Canine microfilaraemia in some regions of Iran.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are vector-borne zoonotic parasites which affect mainly dogs and humans worldwide. In Iran, information about the distribution of those nematodes is scant in several regions. Therefore, we investigated the prevalence of these filarial parasites in stray dogs from five Iranian provinces where no information about these parasites is available. METHODS: Blood samples were collected from 344 stray dogs in five provinces of Iran (i.e. Mazandaran, Gilan, Esfahan, Qazvin and Loresan). The presence of microfilariae was assessed using direct smear, modified Knott's test, molecular detection of filarial DNA (cox1 gene) and Wolbachia endosymbiont of parasitic nematodes (ftsZ gene) by conventional PCR (cPCR). All of the PCR products were sequenced and phylogenetic analysis was performed. RESULTS: In total, 75 dogs (21.8%) were found to be positive for D. immitis by cPCR. Infection was detected in all provinces, with the highest prevalence in Gilan province (22/28; 78.6%). Acanthocheilonema reconditum was diagnosed in five dogs (1.4%) from three provinces (i.e. Esfahan, Mazandaran, Gilan). Two dogs were infected with both parasites and three were only infected with A. reconditum. Dirofilaria repens infection was not found in the examined population. Representative sequences of the D. immitis cox1 gene from dogs from the northern provinces (Mazandaran, Gilan, Qazvin) were grouped together and distinctly separate from the ones from western and central provinces (Lorestan and Esfahan), suggesting that different nematode populations are present in the country. CONCLUSION: The data reported herein fill existing gaps in knowledge about canine filarial infection in two Iranian provinces and record the highest prevalence of D. immitis ever reported in the country (i.e. 78.6%). A geographical review of the literature about Dirofilaria spp. and A. reconditum infections in dogs and humans has also been summarized, indicating that D. immitis and D. repens are distributed in 22 of 31 provinces in Iran, whereas A. reconditum is present in fewer regions. Effective control strategies are advocated for owned dogs, and a national program for the management of stray dogs is needed to minimize the risk of infection in animals and humans.

Echinococcoses in Iran, Turkey, and Pakistan: Old Diseases in the New Millennium.

Echinococcosis is considered a cosmopolitan zoonosis caused by different species of small taeniid tapeworms of the genus Echinococcus and is regarded as a neglected zoonosis. Cystic and alveolar echinococcoses are endemic diseases of Tibetan, Pamir, and Iranian plateaus. All of the countries within the Iranian plateau are affected by echinococcosis. Pakistan, Turkey, and Iran are the three most populous countries of the region, in which echinococcosis is highly endemic. The three neighboring countries share strong cultural and socioeconomic ties. The present study aimed to provide a broad review of the status of cystic and alveolar echinococcosis, summarizing the current knowledge about geographical distribution, molecular epidemiology, and transmission dynamics of Echinococcus granulosus sensu lato and Echinococcus multilocularis in this region. Additionally, we aimed to understand disease burden and risk factors as basic requirements for establishing a surveillance system and planning prevention and control programs. A considerable body of information is available on different aspects of echinococcosis in this region; however, several information and research gaps need to be filled before planning control programs. None of the countries in the region have an elaborate echinococcosis control program. Effective control programs require multi/intersectoral coordination within a One Health approach with a long-term political and administrative commitment and enhanced international collaboration among the three countries.

Phylogenetic Analysis of C Type Lectin from Toxocara canis Infective Larvae and Comparison with the C Type Lectin Family in the Immune System of Mouse and Human.

BACKGROUND: C type lectin (CTL) family is a type of calcium-dependent proteins found in vertebrates and invertebrates. The objective of this study was to perform a comparative analysis and phylogenetic inferring for understanding the similarities and differences of carbohydrate recognition domain (CRD) domain of Toxocara canis CTL and other nematodes, and similar C type lectin involved in the immune system of mouse and human as their host. METHODS: The female T. canis was retrieved from the 2-6 months puppies (Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, 2015). To collect T. canis eggs, the worms were cultured for 5 d until they were embryonated. The hatching process was accelerated for collecting the stage 2 larvae, and the larvae were cultured for a week. A cDNA library was made from the total mRNA of T. canis infective larvae. The PCR amplification for C type lectin gene was performed and the amino acids were analyzed using the alignment method and the construction of phylogenetic tree. RESULTS: The suspension sample maintained at 30 ºC for four weeks could embryonate 90%-100% of eggs. T. canis CTL gene was 657 bp in length and encoded a protein with 219 amino acids. The CTL of species of Strongylida order were closely placed in the tree, whereas the members of Ascaridida orders were located in a separate branch. High levels of similarity (36%-44%) and conservation of C type lectin from T. canis with mouse and human C type lectins. Its C type lectin showed a higher similarity with asialoglycoprotein receptor (ASGPR), macrophage lectin, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), MINCLE receptor of mouse and human. CONCLUSION: Analysis of CRD domain of C type lectin protein could make a better understanding of their role in the interaction of nematode parasite with their hosts.

Design and construction of a new recombinant fusion protein (2b2t+EPC1) and its assessment for serodiagnosis of cystic echinococcosis.

The immunodiagnostic tests for cystic echinococcosis (CE) are mostly serological tests based on ELISA that use hydatid cyst antigens for primary screening because of its simple preparation and availability. The challenge to develop new serological methods (as compared to those based on the hydatid cyst fluid antigens) to meet the gold standard remains. Appropriate sources of antigenic material are necessary for application to improve the efficacy of immunodiagnostic tests at a population level. In the current study, a fusion protein containing the coding sequence of antigen B2t and two sequences of EPC1 antigen with some modifications was reconstructed. Using bioinformatics tools, these sequences were joined together by applying the sequence of a rigid α-helix-forming linker to obtain an appropriate structure of a fusion protein. Synthetic recombinant fusion protein was expressed using pET28a as a vector and evaluated by indirect ELISA test for sera from patients with hepatic CE and other parasitic infections. The sensitivity of the fusion protein was lower (88.46%) than the available ELISA kit (96.15%). However, the differences in sensitivity were not statistically significant as compared to the recombinant fusion peptide with the commercial kit (p = 0.269). The specificity of the recombinant fusion protein (95.45%) was not significantly lower than the commercial kit (96.59%; p = 1.000). Moreover, surprisingly there was no difference in the cross-reactivity values of performance between the recombinant-ELISA and commercial kit. The positive and negative predictive values of the recombinant antigen were achieved as 92% and 93.33%, respectively, while for the commercial kit, they were obtained as 94.33% and 97.70%, respectively. In conclusion, as an early evaluation of these antigens the performance of our recombinant fusion protein in ELISA is relatively promising. Although, it seemed that this peptide with specific antigenic epitopes might be more appropriate for the serological evaluation of CE by use of bioinformatics tools, our findings showed that cross-reactions and a negative reaction could occur in clinical performance. This fusion protein may have utility for diagnosis in humans, but further evaluation is needed using the WHO ultrasound classification for CE.

Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis.

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis. The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 µg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites.

The Taxonomic Position and Phylogenetic Relationship between Digramma interrupta and Ligula intestinalis Based on Morphological and Molecular Diagnosis.

BACKGROUND: The position of Digramma interrupta remains disputable as it was raised by Cholodkovsky from Ligula alternans. This study aimed to survey the evolutionary relationships and the taxonomic position of D. interrupta and L. intestinalis. It also intended to support or reject the validity of D. interrupt as an independent genus and its correlation with L. intestinalis on the basis of their morphological characteristics and a study on molecular data. METHODS: Overall, 1301 fish varieties, including 883 Alburnoides bipunctatus and 418 Abramis brama, were collected from north and north-western parts of Iran. A. bipunctatus samples were obtained from fresh water sources of the Maragheh dam (northwest) and the Ramesar Lake (north). Moreover, samples of A. brama were captured from the Aras Dam (northwest) and the Bandar-e-Anzali lagoon (north). PCR was used to generate a fragment spanning two independent ITS-inclusive parts: ITS1-5.8S and ITS2 with two pairs of primers. RESULTS: Nucleotide variation between L. intestinalis and D. interrupta samples amounts to about 3% to 7%. Between samples of L. intestinalis and GenBank data, and also between D. interrupta specimens and GenBank data, the diversity was seen for about 1% to 3%. Moreover, about 1% to 4% nucleotide variation was seen only in L. intestinalis samples caught from the same host, which could be supplementary to the presence of a species and/or strains in this genus. CONCLUSION: Maybe D. interrupta was just a rare diplogonadic form of the Ligula species, not a different genus and not synonymous with the Ligula genus, but only another species of the Ligula genus.

Molecular and morphological characterization of Parabronema skrjabini of sheep and goats at three different geographical zones in Iran.

Parabronema skrjabini is a spirurid nematode of the family Habronematidae that lives in the abomasum of ruminants such as sheep and goats. The purpose of this study was to investigate the molecular and morphological aspects of Parabronema skrjabini in sheep and goats in Iran. The worms were collected from these animal species from three different regions. An internal transcribed spacer 2 ribosomal DNA (ITS2-rDNA) fragment of Parabronema skrjabini was amplified by polymerase chain reaction (PCR) using a pair of specific primers (Para-Ir-R and Para-Ir-F). Morphological studies based on the body length, the frontal shield, spicules of male and egg dimensions were performed. ITS2-rDNA sequences were between 167 and 299 bp in different isolates. ITS2 homology in different isolates was between 68% and 77% compared with the sequence data in GenBank. Morphological results showed that the average length of male and female worms in sheep were 16.5 mm and 36 mm and in goats 16 mm and 35.5 mm, respectively. The average length of the small and large spicules in sheep were 657.5 µm and 304.07 µm and in goats 653.08 µm and 302.66 µm, respectively. To our knowledge, this is the first study in the world exploring the genetic diversity of Parabronema in sheep and goats. Add this sentence in discussion: the low ITS2-rDNA identity in different isolates from Iran as compared to the reference sequence in GenBank (68-77%) raise questions regarding the species identity of the parasites isolated in Iran.

A Study of Cross-Reactivity Between Recombinant EPC1 Antigen of Echinococcus granulosus in Serum from Patients with Confirmed Cystic Echinococcosis Infection and Other Parasitic Infections.

A standardized test for the serodiagnosis of cystic echinococcosis (CE) remains an important challenge because of the problems in specificity and sensitivity of the available commercial kits and lack of proper evaluation of antigen. Using appropriate sources of antigenic material is crucial in improvement of the serological methods such as enzyme-linked immunosorbent assay (ELISA). This study was conducted to evaluate the performance of protein named Echinococcus protoscolex calcium binding protein EPC1 for the detection of antibodies in sera from patients with CE. Expressed and purified recombinant protein EPC1 (rEPC1) was used as antigen in ELISA method. Characterization of the rEPC1 antigen was evaluated using the serum of 25 patients with both surgical and imaging confirmed CE and 25 healthy donors as negative controls. Also, a panel of sera including chronic toxoplasmosis (IgG positive), strongyloidosis, fascioliasis, toxocariasis, and kala azar were used and patients with related parasites were confirmed by medical laboratories or clinically by research centers using microscopy or specific ELISA. rEPC1 showed relatively promising performance in total IgG ELISA for the detection of antibodies in sera from the negative controls, and the cut off value 0.4 units of optical density at 490 nm was calculated for ELISA. In this study, sensitivity of 100%, specificity of 93.7, positive predictive value of 92.6%, and negative predictive value of 100% were calculated for rEPC1. On the other hand, commercial ELISA kit based on the native antigen B of Echinococcus granulosus had sensitivity of 96.2% and specificity of 96.8%. No significant difference was found for sensitivity or specificity between the rEPC1 and commercial kit. However, rEPC1 may be a valuable antigen for diagnosis of human CE.

Genotype and Phenotype of Echinococcus granulosus Derived from Wild Sheep (Ovis orientalis) in Iran.

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.

Phylogenetic Study of Haemonchus Species from Iran Based On Morpho-Molecular Characterization.

BACKGROUND: Haemonchosis has a negative effect on the farming industry throughout the world, especially in the tropic and sub-tropic countries. The present study was carried out to differentiate Haemonchus species from its main hosts in Iran, including sheep, goat and camel. METHODS: The identification took place based on the morphometrics of the spicules and molecular characters. Two hundred seventy adult male nematodes were collected from the abomasums of different ruminants (90 samples from each animal) at the slaughterhouses from different localities in Iran. Samples were morphologically identified according to the spicules' morphometric measurements. In the section on molecular study, 10 samples of each Haemonchus isolates were genetically examined. A simple PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the second internal transcribed spacer of ribosomal DNA (ITS2-rDNA) were described to confirm the PCR results. RESULTS: PCR-RFLP profile obtained from the restriction enzyme HPa1 in H. contortus and H. longistipes indicated 1 (278 bp) and 2 (113 and 135 bp) different fragments, respectively. The morphological parameters clearly distinguish H. contortus from H. longistipes. Moreover, regarding the ITS2-rDNA, sequences of 295 bp and 314 bp were obtained from H. contortus and H. longistipes, respectively. CONCLUSION: The genotypic results are in agreement with the phenotypic findings of both species.

Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody.

BACKGROUND: Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method. METHODS: Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA. RESULTS: Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA. CONCLUSION: DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis.

Improved serodiagnosis of hydatid cyst disease using gold nanoparticle labeled antigen B in naturally infected sheep.

BACKGROUND: Cystic echinococcosis caused by the metacestode of Echinococcus granulosus is a major problem in both humans and domestic animals health. Therefore, a standardized and approachable diagnostic tool (rapid tests) for the serodiagnosis of cystic echinococcosis (CE) is still needed. METHODS: In the present work, antigen B labeled with gold nanoparticles was used to detect antibodies against hydatid cyst disease. The prepared antigen B was analyzed by SDS-PAGE. Tetra chloroauric acid (HAuCl4) was used to produce colloidal gold and antigen B labeled by gold nanoparticles, then it was tested by using rabbits antisera and sera from naturally infected sheep. The labeled antigen B was evaluated using Dot-immunogold staining (Dot-IGS) method. RESULTS: Electrophoretic pattern of hydatid cyst fluid showed the quality of bands in the condensed fluid is better than crude fluid. SDS-PAGE analysis cyst fluid and antigen B revealed three specific protein bands that were detected at molecular weights of 24, 30 and 40 kDa that all are the subunits of antigen B. Evaluation of antigen B labeled by gold nanoparticles by using Dot-IGS technique showed 1/1 and 1/50 dilutions in comparison with another has the best immunoreaction. In this method, nanoparticles produced a typical purple color, when they binded to the strip at the site of immunoreaction. CONCLUSION: Therefore, using gold nanoparticles is a good candidate for detection of helminthiasis, also as selective tools of early detection, simple and cost-effective, regardless of specific skills and equipment with optimal durability.

Immunoproteomics approach for EPC1 antigenic epitope prediction of G1 and G6 strains of Echinococcus granulosus.

It is important to establish the diagnosis of cystic echinococcosis (CE) infection and begin control management. Currently, it is difficult to make an accurate diagnosis of CE without the availability of an accurate test, which requires the use of sensitive and specific antigens. Using recombinant antigens the sensitivity and specificity of the CE serology assays could be improved considerably. Recently, a highly antigenic protein named EPC1was characterized and isolated from an Echinococcus granulosus protoscoleces. The current study was designed to assess the sequences of EPC1 isolated from different intermediate hosts of E. granulosus. In addition, identification of a highly antigenic linear B cell epitope was found within EPC1 antigen candidate. The EPC1 sequence contains coding and non-coding regions and was compared between two predominant strains (G1 and G6) in Iran. Sequence polymorphism was not found in protein coding regions, suggesting that these regions may be useful for identification of protein expression as an antigen. The average antigenic activity for the whole protein is above 1.1, and hydrophobicity below 0 indicates that it is hydrophilic. Structural analysis showed alpha helical regions in amino acids 6-25, 35-44, 52-62, and 72-78. Nine B cell epitope residues were identified out of 67 total residues. The identity of EPC1 sequence in both G1 and G6 genotypes affects the antigenic efficacy of EPC1and suggests the recombinant protein will be useful in serological assays in the regions where the two strains are prevalent.

Morphological and molecular characterisation of Echinococcus granulosus from goat isolates in Iran.

Hydatidosis is considered to be an important economic and human public health problem in Iran, where a variety of animals act as intermediate hosts. There is limited information about the genotypes of Echinococcus granulosus in goats. In this study, 20 isolates of E. granulosus obtained from goats were characterised by mitochondrial DNA sequencing and morphology of the metacestode. The mitochondrial cytochrome oxidase 1 sequences were evaluated, and the sequence analysis indicated two genotypes within the isolates. 17 samples were identified as G1 strain, and 3 isolates were identified as G6 strain. The results of the morphological studies support the findings of the molecular studies. Two types of rostellar hooks were observed in the goat isolates, in agreement with the strain identification. Type 1 hooks were morphologically similar to those of the common sheep strains, whereas the dimensions of the hooks in type 2 were similar to those normally found in the camel strain. The morphological results suggest that Echinococcus of goat origin is phenotypically similar to either the sheep (G1) or the camel (G6) strains. Further, these results suggest that the transmission of the G1 genotype between sheep and goats seems to be the predominant mode of transmission, but further work is required to verify this.

Mutation in pfmdr1 gene in chloroquine-resistant Plasmodium falciparum isolates, Southeast Iran.

OBJECTIVES: The main objective of the present study was to detect point mutations at positions 86, 184, 1034, 1042, and 1246 of the Plasmodium falciparum multidrug resistance gene (pfmdr1) in blood samples collected from malaria patients in Chabahar, a harbor city located in Southeast Iran. METHODS: Twenty-six blood samples from patients infected with P. falciparum, who had a chloroquine (CQ) response failure, were collected pre-treatment. Following treatment with CQ, drug susceptibility was assessed using an in vivo test. Molecular detection of single nucleotide polymorphisms (SNPs) was carried out using the LightCycler hybridization probe assay. RESULTS: The pfmdr1 N86Y mutation was found in six isolates (23.1%). Mutations at the four other positions were not observed in any isolates. CONCLUSION: The present study showed no mutation at codon positions 184, 1034, 1042, and 1246 of pfmdr1 in any of the Iranian P. falciparum isolates; thus these alleles cannot serve as markers for CQ resistance in Iran.