Thyroid involvement in cystic echinococcosis: a systematic review.

BACKGROUND: Thyroid Hydatid Cyst (THC), a pathological state induced by the larval form of Echinococcus granulosus, represents a multifaceted clinical entity with nonspecific symptoms, making both diagnosis and treatment intricate. The current understanding of THC's attributes is somewhat limited. To gain a broader perspective on the disease's clinical and epidemiological characteristics, we have systematically reviewed the existing literature. METHODS: We performed an extensive review of articles on THC across four key scientific databases: PubMed, Scopus, Web of Science, and Google Scholar. Our study encompassed all patients diagnosed with THC through post-surgical pathology or Fine Needle Aspiration Cytology (FNAC) examinations, extracting clinical, epidemiological, and therapeutic data of THC patients from publications up to October 2023. RESULTS: From 770 articles, 57 met our criteria, detailing 75 THC patients. The gender ratio was 2.36 females per one male. The patients averaged 36.1 years old, with common symptoms including neck mass, hoarseness, shortness of breath, and dysphagia. The left lobe was involved in most patients, and only 21.3% had extrathyroidal involvement. Cysts averaged 36.4 mm in diameter, with cystic nodules being the most frequent imaging finding (91.2%). Serological tests were performed for 42.6% of cases, of which 62.5% were positive. Surgery was undertaken in 71 patients (94.6%). CONCLUSION: Cystic echinococcosis (CE) of the thyroid should be considered as part of the differential diagnosis in patients with cervicofacial mass, especially in endemic countries. The present study provides reliable data to improve our understanding of the features of the disease for a better diagnosis and management.

MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

Genetic diversity and haplotypes of Echinococcus granulosus isolated from cattle and buffaloes and first report of E. ortleppi (G5) in buffaloes in Pakistan based on mitochondrial cytochrome c oxidase subunit-1 gene (mt-CO1) markers.

Cystic echinococcosis (CE) is a parasitic disease that is caused by larval stage of Echinococcus granulosus tapeworm, one of the most important and neglected zoonotic disease. Although the echinococcosis is endemic in the neighboring countries, information regarding circulating genotypes of E. granulosus sensu lato is scarce in Pakistan. Therefore, the main purpose of this report was to contribute in molecular epidemiology and to find genetic variation and haplotypes of E. granulosus s.l. in cattle and buffalo isolates. To identify species circulating in country, parasite samples were collected from different slaughterhouses and butcher shops of two major cities, Rawalpindi and Peshawar located in Punjab and Khyber Pakhtunkhwa (KP) provinces, Pakistan, respectively. A total of 100 CE cyst samples were investigated from buffalo (n = 61), and cattle (n = 39) hosts. After genomic DNA extraction from individual cyst materials, mt-CO1 (875 bp) gene was amplified by PCR. After that, PCR products were electrophoresed on the agarose gel then purified and sequenced using forward primer. The sequences were trimmed (779 bp), aligned and matched with NCBI published sequences. E. granulosus s.s. (G1, G3) (71.4%; n = 20/28) was confirmed as the dominant species in buffalo and cattle. E. ortleppi (G5) (28.6%; n = 8/28) was recorded for the first time in both buffalo and cattle isolates from Rawalpindi. E. granulosus s.l. haplotype network showed single predominant haplotype, which comprised 40% of population. Tajima's D and Fu's Fs were negative and significant for E. ortleppi (G5), suggesting population expansion in Pakistan. Therefore, more studies using isolates of E. granulosus s.l. from various locations and intermediate hosts across Pakistan will add new data on molecular epidemiology and genotyping for effective control strategies of CE in Pakistan.

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.

Molecular identification and phylogenetic analysis of free-living amoeba in the water resources of Arak, Iran.

The aim of the present study was to detect free-living amoeba (FLA) in the water resources of Arak, Iran using molecular tools. A total of 154 samples were collected from different water supplies. Molecular analyses, sequencing, and phylogenetic study were conducted to confirm the species and genotypes of FLA. Fisher exact test was used to determine the significance. Of 154 water samples, 19 (12.3%) samples were tested positive for FLA. Three genotypes of Acanthamoeba including T4, subtype D, and T5 were identified among the isolates. The pathogenicity assay showed that the isolate of Acanthamoeba in drinking water was highly pathogenic. Three species of Naegleria, including N. australiensis, N. pagei, and N. gruberi were found among the samples. Six isolates of Vermamoeba were identified as V. vermiformis. Meanwhile, three other species including Vannella sp., Vahlkampfia avara, and Stenamoeba polymorpha were also recovered from the water samples. Statistical analysis showed a significant difference between the various water resources contaminated with FLA. This is the first study to reveal the presence of S. polymorpha in water sources in Iran. According to the findings of the present study, health officials should be beware of potential public health impacts of FLA in water resources.

Anti-echinococcal effects of sumac, Rhus coriaria, in a murine model of cystic echinococcosis: Parasitological and molecular evaluation.

Sumac has been traditionally used by people as a medicinal plant for the treatment of different disorders. Cystic echinococcosis (CE) is one of the major zoonotic diseases of human with a worldwide distribution. Long term albendazole therapy is usually associated with side effects including impaired liver function and leukopenia. The present study investigated the efficacy of the methanolic extract of Sumac, Rhus coriaria, on the secondary hydatid cyst development in mice and evaluated sumac effects on the expression of a profile of genes with a potential role in parasite development. Thirty-six mice were intraperitoneally inoculated with of with 3000 protoscoleces and six months after induction of infection were divided into three groups that received either oral sumac extract, albendazole or distilled water. The mice were necropsied 45 days later and the volume and weight of cysts were measured. The expression level of five target genes were analyzed using RT-qPCR. The volume and weight of the cysts were significantly lower in the sumac group compared to the controls. Decreased expressions were found in four out of the five genes following sumac administration. While significantly lower expressions in the sumac group were found for the cdk6, b-raf, fgfr and ras genes, no significant difference was found in cdk2 expression as compared with the control groups. Findings of the present study indicate high efficacy of sumac on the size and volume of secondary hydatid cysts in a murine CE model. Further studies are required to explore the most active and effective ingredients of this natural product.

MicroRNA profile of the strobilated worms of Echinococcus granulosus derived from in vivo and in vitro systems by using high-throughput approach.

MicroRNAs are critical gene regulators at the post-transcriptional level and play essential roles in numerous developmental processes in metazoan parasites including the causative agent of cystic echinococcosis, Echinococcus granulosus. The molecular basis of different patterns of E. granulosus development in the canine definitive host and in in vitro culture systems is poorly understood. In the present study, miRNA transcriptomes of the strobilated worms derived from experimental infection in the definitive host were compared with those from diphasic culture system after 60-day protoscoleces cultivation. Total RNA was extracted from in vivo- and in vitro-derived strobilated worms. Small RNA libraries were constructed, and deep sequencing was performed. Subsequently, differential miRNA expressions and target predictions were obtained, and pathway analysis was performed by gene ontology and KEGG. Seven miRNAs were differentially expressed between the in vivo- and in vitro-derived worms. In addition, we reported 13 novel miRNA candidates and 42 conserved miRNAs. Four out of five top miRNAs with the highest read counts were shared between the in vivo and in vitro-derived worms, i.e., egr-miR-10a-5p, egr-let-7-5p, egr-bantam-3p, and egr-miR-71-5p. Target prediction of the differential miRNAs between the two systems showed significant differences in the membrane-enclosed lumen, membrane part, and an intrinsic component of the membrane. Findings of KEGG analysis indicated that differentially expressed miRNAs were involved in hippo, MAPK, and WNT signaling pathways. The study demonstrated a significant difference in miRNA transcriptomes and related signaling pathways between the two systems, suggesting the importance of host-parasite interplay in the fate of protoscoleces development in in vivo and in vitro systems.

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.

In vitro evaluation on the scolicidal effect of Myrtus communis L. and Tripleurospermum disciforme L. methanolic extracts.

Hydatid disease, a zoonotic disease, is still endemic in many developing countries that is caused by the metacestode of Echinococcus (E.) granulosus. Surgical management is one of the best choices for the treatment of the hydatidosis and using effective scolicidal agents during hydatid surgery is essential to prevent the secondary infection. The aim of the present in vitro study was to evaluate the scolicidal effect of the methanolic extract of Myrtus communis and Tripleurospermum disciforme against protoscoleces of hydatid cyst. Protoscoleces of E. granulosus were aspirated aseptically from infected livers. Various concentrations of M. communis and T. disciforme extracts at different exposure times were examined for their scolicidal activity. Normal saline and silver nitrate were used as negative and positive groups, correspondingly. The viability of protoscoleces was evaluated by 0.1% eosin. The result of the current study indicated that the highest scolicidal effect (100%) of M. communis was obtained at 100 and 50 mg/ml concentrations and LC50 in 10, 20 and 30 min were 11.64 mg/ml, 7.62 mg/ml, and 6.47 mg/ml respectively. The scolicidal activity of T. disciforme was negligible even at high concentration. The findings have shown that the scolicidal activity of M. communis against echinococcosis protoscoleces was strong, while the T. disciforme extract showed fewer effects. However, further studies are required for identification of the active ingredients in the extract and its safety on cells in effective concentrations.

Experimental and field investigation of non-biting flies as potential mechanical vectors of Echinococcus granulosus eggs.

Synanthropic fly species can be potential mechanical vectors of many infectious agents. The potential of the flies to carry Echinococcus granulosus eggs is not fully documented. The purpose of the present study was to determine the possible role of non-biting flies to carry taeniid eggs. A total of 210 flies were collected from seven selected sites in areas of Kerman city, southeastern Iran from November 2016 to May 2017. Adult flies were live-caught using sweeping nets. Flies were placed individually in small glass bottles and transported to the laboratory. All the flies were killed by deep freezing and then identified to the species level using both morphological and molecular methods. The flies were homogenized in test tubes and genomic DNA was extracted and amplified by PCR. PCR protocols were used both to identify the live-caught flies to the species level, and for the detection of E. granulosus. The laboratory reared second generation flies were experimentally exposed to dog feces manually spiked by Echinococcus eggs. Two runs of experiments with 1-3 h of exposure were designed. For each experiment 20 flies were selected from the stock colony and were starved for three days. After each experiment, the flies were frozen for further molecular studies. The dominant fly species were Musca domestica and Lucilia sericata. No eggs were found on the body surface and/or guts of live-caught flies. After the first hour of exposure, 60%, of the flies of both species were found to harbor Echinococcus eggs. However, in the case of L. sericata 50% of the flies harbored Echinococcus eggs after 3 h of exposure. Results of the present study indicate the probable role of synanthropic flies in harboring Echinococcus eggs and mechanical transmission of cystic echinococcosis. When the helminth eggs are susceptible to desiccation grooming flies can remove many of eggs from exterior surfaces of them. Despite this result the role of synanthropic flies in the transmission of certain helminthiases should not be discounted because of their vagility and feeding mechanisms.

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.

Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms.

Parts of the natural life cycle of Echinococcus granulosus can be retraced in vitro such as the development of protoscoleces into semiadult worms with three or more proglottids, or the redifferentiation of in vitro cultured protoscoleces into metacestode-like cystic structures. Most in vitro generated samples share-at the microscopical level-high similarities with those naturally grown, but developmental differences have also been documented, such as missing egg production in in vitro grown adults or unusual bladder/vesicle formation in protoscoleces cultured into the metacestode direction. The aim of the present study was to explore how far different in vitro generated stage-specific materials/structures match the natural situation on the transcriptome level, based on testing five exemplarily chosen different genes: the frizzled receptor eg-fz4 (posterior marker), the FGF receptor-like factor eg-fgfrl (anterior association), the cell differentiation protein eg-rcd1 (part of the CCR4-NOT complex, a key regulator of eukaryotic gene expression), the rapidly accelerated fibrosarcoma serin/threonin kinase eg-braf (part of the MAPK pathway involved, e.g., in EGF signaling) and the co-smad eg-smadD (downstream factor of TGFß/BMP2/activin signaling). These genes-tested via qPCR-were selected such as to allow a discussion on their potential role in the development of E. granulosus into the adult stage. Thus, testing took place with three ex vivo isolated samples, namely (i) egg-containing adult worms, (ii) invaginated protoscoleces, and (iii) protoscolex-free germinal layer tissue. Respective data were compared (a) with in vitro generated metacestode-like microcysts developed from protoscolices, and (b) different development stages of protoscoleces in vitro cultured toward adult maturation. As a finding, only eg-smadD and partially eg-fz4 showed high expression similarities between ex vivo harvested and in vitro cultured E. granulosus, thus suggesting a putative role in adult maturation. Conclusively, the fact of using "only" five genes did not allow answering the question if ex vivo and in vitro materials are similar on the transcriptome level. Another experimental restriction arises from different growth conditions of the in vitro cultured materials, and comparing these to the ex vivo harvested ones. Future experiments may solve the problems by using fully standardized E. granulosus sample collection and fully standardized culture conditions.

Differential Expression of Hox and Notch Genes in Larval and Adult Stages of Echinococcus granulosus.

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus.

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.

Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran.

Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica.

Scolicidal effects of black cumin seed (Nigella sativa) essential oil on hydatid cysts.

Surgery remains the preferred treatment for hydatid cyst (cystic echinococcosis, CE). Various scolicidal agents have been used for inactivation of protoscolices during surgery, but most of them are associated with adverse side effects. The present study aimed to evaluate the in vitro scolicidal effect of Nigella sativa (Ranunculaceae) essential oil and also its active principle, thymoquinone, against protoscolices of hydatid cysts. Protoscolices were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the essential oil (0.01-10 mg/ml) and thymoquinone (0.125-1.0 mg/ml) were used for 5 to 60 min. Viability of protoscolices was confirmed by 0.1% eosin staining. Furthermore, the components of the N. sativa essential oil were identified by gas chromatography/mass spectroscopy (GC/MS). Our study revealed that the essential oil of N. sativa at the concentration of 10 mg/ml and its main component, thymoquinone, at the concentration of 1 mg/ml had potent scolicidal activities against protoscolices of Echinococcus granulosus after 10 min exposure. Moreover, thymoquinone (42.4%), p-cymene (14.1%), carvacrol (10.3%), and longifolene (6.1%) were found to be the major components of N. sativa essential oil by GC/MS analysis. The results of this study indicated the potential of N. sativa as a natural source for production of a new scolicidal agent for use in hydatid cyst surgery. However, further studies will be needed to confirm these results by checking the essential oil and its active component in in vivo models.

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.

Trichinella infections in animals and humans of Iran and Turkey.

Trichinellosis is considered as a cosmopolitan zoonosis caused by different species of the small nematodes of the genus Trichinella. The present study aimed to provide a broad review for exploring Trichinella sp. infection in humans and animals of Iran and Turkey. Additionally, we aimed to explore bases for trichinellosis prevention and control. Two reports of human trichinellosis following the consumption of meat of wild boar are available in the northern Iran. A large outbreak of trichinellosis and some other sporadic cases are reported mainly as a result of eating wild boar or pork meat from Turkey, where T. britovi is present. Field studies show that Trichinella sp. infections occur in wild carnivores of Iran, particularly the golden jackal (Canis aureus) as the most frequently infected species. T. britovi has been reported to be present elsewhere in Iran in wild mammals, where wild boar is the main source of Trichinella sp. infection. In Turkey, Trichinella spp. has been reported from animals including both domesticated and wild pigs and gray wolf (Canis lupus). However, current data on the distribution of Trichinella taxa are fragmentary in the Anatolian region.

Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis.

BACKGROUND: Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated. METHODS: To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 µl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I. RESULTS: Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases. CONCLUSIONS: Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.