Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.

Infection of sheep by Echinococcus multilocularis in Gansu,China:evidence from mitochondrial and nuclear DNA analysis

BackgroundIn the normal life cycle of the parasite(Echinococcus multilocularis)that causes alveolar echinococcosis,domestic and wild carnivores act as definitive hosts,and rodents act as intermediate hosts.The presented study contributes to the research on the distribution and transmission pattern of E.multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province,China.MethodsFrom 2020 to 2021,nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination.A near complete mitochondrial(mt)genome and selected nuclear genes were amplified from the cyst-like lesion for identification.To confirm the status of the specimen,comparative analysis with reference sequences,phylogenetic analysis,and network analysis were performed.ResultsThe isolates displayed≥98.87%similarity to E.multilocularis NADH dehydrogenase sub-unit 1(nad1)(894 bp)reference sequences deposited in GenBank.Furthermore,amplification of the nad4 and nad2 genes also confirmed all nine samples as E.multilocularis with>99.30%similarity.Additionally,three nuclear genes,pepck(1545 bp),elp-exons VII and VIII(566 bp),and elp-exon IX(256 bp),were successfully amplified and sequenced for one of the isolates with 98.42%similarity,confirming the isolates were correctly identified as E.multilocularis.Network analysis also correctly placed the isolates with other E.multilocularis.ConclusionsAs a result of the discovery of E.multilocularis in an unusual intermediate host,which is considered to have the highest zoonotic potential,the result clearly demonstrated the necessity for expanded surveillance in the area.

Impact of overgrazing on the transmission of Echinococcus multilocularis in Tibetan pastoral communities of Sichuan Province,China

Background Overgrazing was assumed to increase the population density of small mammals that are the intermediate hosts of Echinococcus multilocularis, the pathogen of alveolar echinococcosis in the Qinghai Tibet Plateau. This research tested the hypothesis that overgrazing might promote Echinococcus multilocularis transmission through increasing populations of small mammal, intermediate hosts in Tibetan pastoral communities. Methods Grazing practices, small mammal indices and dog Echinococcus multilocularis infection data were collected to analyze the relation between overgrazing and Echinococcus multilocularis transmission using nonparametric tests and multiple stepwise logistic regression. Results In the investigated area, raising livestock was a key industry. The communal pastures existed and the available forage was deficient for grazing. Open （common） pastures were overgrazed and had higher burrow density of small mammals compared with neighboring fenced （private） pastures; this high overgrazing pressure on the open pastures measured by neighboring fenced area led to higher burrow density of small mammals in open pastures. The median burrow density of small mammals in open pastures was independently associated with nearby canine Echinococcus multilocularis infection （P=-0.003, OR=1.048）. Conclusion Overgrazing may promote the transmission of Echinococcus multilocularis through increasing the population density of small mammals.

Suppression of acute rejective response following orthotopic liver transplantation in experimental rats infected with Echinococcus multilocularis

Background Hepatic alveolar echinococcosis （AE） is a parasitic disease in humans and caused by the Echinococcus multilocularis （Em）. Orthotopic liver transplantation （OLT） may be the only effective treatment for end-stage hepatic AE. However, in some AE patients, extrahepatic Em can not be completely eliminated after OLT. We aimed to study whether the immunological changes caused by Em evasion may influence the rejective response. Methods Rat modles of AE were established by injecting the Em suspension into abdomen of Brown Norway （BN） rats. Three months later, in the experimental group, the liver was transplanted from Lewis （LEW） rats to Em-infected BN rats. In the control group, transplantation was from LEW rats to healthy BN rats. Liver tissue and peripheral blood （PB） samples were collected on days 1, 3, 5, and 7 after OLT. Liver tissue was analyzed after hematoxylin and eosin （H＆E） staining; numbers of CD4, CD8, and CD28 on peripheral blood cells were detected by flow cytometry; and expression of the chemokine fractalkine （Fkn） was detected by reverse transcription PCR （RT-PCR）. Interleukin-10 （IL-10） was measured in the serum by enzyme-linked immunosorbent assay （ELISA）. In every group, eight BN rats were retained for observing survival time. Results The survival times of recipients in the experimental group were prolonged compared with those in the control group. The rejective response occurred later and was milder in the experimental group, percentage of CD4, CD8, CD28 T-cells and Fkn mRNA expression were lower in the experimental group. While the serum IL-10 levels were higher in the experimental group than those in the control group. Conclusions Acute rejective response after OLT was attenuated in the rats with Em infection, and the recipients＂ survival time was prolonged. Em may play a role in this process by elevating IL-10 secretion, decreasing the effector T cells, inhibiting the expression of Fkn, which lead to reduce the inflammatory cells infiltration into the liver.

A multiplex PCR for differential detection of Echinococcus granulosus sensu stricto,Echinococcus multilocularis and Echinococcus canadensis in China

Background:Echinococcosis caused byEchinococcus is one of the most major infectious diseases in north-west highland of China.E.granulosus sensu strict,E.multilocularis,andE.canadensis are known to be the only three species related to human health transmitting in the areas.To achieve targeted treatment and control of echinococcosis,the accurate identification and discrimination of the species are important.However,currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency.Methods:In the study,a set of primers were designed to aim at the three human-pathogenicEchinococcus species in China.The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity.A total of 73parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method.Results:The multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products.The detection limit of the assay for each of the three species was 5 pg/μl when they were tested separately.When DNA mixtures of the targeted species containing the same concentration were used as templates,the lowest amount of DNA which can be detected was 50 pg/μl,10 pg/μl and 5 pg/μl forE.granulosus s.s.,E.multilocularis,andE.canadensis respectively.No cross-reactivity was observed when DNA from eight genetically close species was used as control templates.The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected withE.shiquicus,which showed negative signals in the developed assay.Of all the tested stool materials,16 were previously found positive forEchinococcus by visual and microscopic examination.Among these 16 samples,13 were confirmed by the multiplex PCR,and the other three tested negative.Additionally,the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms.Conclusions:The developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation,which proven to be a promising tool utilized in clinic and surveillance system.

Lasiopodomys fuscus as an important intermediate host for Echinococcus multilocularis:isolation and phylogenetic identification of the parasite

Background:Echinococcus multilocularis causes alveolar echinococcosis(AE)and is widely prevalent in Qinghai Province,China,where a number of different species have been identified as hosts.However,limited information is available on the Qinghai vole(Lasiopodomys fuscus),which is hyper endemic to Qinghai Province and may represent a potential intermediate host of E.multilocularis.Thus,L.fuscus could contribute to the endemicity of AE in the area.Methods:Fifty Qinghai voles were captured from Jigzhi County in Qinghai Province for the clinical identification of E.multilocularis infection via anatomical examination.Hydatid fluid was collected from vesicles of the livers in suspected voles and subjected to a microscopic examination and PCR assay based on the barcoding gene of cox 1.PCR-amplified segments were sequenced for a phylogenetic analysis.E.multilocularis-infected Qinghai voles were morphologically identified and subjected to a phylogenetic analysis to confirm their identities.Results:Seventeen of the 50 Qinghai voles had E.multilocularis-infection-like vesicles in their livers.Eleven out of the 17 Qinghai voles presented E.multilocularis infection,which was detected by PCR and sequencing.The phylogenetic analysis showed that all 11 positive samples belonged to the E.multilocularis Asian genotype.A morphological identification and phylogenetic analysis of the E.multilocularis-infected Qinghai voles confirmed that all captured animals were L.fuscus.Conclusions:L.fuscus can be infected with E.multilocularis and plays a potential role in the life cycle and epidemiology of E.multilocularis in the Qinghai-Tibetan Plateau of China.

Distribution of the intermediate hosts of Echinococcus multilocularis in Shiqu County, Sichuan, China

Background The rodentia and lagomorpha animals are the intermediate hosts of Echinococcus multilocularis, their distribution and infection of this parasite may facilitate the infection of definitive hosts such as dogs. The aim of this study was to determine the distribution of the intermediate hosts of Echinococcus multilocularis in Shiqu County, Sichuan, China. Methods A systematic sampling method was used to investigate the density of burrows of rodents and lagomorphs at 97 pasture sites in winter and summer pastureland and remote sensing （RS） techno（ogy was used to correlate their densities to the distribution of these animals in different landscape types. Results Based on the densities of Ochotona curzoniae, Microtus fuscus （dependent variable） and their burrow densities （independent variable） in survey points, regression equations were fitted respectively （Ochotona curzoniae, P 〈0.0001, R2=0.8705; Microtus fuscus, P 〈0.0001,R2=0.9736）. Their burrow density in summer pastureland was higher than in winter pastureiand （F=36.65, P 〈0.0001）. The burrow densities of Ochotona curzoniae and Microtus fuscus in bareland and half-bareland are higher than in grassland （F=7.73, P〈0.001）. Conclusions The regression relationship between the densities of Ochotona curzoniae and Microtus fuscus and their burrow densities indicate that the burrow densities could reflect the animal densities and that the burrow density was greater in summer pastureland than in winter pastureland. The main distribution areas of the intermediate hosts were in bareland and half-bareland.

THE FIRST REPORT OF ECHINOCOCCUS MULTILOCULARIS IN DOMESTIC DOGS IN ZHANG COUNTY OF GANSU PROVINCE

In Zhang County of Gansu Province, 13 human cases of alveolar hydatid disease were reported up to 1990. However, the causative organism has not yet been described in animal hosts. In July 1990, postmortem examinations were made on 59 dogs (Canis familiaris) from the Caotan region of Zhang County. Of them, 6 were found to be infected with the adult worms of Echinococcus multilocularis. The morphological characteristics of the adult were compared in detail with those reported from different parts of China. Our adult specimens were closely resembling those of E. multilocularis reported from Ningxia. The role of domestic dogs in the epidemiology of alveolar echinococcosis in Zhang County was discussed.

Characteristics of the imaging diagnosis of alveolar echinococcosis

Alveolar echinococcosis(AE)primarily manifests in the liver and exhibits charac-teristics resembling those of slow-growing malignant tumours.Untreated Echino-coccus multilocularis infection can be lethal.By infiltrating the vascular systems,biliary tracts,and the hilum of the liver,it might lead to various problems.Due to its ability to infiltrate neighbouring tissues or metastasize to distant organs,AE can often be mistaken for malignancies.We present a concise overview of the epi-demiological and pathophysiological characteristics of AE,as well as the clinical manifestations of the disease.This article primarily examines the imaging charac-teristics of AE using various imaging techniques such as ultrasonography,com-puted tomography(CT),magnetic resonance imaging,diffusion-weighted ima-ging,and virtual non-enhanced dual-energy CT.We additionally examined the contribution of radiography in the diagnosis,treatment,and monitoring of the condition.

Long-term follow-up of liver alveolar echinococcosis using echinococcosis multilocularis ultrasound classification

BACKGROUND When Echinococcus multilocularis infects humans as a false intermediate host,alveolar echinococcosis(AE)usually manifests primarily intrahepatically and is initially asymptomatic.If the disease remains undiagnosed and untreated,progressive growth occurs,reminiscent of malignant tumours.The only curative therapy is complete resection,which is limited to localised stages,and palliative drug therapy is used otherwise.Consequently,early diagnosis and reliable detection of AE lesions are important.For this reason,abdominal ultrasonography,as the most common primary imaging for AE,relies on classification systems.AIM To investigate how hepatic AE lesion sonomorphology changes over time in the Echinococcosis Multilocularis Ulm Classification(EMUC)-ultrasound(US)classification.METHODS Based on data from Germany’s national echinococcosis database,we evaluated clinical and US imaging data for 59 patients according to the AE case definition in our preliminary retrospective longitudinal study.There had to be at least two liver sonographies≥6 mo apart,≥1 hepatic AE lesion,and complete documentation in all US examinations.The minimum interval between two separately evaluated US examinations was 4 wk.The AE reference lesion was the largest hepatic AE lesion at the time of the first US examination.To classify the sonomorphologic pattern,we used EMUC-US.In addition to classifying the findings of the original US examiner,all reference lesions at each examination time point were assigned EMUC-US patterns in a blinded fashion by two investigators experienced in US diagnosis.Statistical analysis was performed using SAS version 9.4(SAS Institute Inc.,Cary,NC,United Stated).P values<0.05 were considered statistically significant.RESULTS The preliminary study included 59 patients,38(64.5%)women and 21(35.6%)men.The mean age at initial diagnosis was 59.9±16.9 years.At the time of initial ultrasonography,a hailstorm pattern was present in 42.4%(25/59)of cases,a hemangioma-like pattern in 16.9%(10/59),a pseudocystic pattern in 15.3%(9/59),and a metastasis-like pattern in 25.4%(15/59).For the hailstorm pattern,the average lesion size was 67.4±26.3 mm.The average lesion size was 113.7±40.8 mm with the pseudocystic pattern and 83.5±27.3 mm with the hemangioma-like pattern.An average lesion size of 21.7±11.0 mm was determined for the metastasis-like pattern.Although the sonomorphologic pattern remained unchanged in 84.7%(50/59)of AE reference lesions,15.3%(9/59)showed a change over time.A change in pattern was seen exclusively for AE lesions initially classified as hemangioma-like or pseudocystic.A total of 70%(7/10)of AE lesions initially classified as hemangioma-like showed a relevant change in pattern over time,and 85.7%(6/7)of these were secondarily classified as having a hailstorm pattern,with the remainder(1/7;14.3%)classified as having a pseudocystic pattern.A total of 22.2%(2/9)of AE lesions initially classified as pseudocystic showed a relevant change in pattern over time and were classified as having a hailstorm pattern.For AE lesions initially classified as having a hailstorm or metastatic pattern,no pattern change was evident.All patients with pattern change were on continuous drug therapy with albendazole.CONCLUSION The sonomorphology of hepatic AE lesions may change over time.The hemangioma-like and pseudocystic patterns are affected.

Evaluation of intrahepatic manifestation and distant extrahepatic disease in alveolar echinococcosis

BACKGROUND The main endemic areas of alveolar echinococcosis(AE)are in Central Europe and Western China.Both the infiltration of intrahepatic vascular and bile duct structures as well as extrahepatic disease can lead to further complications and may increase morbidity in patients with AE.AIM To evaluate vascular/biliary involvement in hepatic AE and its distant extrahepatic disease manifestations in an international collective was the aim.METHODS Consecutively,five experienced examiners evaluated contrast-enhanced abdominal computed tomography(CT)scans for 200 patients with hepatic AE of each of four locations(n=50)in Germany,France and China.Therefore,we retrospectively included the 50 most recent abdominal contrast-enhanced CT examinations at each center,performed because of hepatic AE from September 21,2007 to March 21,2018.AE liver lesions were classified according to the echinococcosis multilocularis Ulm classification for CT(EMUC-CT).Distant extrahepatic manifestations were documented either by whole body positron emission tomography–CT or with the addition of thoracic CT and cranial magnetic resonance imaging.Vascular/biliary involvement of the hepatic disease as well as the presence of distant extrahepatic manifestations were correlated with the EMUC-CT types of liver lesion.Statistical analysis was performed using SAS Version 9.4(SAS Institute Inc.,Cary,NC,United States).RESULTS Distant extrahepatic AE manifestations were significantly more frequent in China than in Europe(P=0.0091).A significant relationship was found between the presence of distant extrahepatic disease and AE liver lesion size(P=0.0075).Vascular/biliary structures were involved by the liver lesions significantly more frequently in China than in Europe(P<0.0001),and vascular/biliary involvement depended on lesion size.Different morphological types of AE liver lesions led to varying frequencies of vascular/biliary involvement and were associated with different frequencies of distant extrahepatic manifestations:Vascular/biliary involvement as a function of lesions primary morphology ranged from 5.88%of type IV liver lesions to 100%among type III lesions.Type IV differed significantly in these associations from types I,II,and III(P<0.0001).With respect to extrahepatic disease,the primary morphology types IV and V of liver lesions were not associated with any case of distant extrahepatic disease.In contrast,distant extrahepatic manifestations in types I–III were found to varying degrees,with a maximum of 22%for type III.CONCLUSION Different CT morphological patterns of hepatic AE lesions influence vascular/biliary involvement and the occurrence of distant extrahepatic manifestations.There are intercontinental differences regarding the characteristics of AE manifestation.

多房棘球绦虫在我国自然动物宿主的发现及其形态学研究

本文报告在我国宁夏固原地区野生动物首次发现多房棘球蚴痫的病原自然感染。当地红狐自然感染多房棘球绦虫成虫27.27％,感染强度1,840—4,050条;达乌尔黄鼠citellus dauricus自然感染多房棘球蚴0.2％。成虫孕节人工感染小白鼠后163—424天,在其肝脏内可检到发育含有大量成熟原头节的多房棘球蚴。成虫期与幼虫期的形态特征有详细的描述并同国外代表性地区的报告列表比较。对我国西北地区人体多房棘球蚴病作综合报道。

Proposal of a computed tomography classification for hepatic alveolar echinococcosis

AIM: To establish a computed tomography (CT)-morphological classification for hepatic alveolar echinococcosis was the aim of the study.METHODS: The CT morphology of hepatic lesions in 228 patients with confirmed alveolar echinococcosis (AE) drawn from the Echinococcus Databank of the University Hospital of Ulm was reviewed retrospectively. For this reason, CT datasets of combined positron emission tomography (PET)-CT examinations were evaluated. The diagnosis of AE was made in patients with unequivocal seropositivity; positive histological findings following diagnostic puncture or partial resection of the liver; and/or findings typical for AE at either ultrasonography, CT, magnetic resonance imaging or PET-CT. The CT-morphological findings were grouped into the new classification scheme.RESULTS: Within the classification a lesion was dedicated to one out of five &#x0201c;primary morphologies&#x0201d; as well as to one out of six &#x0201c;patterns of calcification&#x0201d;. &#x0201c;primary morphology&#x0201d; and &#x0201c;pattern of calcification&#x0201d; are primarily focussed on separately from each other and combined, whereas the &#x0201c;primary morphology&#x0201d; V is not further characterized by a &#x0201c;pattern of calcification&#x0201d;. Based on the five primary morphologies, further descriptive sub-criteria were appended to types&#x02005;I-III. An analysis of the calcification pattern in relation to the primary morphology revealed the exclusive association of the central calcification with type IV primary morphology. Similarly, certain calcification patterns exhibited a clear predominance for other primary morphologies, which underscores the delimitation of the individual primary morphological types from each other. These relationships in terms of calcification patterns extend into the primary morphological sub-criteria, demonstrating the clear subordination of those criteria.CONCLUSION: The proposed CT-morphological classification (EMUC-CT) is intended to facilitate the recognition and interpretation of lesions in hepatic alveolar echinococcosis. This could help to interpret different clinical courses better and shall assist in the context of scientific studies to improve the comparability of CT findings.

Estimating the prevalence of Echinococcus in domestic dogs in highly endemic for echinococcosis

Background:Cystic echinococcosis(CE)and alveolar echinococcosis(AE)are highly endemic in Xiji County of Ningxia Hui Autonomous Region(NHAR)in China where the control campaign based on dog de-worming with praziquantel has been undertaken over preceding decades.This study is to determine the current prevalence of Echinococcus granulosus and E.multilocularis in domestic dogs and monitor the echinococcosis transmission dynamics.Methods:Study villages were selected using landscape patterns(Geographic Information System,GIS)for Echinococcus transmission“hot spots”,combined with hospital records identifying risk areas for AE and CE.A survey of 750 domestic dogs,including copro-sampling and owner questionnaires,from 25 selected villages,was undertaken in 2012.A copro-multiplex PCR assay was used for the specific diagnosis of E.granulosus and E.multilocularis in the dogs.Data analysis,using IBM SPSS Statistics,was undertaken,to compare the prevalence of the two Echinococcus spp.in dogs between four geographical areas of Xiji by theχ^(2)test.Univariate analysis of the combinations of outcomes from the questionnaire and copro-PCR assay data was carried out to determine the significant risk factors for dog infection.Results:The highest de-worming rate of 84.0%was found in the northwest area of Xiji County,and significant differences(P<0.05)in the de-worming rates among dogs from the four geographical areas of Xiji were detected.The highest prevalence(19.7%,59/300)of E.multilocularis occurred in northwest Xiji,though the highest prevalence(18.1%,38/210)of E.granulosus occurred in southwest Xiji.There was no significant difference(P>0.05)in the prevalence of E.granulosus in dogs from the northwest,southwest,northeast,and southeast of Xiji,but there were significant differences(P<0.05)between dogs infected with E.multilocularis from the four areas.None of the other independent variables was statistically significant.Conclusions:The results from this study indicate a high prevalence of both E.granulosus and E.muiltilocularis in dogs in Xiji County,NHAR.Transmission of E.multilocularis was more impacted by geographical risk-factors in Xiji County than that of E.granulosus.Dogs have the potential to maintain the transmission of both species of Echinococcus within local Xiji communities,and the current praziquantel dosing of dogs appears to be ineffective or poorly implemented in this area.