Cristian A. Alvarez Rojas

Pilot field trial of the EG95 vaccine against ovine cystic echinococcosis in Rio Negro, Argentina: Early impact and preliminary data

Cystic echinococcosis is endemic in the Rio Negro province of Argentina. After 30 years of control using praziquantel in dogs the transmission rate to humans and sheep has decreased significantly, however transmission persists. The objective of the study is to assess the impact of the inclusion of the EG95 vaccine for sheep in the control programme, including analysis of the vaccines operative feasibility in field conditions. The vaccine was applied in an area comprising four communities of native people including 79 farms with 3146 lambs and 311 dogs in total. Seventy one farms were designated as control areas where no vaccinations were undertaken while vaccinations of lambs undertaken on 91 farms. Lambs received two vaccinations with the EG95 vaccine followed by a single booster injection when the animals were 1-1.5 years of age. Farm locations were defined using GPS coordinates for the houses. Evidence for Echinococcus granulosus transmission was monitored by coproantigen ELISA on samples of dog faeces, by E. granulosus-specific PCR using soil samples, and anti-E. granulosus antibody assessments in sera from 2 to 4 teeth lambs, purgation of dogs to detect E. granulosus worms and necropsy on adult sheep. Before the vaccine was introduced, 26.2% of sheep with 2-4 teeth were positive using ELISA/WB, the prevalence decreased to 7.8% at the third year following use of the vaccine. Necropsy of animals older than 6 years (not vaccinated) showed that 66.1% of animals were infected with E. granulosus. In dogs, 4% was found positive for E. granulosus using arecoline purgation and 24.7% of the farms were infected using coproELISA/WB. During the first year of vaccination 2721 lambs received the first vaccine dose and 2448 received a booster. In the second year 2138 lambs were initially vaccinated and 1745 received a booster, and 1308 animals received the third dose. During the third year 1110 lambs received the first dose from which 539 received a booster and 723 animals received the third dose. An analysis of advantages and limitations of the diagnostic techniques used and the ability of the geospatial analysis to detect risk area are included. Based in the immunodiagnostic techniques, the EG95 vaccine has been able to prevent the infection in animals up to 3 years old. Also, the difficulties in the field for the correct vaccine administration and the social features and habits that may impact on echinococcosis control are included in the analysis.

Transcriptional analysis identifies key genes involved in metabolism, fibrosis/tissue repair and the immune response against Fasciola hepatica in sheep liver

BackgroundAlthough fascioliasis has been relatively well studied, little is known about the molecular basis of this disease. This is particularly relevant, considering the very different response that sheep have to Fasciola hepatica relative to cattle. The acute phase of this disease is severe in sheep, whereas chronic fascioliasis is more common in cattle.MethodsTo begin to explore the host-response to Fasciola in sheep and improve the understanding of the host-pathogen interactions during the parasite’s migration through liver parenchyma to the bile duct, we used RNA sequencing (RNA-seq) to investigate livers from sheep infected for eight weeks compared with those from uninfected controls.ResultsThis study identified 572 and 42 genes that were up- and down-regulated, respectively, in infected livers relative to uninfected controls. Our molecular findings provide significant new insights into the mechanisms linked to metabolism, fibrosis and tissue-repair in sheep, and highlight the relative importance of specific components of immune response pathways, which appear to be driven toward a suppression of inflammation.ConclusionsThis study is, to our knowledge, the first detailed investigation of the transcriptomic responses in the liver tissue of any host to F. hepatica infection. It defines the involvement of specific genes associated with the host’s metabolism, immune response and tissue repair/regeneration, and highlights an apparent overlapping function of many genes involved in these processes.

Time-Course Study of the Transcriptome of Peripheral Blood Mononuclear Cells (PBMCs) from Sheep Infected with Fasciola hepatica

Fasciola hepatica is a parasitic trematode that infects a wide range of mammalian hosts, including livestock and humans, in temperate and tropical regions globally. This trematode causes the disease fascioliasis, which consists of an acute phase (≤ 12 weeks) during which juvenile parasites migrate through the host liver tissues, and a chronic phase (> 12 weeks) following the establishment of adult parasites in the liver bile ducts. Few studies have explored the progression of the host response over the course of Fasciola infection in the same animals. In this study, we characterized transcriptomic changes in peripheral blood mononuclear cells (PBMCs) collected from sheep at three time points over the first eight weeks of infection relative to uninfected controls. In total, 183 and 76 genes were found to be differentially transcribed at two and eight weeks post-infection respectively. Functional and pathway analysis of differentially transcribed genes revealed changes related to T-cell activation that may underpin a Th2-biased immune response against this parasite. This first insight into the dynamics of host responses during the early stages of infection improves the understanding of the pathogenesis of acute fascioliasis, informs vaccine development and presents a set of PBMC markers with diagnostic potential.

Techniques for the Diagnosis of Fasciola Infections in Animals: Room for Improvement

The common liver fluke, Fasciola hepatica, causes fascioliasis, a significant disease in mammals, including livestock, wildlife and humans, with a major socioeconomic impact worldwide. In spite of its impact, and some advances towards the development of vaccines and new therapeutic agents, limited attention has been paid to the need for practical and reliable methods for the diagnosis of infection or disease. Accurate diagnosis is central to effective control, particularly given an emerging problem with drug resistance in F. hepatica. Traditional coprological techniques have been widely used, but are often unreliable. Although there have been some advances in establishing immunologic techniques, these tools can suffer from a lack of diagnostic specificity and/or sensitivity. Nonetheless, antigen detection tests seem to have considerable potential, but have not yet been adequately evaluated in the field. Moreover, advanced nucleic acid-based methods appear to offer the most promise for the diagnosis of current infection. This chapter (i) provides a brief account of the biology and significance of F. hepatica/fascioliasis, (ii) describes key techniques currently in use, (iii) compares their advantages/disadvantages and (iv) reviews polymerase chain reaction-based methods for specific diagnosis and/or the genetic characterization of Fasciola species.

Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus

Cystic echinococcosis in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. A number of genotypes of the parasite (designated G1-G10) are known to exist, with the genotype cluster G1-G3 and genotype G6 being responsible for the majority of humans infections. A recombinant vaccine has been developed for use in livestock to prevent infection with E. granulosus. The vaccine is based on the antigen EG95 which is expressed in the early larval stage (oncosphere) of the parasite. The EG95 antigen was originally cloned from the G1 genotype of E. granulosus and the protein has been found to be encoded by members of a small family of related genes in this genotype. Reliable information has not been available about the likely efficacy of the EG95 vaccine against genotypes other than G1. In this study, genomic DNA cloning techniques were used to characterize seven eg95-related gene fragments from the G6 genotype of E. granulosus. Three proteins appear to be encoded by these genes. Considerable differences were found between the EG95 related proteins from the G6 genotype compared with the EG95 protein from the G1 genotype. These differences suggest that the EG95-related proteins from the G6 genotype may have different antigenic epitopes compared with the current vaccine antigen. Data presented in this study have implications for future vaccine design and provide the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of cystic echinococcosis transmission.

High intraspecific variability of Echinococcus granulosus sensu stricto in Chile

Echinococcus granulosus sensu stricto is the major cause of cystic echinococcosis in most human and animal cases in the world and the most widespread species within the E. granulosus sensu lato complex. E. granulosus s.s. remains endemic in South America together with other species of the Echinococcus genus, especially in some areas in Argentina, Brazil, Chile and Peru. Except for a single human case caused by E. canadensis (G6) described in the literature, only E. granulosus s.s. has been found in the Chilean territory. In the current study 1609bp of the cox1 gene from 69 Chilean isolates of E. granulosus s.s. from humans and animals were analysed. In total, 26 cox1 haplotypes were found, including the widespread haplotype EG01 (22 isolates) and also EGp1 (5), EgRUS7 (1), EgAus02 (1) and EgAus03 (2). Twenty-one different haplotype not previously described were identified from 38 Chilean isolates designated EgCL1-EgCL21. Previous work had described low variability of E. granulosus s.s. in South America, based on isolates from Peru. Results obtained in this work challenge the previously described idea of the low diversity of the parasite in South America, and warrant future investigation on the origin and spread of the parasite in the continent after the Spanish arrival.

Time-Dependent Transcriptional Changes in Axenic Giardia duodenalis Trophozoites

Giardia duodenalis is the most common gastrointestinal protozoan parasite of humans and a significant contributor to the global burden of both diarrheal disease and post-infectious chronic disorders. Although G. duodenalis can be cultured axenically, significant gaps exist in our understanding of the molecular biology and metabolism of this pathogen. The present study employed RNA sequencing to characterize the mRNA transcriptome of G. duodenalis trophozoites in axenic culture, at log (48 h of growth), stationary (60 h), and declining (96 h) growth phases. Using ~400-times coverage of the transcriptome, we identified 754 differentially transcribed genes (DTGs), mainly representing two large DTG groups: 438 that were down-regulated in the declining phase relative to log and stationary phases, and 281 that were up-regulated. Differential transcription of prominent antioxidant and glycolytic enzymes implicated oxygen tension as a key factor influencing the transcriptional program of axenic trophozoites. Systematic bioinformatic characterization of numerous DTGs encoding hypothetical proteins of unknown function was achieved using structural homology searching. This powerful approach greatly informed the differential transcription analysis and revealed putative novel antioxidant-coding genes, and the presence of a near-complete two-component-like signaling system that may link cytosolic redox or metabolite sensing to the observed transcriptional changes. Motif searching applied to promoter regions of the two large DTG groups identified different putative transcription factor-binding motifs that may underpin global transcriptional regulation. This study provides new insights into the drivers and potential mediators of transcriptional variation in axenic G. duodenalis and provides context for static transcriptional studies.

First meeting "cystic echinococcosis in Chile, update in alternatives for control and diagnostics in animals and humans"

This report summarizes the outcomes of a meeting on cystic echinococcosis (CE) in animals and humans in Chile held in Santiago, Chile, between the 21st and 22nd of January 2016. The meeting participants included representatives of the Departamento de Zoonosis, Ministerio de Salud (Zoonotic Diseases Department, Ministry of Health), representatives of the Secretarias Regionales del Ministerio de Salud (Regional Department of Health, Ministry of Health), Instituto Nacional de Desarrollo Agropecuario (National Institute for the Development of Agriculture and Livestock, INDAP), Instituto de Salud Pública (National Institute for Public Health, ISP) and the Servicio Agrícola y Ganadero (Animal Health Department, SAG), academics from various universities, veterinarians and physicians. Current and future CE control activities were discussed. It was noted that the EG95 vaccine was being implemented for the first time in pilot control programmes, with the vaccine scheduled during 2016 in two different regions in the South of Chile. In relation to use of the vaccine, the need was highlighted for acquiring good quality data, based on CE findings at slaughterhouse, previous to initiation of vaccination so as to enable correct assessment of the efficacy of the vaccine in the following years. The current world’s-best-practice concerning the use of ultrasound as a diagnostic tool for the screening population in highly endemic remote and poor areas was also discussed.

First description of Echinococcus ortleppi and cystic echinococcosis infection status in Chile

Cystic echinococcosis (CE), a parasitic disease caused by the cestode Echinococcus granulosus sensu lato (s.l.), is a worldwide zoonotic infection. Although endemic in Chile, information on the molecular characteristics of CE in livestock remains scarce. Therefore we aimed to describe the status of infection with E. granulosus s.l. in cattle from central Chile and also to contribute to the study of the molecular epidemiology of this parasite. According to our results, the prevalence of CE is 18.84% in cattle, similar to previous reports from Chile, suggesting that the prevalence in Santiago Metropolitan area has not changed in the last 30 years. Most of the cysts were found only in lungs (51%), followed by concurrent infection in liver and lungs (30%), and only liver (19%). Molecular characterization of the genetic diversity and population structure of E. granulosus s.l. from cattle in central Chile was performed using a section of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. E. granulosus sensu stricto (s.s.) (G1-G3 genotypes) was confirmed by RFLP-PCR to be the dominant species affecting cattle (284 samples/290 samples); we also report for the first time in Chile the presence of E. ortleppi (G5 genotype) (2 samples/61 samples). The Chilean E. granulosus s.s. parsimony network displayed 1 main haplotype. Additional studies using isolates from many locations across Chile and different intermediate hosts will provide more data on the molecular structure of E. granulosus s.s. within this region. Likewise, investigations of the importance of E. ortleppi in human infection in Chile deserve future attention.

Limitations of the Echinococcus granulosus genome sequence assemblies for analysis of the gene family encoding the EG95 vaccine antigen

Echinococcus granulosus is an important zoonotic parasite that is distributed worldwide. The EG95 vaccine was developed to assist with control of E. granulosus transmission through the parasites livestock intermediate hosts. The vaccine is based on a recombinant antigen encoded by a gene which is a member of a multi-gene family. With the recent availability of two E. granulosus draft genomes, we sought to map the eg95 gene family to the genomes. We were unable to map unequivocally any of the eg95 gene family members which had previously been characterized by cloning and sequencing both strands of genomic DNA fragments. Our inability to map EG95-related genes to the genomes has revealed limitations in the assembled sequence data when utilized for gene family analyses. This study contrasts with the expectations expressed in often high-profile publications describing draft genomes of parasitic organisms, highlighting deficiencies in currently available genomic resources for E. granulosus and provides a cautionary note for research which seeks to utilize these genome datasets.