Morphological Description, Phylogenetic and Molecular Analysis of Dirofilaria immitis Isolated from Dogs in the Northwest of Iran

Background: Dirofilariasis is a globally distributed arthropod-borne parasitic disease of mainly canids and felids. We evaluated to extend the knowledge of morpho-molecular characteristics and outer ultrastructure of Dirofilaria immitis isolated from Northwest of Iran. Methods: Overall, 67 filarial worms including 41 females and 26 males parasites were collected from the cardiovascular system of the 43 stray dogs in Meshkinshar, Ardebil Province, Northwest of Iran in 2017, and subjected to light and scanning electron microscopy (SEM) as well as carmine alum staining for morpho-molecular and identification. Molecular methods were used for confirmation of morphological findings by sequencing of Cyto-chrome c oxidase subunit I (cox1) gene. Results: The partial DNA sequencing of cox1 gene of adult parasites showed considerable homology and close proximity to the previously isolated from Kerman and Meshkinshahr, Iran. The lowest genetic variation and the highest intra-species variability was found in D. immitis and Dirofilaria repens, respectively. No similarity was identified between D. immitis nucleotide sequence and Wolbachia species as its endosymbiont bacteria. Conclusion: The SEM technique is an excellent tool for differential recognition of the parasite surface morphology and molecular techniques could differentiate and identify Dirofilaria spp. keywords: Dirofilaria immitis, Homology, Iran, Scanning electron microscopy (SEM

Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens

In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity

Fatty acid and retinol-binding protein: A novel antigen for immunodiagnosis of human strongyloidiasis

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity

Co-infection of Phlebotomus papatasi (Diptera: Psychodidae) gut bacteria with Leishmania major exacerbates the pathological responses of BALB/c mice

Clinical features and severity of the leishmaniasis is extremely intricate and depend on several factors, especially sand fly-derived products. Bacteria in the sand fly’s gut are a perpetual companion of Leishmania parasites. However, consequences of the concomitance of these bacteria and Leishmania parasite outside the midgut environment have not been investigated in the infection process. Herein, a needle infection model was designed to mimic transmission by sand flies, to examine differences in the onset and progression of L. major infection initiated by inoculation with “low” or “high” doses of Enterobacter cloacae and Bacillus subtilis bacteria. The results showed an alteration in the local expression of pro- and anti-inflammatory cytokines in mice receiving different inoculations of bacteria. Simultaneous injection of two bacteria with Leishmania parasites in the low-dose group caused greater thickness of ear pinna and enhanced tissue chronic inflammatory cells, as well as resulted in multifold increase in the expression of IL-4 and IL-1β and a decrease in the iNOS expression, without changing the L. major burden. Despite advances in scientific breakthroughs, scant survey has investigated the interaction between micro and macro levels of organization of leishmaniasis that ranges from the cellular to macro ecosystem levels, giving rise to the spread and persistence of the disease in a region. Our findings provide new insight into using the potential of the vector-derived microbiota in modulating the vertebrate immune system for the benefit of the host or recommend the use of appropriate antibiotics along with antileishmanial medicines

Detection of Echinococcus multilocularis in Carnivores in Razavi Khorasan Province, Iran Using Mitochondrial DNA

Echinococcus multilocularis causes alveolar echinococcosis, a serious zoonotic disease present in many areas of the world. The parasite is maintained in nature through a life cycle in which adult worms in the intestine of carnivores transmit infection to small mammals, predominantly rodents, via eggs in the feces. Humans may accidentally ingest eggs of E. multilocularis through contact with the definitive host or by direct ingestion of contaminated water or foods, causing development of a multivesicular cyst in the viscera, especially liver and lung. We found adult E. multilocularis in the intestine and/or eggs in feces of all wild carnivores examined and in some stray and domestic dogs in villages of Chenaran region, northeastern Iran. The life cycle of E. multilocularis is being maintained in this area by wild carnivores, and the local population and visitors are at risk of infection with alveolar echinococcosis. Intensive health initiatives for control of the parasite and diagnosis of this potentially fatal disease in humans, in this area of Iran, are needed

Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis

Laboratory diagnosis of sheep fasciolosis is commonly performed by coprological examinations; however, this method may lead to false negative results during the acute phase of the infection. Furthermore, the poor sensitivity of coprological methods is considered to be a paradox in the chronic phase of the infection. In this study, we compared the immunoreactivity of native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined their capabilities for the development of F. hepatica-specific immunoassays. Immunoreactivity and specificity of recombinant and native forms of F. hepatica antigens, including fatty acid binding protein (FABP), glutathione-S-transferase (GST), and cathepsin L-1 (CL1), in parallel with native forms of FABP and GST, were studied for serodiagnosis of the chronic form of sheep fasciolosis, individually or in combination with each other by enzyme-linked immunosorbent assays (ELISA). The correlation of the findings was assessed by receiver-operator characteristic (ROC); furthermore, the specificity and sensitivity were assessed by Youden's J. Serologic cross-reactivity was evaluated using samples from healthy sheep (n = 40), Fasciola-infected sheep (n = 30), and sheep with other parasitic infections (n = 43). The FABPs were determined to be greater than 95% sensitive for F. hepatica serodiagnosis. The most desirable diagnostic recombinant antigen was rCL1, which showed 100% sensitivity and 97% specificity in ELISA and was capable of discriminating the positive and negative samples by maximum Youden's J results. We conclude that rCL1 can be used for routine serodiagnosis of chronic fasciolosis. Thus, it could be advantageous in development of immunoassays for screening of ovine herds in fasciolosis-endemic areas and as a reliable agent for detection of fasciolosis in non-endemic regions

Detection and genetic characterization of Echinococcus granulosus mitochondrial DNA in serum and formalin-fixed paraffin embedded cyst tissue samples of cystic echinococcosis patients.

Cystic echinococcosis (CE) is a worldwide zoonotic disease caused by the larval stage of Echinococcus granulosus. We investigated the presence of E. granulosus-specific DNA in the serum of CE patients by detecting the cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) mitochondrial genes. Serum and formalin-fixed paraffin embedded (FFPE) cyst tissue samples of 80 CE patients were analyzed. The extracted DNA of samples was submitted to PCR amplification of cox1 and nad1 genes, and products were sequenced and genotyped. Nineteen (23.8%; 95% CI 15.8-34.1) serum and 78 (97.5%; 95% CI 91.3-99.3) FFPE cyst tissue samples were successfully amplified with at least one gene. Echinococcus DNA was detected in the sera of 15.0% (95% CI: 8.8-24.4) and 10.0% (95% CI: 5.2-18.5) and in cyst tissue of 91.3% (95% CI: 83.0-95.7) and 83.8% (95% CI: 74.2-90.3) of 80 patients by cox1 and nad1 gene, respectively. Four genotypes of E. granulosus were distinguished in the CE patients, with predominance of genotype G1, followed by G3, G2, and G6. The finding of E. granulosus DNA in 23.8% of serum samples from CE patients confirmed that E. granulosus releases cell-free DNA into the circulatory system, but quantities may be inadequate for the diagnosis of CE. Genotype G1 predominance suggests the sheep-dog cycle as the primary route of human infection

Frequency of Toxoplasma gondii in HIV Positive Patients from West of Iran by ELISA and PCR

Background: Toxoplasma gondii, the obligate intracellular parasite is life threatening in AIDS patients. Diagnosis of toxoplasmosis is based on serological methods especially increasing of IgM and IgG titers, but finding of parasite or its components (antigenemia) may be beneficial method in order to detection of acute toxoplasmosis in immunocompromised patients. Methods: Ninety-four serum samples from HIV positive patients were collected from Sanandaj, Kordistan west of Iran. These patients were lived in Sanandaj of whom 26 were prisoners infected with HIV virus in prison. Toxoplasma gondii antibodies were determined by IgG ELISA. T. gondii antigen was identified by capture-ELISA. PCR was performed on samples with T. gondii antigenemia. CD4+ T cellscounts had been determined by flowcytometry and were obtained from records of each patient. Results: Among the examined HIV seropositive individuals, 19.1% (18/94) and 5.3% (5/94) were positive for Toxoplasma-IgG and antigenemia, respectively. Besides,one of the samples was positively detected by PCR method. Mean age of participants was 37.9 ± 9.5 year. Prevalence of IgG antibody and antgenemia was higher in age group of 40-50 years old. The Mean of CD4+ T cells counts of participants (total of HIV+ patients, IgG positive patients and patients with antigenemia) was 699.2 ± 345.2, 655.1 ± 237.9 and 620.2 ± 215.1 respectively. Conclusion: Capture-ELISA and PCR could confirm the T. gondii acute infection in HIV positive patients. For precise diagnosis of acute toxoplasmosis in HIV positive patient, performance of more studies based on more sensitive types of PCR is suggested.

Fatty acid and retinol-binding protein: A novel antigen for immunodiagnosis of human strongyloidiasis.

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity