopic retrograde cholangiopancreatography (ERCP) was performed and live parasites were diagnosed and successfully managed into the duodenal lumen. The clinical findings of the patient improved after the procedure. This report emphasizes that the prevalence of Fasciola in Ardabil is predictable and this infection could be simultaneously diagnosed and treated by the ERCP.

Postpartum Spontaneous Rupture of a Primary Splenic Hydatid Cyst: A Case Report

Aabid Ashraf, Ritika Mittal — Sun, 09 Jun 2024 20:11:43 +0430

Hydatid disease is endemic in some regions of the world. Even in endemic regions, splenic hydatid cysts are rare, especially in pregnancy. The most serious presentation is intra peritoneal rupture, which is a surgical emergency. Exploration with splenectomy is the favoured management. We present a case of splenic hydatid cyst, detected late in pregnancy, with intraperitoneal rupture in the postpartum period. Cystic lesions located anywhere in the body in endemic regions could be hydatid cysts. Prompt treatment should be planned immediately on detection in order to prevent potentially serious complications like rupture.

The Hepatic Phase of Fascioliasis Complicated with Liver Bleeding: Presentation of Three Cases

Feyzullah Uçmak, Muhsin Kaya — Sun, 09 Jun 2024 20:12:39 +0430

Bleeding is a very rare complication of Fasciola species (F. hepatica or F. gigantica) infection. We present here three cases of subcapsular liver bleeding caused by the hepatic phase of Fasciola spp. infection in patients, two of whom were women, aged 22, 66, and 84 years in Diyarbakir Province, southeastern Turkey. They had symptoms of right upper quadrant pain (n = 3), nausea (n = 1), and vomiting (n = 2) for periods ranging from 6 hours to 15 days. All patients with clinical presentations ranging from moderate abdominal pain to hypovolemic shock and ischemic hepatitis were improved with supportive treatment without the need for surgery. They showed complete clinical and laboratory recovery after triclabendazole administration in their follow-up. In conclusion, Fasciola spp. infection should be considered in the etiology of bleeding from liver disease.

Ruptured Lung Hydatid Cyst Masquerading as a Transudative Parapneumonic Effusion: A Case Report

Brahmansh Singh, Kundan Nikit Mehta — Sun, 09 Jun 2024 20:20:44 +0430

Hydatid disease can virtually involve any organs, liver being the most common followed by lungs. Pleural effusion as a complication of pulmonary hydatid cyst is exceptionally rare and its diagnosis and treatment pose significant challenges. We present an intriguing case managed in Dr D Y Patil medical college and Hospital in west India in June 2023 involving a 70-year-old female who presented with symptoms of right-sided chest pain and acute-onset dyspnoea. Referred from a local hospital, a chest radiograph revealed the presence of right pleural effusion. Subsequent radiological investigations including a contrast enhanced CT at our centre exposed two large, well-defined hypodense lesions with fluid density, encased by thick enhancing walls, along with right-sided pleural effusion and hence a diagnosis of lung abscess with right pleural effusion (right parapneumonic effusion) was established. Despite ongoing care, clinical improvement eluded us. Thoracocentesis yielded a surprising revelation – the pleural fluid was transudative with visible hooklets and protoscolices, indicating a ruptured pulmonary hydatid cyst. The patient began albendazole treatment and received a CVTS consultation. They recommended a right lower lobe lobectomy, now scheduled for the near future.