Cystic Echinococcosis of Camels: 12S rRNA Gene Variation Revealed Changing Pattern of Genetic Diversity Within Echinococcus granulosus sensu lato in the Middle East and North/Sub-Saharan Africa.

Cystic echinococcosis (CE) is one of the most widespread zoonotic diseases, with considerable public health and economic importance. Camels play a significant role in transmission cycle of Echinococcus granulosus especially, in the Middle East and North Africa (MENA). The present study aimed to identify the genetic variation and haplotype distribution of camel isolates of E. granulosus sensu lato using all existing E. granulosus mitochondrial DNA data from camels in different parts of the world. Sequence data from 1,144 camel isolates of E. granulosus s.l. available in the NCBI GenBank including 57 camel hydatid cysts collected in central Iran were used to analyze the nature of genetic variation within the camel isolates of E. granulosus s.l. in MENA region. Fifty-seven camel isolates were also PCR-sequenced on mitochondrial 12S rRNA gene. Haplotype network analysis revealed seven different haplotypes clustered into four major groups. E. intermedius G6 was identified as the most commonly represented genotype in camels followed by G1. Mitochondrial 12S rRNA gene sequence analysis on 57 camel isolates identified three different genotypes, including E. intermedius/G6 (35/57, 61.4%), E. granulosus sensu stricto/G1-G3 (21/57, 36.8%) as well as one isolate identified as E. ortleppi/G5 (1/57, 1.8%). The number of base substitutions per site over 420 positions of partial 12S rRNA gene sequences were shown as 0.000 and 0.004 for E. intermedius (G6) corresponding to the Middle East and sub-Saharan isolates, respectively. Camel isolates of E. granulosus in the MENA region present moderate genetic diversity (Hd = 0.5540-0.6050). The Middle East isolates demonstrated a more diverse population than the North/sub-Saharan isolates, where six out of seven 12S rRNA haplotypes were identified in the former region. E. intermedius (G6 genotype) was shown to be the most common species in the world camel population. In conclusion, camels showed to be an important intermediate host species in the MENA region with different patterns of genetic variation between the Middle East and Africa.

Genetic Profile of Hydatid Cysts in Patients with Multi-Organ Involvement: Mixed Infections by Different Strains.

Our knowledge on the susceptibility of humans to different genotypes of the zoonotic tapeworm Echinococcus granulosus and the genetic make-up of the cysts in multi-organ involvements is limited. This study aimed to identify the genotype profile of E. granulosus in patients undergoing hydatid surgery in an endemic area for cystic echinococcosis (CE) in southeastern Iran. Individuals who underwent hydatid cyst surgery were included in this study. Protoscoleces/germinal layers from each individual isolate were washed and kept in -20°C until use. Genotyping was carried out using PCR-sequencing of two mitochondrial CO1 and ND1 genes. Molecular phylogeny and haplotype network analysis of the human isolates were carried out using sequence data obtained from this study and National Center for Biotechnology Information (NCBI) databases. Forty-two patients (23 women and 19 men) participated in the study; the mean age was 43 years. Eighteen (42.9%) and 24 (57.1%) patients were infected by E. granulosus sensu stricto (G1-G3) and Echinococcus intermedius (G6 genotype), respectively. Molecular study showed mixed infection of G1 (in the liver and right lung) and G6 (in left lung) in a patient. The study showed a significantly high proportion of CE patients infected with the G6 genotype particularly in the southern parts of the province. In the present study a human CE patient infected by two species/genotypes of E. granulosus sensu lato is documented.

Genetic and morphometric categorization of Taenia ovis from Sheep in Iran.

Little is known about the genetic and morphological characters of Taenia ovis. The purpose of the present study was to characterize sheep isolates of T. ovis using rostellar hook morphometry as well as mitochondrial genes sequence analysis. Ninety sheep specimens of Cysticercus ovis were collected from 18 slaughterhouses in Iran. The mean ± s.d. for total length of large and small hooks were 174.1 ± 6.4 and 116.7 ± 5.4 µm, respectively. CO1 and 12S rRNA sequence analysis showed 11 and nine haplotypes, respectively. The level of pairwise nucleotide variations between individual haplotypes of CO1 and 12S rRNA genes were 0.3-1.1 and 0.2-1.0%, respectively. Level of nucleotide variation in CO1 and 12S rRNA between T. ovis haplotypes from present study and eight other Taenia species was found to be 11.3-17.8 and 5.3-16.3%, respectively. Phylogenetic analysis clustered all T. ovis isolates into a single clade comprised of the all CO1 and 12S rRNA haplotypes. CO1 nucleotide difference between T. ovis ovis and T. asiatica was 13.6% that is lesser than the corresponding difference between T. ovis ovis and T. ovis krabbei, warranting the designation of two separate species as T. ovis and T. krabbei. Interclass correlation coefficients showed that there was no significant association between rostellar hook length variation and the variability of the mitochondrial genes.

A conventional PCR for differentiating common taeniid species of dogs based on in silico microsatellite analysis.

Canine taeniids are among the major tapeworms with remarkable medical and economic significance. Reliable diagnosis and differentiation of dog taeniids using simple and sensitive tools are of paramount importance for establishing an efficient surveillance system. Microsatellites as abundant unique tandem repeats of short DNA motifs are useful genetic markers for molecular epidemiological studies. The purpose of the present study was to find a primer pair for rapid differentiation of major tapeworms of dogs, Taenia hydatigena, T. multiceps, T. ovis and Echinococcus granulosus, by screening existing nucleotide data. All the mitochondrial genome records as well as non-coding ITS1 sequences of Taeniidae species were downloaded from Nucleotide database from NCBI. For prediction and analysis of potential loci of STR/SSR in ITS1 as well as mitochondrial regions, we used ChloroMitoSSRDB 2.0 and GMATo v1.2. software. Different tapeworm species were categorized according to different motif sequences and type and size of each microsatellite locus. Three primer sets were designed and tested for differentiating taeniid species and evaluated in a conventional PCR system. Four taeniid species were successfully differentiated using a primer pair in a simple conventional PCR system. We predicted 2-19 and 1-4 microsatellite loci in ITS1 and mitochondrial genome, respectively. In ITS1, 41 Di and 21 Tri motifs were found in the taeniids while the majority of the motifs in the mitochondrial genome were Tetra (89) and Tri (70). It is documented that the number and diversity of microsatellite loci is higher in nuclear ITS1 region than mostly coding mitochondrial genome.

Evaluation of Microsatellites Markers to Discriminate Four Main Taeniid Tapeworms of Dogs.

BACKGROUND: Echinococcus granulosus, Taenia multiceps, Taenia ovis and Taenia hydatigena are among the most prevalent taeniid species of dogs. These tapeworms infect ruminant and humans as intermediate hosts and domestic/wild carnivores as the definitive hosts. Molecular tools using hypervariable microsatellite regions might provide more information about parasite variation. Highly variable and specific tools are needed for transmission tracking studies of canine echinococcosis as an essential element for implementation of hydatid control programs. Suitable microsatellite markers used so far are EmsJ, EmsK, EmsB, EMms1, Egmsca1, Egmsga1, U1 snRNA. The purpose of the present study was to determine the microsatellite variability of EmsB as well as six other microsatellites in major taeniid species infecting dogs in Iran. METHODS: Twenty isolates of each of the four Taeniidae tapeworms were collected from sheep during routine veterinary inspection in Tehran, Alborz and Kerman provinces from October 2010 to May 2011. After DNA extraction, PCR was set up with optimum conditions using specific primers for each individual microsatellite marker. All the PCR products were evaluated by agarose gel electrophoresis. We used SDS-PAGE for evaluating patterns of PCR products in the tapeworms. RESULTS: E. granulosus as well as Taenia species could be differentiated based on EmsB microsatellite patterns. The electrophoresis patterns of two taeniid genera were readily distinguishable. EmsB could be specifically used in epidemiological studies of canine echinococcosis. CONCLUSION: Different patterns of EmsB proved this microsatellite marker as a reliable tool for epidemiological studies on canine echinococcosis.

Morphometric and Molecular Study of Fasciola Isolates from Ruminants in Iran.

OBJECTIVE: The purpose of the present study was morphometric and molecular characterization of Fasciola isolates from ruminants in Iran. METHODS: Flukes were collected from the livers of 54 naturally infected sheep and cattle. The proportion of body length to width (L/W) of each fresh fluke was measured using a digital caliper. We employed receiver operating characteristic (ROC) curve analysis to explore the reliability of L/W for differentiating the two species. Polymerase chain reaction (PCR)-sequencing was performed on ribosomal Internal Transcribed Spacers (ITS) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes. The sequences were then analyzed and phylogenetic relationships were investigated. RESULT: Forty-eight out of 54 isolates (88.9%) were identified as F. hepatica and four isolates (7.4%) as F. gigantica. All the sheep isolates were F. hepatica, while 4 out of 10 cattle were infected with F. gigantica. The morphometric study revealed an L/W ratio of 1.2 to 6.5 in Fasciola isolates with significantly higher L/W ratio in F. gigantica (p<0.00). According to the ROC curve analysis, the L/W value of 3.55 was regarded as the critical value to discriminate between the two species. CONCLUSIONS: Findings of the present study indicate the presence of both Fasciola species in southeastern Iran. The phylogenetic analysis revealed two different clades representing F. hepatica and F. gigantica. The two isolates in this study were described as Fasciola sp. The mitochondrial DNA of these isolates were similar to F. hepatica, while their ITS fragments were identical to F. gigantica.

Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN.

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene.

Genetic variability of Taenia saginata inferred from mitochondrial DNA sequences.

Taenia saginata is an important tapeworm, infecting humans in many parts of the world. The present study was undertaken to identify inter- and intraspecific variation of T. saginata isolated from cattle in different parts of Iran using two mitochondrial CO1 and 12S rRNA genes. Up to 105 bovine specimens of T. saginata were collected from 20 slaughterhouses in three provinces of Iran. DNA were extracted from the metacestode Cysticercus bovis. After PCR amplification, sequencing of CO1 and 12S rRNA genes were carried out and two phylogenetic analyses of the sequence data were generated by Bayesian inference on CO1 and 12S rRNA sequences. Sequence analyses of CO1 and 12S rRNA genes showed 11 and 29 representative profiles respectively. The level of pairwise nucleotide variation between individual haplotypes of CO1 gene was 0.3-2.4% while the overall nucleotide variation among all 11 haplotypes was 4.6%. For 12S rRNA sequence data, level of pairwise nucleotide variation was 0.2-2.5% and the overall nucleotide variation was determined as 5.8% among 29 haplotypes of 12S rRNA gene. Considerable genetic diversity was found in both mitochondrial genes particularly in 12S rRNA gene.

Genetic characterization of Echinococcus granulosus from a large number of formalin-fixed, paraffin-embedded tissue samples of human isolates in Iran.

Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, presents an important medical and veterinary problem globally, including that in Iran. Different genotypes of E. granulosus have been reported from human isolates worldwide. This study identifies the genotype of the parasite responsible for human hydatidosis in three provinces of Iran using formalin-fixed paraffin-embedded tissue samples. In this study, 200 formalin-fixed paraffin-embedded tissue samples from human CE cases were collected from Alborz, Tehran, and Kerman provinces. Polymerase chain reaction amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 gene were performed for genetic characterization of the samples. Phylogenetic analysis of the isolates from this study and reference sequences of different genotypes was done using a maximum likelihood method. In total, 54.4%, 0.8%, 1%, and 40.8% of the samples were identified as the G1, G2, G3, and G6 genotypes, respectively. The findings of the current study confirm the G1 genotype (sheep strain) to be the most prevalent genotype involved in human CE cases in Iran and indicates the high prevalence of the G6 genotype with a high infectivity for humans. Furthermore, this study illustrates the first documented human CE case in Iran infected with the G2 genotype.

High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato.

Reliable and rapid genotyping of large number of Echinococcus granulosus sensu lato isolates is crucial for understanding the epidemiology and transmission of cystic echinococcosis. We have developed a method for distinguishing and discriminating common genotypes of E. granulosus s.l. (G1, G3, and G6) in Iran. This method is based on polymerase chain reaction coupled with high resolution melting curve (HRM), ramping from 70 to 86 °C with fluorescence data acquisition set at 0.1 °C increments and continuous fluorescence monitoring. Consistency of this technique was assessed by inter- and intra-assays. Assessment of intra- and inter-assay variability showed low and acceptable coefficient of variations ranging from 0.09 to 0.17 %. Two hundred and eighty E. granulosus s.l. isolates from sheep, cattle, and camel were used to evaluate the applicability and accuracy of the method. The isolates were categorized as G1 (93, 94, and 25%), G3 (7, 4, and 4%), and G6 (0, 2, and 71%) for sheep, cattle, and camel, respectively. HRM results were completely compatible with those obtained from sequencing and rostellar hook measurement. This method proved to be a valuable screening tool for large-scale molecular epidemiological studies.

Cytochrome c oxidase subunit 1 and 12S ribosomal RNA characterization of Coenurus cerebralis from sheep in Iran.

Taenia multiceps is a widely distributed zoonotic tapeworm of canids. The larval stage of the parasite (Coenurus) occurs in sheep, goat and cattle and has been rarely reported from humans. This study investigated genetic variability of two mitochondrial genes in 102 isolates of T. multiceps. Metacestodes were collected from brains and hearts of sheep in Tehran and Qom provinces of Iran. DNA of each isolate was extracted and used for PCR amplification of cytochrome c oxidase subunit I (CO1) and 12S ribosomal DNA (12S rRNA) genes. All amplicons were sequenced and the sequence data were analyzed using NCBI Blast and BioEdit. Phylogenetic trees and pairwise calculations were obtained by using Mega5 software. In total 7 and 25 representative haplotypes were differentiated for CO1 and 12S rRNA genes, respectively. For CO1 sequences 11 segregation sites within 7 haplotypes were observed. For 12S rRNA sequences a total of 32 segregation sites were observed in 25 haplotypes. CO1 gene displayed lower diversity than 12S rRNA gene with an overall nucleotide variation of 3.0% for CO1 vs. 7.2% for 12S rRNA. Pairwise comparisons among 7 haplotypes in CO1 and 12S rRNA genes showed the level of nucleotide differences 0.3-2.5% and 0.2-4.0%, respectively. A high degree of genetic variation was found in the isolates of T. multiceps in Iran. Additional molecular studies are required on the parasite from other intermediate hosts.

Morphometric Analysis of Larval Rostellar Hooks in Taenia multiceps of Sheep in Iran and Its Association with Mitochondrial Gene Variability.

BACKGROUND: The purposes of the present study were morphometric characterization of rostellar hooks of Taenia multiceps and to investigate the association of hook length variation and the variability within two mitochondrial genes of sheep isolates of the parasite. METHODS: Up to 4500 sheep brains were examined for the presence of C. cerebralis. Biometric characters based on the larval rostellar hook size were measured for each individual isolate. Representative mitochondrial CO1 and 12S rRNA gene sequences for each of the isolates were obtained from NCBI GenBank. Morphometric and genetic data were analyzed using cluster analysis, Interclass Correlation Coefficient (ICC) and random effects model. RESULTS: One hundred and fourteen sheep (2.5%) were found infected with the coenuri. The minimum and maximum number of scoleces per cyst was 40 and 550 respectively. Each scolex contained 22-27 hooks arranged in two rows of large and small hooks. The average total length of the large and small hooks was 158.9 and 112.1 µm, respectively. Using ICC, statistically significant clusters of different hook sizes were identified within the isolates. The length of the large and small hooks was significantly associated with the variability in mitochondrial 12S rRNA gene. CONCLUSION: Taenia multiceps, is a relatively important zoonotic infection in Iranian sheep with the prevalence rate of 2.5%. Hook length analysis revealed statistically significant difference among individual isolates. Associations between the rostellar hook length and variability in the mitochondrial 12S rRNA was documented.

The monetary burden of cystic echinococcosis in Iran.

Cystic echinococcosis (CE) is a globally distributed parasitic infection of humans and livestock. The disease is of significant medical and economic importance in many developing countries, including Iran. However, the socioeconomic impact of the disease, in most endemic countries, is not fully understood. The purpose of the present study was to determine the monetary burden of CE in Iran. Epidemiological data, including prevalence and incidence of CE in humans and animals, were obtained from regional hospitals, the scientific literature, and official government reports. Economic data relating to human and animal disease, including cost of treatment, productivity losses, and livestock production losses were obtained from official national and international datasets. Monte Carlo simulation methods were used to represent uncertainty in input parameters. Mean number of surgical CE cases per year for 2000-2009 was estimated at 1,295. The number of asymptomatic individuals living in the country was estimated at 635,232 (95% Credible Interval, CI 149,466-1,120,998). The overall annual cost of CE in Iran was estimated at US$232.3 million (95% CI US$103.1-397.8 million), including both direct and indirect costs. The cost associated with human CE was estimated at US$93.39 million (95% CI US$6.1-222.7 million) and the annual cost associated with CE in livestock was estimated at US$132 million (95% CI US$61.8-246.5 million). The cost per surgical human case was estimated at US$1,539. CE has a considerable economic impact on Iran, with the cost of the disease approximated at 0.03% of the country's gross domestic product. Establishment of a CE surveillance system and implementation of a control program are necessary to reduce the economic burden of CE on the country. Cost-benefit analysis of different control programs is recommended, incorporating present knowledge of the economic losses due to CE in Iran.

Genotyping Echinococcus granulosus from dogs from Western Iran.

Cystic echinococcosis is a zoonotic infection caused by the dog tapeworm, Echinococcus granulosus. In the present study, adults of E. granulosus (n=20) were collected from 71 dogs from Western Iran and were genetically characterized using DNA sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1). Consensus sequences were obtained for cox1 (366) and nad1 (471) genes. Phylogenetic analysis of concatenated nad1 and cox1 nucleotide sequence data was performed using Bayesian Inference approach. Overall, the dog isolates indicated nine different sequences in cox1 and seven in nad1 genes. Three genotypes (G1 [75%], G2 [10%] and G3 [15%]) were identified from the isolates. The G2 sequences indicated 100% homology with reference G2 sequence in both cox1 (Genbank accession number M84662) and nad1 (AJ237633) genes. G3 sequences showed 100% homology with G3 reference sequence in nad1 (AJ237633), but displayed two different cox1 profiles, each having 99% homology with reference G3 sequence (M84663). In the phylogenetic tree all of the isolates were grouped into a distinct cluster corresponding to the G1-G3 complex with relevant reference sequences. The presence of G1 genotype (sheep strain) of E. granulosus sensu stricto as dominant genotype in dogs is emphasized. To the best of our knowledge, this study established the first record of E. granulosus sensu stricto, G2 genotype in Iran.

The effects of cyclooxygenase inhibitors on the brain inflammatory response following traumatic brain injury in rats.

OBJECTIVES: Cytokines such as IL-1ß are involved in inflammatory responses. This study evaluated the role of two different kinds of drugs (ibuprofen and celecoxib) on brain IL-10 and IL-1ß after traumatic brain injury (TBI) in male rats. MATERIALS AND METHODS: Rats were assigned into 6 groups: intact, sham, TBI, and treated rats with vehicle, celecoxib or iboprophen. Cytokine concentrations were quantified by ELISA kits. RESULTS: Groups showed no significant difference in brain IL-10 either after TBI induction or after treatment with ibuprofen or celecoxib. Serum IL-10 in vehicle or ibuprofen treated animals was lower than in sham groups (P< 0.01). Brain IL-1ß decreased after treatment by ibuprofen or celecoxib (P< 0.001). There was no statistical difference in serum IL-1ß in TBI and intact. Serum IL-1ß significantly decreased in rats that received celecoxib compared to TBI group (P< 0.01). CONCLUSION: Based on our study IL-1ß can decrease through both cyclooxygenase 1 (COX-1) and COX-2 pathway but serum IL-1ß can decrease only by COX-2 pathway.