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The EG95 recombinant vaccine is protective against cystic echinococcus in animal intermediate hosts. Preparation of the existing, registered EG95 vaccines involves semi-purification of the vaccine protein, adding to the cost of production. Truncation of the EG95 cDNA, shortening both the amino and carboxy-termini of the protein, leads to high levels of recombinant protein expression. The recombinant EG95 protein was prepared as a bacterin from clarified, whole bacterial lysate, and used in a vaccine trial in sheep against an experimental challenge infection with Echinococcus granulosus eggs. The EG95 bacterin was found to induce 98% protection. Use of this in a new generation EG95 vaccine would simplify production, facilitate new sources of the vaccine and potentially enhance uptake of vaccination in control of E. granulosus transmission.

This review is aimed to (i) appraise the literature on the use of molecular techniques for the detection, quantification and differentiation of gastrointestinal helminths (GIH) of equids, (ii) identify the knowledge gaps and, (iii) discuss diagnostic prospects in equine parasitology. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we retrieved 54 studies (horses: 50/54; donkeys and zebras: 4/54) from four databases. Polymerase chain reaction (PCR) was employed in all of the studies whereas PCR amplicons were sequenced in only 18 of them. Other techniques used (including modifications of PCR) were reverse line blot, quantitative (q)PCR, restriction fragment length polymorphism, nested-PCR, PCR-directed next-generation sequencing, Southern blotting, single strand conformation polymorphism, PCR-enzyme linked immunosorbent assay, matrix-assisted laser desorption/ionisation-time of flight and random amplification of polymorphic DNA. Most of the studies (53/54) used nuclear ribosomal RNA (including the internal transcribed spacers, intergenic spacer, 5.8 S, 18 S, 28 S and 12 S) as target loci while cytochrome c oxidase subunit 1 and random genomic regions were targeted in only three and one studies, respectively. Overall, to date, the majority of molecular studies have focused on the diagnosis and identification of GIHs of equids (i.e. species of Anoplocephala, Craterostomum, cyathostomins, Oesophagodontus, Parascaris, Strongylus, Strongyloides and Triodontophorus), with a recent shift towards investigations on anthelmintic resistance and the use of high-throughput nemabiome metabarcoding. With the increasing reports of anthelmintic resistance in equid GIHs, it is crucial to develop and apply techniques such as advanced metabarcoding for surveillance of parasite populations in order to gain detailed insights into their diversity and sustainable control. To the best of our knowledge, this is the first systematic review that evaluates molecular investigations published on the diagnosis and quantification of equid GIHs and provides useful insights into important knowledge gaps and future research directions in equid molecular parasitology.

Three recombinant Taenia ovis antigens (To45, To16, To18) each induce protective immunity in lambs or ewes against infection with T. ovis metacestodes. The degree and duration of immunity were assessed in lambs born from vaccinated ewes. Treatment group sizes varied, typically not fewer than 5 animals per group. Ewes were immunised with one T. ovis recombinant protein prior to lambing and the degree and duration of passive immunity in their lambs was assessed by challenge infection up to 18 weeks. Lambs were fully protected up to 6 weeks of age but immunity waned from 6 to 12 weeks and there was no protection when lambs were challenged at 15 weeks. Immunisation of lambs with the homologous recombinant antigen was not effective when vaccinations were given when maternal antibody was high. Lambs were effectively immunised in the presence of passively protective antibody when vaccinated with an antigen that was different to that given to ewes. Vaccination of lambs with a combination of two proteins, To16 and To18, was more effective than giving these single antigens and gave a significant reduction of cyst numbers when lambs were challenged 12 months after immunisation. These results indicate that the use of combinations of T. ovis recombinant antigens could enable complete protection of lambs against infection, if a delivery system becomes available that will maintain antibody at protective levels for 12 months. Alternatively, a third injection given at 6 months may promote the anamnestic response to give long lasting protection.

We genetically characterised larval and adult specimens of species of Echinocephalus Molin, 1858 (Gnathostomatidae) collected from various hosts found within Australian waters. Adult specimens of Echinocephalus were collected from a dasyatid stingray [Pastinachus ater (Macleay); n = 2] from Moreton Bay, Queensland and larvae from a hydrophiine sea snake [Hydrophis peronii (Duméril); n = 3] from Cape York Peninsula, Queensland, from an octopus (Octopus djinda Amor & Hart; n = 3) from Fremantle, Western Australia and from a lucinid bivalve [Codakia paytenorum (Iredale); n = 5] from Heron Island, Queensland Australia. All nematode samples were identified morphologically and genetically characterised using the small subunit nuclear ribosomal DNA (SSU). Some morphological differences were identified between previous studies of Echinocephalus spp. and those observed herein but the significance of these differences remains unresolved. Molecular phylogenetic analyses revealed that larval Echinocephalus sp. from H. peronii and C. paytenorum in Australia were very similar (with strong nodal support) to larval Echinocephalus sp. infecting two fish species from Egypt, Saurida undosquamis (Richardson) (Synodontidae) and Pagrus pagrus (Linnaeus) (Sparidae). The SSU sequences of larval Echinocephalus sp. from O. djinda and adults from P. ater formed a well-supported clade with that of adult E. overstreeti Deardorff and Ko, 1983 from the Port Jackson shark, Heterodontus portusjacksoni (Meyer), as well as that of the larval Echinocephalus sp., from the common carp (Cyprinus carpio Linnaeus) from Egypt. This study extends the intermediate host range of Echinocephalus larvae by including a sea snake for the first time. Findings of this study highlight the importance of genetic characterisation of larval and adult specimens of Echinocephalus spp. to resolve the current difficulties in the taxonomy of this genus., Graphical abstract Image 1, Highlights • Historical morphological descriptions of larval forms of Echinocephalus provide inadequate species identification. • Larval stages of Echinocephalus species from various hosts found within Australian waters were characterised. • Intermediate host range of Echinocephalus larvae was extended to include a reptile. • Future studies should include genetic characterisation of Echinocephalus spp. to resolve taxonomic inaccuracies.

Ticks and tick-borne diseases (TTBDs) substantially affect the health and production of ruminants, particularly in resource-poor, small-scale farming systems worldwide. However, to date, there has been no critical appraisal of the current state of knowledge of TTBDs in such farming systems. In this article, we systematically reviewed the situation in Pakistan-as an example of a country that is highly reliant on agriculture to sustain its economy, particularly smallholder livestock farms, which are continually faced with challenges associated with TTBDs. The main aims of this review were to gain improved insights into the current status of TTBDs in small-scale farming systems, and to identify knowledge gaps, through the systematic evaluation of published literature on this topic from Pakistan, and to recommend future research directions. We searched publicly available literature from three databases (i.e. Web of Science, Google Scholar, and PubMed) on bovine TTBDs in Pakistan. Of 11,224 published studies identified, 185 were eligible for inclusion; these studies were published between August 1947 and June 2021. A critical analysis of these 185 studies revealed that the diagnosis of ticks and tick-borne pathogens (TBPs) in Pakistan has been based largely on the use of traditional methods (i.e. 'morpho-taxonomy'). At least 54 species of tick have been recorded, most of which belong to the genera Haemaphysalis, Hyalomma and Rhipicephalus. The prevalence of ticks was higher, particularly in young, exotic and crossbred female cattle, during the summer season. Major TBPs include species of Anaplasma, Babesia and Theileria, with prevalences being higher in cattle than buffaloes. Additionally, pathogens of zoonotic potential, including species of Anaplasma, Borrelia, the Crimean-Congo haemorrhagic fever virus, Coxiella, Ehrlichia and Rickettsia, have been recorded in both tick and bovine populations. Information on risk factors, spatial-temporal distribution, genetic diversity, and control of ticks and TBPs is limited, the vector potential of ticks and the distribution patterns of ticks and TBPs in relation to climate remains largely unexplored. Future research should focus on addressing these knowledge gaps and the key challenges of poverty, food security and disease outbreaks in a small-scale livestock farming context in order to provide sustainable, environment-friendly control measures for TTBDs.

Background Strongyloides westeri is found in the small intestine of young horses, mainly in foals up to about 16 weeks of age. The main source of infection for foals is through transmammary transmission, and foals can develop acute diarrhoea, weakness, dermatitis and respiratory signs. The epidemiology of S. westeri in Australia is largely unknown. Further, molecular techniques have never been employed for detection of S. westeri in horses. This pilot study aimed to assess the utility of a molecular phylogenetic method for the detection of S. westeri in the faeces of foals. Methods Faecal samples were collected from a foal of less than 2 months of age, and eggs of Strongyloides sp. were detected using the modified McMaster technique. DNA was extracted from purified eggs, and a partial fragment of the small subunit of the nuclear ribosomal DNA (18S) was characterised using polymerase chain reaction, DNA sequencing and phylogenetic methods. Results Microscopic examination of faeces revealed small ellipsoidal eggs typical of Strongyloides sp. The 18S sequence generated by PCR in this study revealed 98.4% identity with that of a reference sequence of S. westeri available from GenBank. Phylogenetic analyses revealed a polyphyletic clustering of S. westeri sequences. Conclusion This is the first study reporting the detection of DNA of Strongyloides sp. in faeces of a foal using a molecular phylogenetic approach targeting the variable region of 18S rDNA. It is anticipated that this study will allow future molecular epidemiological studies on S. westeri in horses. Graphical abstract

Background Echinococcus tapeworms cause a severe helminthic zoonosis called echinococcosis. The genus comprises various species and genotypes, of which E. granulosus (sensu stricto) represents a significant global public health and socioeconomic burden. Mitochondrial (mt) genomes have provided useful genetic markers to explore the nature and extent of genetic diversity within Echinococcus and have underpinned phylogenetic and population structure analyses of this genus. Our recent work indicated a sequence gap (> 1 kb) in the mt genomes of E. granulosus genotype G1, which could not be determined by PCR-based Sanger sequencing. The aim of the present study was to define the complete mt genome, irrespective of structural complexities, using a long-read sequencing method. Methods We extracted high molecular weight genomic DNA from protoscoleces from a single cyst of E. granulosus genotype G1 from a sheep from Australia using a conventional method and sequenced it using PacBio Sequel (long-read) technology, complemented by BGISEQ-500 short-read sequencing. Sequence data obtained were assembled using a recently-developed workflow. Results We assembled a complete mt genome sequence of 17,675 bp, which is > 4 kb larger than the complete mt genomes known for E. granulosus genotype G1. This assembly includes a previously-elusive tandem repeat region, which is 4417 bp long and consists of ten near-identical 441–445 bp repeat units, each harbouring a 184 bp non-coding region and adjacent regions. We also identified a short non-coding region of 183 bp, which includes an inverted repeat. Conclusions We report what we consider to be the first complete mt genome of E. granulosus genotype G1 and characterise all repeat regions in this genome. The numbers, sizes, sequences and functions of tandem repeat regions remain to be studied in different isolates of genotype G1 and in other genotypes and species. The discovery of such ‘new’ repeat elements in the mt genome of genotype G1 by PacBio sequencing raises a question about the completeness of some published genomes of taeniid cestodes assembled from conventional or short-read sequence datasets. This study shows that long-read sequencing readily overcomes the challenges of assembling repeat elements to achieve improved genomes.

A three year old female water buffalo was slaughtered for human consumption on a dairy buffalo farm in eastern New South Wales, Australia. Gross examination of the offal revealed four small, superficial hydatid cysts in the liver and two larger superficial cysts in one lung. All organs were sliced and no other cysts were found. Histology and PCR confirmed the cysts to be cysts of Echinococcus granulosus senso stricto. None of the cysts contained protoscoleces. The source ofinfection is equivocal, but it is most likely from E. granulosus eggs passed in the faeces of wild dogs (dingoes and dingo-wild dog hybrids). Wild dogs are resident in the bush that abuts the farm boundary and from time to time wild dogs are seen in the buffalo paddocks on the farm. Sylvatic transmission of E. granulosus occurs commonly in eastern Australia through a predator/prey interaction between wild dogs and macropod marsupials., Graphical abstract Image 1, Highlights • Farmed Australian water buffalo can become naturally infected with hydatid disease. • The strain of Echinococcus causing hydatid disease in Australian farmed water buffalo is E. granulosus senso stricto. • Wild buffalo in northern Australia should be considered as an additional potential wildlife intermediate host for Echinococcus granulosus in Australia.

Cystic echinococcosis (CE) 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 was to assess the inclusion of the EG95 for sheep in the control program and to determine the vaccine's operative feasibility in field conditions. An intervention study was defined in Rio Negro Province in Argentina comprising, in total, an area of 5820 Km2. Lambs received two vaccinations with the EG95 vaccine followed by a single booster injection when the animals were 1-1.5 years of age. Vaccination of lambs born into one trial site was introduced and continued for 8 years. Evidence for Echinococcus granulosus transmission was monitored before and after vaccination by coproantigen ELISA in faecal samples of dog, purgation of dogs to detect E. granulosus worms, necropsy on adult sheep and by ultrasound screening in children of 6-14 years old. 29,323 doses of vaccine were applied between 2009 and 2017, which a vaccination coverage of 80.1%/85.7% (57.3% average for fully vaccinated). Before the introduction of the vaccine 56.3% of the 6-year-old sheep were infected with E. granulosus at necropsy and 84.2% of the farms had infected sheep; 4.3% of the dogs were positive for E. granulosus infection using the arecoline test, and with coproELISA 9.6% of dog fecal samples were positive and 20.3% of the farms had infected dog.After the vaccine was introduced, 21.6% of sheep older than 6 years were found to be infected at necropsy and 20.2% of the farms were found to be infected; in dogs, 4.5% were found positive for E. granulosus using arecoline purgation and with coproELISA 3.7% of samples were positive, with 8.9% of farms having a positive dog. In 2016 only one case of E. granulosus infection was diagnosed by US screening in a 6-14 years old child. Included in the analysis are discussions of difficulties experienced in the field which affected correct vaccine administration as well as social features and practices that may impact on echinococcosis control and the EG95 vaccination program in Rio Negro. Vaccination of sheep with the EG95 vaccine provides a valuable new tool which improves the effectiveness of CE control activities. Vaccination was effective even in a difficult, remote environment where only approximately half the lambs born into the communities were fully vaccinated.

Faecal egg counting techniques (FECT) form the cornerstone for the detection of gastrointestinal parasites in equines. For this purpose, several flotation, centrifugation, image- and artificial intelligence-based techniques are used, with varying levels of performance. This review aimed to critically appraise the literature on the assessment and comparison of various coprological techniques and/or modifications of these techniques used for equines and to identify the knowledge gaps and future research directions. We searched three databases for published scientific studies on the assessment and comparison of FECT in equines and included 27 studies in the final synthesis. Overall, the performance parameters of McMaster (81.5%), Mini-FLOTAC® (33.3%) and simple flotation (25.5%) techniques were assessed in most of the studies, with 77.8% of them comparing the performance of at least two or three methods. The detection of strongyle, Parascaris spp. and cestode eggs was assessed for various FECT in 70.4%, 18.5% and 18.5% studies, respectively. A sugar-based flotation solution with a specific gravity of ≥1.2 was found to be the optimal flotation solution for parasitic eggs in the majority of FECT. No uniform or standardised protocol was followed for the comparison of various FECT, and the tested sample size (i.e. equine population and faecal samples) also varied substantially across all studies. To the best of our knowledge, this is the first systematic review to evaluate studies on the comparison of FECT in equines and it highlights important knowledge gaps in the evaluation and comparison of such techniques.

Taenia solium oncosphere protein TSOL18 is the host-protective antigen against porcine cysticercosis. Little attention has been given to use it as target molecule in immunodiagnostic tests. The objective of this paper is to describe the immunodiagnostic potential of monoclonal antibodies (MoAbs) raised against conformational epitopes of TSOL18. Three murine IgG1 MoAbs (25D12C1, 21C2D2, 10H1F2) against three different conformational epitopes of TSOL18 were produced and evaluated with an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. Serum samples from pigs immunized with TSOL18 inhibited the binding of the three MoAbs to TSOL18 antigen in i-ELISA. The highest inhibition of anti-TSOL18 antibodies in immunized pigs was observed with MoAb 25D12C1. Ten field sera (12.19%) from 82 non-vaccinated and non-infected pigs showed anti-oncosphere antibodies inhibiting the binding of MoAb 25D12C1. Anti-oncosphere antibodies in pigs experimentally infected with T. solium eggs inhibited the binding of MoAb 25D12C1 from 2 to 8 week-post infection. It is concluded that MoAb 25D12C1 has excellent immunodiagnostic potentials to detect anti-oncosphere antibodies in the intermediate hosts at early exposure to T. solium eggs. Further investigations on potential use of MoAb 25D12C1 in a capture antigen ELISA for the detection of post-oncospheral antigens in infected pigs cannot be overemphasized., Graphical abstract Unlabelled Image, Highlights • We describe the potential usefulness of the monoclonal antibody 25D12C1 to TSOL18 protein. • Serum samples from pigs immunized with TSOL18 inhibited the binding of 25D12C1 in an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. • Anti-oncosphere antibodies in pigs infected with T. solium eggs inhibited also the binding of 25D12C1. • It is concluded that MoAb i-ELISA using 25D12C1 has the potential to identify animals exposed to T. solium infection.

Background Cystic echinococcosis (CE) is an important cause of human morbidity and mortality worldwide, particularly in Morocco and other North African countries. Methodology/Principal findings We 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, Author summary Cystic echinococcosis remains a major public health problem in Morocco. It is a major zoonosis affecting humans and animals caused by the larval stage of the cestode Echinococcus granulosus. The dog (final host), plays an essential role in the dissemination of eggs in the environment via its feces. The rural and poorest regions in the Middle Atlas that practice extensive sheep farming (intermediate host) are severely affected. Women and children are particularly affected by this zoonosis. Despite previous efforts done by the Moroccan authorities to reduce the incidence of infestation, these measures have been insufficient to control the disease. Through our study protocols in natural field conditions, we have shown that vaccination of the intermediate host is an effective control option in the Moroccan context, with an immune protection rate of 97%. Regular chemotherapy (4 months intervals) of owned dogs only proved to have little efficiency on incidence in sheep.

Ticks and tick-borne diseases (TTBDs) substantially affect the health and production of ruminants, particularly in resource-poor, small-scale farming systems worldwide. However, to date, there has been no critical appraisal of the current state of knowledge of TTBDs in such farming systems. In this article, we systematically reviewed the situation in Pakistan-as an example of a country that is highly reliant on agriculture to sustain its economy, particularly smallholder livestock farms, which are continually faced with challenges associated with TTBDs. The main aims of this review were to gain improved insights into the current status of TTBDs in small-scale farming systems, and to identify knowledge gaps, through the systematic evaluation of published literature on this topic from Pakistan, and to recommend future research directions. We searched publicly available literature from three databases (i.e. Web of Science, Google Scholar, and PubMed) on bovine TTBDs in Pakistan. Of 11,224 published studies identified, 185 were eligible for inclusion; these studies were published between August 1947 and June 2021. A critical analysis of these 185 studies revealed that the diagnosis of ticks and tick-borne pathogens (TBPs) in Pakistan has been based largely on the use of traditional methods (i.e. 'morpho-taxonomy'). At least 54 species of tick have been recorded, most of which belong to the genera Haemaphysalis, Hyalomma and Rhipicephalus. The prevalence of ticks was higher, particularly in young, exotic and crossbred female cattle, during the summer season. Major TBPs include species of Anaplasma, Babesia and Theileria, with prevalences being higher in cattle than buffaloes. Additionally, pathogens of zoonotic potential, including species of Anaplasma, Borrelia, the Crimean-Congo haemorrhagic fever virus, Coxiella, Ehrlichia and Rickettsia, have been recorded in both tick and bovine populations. Information on risk factors, spatial-temporal distribution, genetic diversity, and control of ticks and TBPs is limited, the vector potential of ticks and the distribution patterns of ticks and TBPs in relation to climate remains largely unexplored. Future research should focus on addressing these knowledge gaps and the key challenges of poverty, food security and disease outbreaks in a small-scale livestock farming context in order to provide sustainable, environment-friendly control measures for TTBDs.

The COVID-19 pandemic has disrupted parasitology curricula worldwide, which is expected to lead to the reshaping of parasitology education. Here, we share our experiences of remote teaching and learning of veterinary parasitology and discuss opportunities offered by remote teaching during COVID-19 lockdowns, enabling the development of interactive online parasitology courses.

This study investigated ticks and tick-borne microorganisms of small ruminants from five districts of the Federally Administered Tribal Area (FATA) of Pakistan. Morphological (n = 104) and molecular (n = 54) characterization of the ticks revealed the presence of six ixodid ticks: Rhipicephalus (Rh.) haemaphysaloides, Rh. microplus, Rh. turanicus, Haemaphysalis (Hs.) punctata, Hs. sulcata and Hyalomma anatolicum. Phylogenetic analyses of nucleotide sequence data for two mitochondrial (16S and cytochrome c oxidase 1) and one nuclear (second internal transcribed spacer) DNA regions provided strong support for the grouping of the six tick species identified in this study. Microfluidic real-time PCR, employing multiple pre-validated nuclear and mitochondrial genetic markers, detected 11 potential pathogens and endosymbionts in 72.2% of the ticks (n = 54) tested. Rickettsia (R.) massiliae was the most common pathogen found (42.6% of ticks) followed by Theileria spp. (33.3%), Anaplasma (A.) ovis and R. slovaca (25.9% each). Anaplasma centrale, A. marginale, Ehrlichia spp., R. aeschlimannii, R. conorii and endosymbionts (Francisella- and Coxiella-like) were detected at much lower rates (1.9&ndash, 22.2%) in ticks. Ticks from goats (83.9%) carried significantly higher microorganisms than those from sheep (56.5%). This study demonstrates that ticks of small ruminants from the FATA are carrying multiple microorganisms of veterinary and medical health significance and provides the basis for future investigations of ticks and tick-borne diseases of animals and humans in this and neighboring regions.

Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries. In this study, we expressed the modified antigen, EG95NC-GST, in Escherichia coli for use as a vaccine against Echinococcus granulosus in ruminants. We tested three different media formulations for E. coli culture and established for each culture conditions for optimal levels of soluble EG95 expression. The results demonstrate that SOC and TB media provided high yields in cell density and EG95 protein expression. Purification of the recombinant protein with affinity chromatography (using FPLC) was also performed to increase the purity of the EG95NC−-GST antigen.

Cystic echinococcosis is endemic in the Rio Negro province of Argentina and, for this, a control program using praziquantel in dogs was developed from 1980. The transmission rate to humans and sheep has decreased significantly, however transmission persists. In 2009 the vaccination of sheep with EG95 was incorporated in some areas of the province. The objective of the study was to evaluate the humoral responses to the vaccine EG95. Lambs received two vaccinations with the EG95 vaccine followed by a single booster injection when the animals were 1-1.5 years of age. Blood samples from 6 vaccinated groups and 4 no vaccinated sheep were obtained for determination of antibody titles against EG95 protein. Anti-EG95 responses were determined as described by Heath and Koolaard, 2012. Responses were evaluated from 331 animals. Median ELISA absorbance values in vaccinated group was 0.828, and in non-vaccinated groups was 0.218. EG95 antibody responses in sheep from different cohorts of non-immunized control groups and vaccinated groups reveal the sustained increase in response seen in animals following the third immunization. (p< 0.0001). Significant differences are also evidenced in ANOVA test (p< 0.001). An anti-EG95 antibody response was induced in all groups of immunized sheep, 4 times higher (0.828) than the mean observed in the control groups (0.218). Data described here indicate that following a third vaccination with the EG95 vaccine at 1 year of age the specific IgG responses detected in the serum of sheep increased to a level greater than that seen following the second immunization and that this response was maintained longitudinally over time, for at least 5 years. Fil: Larrieu, Edmundo Juan. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina. Universidad Nacional de Río Negro; Argentina Fil: Poggio, Thelma Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; Argentina Fil: Mujica, Guillermo. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Gauci, Charles G.. University of Melbourne; Australia Fil: Labanchi, Jose Luis. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Herrero, Eduardo. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Araya, Daniel. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Grizmado, Claudia. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Calabro, Arnoldo. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Talmon, Gabriel. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Crowley, Pablo Ernesto. Universidad Nacional de Río Negro; Argentina Fil: Santillán, Graciela. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Departamento de Parasitología; Argentina Fil: Vizcaychipi, Katherina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Departamento de Parasitología; Argentina Fil: Seleiman, Marcos. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Sepulveda, Luis. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: Arezo, Marcos. Provincia de Río Negro. Ministerio de Salud; Argentina Fil: García Cachau, Mariela Alejandra. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina Fil: Lamberti, Roberto. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina Fil: Molina, Leonardo. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina Fil: Gino, Lilia Mabel. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias; Argentina Fil: Donadeu, Meritxell. University of Melbourne; Australia Fil: Lightowlers, Marshall W.. University of Melbourne; Australia

Detailed post mortem analyses of 68 free-ranging, slaughter-age pigs from two sites in the Banke District of Nepal identified 36% as being infected with Echinococcus granulosus. The cysts ranged from infertile, immature cysts a few millimetres in diameter to fertile cysts >10 cm in diameter. PCR RFLP and DNA sequencing identified the cysts as being E. granulosus sensu stricto. The Banke district has recently been identified as having a high prevalence of porcine cysticercosis. These data suggest that cestode zoonoses in this, and possibly other parts of Nepal may be a serious concern for human health. An assessment of the level of human cystic echinococcosis and neurocysticercosis, in the region is warranted and the introduction of control measures are required to limit the parasites' transmission.

Following confirmation that a remote village of approximately 300 inhabitants in northern Lao PDR was hyperendemic for the Neglected Tropical Disease Taenia solium, a pilot human-porcine therapeutic control intervention was implemented between October 2013 and November 2014. Mass drug administration with a three day albendazole 400mg protocol was offered to all eligible humans in October 2013 and March 2014. At these times, and again in October 2014, eligible village pigs received the anti-cysticercosis TSOL18 vaccination and an oral dose of oxfendazole anthelmintic at 30mg/kg, both repeated one month later. Community and individual human taeniasis prevalences were estimated via copro-antigen ELISA of volunteered human faecal samples prior to October 2013, and again in January 2015, in order to examine the short term impact of the intervention.

Summary There is an increasing interest in reducing the incidence of human neurocysticercosis, caused by infection with the larval stage of Taenia solium. Several intervention trials are currently assessing various options for control of T. solium transmission. A critical aspect of these trials will be the evaluation of whether the interventions have been successful. However, there is no consensus about the most appropriate or valuable methods that should be used. Here, we undertake a critical assessment of the diagnostic tests which are currently available for human T. solium taeniasis and human and porcine cysticercosis, as well as their suitability for evaluation of intervention trial outcomes. Suggestions are made about which of the measures that are available for evaluation of T. solium interventions would be most suitable, and which methodologies are the most appropriate given currently available technologies. Suggestions are also made in relation to the most urgent research needs in order to address deficiencies in current diagnostic methods.

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.

Taenia solium is a zoonotic cestode parasite which causes human neurocysticercosis. Pigs transmit the parasite by acting as the intermediate host. An intervention was implemented in pigs to control transmission of T. solium in Dalit communities of Banke District, Nepal. Every 3 months, pigs were vaccinated with the TSOL18 recombinant vaccine (Cysvax™, IIL, India)) and, at the same time, given an oral treatment with 30mg/kg oxfendazole (Paranthic 10%™, MCI, Morocco). The prevalence of porcine cysticercosis was determined in both an intervention area as well as a similar no intervention control area, among randomly selected, slaughter-age pigs. Post mortem assessments were undertaken both at the start and at the end of the intervention. Participants conducting the post mortem assessments were blinded as to the source of the animals being assessed. At the start of the intervention the prevalence of porcine cysticercosis was 23.6% and 34.5% in the control and intervention areas, respectively. Following the intervention, the prevalence of cysticercosis in pigs from the control area was 16.7% (no significant change), whereas no infection was detected after complete slicing of all muscle tissue and brain in animals from the intervention area (P=0.004). These findings are discussed in relation to the feasibility and sustainability of T. solium control. The 3-monthly vaccination and drug treatment intervention in pigs used here is suggested as an effective and practical method for reducing T. solium transmission, thereby reducing the incidence of human neurocysticercosis.Author summaryNeurocysticercosis is a disease caused by a parasitic infection of the brain. The parasite responsible, Taenia solium, is transmitted by pigs where human sanitation is poor and pigs roam freely. Neurocysticercosis is responsible for many cases of epilepsy in people living in poor, developing countries. The feasibility and sustainability of implementing control measures have been major impediments to reducing the incidence of neurocysticercosis. Recently, two new commercial products have become available for pigs which together offer the possibility of interrupting the parasite’s transmission the TSOL18 vaccine (Cysvax™, IIL, India) and an oxfendazole formulation (Paranthic 10%™, MCI, Morocco) licensed for use in pigs for the treatment of cysticercosis. Here we describe the impact of implementing vaccination plus drug treatment of pigs in the Banke district of Nepal. The intervention eliminated the risk of transmission of T. solium by the animals vaccinated and treated during the trial. Application of the vaccination and drug treatment program used here, possibly with strategic use of anthelmintics also in the human population, is an effective option for reducing the incidence of neurocysticercosis in Nepal and elsewhere.

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 parasite's 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.

Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

Cystic echinococcosis caused by Echinococcus granulosus remains an important and neglected issue in public health. The study of the likely efficacy of the currently available EG95 vaccine against other genotypes of the parasite is important to improve the vaccine as a potential tool to be used in control programmes. The recombinant vaccine EG95-1G1 was developed based on the G1 genotype of E. granulosus. Characterization of the eg95 gene family in the G6 genotype by genomic DNA cloning previously produced the first unequivocal information about the composition of the gene family in a different genotype. The information was used in this study to predict and express two EG95-related proteins from the G6 genotype as recombinants, for assessment of their capacity to bind antibodies raised in sheep vaccinated with the EG95-1G1 vaccine. The proteins (EG95-1G6 and EG95-5G6) from the G6 genotype of E. granulosus were unable to bind all the antibodies raised by sheep vaccinated with EG95-1G1. Differences in the amino acid sequence of EG95-related proteins from G6 and likely the differences in the encoded FnIII domain may be responsible for changes in the conformation of these epitopes.

SUMMARYEchinococcus granulosus (sensu lato) is now recognized as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. One of these species, E. granulosus sensu stricto (s.s.), is now clearly identified as the principal agent causing cystic echinococcosis in humans. Previous studies of a small section of the cox1 and nadh1 genes identified two variants of E. granulosus s.s. to be present in Australia; however, no further work has been carried out to characterize the microdiversity of the parasite in its territory. We have analysed the sequence of the full length of the cox1 gene (1609 bp) from 37 isolates of E. granulosus from different hosts and geographic regions of Australia. The analysis shows that seven haplotypes of E. granulosus s.s. not previously described were found, together with five haplotypes known to be present in other parts of the world, including the haplotype EG01 which is widespread and present in all endemic regions. These data extend knowledge related to the geographical spread and host range of E. granulosus s.s. in a country such as Australia in which the parasite established around 200 years ago.

SUMMARYSpecific antibody responses were assessed in pigs immunized with the Taenia solium vaccine TSOL18. Anti-TSOL18 responses were compared 2 weeks after secondary immunization, where the interval between primary and secondary immunization was 4, 8, 12, 16 or 20 weeks. All animals responded to the vaccine and there was no diminution in antibody responses in animals receiving their second injection after an interval up to 20 weeks. Pigs receiving vaccinations at an interval of 12 weeks developed significantly increased antibody responses compared with animals receiving immunizations 4 weeks apart (P = 0·046). The ability to deliver TSOL18 vaccination effectively where the revaccination schedule can be delayed for up to 12–16 weeks in pigs increases the options available for designing T. solium control interventions that incorporate TSOL18 vaccination.

Recombinant antigens cloned from the oncosphere life cycle stage of the cestode parasite Taenia solium (T. solium) have been proven to be effective as vaccines for protecting pigs against infections with T. solium. Previous studies have defined three different host protective oncosphere antigens, TSOL18, TSOL16 and TSOL45. In this study, we evaluated the potential for combining the antigens TSOL16 and TSOL18 as a practical vaccine. Firstly, in a laboratory trial, we compared the immunogenicity of the combined antigens (TSOL16/18) versus the immunogenicity of the antigens separately. Secondly, in a field trial, we tested the ability of the TSOL16/18 vaccine to induce detectable antibody responses in animals living under environmental stress and traditionally reared in areas where T. solium cysticercosis is endemic; and finally, we characterised the immune response of the study population. Pigs of 8-16 weeks of age were vaccinated with 200 μg each of TSOL16 and TSOL18, plus 5mg of Quil-A. Specific total IgG, IgG(1) and IgG(2) antibody responses induced by TSOL16 and TSOL18 were determined with ELISA. The immunogenicity of both antigens was retained in the combined TSOL16/18 vaccine. The combined vaccine TSOL16/18 induced detectable specific anti-TSOL18 antibody responses in 100% (113/113) and specific anti-TSOL16 in 99% (112/113) of the vaccinated animals measured at 2 weeks following the booster vaccination. From the two IgG antibody subtypes analysed we found there was stronger response to IgG(2).

Taenia solium causes cysticercosis in pigs and taeniasis and neurocysticercosis in humans. Oncosphere antigens have proven to be effective as vaccines to protect pigs against an experimental infection with T. solium. A pair-matched vaccination trial field, using a combination of two recombinant antigens, TSOL16 and TSOL18, was undertaken in rural villages of Peru to evaluate the efficacy of this vaccine under natural conditions. Pairs of pigs (n=137) comprising one vaccinated and one control animal, were allocated to local villagers. Animals received two vaccinations with 200μg of each of TSOL16 and TSOL18, plus 5mg Quil-A. Necropsies were performed 7 months after the animals were distributed to the farmers. Vaccination reduced 99.7% and 99.9% (p

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.

Highlights ► Experimental Taenia solium challenge of piglets vaccinated with recombinant antigens. ► Protection of piglets immunized with the TSOL16 recombinant antigen. ► Assessment of antibody responses in pigs vaccinated with recombinant antigens., Recombinant antigens from the oncosphere stage of the parasite Taenia solium were expressed in Escherichia coli. The TSOL16, TSOL45-1A and TSOL45-1B recombinant antigens, each consisting of fibronectin type III (FnIII) domain S, were produced as fusion proteins with glutathione S-transferase (GST) and maltose binding protein (MBP). Groups of pigs were immunized twice with the GST fusions of the antigens and boosted a third time with the MBP fusions prior to receiving a challenge infection with T. solium eggs. The TSOL16 antigen was found to be capable of inducing high levels of immunity in pigs against a challenge infection with T. solium. Immunological investigations identified differences in immune responses in the pigs vaccinated with the various antigens. The results demonstrate that the TSOL16 antigen could be a valuable adjunct to current porcine vaccination approaches and may allow the further development of new vaccination strategies against T. solium cysticercosis.

Taenia ovisis a cestode parasite infecting primarily sheep as intermediate hosts and dogs as definitive hosts. The first highly effective, recombinant vaccine against a parasitic organism was developed againstT. ovisinfection in sheep. Three separate host-protective antigens (To16, To18, and To45W) have been cloned from the oncosphere of the parasite. We localize these antigens in the oncosphere by using quantitative immunogold labeling and transmission electron microscopy. The three antigens were uniquely associated with penetration gland cells. The cytoplasm and secretory granules of both penetration gland type 1 and type 2 cells exhibited statistically significant levels of staining for each of the three antigens. The intensity of labeling of the penetration gland type 1 cell was approximately three to five times greater (P< 0.01) compared to the level of staining intensity seen in the penetration gland type 2 cell. In activated oncospheres, secretory blebs were found to contain granules with a structure similar to those observed in the penetration gland cells. The granules within the secretory blebs were shown to stain specifically for the presence of each of the three host-protective antigens. The absence of surface location of theT. ovisantigens suggests that the parasite may not be susceptible to vaccine-induced antibody- and complement-mediated attack until some postoncospheral development has occurred after infection of the intermediate host.

SUMMARYImmunohistochemistry and immunogold labelling techniques were used to localize the EG95 vaccine antigen in Echinococcus granulosus oncospheres. In non-activated oncospheres, the cytoplasm of 2 pairs of bilateral cells exhibited specific positive labelling for the presence of EG95. No surface localization was seen in non-activated or recently activated oncospheres. Besides the staining of 2 pairs of bilateral cells, there was also a generalized distribution of specific staining for EG95 throughout the parenchyma of activated oncospheres. Immunogold labelling of non-activated oncosphere revealed specific reactivity for EG95 involving 2 pairs of bilateral cells and the ultrastructural characteristics of these cells were consistent with them being penetration gland cells. No other oncospheral structures stained specifically for the presence of EG95. The absence of surface location of EG95 in oncospheres suggests that the parasite may not be susceptible to vaccine-induced antibody and complement mediated attack until some post-oncospheral development has occurred. Further studies would be required to determine when the EG95 antigen associates with the parasite's surface, thus making them susceptible to immune attack.

Graphical abstract, A pilot field trial of the TSOL18 vaccine was undertaken in Cameroon. Two hundred and forty, 2–3 month-old piglets were distributed to 114 individual households in pairs. Vaccinated animals received three immunisations with 200 μg TSOL18 plus 5 mg Quil A and 30 mg/kg oxfendazole at the time of the second immunisation. Necropsies were undertaken when the pigs were approximately 12 months of age. Viable Taenia solium cysticerci were identified in 20 control pigs (prevalence 19.6%); no cysticerci were found in any of the vaccinated animals (P

Immunohistochemistry, confocal immunofluorescence and immunogold labelling were used to determine the localisation of the host-protective antigens To16, To18 and To45W in Taenia ovis oncospheres. During maturation of the adult tapeworm the antigens were initially seen as diffuse staining in the developing oncospheres but in mature oncospheres four distinct cells stained positively for the antigens. Confocal fluorescence microscopy using different fluorophores revealed that each of the antigens co-localises within the same cells in the oncosphere. No surface localisation was seen in non-activated or recently activated parasites. Immunogold labelling of non-activated oncosphere sections viewed in transmission electron microscopy revealed labelling of bilateral cells, however the identities of these cells was unclear due to deficiencies in the current level of understanding of oncosphere ultrastructure. Localisation of all the antigens changed dramatically after oncospheres were activated in vitro with each of the antigens being dispersed more generally throughout the parasite parenchyma. During development of the parasites in in vitro culture, surface localisation of the proteins was seen in parasites after 3 or more days in culture. All three antigens were found to be completely absent in parasites by 15 days of culture. The location of the host-protective antigens suggests that initially the invading oncospheres are not susceptible to vaccine-induced antibody and complement mediated attack, but that as the parasites mature, the host-protective antigens come to be associated with the parasite's surface, rendering them susceptible to immune attack.

TSOL18 is a recombinant protein that has been shown in repeated experimental trials to be capable of protecting pigs against challenge infection with the cestode parasite Taenia solium. Antibodies raised by the vaccine are capable of killing the parasite in an in vitro culture and it is believed that antibody and complement-mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. Investigations were undertaken to characterize whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N(-)) and the two truncations (TSOL18N(-)-1 and TSOL18N(-)-2) were used in inhibition ELISA. TSOL18N(-) was shown to be capable of completely inhibiting the binding of pig anti-TSOL18N(-) antibodies to TSOL18N(-) in ELISA. However, neither TSOL18N(-)-1 nor TSOL18N(-)-2, either alone or when combined together, was capable of inhibiting any detectable amount of reactivity of pig anti-TSOL18N(-) antibodies with TSOL18N(-). It is concluded that the dominant antibody specificities, and probably the host-protective specificities, of TSOL18 are conformational epitopes.

The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95.