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 vaccine's 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.

Maternal antibody parameters of cattle and calves receiving EG95 vaccine to protect against Echinococcus granulosus.

Cattle may act as hosts for the transmission of the cestode parasite Echinococcus granulosus and play a role in transmission of the parasite leading to human cystic echinococcosis (CE). The recombinant EG95 vaccine has been shown to be able to protect cattle and other intermediate host species against CE. Ideally the immunisation of bovines against E. granulosus, using EG95 vaccine, should occur early in life so as to provide maximum protection against the establishment of hydatid cysts. Maternally derived antibody from vaccinated cows may provide some protection for the neonate, but may also interfere with the active response to vaccination. Experiments were undertaken to determine the optimal regime for protection of young cattle against CE. One group of pregnant cattle received 2 vaccinations of EG95 antigen+Quil A adjuvant two months and one month prior to calving. The control group of pregnant cattle were not vaccinated. Calves were either challenged with E. granulosus eggs at 4, 9, 13 or 17 weeks post-birth or were given their first vaccination at 8, 12 or 16 weeks post-birth. Sera obtained at regular intervals were tested by ELISA to assess the immunological response. All calves were experimentally challenged with E. granulosus eggs and subsequent necropsy confirmed the levels of protection. Maternal antibody was shown to protect calves to some extent for at least 17 weeks. Calves from vaccinated cows responded well serologically if the first vaccination was given at 8 or 12 weeks, but full protection against a challenge infection was achieved only if the first vaccination was delayed until 16 weeks after birth. Calves from non-vaccinated cattle also were not fully protected if the first vaccination was at 8 or 12 weeks, but were fully protected if the first vaccination was given when they were 16 weeks old. This suggests that immunological maturity is not acquired in calves until 4 or 5 months of age. No safety problems were observed following two vaccinations of 40 pregnant cows or 30 suckling calves.

Vaccination of bovines against Echinococcus granulosus (cystic echinococcosis).

Hydatid disease is an important human zoonosis. Humans become infected from carnivores that are infected with the Echinococcus granulosus tapeworm. Carnivores become infected after consuming hydatid cysts from grazing animals, which are generally sheep, goats and cattle. A vaccine, known as EG95, can protect sheep and goats against cystic echinococcosis. This paper describes the adaptation of the EG95 vaccine for use in cattle. The monitoring of results used serology and also infection with E. granulosus eggs, followed by necropsy. Immunisation with living E. granulosus oncospheres showed that cattle could be immunised against E. granulosus. Immunisation of cattle with EG95 plus QuilA was also successful. A dose-response and adjuvant trial showed best results were achieved with 250 µg of antigen and 5mg of the adjuvant QuilA, which was 5 times the recommended sheep dose. After two vaccinations given one month apart, 90% protection was maintained for 12 months. At 12 months a third vaccination boosted protection to 99% which was maintained for a further 11 months.

Echinococcus granulosus infection and options for control of cystic echinococcosis in Tibetan communities of Western Sichuan Province, China.

BACKGROUND: Human cystic echinococcosis (CE) is highly endemic in the Tibetan regions of Sichuan where most families keep guard dogs and where there are considerable numbers of ownerless/stray dogs. Strong Buddhist beliefs do not allow for elimination of stray dogs, and many strays are actually fed and adopted by households or monasteries. On account of the high altitude (3900-5000 m), pasturage is the major agricultural activity in this area. The harsh mountainous climate often leads to many grazing animals dying on the pasture at the end of a hard winter. The skin and some meat are taken, and the rest of the animal is left for scavenging birds and animals. The poor sanitation and hygiene, the Buddhist doctrine of allowing old livestock to die naturally, plus the unrestricted disposal of animal viscera post-slaughter may be responsible for the high prevalence of human CE in this setting. METHODS AND FINDINGS: As part of a large collaborative control program for CE in Ganzi County, situated in the west of Sichuan Province, surveillance for Echinococcus infection in domestic dogs using a coproantigen method and necropsy of unwanted dogs was carried out prior to (in 2000) and after (in 2005) dog anthelminthic treatment (5 mg/kg oral praziquantal at 6 month intervals) to determine the efficacy of the treatment for control. The prevalence of E. granulosus only in dogs by necropsy was 27% and 22%, and prevalence of both Echinococcus spp. by necropsy was 63% and 38%; prevalence of both Echinococcus spp. by coproantigen analysis was 50% and 17%. Necropsy of sheep/goats (age <1 to 12 years) (prevalence of E. granulosus in 1-6-year-old animals was 38% and in 10-12-year-old animals was 70%) and yaks (age 4 years) (prevalence of E. granulosus was 38%) was undertaken to determine the baseline transmission pressure. Protoscoleces were only found in very old sheep/goats and yaks. Necropsy of dogs in the Datangma district indicated that there was no apparent significant change in the overall prevalence of E. granulosus in unwanted dogs after 5 years of 6-month praziquantel treatment. However, this was likely due to the number of dogs available for necropsy being too small to reflect the real situation prevailing. There was a highly significant decrease in Echinococcus prevalence after the 5-year treatment program shown by coproantigen-ELISA. This indicated a decreasing but continuing risk for re-infection of domestic and stray dogs. Genotyping of E. granulosus samples obtained from necropsied sheep/goats and yaks and from locally infected humans at surgery was carried out to determine the strain of parasite responsible for human infection. DNA genotyping indicated that only the sheep strain (G1) of E. granulosus was present in the study area. CONCLUSIONS: Considerable re-infection rates of E. granulosus among dogs indicated a high infection pressure from infected livestock in this region, most likely from older animals dying on the pasture. A combination of livestock vaccination with the Eg95 vaccine, which is effective against the sheep strain of E. granulosus, and dog anthelmintic treatment, thus targeting two critical points of the parasite life-cycle, would avoid the conflicts of religion or local culture and could achieve the goal of hydatid control in the long term.

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.

Heritable transgenesis of Parastrongyloides trichosuri: a nematode parasite of mammals.

Germline transformation of a parasitic nematode of mammals has proven to be an elusive goal. We report here the heritable germline transformation of Parastrongyloides trichosuri, a nematode parasite whose natural hosts are Australian possums of the genus Trichosurus. This parasite can undergo multiple free-living life cycles and these replicative cycles can be maintained indefinitely in the laboratory. Transformation was achieved by microinjection of DNA into the ovary syncytium of either free-living or parasitic adult females. By selecting for the transgenic progeny of successive free-living life cycles, it was possible to establish and maintain transgenic lines. All three transgenic lines tested were shown capable of establishing patent infections in possums and to transmit the functional transgene to their progeny. The transgene, driven by the Pt hsp-1 promoter, was constitutively expressed in intestinal cells at all stages of both parasitic and free-living life cycles, although gene silencing appears to occur in some transgenic progeny. This is the first report of heritable transgenesis in a parasitic nematode of a mammal and we discuss a variety of previously inaccessible experimental avenues that will now be possible with this powerful model system.

Short report: Inadequacy of yaks as hosts for the sheep dog strain of Echinococcus granulosus or for E. Multilocularis.

Hydatid disease (echinococcosis) has a two-host cycle involving the domestic dog and grazing animals. Humans are also infected by the dog. Both unilocular (Echinococcus granulosus in yaks, sheep, and goats) and multilocular (alveolar) (E. multilocularis in hares and rodents) hydatids are common in western Sichuan in the People's Republic of China. Humans and dogs are equally infected with both species. Many yaks (Bos grunniens) were found with multilocular cysts that visually were deemed to be E. multilocularis. However, a histologic and molecular study showed that they were actually E. granulosus. No infective cysts were found in 125 necropsied yaks. We conclude that the yak is an inadequate and dead-end host for the sheep dog (G1) strain of Echinococcus granulosus and also for E. multilocularis.

Control of echinococcosis and cysticercosis: a public health challenge to international cooperation in China.

Echinococcosis, both cystic and alveolar, and Taenia solium cysticercosis are the most serious zoonotic cestodoses worldwide. Because of the emerging importance of these diseases in China, several international workshops and meetings were held in this country from 1998 to 2001. Based on round table discussions in Chengdu 2000, the proposal of a strategy to control echinococcosis and cysticercosis has been prepared in China. It includes a comprehensive approach based on a careful analysis of the local situations (particularly concerning the particularities of the cycle, ecology, and ethology of the animal hosts, and behavioral characteristics of the population at risk), the use of newly developed tools both in animals and human (immunology, molecular biology, and imaging), and the association of the traditional control measures (control of slaughtering, antiparasitic treatment and control of the definitive hosts, and health education) with more recent developments such as vaccination of the intermediate hosts. Plans on for the control of echinococcosis and cysticercosis in China are summarized.

Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus.

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.