Mouse model of secondary cystic echinococcosis.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of the cestode Echinococcus granulosus sensu lato (s. l.), a genetic complex composed of five species: E. granulosus sensu stricto (s. s.), E. equinus, E. ortleppi, E. canadensis, and E. felidis. The parasite requires two mammalian hosts to complete its life cycle: a definitive host (mainly dogs) harboring the adult parasite in its intestines, and an intermediate host (mostly farm and wild ungulates) where hydatid cysts develop mainly in the liver and lungs. Humans are accidental intermediate hosts, being susceptible to either primary or secondary forms of CE; the first one due to the ingestion of oncospheres, and the second one because of the spillage of protoscoleces (PSC) contained within a primary cyst. Secondary CE is a serious medical problem, and can be modeled in immunocompetent mice (a non-natural intermediate host) through the intraperitoneal inoculation of viable PSC from E. granulosus s. l. This model is useful to study not only the immunobiology of CE, but also to test new chemotherapeutics or therapeutical protocols, to explore novel vaccine candidates, and to evaluate alternative diagnostic and/or follow-up tools. The mouse model of secondary CE involves two sequential stages: an early stage of parasite pre-encystment (PSC develop into hydatid cysts in the peritoneal cavity of mice), and a late or chronic stage of parasite post-encystment (already differentiated cysts slowly grow during the whole host lifespan). This model is a time-consuming infection, whose outcome depends on several factors like the parasite infective dose, the mouse strain, and the parasite species/genotype. Thus, such variables should always be adjusted according to the research objectives. Herein, the general materials and procedures needed to establish secondary CE in mice are described, as well as several useful tips and recommendations.

Detection of circulatory E. granulosus-derived cell-free DNA in the plasma and urine of human cystic echinococcosis using an in-house PCR: a potential promising diagnostic biomarker.

BACKGROUND: The diagnostic tool for identifying cystic echinococcosis (CE) patients at an early stage is currently lacking. However, circulatory cell-free DNA (cfDNA) has shown potential as a biomarker for parasitic infections and could be used for diagnosing CE. RESEARCH DESIGN AND METHODS: The plasma and urine samples were collected from 39 patients with confirmed CE through imaging and histopathological techniques. All plasma samples were tested for anti-echinococcal antibodies using a commercial ELISA test. Total plasma and urine cfDNA were extracted and an in-house PCR assay was developed to detect E. granulosus specific cfDNA in the samples of CE patients. RESULTS: Out of the 39 patients, 30 tested positive for E. granulosus using serology, with a sensitivity of 76.9%. Moreover, the detection rates for the cfDNA were 79.5% in plasma samples and 58.97% in urine samples using the 80 bp COX1 gene. The plasma-based PCR and serology test showed the highest agreement (Kappa = 0.53). CONCLUSIONS: Plasma-based PCR has been found to be a reliable diagnostic tool for identifying CE patients at different cyst stages. It offers validity, speed, and sufficient sensitivity, making it an alternative to serology in diagnosing CE in endemic areas.

Understanding the role of pigs in the transmission of zoonotic Echinococcus ortleppi in Haryana, India.

The etiological agents of zoonotic cystic echinococcosis comprise the Echinococcus granulosus sensu lato (s.l.) species complex. The present study was aimed at investigating the zoonotic genotypes of Echinococcus granulosus s.l. circulating in the pig population of Haryana, India. Out of 253 slaughtered pigs screened, 5 showed the presence of hydatid cysts. The amplification of the partial mitochondrial NADH dehydrogenase subunit 1 (nad1) gene for the molecular confirmation and phylogenetics of the retrieved metacestodes (n = 2) revealed the presence of E. ortleppi. The sequences generated herein exhibited 99.80% homology to the GenBank archived E. ortleppi sequences. Cladistics targeting genetic diversity and haplotype network analysis involved 37 E. granulosus s.l. GenBank archived sequences from India corresponding to different hosts (large and small ruminants and humans) along with the sequences (n = 2) generated in the present study. Overall, 14 haplotypes with high haplotype (0.780 ± 0.059) and low nucleotide (0.033 ± 0.010) diversities were recorded for the overall data set, which evinced a population expansion. The median-joining haplotype network revealed a stellate shape of E. granulosus sensu stricto (s.s.) sequences, which was indicative of rapid population expansion. High genetic differentiation (FST = 0.840 - 0.983) and low gene flow (Nm = 0.003 - 0.047) were recorded between the pig intermediate hosts infected with E. ortleppi and other hosts infected with E. granulosus s.s. The findings are of paramount significance for the formulation of effective control strategies considering the public health and economic impact of cystic echinococcosis.

Molecular discrimination of G1 and G3 genotypes of Echinococcus granulosus sensu stricto obtained from human, cattle, and sheep using the mitochondrial NADH dehydrogenase subunit 5 marker.

Cystic echinococcosis (CE) is a common zoonotic disease caused by the larval form of Echinococcus granulosus sensu lato. This study determined the genotype and haplotype differences using the NADH dehydrogenase subunit 5 gene in hydatid cyst samples. Human (n = 12), cattle (n = 28), and sheep (n = 31) hydatid cyst isolates were included. Seventy-one genomic DNA samples were successfully extracted, and a 759 bp mitochondrial NADH dehydrogenase subunit 5 gene fragment was amplified by PCR. Following the sequence analysis, E. granulosus sensu stricto isolates were identified as G1 (n = 61) and G3 (n = 10). A total of 23 haplotypes were obtained from the 71 E. granulosus s.s. G1 and G3 samples. The main haplotype was Hap01 (60.56 %), which consisted of the G1 genotype. The second largest haplotype was Hap04, which consisted entirely of the G3 genotype. Hap14 acted as a bridge between the G1 and G3 genotypes. This study identifies G1 as the dominant genotype in humans and farm animals in Turkey. High haplotype and nucleotide diversity in genotypes were observed. Additionally, this is the first report on the phylogeography and gene flow models of the E. granulosus s.s. population in Turkey using the NADH dehydrogenase subunit 5 gene, the best marker distinguishing between G1 and G3 genotypes.

High genetic diversity of Echinococcus canadensis G10 in northeastern Asia: is it the region of origin?

Echinococcus canadensis consists of 4 genotypes: G6, G7, G8 and G10. While the first 2 predominantly infect domestic animals, the latter are sylvatic in nature involving mainly wolves and cervids as hosts and can be found in the northern temperate to Arctic latitudes. This circumstance makes the acquisition of sample material difficult, and little information is known about their genetic structure. The majority of specimens analysed to date have been from the European region, comparatively few from northeast Asia and Alaska. In the current study, Echinococcus spp. from wolves and intermediate hosts from the Republic of Sakha in eastern Russia were examined. Echinococcus canadensis G10 was identified in 15 wolves and 4 cervid intermediate hosts. Complete mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences were obtained from 42 worm and cyst specimens from Sakha and, for comparison, from an additional 13 G10 cysts from Finland. For comparative analyses of the genetic diversity of G10 of European and Asian origin, all available cox1 sequences from GenBank were included, increasing the number of sequences to 99. The diversity found in northeast Asia was by far higher than in Europe, suggesting that the geographic origin of E. canadensis (at least of G10) might be northeast Asia.

miRNA based biomarkers for the early diagnosis of Echinococcus granulosus in experimentally infected dogs.

Cystic echinococcosis, which is caused by the Echinococcus granulosus. Carnivores, as final hosts, contain adult tapeworms in the small intestine, and a variety of mammals, including humans, harbor the metacestod. This study was designed to investigate the miRNA-based biomarkers for early and accurate diagnosis of E. granulosus in experimentally infected dogs. A liver with an obvious hydatid cyst was obtained from a slaughterhouse and then protoscoleces were collected. Following, viable protoscoleces were administred to three experimental dogs (ED1, ED2 and ED3) and another uninfected control dog (UCD) was kept as control without infection. Stool samples of all groups were collected during 50 days from the beginning of the experimental infection and stored at - 80 °C till work. Total miRNA was isolated from all individual stool samples. The qRT-PCR method was used to determine the differences in the expression levels of E. granulosus specific miRNAs which were egr-let-7-5p, egr-miR-2b-5p, egr-miR-71-5p and egr-miR-125-5p. All miRNAs were found to be expressed from the first day in all infected dogs. In the stool samples of the UCD, the egr-miR-71-5p was detected, while the other miRNAs (egr-let-7-5p, egr-miR-2b-5p, egr-miR-125-5p) were not expressed. The expression of egr-let-7-5p and egr-miR-125-5p was significantly increased in ED1 compared to UCD on all days. In particular, for the first time, the expression levels of egr-let-7-5p and egr-miR-125-5p increased significantly between days 15 and 19. Similarly, the increase in let-7-5p and miR-125-5p expression was statistically significant in ED2. In ED3, egr-let-7-5p, egr-miR2b-5p and egr-miR-125-5p expressions were significantly increased on all days. In particular, egr-let-7-5p expression levels increased significantly for the first time between days 15 and 19. In addition, egr-mir-125-5p expression levels were found to increase at a high level for the first time on day 16. In conclusion, especially egr-let-7-5p and egr-miR-125-5p can be used as early diagnostic biomarkers in dogs infected with E. granulosus.

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.

Cystic echinococcosis microRNAs as potential noninvasive biomarkers: current insights and upcoming perspective.

INTRODUCTION: Echinococcosis, also known as hydatidosis, is a zoonotic foodborne disease occurred by infection with the larvae of Echinococcus spp. which can lead to the development of hydatid cysts in various organs of the host. The diagnosis of echinococcosis remains challenging due to limited diagnostic tools. AREAS COVERED: In recent years, microRNAs (miRNAs) have emerged as a promising biomarker for various infectious diseases, including those caused by helminths. Recent studies have identified several novel miRNAs in Echinococcus spp. shedding light on their essential roles in hydatid cyst host-parasite interactions. In this regard, several studies have shown that Echinococcus-derived miRNAs are present in biofluids such as serum and plasma of infected hosts. The detection of these miRNAs in the early stages of infection can serve as an early prognostic and diagnostic biomarker for echinococcosis. EXPERT OPINION: The miRNAs specific to Echinococcus spp. show great potential as early diagnostic biomarkers for echinococcosis and can also provide insights into the pathogenesis of this disease. This review attempts to provide a comprehensive overview of Echinococcus-specific miRNAs, their use as early diagnostic biomarkers, and their function in host-parasite interactions.

Application and evaluation of native antigen B from Echinococcus granulosus sensu stricto and E. canadensis alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting.

Cystic echinococcosis (CE) is one of the most important helminthic diseases in the world with different genotypes distribution. The application of specific genotype antigens together with sera from patients with specific cyst genotypes have not been reported, so far. The present study aimed to apply and evaluate native AgB from Echinococcus granulosus sensu stricto (Eg) and Echinococcus canadensis (Ec) alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting. A total of 47 human sera along with 47 human CE cysts were collected. CE genotypes were determined. Native AgB were prepared from E. granulosus s.s and E. canadensis genotypes. ELISA and Western blot were performed on human specific G1-G3 and G6/G7 genotypes sera. Species specific native AgB were used alone or mixed. The sensitivity of ELISA using alone and mixed 1Eg-1Ec, 1Eg-2Ec, and 2Eg-1Ec of native AgB from E. granulosus s.s and E. canadensis genotypes for human G1-G3 sera were 92.10, 89.47, 97.37, 100, and 100%, respectively; while using AgBs, alone and mixed for human G6/G7 sera were 100%. The sensitivity of Western blotting using native AgB of E. granulosus s.s and E. canadensis genotypes alone and mixed 2Eg-1Ec were 78.95% and 100% for human G1-G3 and G6/G7 genotypes sera, respectively. The mixture of AgB from Echinoccus granulosus sensu stricto and Echinococcus canadensis genotypes increased ELISA sensitivity for the diagnosis of human CE. Preparation and application of native AgB from specific and prevalent genotypes of CE in endemic regions is recommended.

Cystic echinococcosis in Iceland: a brief history and genetic analysis of a 46-year-old Echinococcus isolate collected prior to the eradication of this zoonotic disease.

Cystic echinococcosis (CE) is considered the most severe parasitic disease that ever affected the human population in Iceland. Before the start of eradication campaign in the 1860s, Iceland was a country with very high prevalence of human CE, with approximately every fifth person infected. Eradication of CE from Iceland by 1979 was a huge success story and served as a leading example for other countries on how to combat such a severe One Health problem. However, there is no genetic information on Echinococcus parasites before eradication. Here, we reveal the genetic identity for one of the last Echinococcus isolates in Iceland, obtained from a sheep 46 years ago (1977). We sequenced a large portion of the mitochondrial genome (8141 bp) and identified the isolate as Echinococcus granulosus sensu stricto genotype G1. As G1 is known to be highly infective genotype to humans, it may partly explain why such a large proportion of human population in Iceland was infected at a time . The study demonstrates that decades-old samples hold significant potential to uncover genetic identities of parasites in the past.

Molecular characterization and serodiagnostic evaluation of the Echinococcus ortleppi recombinant glutaredoxin 1 protein for cystic echinococcosis in buffalo (Bubalus bubalis).

Cystic echinococcosis (CE), caused by the metacestode of Echinococcus granulosus sensu lato (s.l.), adversely affects the physiology of the vital organs in which they grow. Condemnation of meat causes substantial economic loss to the livestock industry. Conventionally the infection is detected by necropsy as serological diagnosis of the infection in livestock is ambiguous. Identification of specific diagnostic antigens would be a substitute for the cyst fluid antigens which lack adequate diagnostic sensitivity and specificity. BLAST analysis supported by the negligible pairwise nucleotide distance of the 389 nt COX1, 489 nt NAD1, and 425 nt ITS1 with the related sequences of E. ortleppi ascertained the association of E. ortleppi with CE in buffaloes. Given the extensive distribution of glutaredoxin 1 in every developmental stage of Echinococcus granulosus s.l that makes it an ideal serodiagnostic antigen for CE, we expressed the 14 kDa E. ortleppi glutaredoxin 1 (rEoGrx1) protein in E. coli BL21 (DE3) and tested a total of 225 sera samples, including 126 sera samples from the necropsy-positive buffalo, by the rEoGrx1 IgG-ELISA. The ELISA could detect a total of 82/126 sera samples as positive. The diagnostic sensitivity and specificity of the rEoGrx1 IgG-ELISA were 65.1 % and 51.5 %, respectively. The protein showed serological cross-reaction against Fasciola gigantica, Toxoplasma gondii, and Sarcocystis sp. The in silico bioinformatics analysis of the E. ortleppi, F. gigantica, and T. gondii glutaredoxin sequences revealed fully conserved amino acids at positions 11 and 21, the substitution of conserved amino acids at positions 14 and 6, and semi-conserved substitutions at positions 3 and 4, respectively. The findings partly explain the molecular basis of the serological cross-reactivity of the protein.

Genetic characterization of human echinococcosis in Southern Punjab, Pakistan.

Introduction: Echinococcosis is a neglected tropical zoonotic infection that affects both the human and livestock populations. In Pakistan, the infection is long-standing, but data on its molecular epidemiology and genotypic characterization in the southern Punjab region are limited. The aim of the current study was the molecular characterization of human echinococcosis in southern Punjab, Pakistan. Methods: Echinococcal cysts were obtained from a total of 28 surgically treated patients. Patients' demographic characteristics were also recorded. The cyst samples were subjected to further processing to isolate DNA in order to probe the Nad1 and Cyt-b genes, followed by DNA sequencing and phylogenetic analysis for genotypic identification. Results: The majority of the echinococcal cysts were from male patients (60.7%). The liver was the most commonly infected organ (60.71%), followed by the lungs (25%), spleen (7.14%), and the mesentery (7.14%). Molecular and genotypic identification through sequencing and phylogenetic tree analysis showed that most of the cysts (24/28, 85.7%) were caused by the species Echinococcus granulosus sensu stricto (E. granulosus s.s.) (G1 and G3), followed by Echinococcus multilocularis (E. multilocularis) and Echinococcus canadensis (E. canadensis) (G6/G7) (3/28, 10.8%, and 1/28, 3.5%, respectively). Conclusion: The current study concluded that the majority of human infections were caused by E. granulosus s.s., followed by the E. multilocularis and E. canadensis species (G6/G7). Genotypic characterization among both human and livestock populations is needed to explore the genetic diversity of echinococcosis.

Molecular characterization of cattle and sheep isolates of Echinococcus granulosus from Elazig province in Turkey and expression analysis of the non-coding RNAs, egr-miR-7, egr-miR-71 and egr-miR-96.

Cystic Echinococcosis (CE) is a common zoonotic disease seen in human and animals worldwide, caused by the larval form of Echinococcus granulosus. In this study, E. granulosus s.l. species and haplotypes were determined in hydatid cysts isolated from cattle and sheep, and the expression levels of egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were compared in different cyst structures. A total of 82 (cattle, n = 41; sheep, n = 41) hydatid cyst isolates (germinal membranes and/or protoscoleces) were collected from a slaughterhouse in Elazig province of Turkey. After mt-CO1 gene sequences were made, 81 out of 82 hydatid cyst isolates were determined as E. granulosus s.s. (G1 and G3), while an isolate of cattle origin was determined as Echinococcus canadensis (G6/7). A total of 26 nucleotide polymorphisms and 29 haplotype groups were identified in the samples. miRNA expressions in germinal membranes of sterile cysts and germinal membrane and protoscoleces of fertile cysts were investigated by qRT-PCR and Real Time PCR analyses. It was determined that miRNAs were expressed at high levels in 79.31% of the 29 haplotype groups and at low levels in the remaining 10.34%. In 10 fertile samples of sheep origin, egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were found to be 44, 168, and 351-fold higher in expression, respectively, in the germinal membrane compared to the protoscoleces. Especially egr-miR-96 may have the potential to be used as biomarkers in the diagnosis of active CE.

First report of Echinococcus ortleppi and genotype G6 of E. canadensis cluster from southern Punjab, Pakistan and a global overview on genetic structure and host adaptation of E. ortleppi.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected tropical disease known mainly for its zoonotic nature. CE is endemic to Pakistan, however, the disease is not given due consideration and millions of people remain at health risk. This study was undertaken to assess the species and genotypes of E. granulosus sensu lato in sheep, buffaloes and cattle, brought to slaughterhouses of two major cities (Multan and Bahawalpur) of south Punjab, Pakistan. A total of 26 hydatid cyst specimens were characterized through complete cox1 mitochondrial gene (1609 bp) sequencing. Species and genotypes of E. granulosus sensu lato discovered in the southern Punjab consisted of E. granulosus sensu stricto (n =21), E. ortleppi (n=4) and genotype G6 of the E. canadensis cluster (n=1). Of E. granulosus s.s. isolates, the genotype G3 was predominantly involved in causing infections to the livestock of this region. Since all of these species are zoonotic, wide and effective surveillance studies are required to ascertain the risks to human population in Pakistan. Additionally, a global overview on cox1 phylogenetic structure of E. ortleppi was carried out. Despite widespread occurrence, the species is mostly limited to the southern hemisphere. The highest burden has been reported in South America (62.15%) and Africa (28.44%) and by far the most common host is cattle, accounting for >90% of cases.

Exploring the genetic diversity of genotypes G8 and G10 of the Echinococcus canadensis cluster in Europe based on complete mitochondrial genomes (13 550-13 552 bp).

Echinococcus granulosus sensu lato is a group of tapeworm species known to cause cystic echinococcosis. Within this group, the Echinococcus canadensis cluster includes genotypes G8 and G10 that have a predominantly sylvatic life cycle ­ transmission occurs between wild cervids and wolves. Relatively few studies have explored the genetic variation of the elusive G8 and G10, and their extent of genetic variation is yet to be investigated at the complete mitochondrial (mt) genome level. The aim was to explore the genetic variation of these 2 genotypes in Europe using complete mtDNA sequences and provide a high-quality reference dataset for future studies. Sequences of complete mt genomes were produced for 29 samples of genotype G8 and G10 from wolves, moose, reindeer and roe deer, originating from Finland, Sweden, Russia, Poland, Latvia and Estonia. Genetic variation was explored based on phylogenetic network analysis, revealing marked differences between G8 and G10 (over 400 mutations), and more detailed patterns of variability within the 2 genotypes than previously observed. Understanding the mt genetic composition of a species provides a baseline for future studies aiming to understand whether this mt distinctiveness is mirrored in the nuclear genome and whether it has any impact on any phenotypic traits or parasite transmission.

[Isolation of Cystic Echinococcosis Causative Agents of Echinococcus equinus and Echinococcus ortleppi Species from Humans in the Central Anatolia Region of Türkiye]. / Türkiye'nin Iç Anadolu Bölgesindeki Insanlardan Kistik Ekinokokkoz Etkeni Echinococcus equinus ve Echinococcus ortleppi Türlerinin Izole Edilmesi.

Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu strico is neglected in Türkiye despite being one of the common diseases due to agriculture being at the forefront, low socioeconomic status and unhygienic animal slaughter. Considering the morbidity, mortality, and difficulties in treatment, more studies and precautions are needed regarding this disease. In this study, it was aimed to genotype Echinococcus isolated from CE patients in the Central Anatolia region. DNA isolation from tissue samples taken from 60 CE patients was performed using the QIAamp DNA FFPE tissue kit. Cytochrome c oxidase subunit gene region of Echinococcus was targeted and JB3/JB4.5 primers were used for genotyping. Polymerase chain reaction (PCR) products were purified according to the instructions for use of the QIAquick PCR purification kit. PCR products were prepared using the ABI Prism BigDye Terminator V3.1 Cycle sequencing kit and the nucleotide sequences in the samples were evaluated with the ABI 3100 sequencing device. The nucleotide sequences obtained in the study were analyzed using MetaPIGA2, MRBAYES v.3.1.2, phyogenetic analysis using parsimony, Unigen programs, maximum likelihood, Bayesian and parsimony methods. It has been found that 88.4% (53/60) of Echinococcus isolates were E.granulosus s.s. in this study. It has been genotyped as 41.7% (25/60) G1, 30.0% (18/60) G3 and 16.7% (10/60) G2 genotype. It has been determined that 6.6% (4/60) of the other Echinococcus isolates were E.equinus and 5.5% (3/60) were E.ortleppi. It was observed that E.equinus and E.ortleppi were isolated from atypically located cysts and from those living in rural areas. The E.equinus and E.ortleppi species were not found in CE patients living in urban areas. CE cases are common in the Central Anatolia region due to dog and cattle breeding, and the disease agent Echinococcus species vary. Genotyping of Echinococcus species is effective in the development of CE treatment and control strategies. Study results can play an active role in the fight against CE, which has formed the basis of the "one health" approach in the world and in Türkiye in recent years.

The potential role of roaming dogs in establishing a geographically novel life cycle of taeniids (Echinococcus spp. and Taenia spp.) in a non-endemic area.

INTRODUCTION: Cystic Echinococcosis (CE) is endemic in humans and livestock in many pastoral communities in Kenya. The distribution of the disease is enhanced by several factors, including livestock trade, which has allowed for the spread of CE to non-endemic areas such as western Kenya. Dogs' roaming behaviour, with consequent contamination of the environment with intestinal parasites, could then lead to parasite establishment. This study examined dogs' infection levels with taeniid eggs and their potential role in contaminating the environment with intestinal parasites. METHODOLOGY: We selected sixteen ruminant slaughterhouses in Busia and Bungoma Counties, and around each slaughterhouse we identified ten homesteads owning free-roaming dogs. We administered a questionnaire on dog management practices to the homestead owner and collected a faecal sample from the dog's rectum. In homesteads around 8 of the 16 slaughterhouses, we collared dogs with a GPS tracker to assess their movement patterns. The faecal samples were examined microscopically following zinc-chloride sieving-floatation technique for the presence of taeniid eggs and other canine intestinal parasites. Polymerase Chain Reaction - Restriction Fragment Length Polymorphism of NADH dehydrogenase subunit 1 gene and sequencing were used to confirm taeniid eggs identified during microscopy. Additionally, the Coproantigen-ELISA was used to detect the presence of taeniid antigen in a sub-set of the faecal samples. RESULTS: Helminths detected in the 155 dogs sampled included hookworms (n = 92; 59.4%), ascarids (n = 15; 9.7%), and taeniids (n = 1; 0.6%). Through Copro-PCR, 13 eggs extracted from the sample of the only taeniid infected dog were sequenced and identified as E. canadensis (G6/7) [n = 1], Taenia multiceps [n = 1], and Taenia serialis [n = 6]; the remaining were indeterminate. Of the 77 faecal samples tested for E. granulosus sensu lato (s. l.) with the Copro-ELISA, 64 (83.1%) were negative, 12 (15.6%) were positive, while 1 (1.3%) was suspicious. The dogs travelled a median of 13.5 km daily, and 28 dogs visited the slaughterhouses during the 5-day recording period. CONCLUSION: The results indicate a relatively high carriage of zoonotic parasites by free-roaming domestic dogs in western Kenya, which poses a risk to human and livestock populations. We report for the first time a domestic lifecycle of Echinococcus canadensis and Taenia multiceps in western Kenya, as well as a presumptive sylvatic cycle of coenurosis by T. serialis. We recommend an extensive and ongoing Copro-antigen survey of dog faeces, broader assessment of dog parasites with zoonotic potential, adherence to slaughterhouse management practices, and dog-ownership programmes to highlight the importance of deworming and restricted dog movements.

Cystic echinococcosis in donkeys in eastern Africa.

Cystic echinococcosis (CE) is endemic in humans and domestic animals in eastern Africa. All the species of the Echinococcus granulosus sensu lato complex have been reported in this region except for E. equinus, possibly due to the small number of studies involving equids. This study reports the frequency of different Echinococcus species in donkeys from eastern Africa. A total of 5961 donkeys were examined during meat inspection in 3 slaughterhouses in Kenya. Identification of Echinococcus spp. was achieved through polymerase chain reaction-restriction fragment-length polymorphism and sequencing of the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 gene. The prevalence of CE was 5.7% (337/5961). The 263 genotyped cysts belonged to E. equinus (n = 163), E. granulosus sensu stricto (n = 70), E. canadensis (G6/7) (n = 26) and E. ortleppi (n = 4). One donkey harboured a metacestode of Spirometra theileri. All E. equinus cases, except 2, originated from southern Ethiopia, whereas the other species were more evenly distributed across the study area. Most of the cysts belonging to E. equinus were fertile (111/163), while those of the other species were non-fertile. This is the first report of Echinococcus spp. in donkeys from sub-Saharan Africa and the first confirmation of E. equinus in East Africa. The frequent fertility of E. equinus cysts in donkeys affirms their suitability as intermediate hosts of this species, while low frequency and cyst fertility suggest a marginal role of donkeys in the transmission of E. granulosus s. s., E. canadensis (G6/7) and E. ortleppi.

Optimization of single-tube nested PCR for the detection of Echinococcus spp.

Echinococcosis is a serious zoonotic life-threatening parasitic disease caused by metacestodes of Echinococcus spp., and appropriate sensitive diagnosis and genotyping techniques are required to detect infections and study the genetic characterization of Echinococcus spp. isolates. In this study, a single-tube nested PCR (STNPCR) method was developed and evaluated for the detection of Echinococcus spp. DNA based on the COI gene. STNPCR was 100 times more sensitive than conventional PCR and showed the same sensitivity to common nested PCR (NPCR); but with a lower risk of cross-contamination. The limit of detection of the developed STNPCR method was estimated to be 10 copies/µL of the recombinant standard plasmids of Echinococcus spp. COI gene. In clinical application, 8 cyst tissue samples and 12 calcification tissue samples were analysed by conventional PCR with outer and inner primers and resulted in 100.00% (8/8) and 8.33% (1/12), 100.00% (8/8) and 16.67% (2/12) positive reactions, respectively, while STNPCR and NPCR were all able to identify the presence of genomic DNA in 100.00% (8/8) and 83.33% (10/12) of the same samples. Due to its high sensitivity combined with the potential for the elimination of cross-contamination, the STNPCR method was suitable for epidemiological investigations and characteristic genetic studies of Echinococcus spp. tissue samples. The STNPCR method can effectively amplify low concentrations of genomic DNA from calcification samples and cyst residues infected with Echinococcus spp. Subsequently, the sequences of positive PCR products were obtained, which were useful for haplotype analysis, genetic diversity, and evolution studies of Echinococcus spp., and understanding of Echinococcus spp. dissemination and transmission among the hosts.

[Prevalence of Echinococcus infection in small mammals captured from Shiqu County, Sichuan Province from 2015 to 2020].

OBJECTIVE: To investigate the prevalence of Echinococcus infection in small mammals in Shiqu County, Sichuan Province from 2015 to 2020, so as to provide insights into echinococcosis control in Shiqu County. METHODS: One setting with frequent activity of small mammals was sampled as the survey site from each of 9 townships where human alveolar echinococcosis was hyperendemic, in Shiqu County, Sichuan Province from 2015 to 2020. Two quadrats measuring 50 m × 50 m were assigned in each survey site during the period between July and August from 2015 to 2020 to capture all small mammals in quadrats, and the species of small mammals were identified by morphological characteristics. All captured small mammals were dissected in the field and Echinococcus infection was identified by visual examinations. The affected organs of Echinococcus - infected small mammals were collected, and Echinococcus infection was detected using PCR assay, with Echinococcus species characterized. The prevalence of Echinococcus infection was calculated in small mammals, and the trends in the prevalence of Echinococcus infection were analyzed during the period from 2015 to 2020. In addition, the prevalence of Echinococcus infection was compared in small mammals using visual examinations and PCR assay. RESULTS: A total of 2 692 small mammals were captured in the survey sites of Shiqu County from 2015 to 2020, and morphology characterized 1 360 Microtus fuscus (50.52%) and 1 332 Plateau pika (49.48%). The prevalence rates of Echinococcus infection were 35.63%, 19.16%, 21.41%, 8.40%, 7.68% and 4.44% by visual examinations and 18.96%, 5.36%, 5.61%, 4.58%, 3.30% and 0.37% by PCR assay in small mammals in Shiqu County from 2015 to 2020, both showing a tendency towards a decline year by year (χ2 = 215.024 and 117.045, both P values < 0.001). The prevalence of Echinococcus infection was significantly higher in small mammals by visual examinations than by PCR assay during the period from 2015 to 2020 except in 2018 (χ2= 33.597, 21.815, 51.373, 17.268 and 9.537, all P values < 0.01). PCR assay detected a reduction in the prevalence of E. multilocularis infection from 10.21% to 0.37% and a reduction in the prevalence of E. shiquicus infection from 8.75% to 0 in small mammals in Shiqu County from 2015 to 2020, both appearing a tendency towards a decline year by year (χ2 = 117.045 and 43.436, both P values < 0.001). In addition, the prevalence of E. multilocularis and E. shiquicus infections reduced from 15.19% to 0.45% and from 8.23% to 0 in M. fuscus, and the prevalence of E. multilocularis and E. shiquicus infections reduced from 7.76% to 0 and from 9.01% to 0 in P. pika in Shiqu County from 2015 to 2020. CONCLUSIONS: M. fuscus and P. pika were dominant species of small mammals in Shiqu County, Sichuan Province from 2015 to 2020, and E. multilocularis infection was mainly found in M. fuscus and E. shiquicus infection mainly found in P. pika. The prevalence of Echinococcus infection appeared a tendency towards a decline in both M. fuscus and P. pika year by year during the period from 2015 to 2020.