Immunization of dogs against Echinococcus granulosus using worm secretory antigens.

A new approach to the immunization of dogs against Echinococcus granulosus, using secretory antigens derived from adult tapeworms cultivated in vitro, resulted in a highly significant suppression of egg production by Echinococcus granulosus in immunized dogs. There was also a significant reduction in the number of proglottids per worm in immunized dogs. It appeared that the suppression of egg production was due to an immune effect on both somatic growth and germinal development. The implications of this work with respect to hydatidosis control programmes are discussed.     

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.     

Moniezia expansa: Subcellular distribution,kinetic properties of acetylcholinesterase and effects of inhibitors and anthelmintics

Acetylcholinesterase (EC 3:1:1:7) has been demonstrated biochemically within partially purified whole worm homogenates of Moniezia expansa. Linear activity occurred with temperature, enzyme concentration, and time. The pH optimum was 8.5 and the Michaelis constant 2.8 mM with inhibition by excess substrate. Inhibitor and specific substrate studies indicated that butyrylcholinesterase was probably absent. The molecular weight of AChE was in excess of 300,000. Greatest activity occurred in the 22,000 and 100,000g particulate fractions. Ultrastructural staining showed that activity was restricted to the ribosomes and cisternae of the rough endoplasmic reticulum. Quinacrine hydrochloride caused 48% inhibition of AChE at 10^?3M and haloxon (di(2-chloroethyl)-3-chloro-4-methyl-7-coumarinyl phosphate) caused 97% inhibition at 10^?4M. No appreciable inhibition (< 25%) occurred with 10^?4M bunamidine hydroxynaphthoate, bephenium hydroxynaphthoate, pyrantel tartrate, p-toluoyl phenyl hydrazone, dichlorphen, thiabendazole, mebendazole, fenbendazole, cambendazole, albendazole, parbendazole, oxibendazole, oxfendazole, praziquantel, piperazine adipate, arecoline hydrobromide, and sodium acetarsol.     

Control of cystic echinococcosis in the Middle Atlas,Morocco: Field evaluation of the EG95 vaccine in sheep and cesticide treatment in dogs

BackgroundCystic echinococcosis (CE) is an important cause of human morbidity and mortality worldwide, particularly in Morocco and other North African countries.Methodology/Principal findingsWe investigated the potential of three strategies to reduce Echinococcus granulosus transmission: (1) 4-monthly treatment of dogs with praziquantel, (2) vaccination of sheep with the EG95 vaccine and (3) a combination of both measures. These measures were implemented during four consecutive years in different areas of the Middle Atlas Mountains in Morocco. The outcome of the interventions was assessed through hydatid cyst (viable and non-viable) counts in liver and lungs using necropsy or in vivo ultrasound examination of the liver. A total of 402 lambs were recruited for annual vaccination with the EG95 anti-E. granulosus vaccine and 395 similar lambs were selected as non-vaccinated controls.At approximately four years of age the relative risk (estimated as odds ratio) for vaccinated sheep to have viable hydatid cysts compared with non-vaccinated controls was 3% (9.37% of the vaccinated sheep were found infected while 72.82% of the controls were infected; p = 0.002). The number of viable cysts in vaccinated animals was reduced by approximately 97% (mean counts were 0.28 and 9.18 respectively; p<0.001). An average of 595 owned dogs received 4-monthly treatment during the 44 months trial, corresponding to 91% of the owned dog population. Approximately, 5% of them were examined for E. granulosus adult worms by arecoline purge or eggs in feces (confirmed by PCR). The proportion of infected dogs significantly decreased after treatment (12% versus 35%; p<0.001). Post-treatment incidence of re-infestation corresponded to a monthly risk of 4% (95% CI: 3–6%). Treatment of owned dogs on a 4-monthly basis did not reduce the level of transmission of E. granulosus to sheep, nor did it enhance the level of control generated by vaccination of sheep with EG95, possibly because of unowned dogs and wild canids were not treated.Conclusions/SignificanceThese data suggest that vaccination of sheep with EG95 has the potential to reduce the level of CE in Morocco and in other parts of the world with similar transmission dynamics. Under the epidemiological circumstances existing in the trial area, 4-monthly treatment of owned dogs with praziquantel was insufficient to have a major impact of E. granulosus transmission to sheep.     

Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis In vitro

Herd R. P., Ko L., Weisbrode S. E. and Heath D. D. 1984. Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis in vitro. International Journal for Parasltology14:141–149. Sequential changes (5,10, 20, 30,40, 50 min, 1, 2, 3, 4, 5, 6, 7, 8, h) were observed by light, scanning and transmission electron microscopy after 38-day-old adult Echinococcus granulosus were exposed to 50% guinea pig serum in vitro. Early changes within 3 h included contraction of worms, fusion of microtriehes, vacuolization and vesiculization of the distal cytoplasm, followed by rupture of vesicles leading to erosion and loss of the distal cytoplasm. This was most marked in the terminal proglottid but ultimately there was complete erosion of the distal cytoplasm of all proglottids and the scolex. After 3 h there was loss of definition of organelles, apparent edema of the perinuclear cytoplasm and, in some instances, rupture of the circular muscle layer with extrusion of parenchyma. Adult tape-worms exposed to heat-inactivated complement showed none of these changes. Lysis and death of the parasite was attributed to osmotic changes subsequent to the formation of trans-membrane channels induced by complement-mediated attack of the tegument after activation of the alternate pathway by factors present in the cestode tegument.     

Anomalous segmentation of Diphyllobothrium nihonkaiense

An anomalous tapeworm with abnormal segmentation was obtained from a 6-year-old boy in Japan. The tapeworm consisted of proglottids with slanted anterior and posterior margins of proglottids and 4–6 sets of reproductive organs arranged between the margins. The morphology of the tapeworm did not correspond to any of the described cestodes. However, molecular identification based on nuclear and mitochondrial genes clearly showed the tapeworm was Diphyllobothrium nihonkaiense.     

First Clinical Cases of Spirometrosis in Two Cats in Korea

This study reports the first two clinical cases of spirometrosis caused by Spirometra sp. in cats in Korea. In these two cases, the cats vomited, and long proglottids of tapeworm were recovered. The sick cats presented with anorexia and lethargy. However, they unexpectedly showed no diarrhea, which is the main symptom of spirometrosis. Based on a fecal floatation test as well as morphological and molecular analyses, the parasite was diagnosed as Spirometra sp. The 2 cases were treated with praziquantel. This study suggests regular monitoring of health and deworming in companion animals, even when animals are well cared for, with regular preventive medication. Additionally, spirometrosis should be considered in the differential diagnosis in cases of gastrointestinal symptoms in Spirometra endemic areas.     

Natural Infection of the Ground Squirrel (Spermophilus spp.) with Echinococcus granulosus in China

Background

Echinococcus granulosus is usually transmitted between canid definitive hosts and ungulate intermediate hosts.

Methodology/Principal Findings

Lesions found in the livers of ground squirrels, Spermophilus dauricus/alashanicus, trapped in Ningxia Hui Autonomous Region, an area in China co-endemic for both E. granulosus and E. multilocularis, were subjected to molecular genotyping for Echinococcus spp. DNA. One of the lesions was shown to be caused by E. granulosus and subsequently by histology to contain viable protoscoleces.

Conclusions/Significance

This is the first report of a natural infection of the ground squirrel with E. granulosus. This does not provide definitive proof of a cycle involving ground squirrels and dogs or foxes, but it is clear that there is active E. granulosus transmission occurring in this area, despite a recent past decline in the dog population in southern Ningxia.     

The migration of oncospheres of Taenia pisiformis, T. serialis and Echinococcus granulosus within the intermediate host

Heath D. D., 1971. The migration of oncospheres of Taenia pisiformis, T. serialis and Echinococcus granulosus within the intermediate host. International journal for Parasitology, 1: 145–152. The oncospheres of Taenia pisiformis and T. serialis hatched, became activated and penetrated the tips of the villi in the jejunal area of the rabbit small intestine. Similar results were obtained forE. granulosus, T. hydatigena and T. ovis in sheep. Most species of oncospheres appeared to progress down the villus beneath the columnar epithelium until a venule of diameter sufficient to allow passive transport to the liver was penetrated. The relatively large villus lacteal of ruminants, and the large diameter ofE. granulosus oncospheres, appeared to provide an opportunity for these organisms to penetrate the lacteal and translocate in the lymph. Parenteral inoculations of activated oncospheres indicated that T. pisiformis and E. granulosus oncospheres probably reach the liver in the portal vein. E. granulosus oncospheres infecting the lung may reach that organ in the lymph. T. serialis oncospheres were able to pass through both the liver and lungs in order to reach the muscles. A stimulus may exist in the liver causing cessation of movement and initiation of postoncospheral development for T. pisiformis and E. granulosus, but not T. serialis.     

Echinococcus granulosus cysts: Early development in vitro in the presence of serum from infected sheep

When cultured in vitro in the presence of serum from a number of sheep infected with Echinococcus granulosus cysts, varying proportions of oncospheres died within 24 h. Of the survivors, some died during reorganization into cysts; others were able to develop normally but showed evidence of precipitates in the outer layers of the cyst. The lethal effects were removed by heating the serum to 56°C for 30 min and could be restored by the addition of freshly-collected normal sheep serum. In the presence of serum from sheep immunized against E. granulosus, most oncospheres were dead within 24 h, and few or none of the survivors were able to reorganize into cysts.     

Echinococcus granulosus protoscoleces promotes proliferation and invasion of hepatocellular carcinoma cells

There may exist a connection between Echinococcus granulosus infection and cancer development. Here, it is aimed to investigate specific effects of E. granulosus protoscoleces (PSCs) on the proliferation and invasion capacities of hepatocellular carcinoma (HCC) cells in vitro and ex vitro. HepG2 cells were cultured with different quantities of E. granulosus PSCs in vitro. MTT analysis was used to evaluate effects of E. granulosus PSCs on the proliferation of HepG2 cells. Besides, scratch and transwell assays were respectively used for the detection of HepG2 cells migration and invasion capacities after co-culture with E. granulosus PSCs. Then, HepG2 cells were subcutaneously transplanted into nude mice with or without E. granulosus PSCs. From the 25th day of transplantation, the volume of subcutaneous lesions was measured every four days. At the 37th day, subcutaneous lesions were removed and their weight was evaluated. H&E staining was used for detecting basic pathological changes. HepG2 cells grew well without obvious morphological changes. Proliferation rate and migration capacity of HepG2 cells were higher in the co-culture group than the control group, which was closely associated with quantities of E. granulosus PSCs and co-culture time length. Moreover, HepG2 cells co-cultured with E. granulosus PSCs had stronger invasion ability than the control HepG2 cells. Importantly, there existed significant differences in the volume and weight of subcutaneous lesions after transplanting HepG2 cells with E. granulosus PSCs than the control group. HepG2 cells were also more pathologically heterogeneous in morphology after transplantation with E. granulosus PSCs. Thus, E. granulosus PSCs may promote proliferation and invasion of HCC cells.     

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.     

A Pilot Study for Control of Hyperendemic Cystic Hydatid Disease in China

Background

Cystic hydatid disease (CHD) is a global parasitic zoonosis caused by the dog tapeworm, Echinococcus granulosus. The disease is hyperendemic in western China because of poor economic development; limited community knowledge of CHD; widespread, small-scale household animal production; home killing of livestock; and the feeding of dogs with uncooked offal.

Methodology/Principal Findings

A control program focusing on monthly praziquantel (PZQ) treatment of all registered dogs and culling unwanted and stray dogs has been designed to control CHD in hyperendemic areas in China. A pilot field control project in two counties (Hutubi and Wensu) in Xinjiang, China showed that after 4 years of treatment, the prevalence of dogs with E. granulosus was reduced from 14.7% and 18.6%, respectively, to 0%, and this caused a 90%–100% decrease of CHD in sheep born after commencement of the control program.

Conclusions/Significance

The strategy aimed at preventing eggs being released from dogs into the environment by treating animals before adult tapeworms are patent can decrease E. granulosus transmission and considerably reduce hyperendemic CHD. Monthly treatment of dogs with PZQ and culling unwanted and stray dogs have been shown to be an efficient, highly cost-effective and practicable measure for implementation in rural communities. As a result, the Chinese Ministry of Health has launched an extensive CHD control program in 117 counties in western China using this control strategy.     

Molecular Characterization of Echinococcus granulosus Cysts in North Indian Patients: Identification of G1, G3, G5 and G6 Genotypes

Background

Cystic echinococcosis (CE) caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s) of the parasite responsible for human hydatidosis in North India.

Methodology/Principal Findings

To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE) patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1) sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32) were found to be infected with hydatid cyst of either G3 (53.1%) or G1 (40.62%) genotype and one each of G5 (cattle strain) and G6 (camel strain) genotype.

Conclusions/Significance

These findings demonstrate the zoonotic potential of G1 (sheep strain) and G3 (buffalo strain) genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain) in an Asian country and presence of G6 genotype (camel strain) in India. The results may have important implications in the planning of control strategies for human hydatidosis.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Immunological responses and potency of the EG95NC− recombinant sheep vaccine against cystic echinococcosis

Cystic echinococcosis (CE) is a zoonotic disease caused by the cestode parasite Echinococcus granulosus. The disease has an important impact on human health as well as economic costs including the cost of treatment as well as loss of productivity for the livestock industry. In many parts of the world where the disease is endemic, sheep and other livestock play an important role in the parasite's transmission. A vaccine to protect livestock against CE can be effective in reducing transmission and economic costs of the disease. A recombinant antigen vaccine has been developed against infection with E. granulosus (EG95) which could potentially be used to reduce the level of E. granulosus transmission and decrease the incidence of human infections. Further development of the EG95 recombinant vaccine as a combined product with clostridial vaccine antigens is one potential strategy which could improve application of the hydatid vaccine by providing an indirect economic incentive to livestock owners to vaccinate against CE. In this study we investigated the efficacy of the EG95 recombinant vaccine produced in Morocco by vaccination of sheep, including a combined vaccine incorporating EG95 and clostridia antigens. Vaccination with EG95 either as a monovalent vaccine or combined with clostridia antigens, protected sheep against a challenge infection with E. granulosus eggs and induced a strong, long lasting, and specific antibody response against the EG95 antigen.     

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

Background

Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved.

Methodology/Principal Findings

Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus.

Conclusions/Significance

This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.     

A biochemical and ultrastructural study of the mode of action of bunamidine against Hymenolepis nana.

In the presence of bunamidine the cestode Hymenolepis nana shows a decrease in the rate of glucose uptake and an increase in the rate of glucose efflux. The surface phosphatase activity is stimulated many fold and much of the enzyme activity is released into the incubation medium. It is suggested that disruption of the integument by bunamidine is the cause of these effects and the mode of action by which death of the worm is caused.These biochemical indications of integumental damage are confirmed by ultrastructural studies which show that there is complete loss of the surface tissue down to the level of the fibrous basal lamina.     

Protein undernutrition reduces the efficacy of praziquantel in a murine model of Schistosoma mansoni infection

BackgroundUndernutrition and schistosomiasis are public health problems and often occur in low and middle-income countries. Protein undernutrition can alter the host-parasite environment system and aggravate the course of schistosomiasis. This study aimed to assess the impact of a low-protein diet on the efficacy of praziquantel.Methodology/Principal findingsThirty-day-old mice were fed with a low-protein diet, and 40 days later, they were individually infected with fifty Schistosoma mansoni cercariae. A 28-day-treatment with praziquantel at 100 mg/kg for five consecutive days followed by distilled water begins on the 36^th day post-infection. Mice were sacrificed on the 64^th day post-infection. We determined the parasitological burden, liver and intestine histomorphometry, liver injury, and immunomodulation parameters. Praziquantel treatment of infected mice fed with a standard diet (IN-PZQ) resulted in a significant reduction of worm and egg burdens and a normalization of iron and calcium levels. The therapy also improved schistosomiasis-induced hepatopathy and oxidative stress. The anti-inflammatory and immunomodulatory activities of praziquantel were also significant in these mice. When infected mice receiving the low-protein diet were treated with praziquantel (ILP-PZQ), the body weight loss and hepatomegaly were not alleviated, and the worm and liver egg burdens were significantly higher than those of IN-PZQ mice (P < 0.001). The treatment did not reduce the increased activities of ALT and γ-GGT, the high malondialdehyde concentration, and the liver granuloma volume. The iron and calcium levels were not ameliorated and differed from those of IN-PZQ mice (P < 0.001 and P < 0.05). Moreover, in these mice, praziquantel treatment did not reverse the high level of IL-5 and the low mRNA expression of CCL3/MIP-1α and CXCL-10/IP-10 induced by S. mansoni infection.Conclusion/SignificanceThese results demonstrated that a low-protein diet reduced the schistosomicidal, antioxidant, anti-inflammatory, and immunomodulatory activities of praziquantel.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.