Achievements of the HERACLES Project on Cystic Echinococcosis.

The FP7 project 'Human Cystic Echinococcosis ReseArch in CentraL and Eastern Societies' (HERACLES), developed between 2013 and 2018 by nine partners in five countries, is one of the largest projects on cystic echinococcosis. Here we present the core HERACLES achievements, which should help to foster the translation of scientific investigations on health policies.

Variability in the Echinococcus granulosus cytochrome C oxidase 1 mitochondrial gene sequence from livestock in Turkey and a re-appraisal of the G1-3 genotype cluster.

Investigations into the genetic strains of Echinococcus granulosus parasites occurring in sheep and cattle in Turkey were undertaken. A total of 112 hydatid cysts were investigated from sheep (100 isolates) derived from widely distributed sites within Turkey as well as from cattle (12 isolates) from the Turkish province of Kars. The parasite genotypes in these isolates were determined by DNA sequencing of part of the mitochondrial Cytochrome C oxidase 1 (cox1) gene. Haplotypes were identified which corresponded clearly to the previously described strain G1 in a total of 107 isolates, including 98 isolates from sheep and 9 isolates from cattle. Five isolates, including 2 sheep and 3 cattle, were determined to belong to the G3 genotype. Parasites of the G3 genotype were identified only in isolates derived from animals in the eastern regions of Turkey. While the majority of the isolates described here had haplotypes corresponding to the G1 genotype, none matched exactly the G1 sequence that was defined in previous studies. Analysis of all GenBank entries for E. granulosus cox1 sequences representing G1, G2 and G3 genotypes identified substantial microsequence variability. G1 and G3 could be distinguished as separate strains, however, the existence of G2 as a separate strain could not be supported. Rather, this can be regarded as a microsequence variation of G3.

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.

Vaccination with recombinant oncosphere antigens reduces the susceptibility of sheep to infection with Taenia multiceps.

Taenia multiceps is a cestode parasite, the larval stage of which encysts in the brain of sheep, goats and cattle causing an often fatal condition. The parasite also causes zoonotic infections in humans. Homologues of the recombinant oncosphere vaccine antigens from Taenia ovis and other Taenia species were identified in T. multiceps. Sequencing of the associated T. multiceps genes and cloning of the encoding mRNA has revealed conserved features in the genes and proteins. The T. multiceps oncosphere proteins, designated Tm16 and Tm18, contain a predicted secretory signal and fibronectin type III domain. The recombinant Tm16 and Tm18 proteins were successfully expressed in Escherichia coli as fusion proteins with GST. The antigens, formulated with Quil A adjuvant, were tested in a vaccine trial in sheep. The antigens stimulated immunity in sheep against challenge infection with T. multiceps eggs. Five of nine control sheep died due to a challenge infection with T. multiceps whereas none of 20 vaccinated animals died as a result of the parasite challenge (P=0.001). In addition, vaccination with the Tm16 protein, or Tm16 plus Tm18, induced significant protection against the number of parasites encysting in the brain as a result of the challenge infection (P=0.023, P=0.015, respectively). No clear relationship was apparent between the level of specific serum antibody in vaccinated animals and either the presence or absence of parasites or the number of parasites that occurred in some of the vaccinated animals. We believe this study is the first description of recombinant vaccine-related investigations for T. multiceps. The recombinant oncosphere antigens identified may allow development of effective vaccination strategies against T. multiceps infection in sheep. They raise the potential for the development of a combined vaccine with the Echinococcus granulosus EG95 antigen for prevention of T. multiceps as well as preventing the transmission of cystic hydatid disease.

Detection of Babesia (Theileria) equi (Laveran, 1901) in horses in the Kars province of Turkey.

This study was carried out in order to detect antibodies to Babesia (Theileria) equi in the local breed of horses in the province of Kars, Turkey. A total of 108 serum samples from apparently healthy horses in eight villages were examined for B. equi antibodies by an indirect immunofluorescent antibody test (IFAT). Of the 108 samples tested, 27 (25%) were found to be seropositive. The horses sampled in Aydinalan village had the highest prevalence (50.0%) of Babesia equi infection while the lowest prevalence was found among horses from Bayraktar village (12.5%). Statistically significant differences in seroprevalence were observed between these two villages (P < 0.05). This study is the first report on the status of B. equi infection in Kars.

Detection of Toxoplasmosis gondii seropositivity in sheep in Yalova by Sabin Feldman Dye Test and Latex Agglutination Test.

Sera collected from 63 sheep older than one year of age in two regions of Yalova were tested for anti- Toxoplasma gondii antibodies using the Sabin-Feldman Dye Test (SFDT) and Latex Agglutination Test (LAT). Of the 63 samples tested, 42 (66.66%) and 41 (65.08 %) were determined to be seropositive by SFDT and by LAT, respectively. Of the positive sheep serum samples, 23 were positive at a dilution of 1/16; 13, at a dilution of 1/64; and 6, at a dilution of 1/256. SFDT was accepted as a reference test. The sensitivity and specificity of LAT were 78.57% and 61.90 %, respectively. The correlation between these two tests was determined to be 73.01%.