Background: The main objective of the current study was to investigate on the cryopreservation of protoscoleces of ECHINOCOCCUS GRANULOSUS, a causative agent of cystic hydatidosis in man. Methods: This study was conducted on isolated protoscoleces from hydatid cysts infected livers collected from slaughterhouse of Tehran, Iran in 2016. Viability of protoscoleces was evaluated by dye test. Cryopreservation of isolated protoscoleces in the presence of Dimethylsulfoxide (DMSO) and glycerol using a three-step cooling protocol involving an initial period at-20 ° C,-80 ° C and liquid nitrogen was performed. Results: The mean viability rate of 10% DMSO and 15% glycerol were 9% and 8% respectively. The protoscoleces of ECHINOCOCCUS GRANULOSUS have been successfully thawed and recovered after 6 months storage in liquid nitrogen. Conclusion: Cryopreservation method needs to be improved for each species of helminthes and can be useful for other immunological and laboratorial studies.

We conducted this study to determine the level of immunity after vaccination of lambs with whole body ECHINOCOCCUS GRANULOSUS. To do so, 200 mature E. GRANULOSUS parasites, which were kept in 10% formaline for 8 months, were obtained from the Department of Parasitology, School of Veterinary Medicine, Ferdowsi University of Mashhad. The soluble protein of the parasite was prepared. The sample was homogenized in a blender, sonicated on ice and then centrifugated for 15 min at 10,000 g. Final yield was kept at -20ºC until used. Eight 4–6-month-old lambs of mixed sex, were divided into 2 equal groups; each lamb in the test group was vaccinated subcutaneously in the neck with 2 ml of the vaccine (1 mg of whole body of E. GRANULOSUS protein dissolved in 1 ml of PBS plus 1 ml of Freund’s complete adjuvant (FCA)). The control lambs were vaccinated only with adjuvant in PBS. Lambs were re-vaccinated four weeks after the first vaccination with the same preparation except that FCA was replaced by Freund’s incomplete adjuvant (FIA). Three weeks later, each lamb was administered a challenge infection dose of 2000 protoscolices intraperitoneally and 10 adult E. GRANULOSUS. After 7 months, all lambs were killed and examined for hydatid cysts. We found two cysts in the liver and and one in the lung of only one of the vaccinated lambs. The number of cysts in vaccinated lambs were significantly lower than that in the control group (P<0.001). This means that the protective immunity in lambs with whole body of E. GRANULOSUS was approximately 90%.    

In the present study, 35 stray dogs (Mixed breed), 3 red foxes (Vulpes vulpes) were examined for ECHINOCOCCUS GRANULOSUS infection as well as 19425 sheep, 5938 goats and 11883 cattle for hydatid infection in Kordestan province in West Iran during three years (1997 - 2000). Meanwhile fertility rate of different types and forms of cysts isolated from infected animals and the viability of protoscoleces were also determined. Our results indicated that, 11.43 % of the dogs were infected, with ECHINOCOCCUS GRANULOSUS. 5.33% of the sheep, 3.06% of the goats and 9.49% of the cattle were also found to be infected with hydatid cyst. In infected sheep, 75.35% of the cysts were found in lungs, 10.53% in liver and 13.93% in both liver and lungs. In infected goats, 80.76% of the cysts were found in lungs. 6.91%in liver and 12.08% in both liver and lungs. In infected cattle, 79.24% of the cysts were found in lungs, 5.49% in liver and 13.12% in both liver and lungs. The cysts isolated from liver and lungs of the sheep show higher fertility rate of cysts isolated from sheep, goats and cattle were 33.82%, 30.95% and 2.52% respectively. The viability rates of protoscoleces of the fertile cysts isolated from the slaughtered animals were found mostly higher than 70%.

Despite the establishment of extensive and successful control programmes in some countries or regions, ECHINOCOCCUS GRANULOSUS still has a very wide geographical distribution. Mature E. GRANULOSUS (n = 120) obtained from Parasitology Department, School of Veterinary Medicine, Ferdowsi University of Mashhad (Iran). Soluble protein of whole body of parasite was prepared by freeze-thawing in liquid nitrogen and at 42oC for three times. The sample was homogenized in a blender, sonicated at 110 V, 170 W for 3 times for 15 s each and then centrifuged at 10,000 xg for 15 min. Final yield was kept in -20oC. Ten mice were randomly divided into 2 groups of 5. Each mouse in test group was vaccinated subcutaneously with 100 µg (100 µl) of whole body of E. GRANULOSUS plus 100 µl of Freund"s complete adjuvant (FCA), respectively. Mice in control group were vaccinated with adjuvant in PBS. Second vaccination was conducted after four weeks with the same preparation except that FCA was replaced by Freund"s incomplete adjuvant (FIA). Three weeks after the second vaccination, each mouse was challenged with 2000 protoscolices, intraperitoneally. Mouse autopsy was carried out eight months post challenge. Our results show that none of the vaccinated mice with the whole body of E. GRANULOSUS had cysts that indicate 100% protective immunity.

Background: Cystic echinococcosis is a zoonotic infection and considered as a major economic and public health concern worldwide. This research was conducted to determine genotypic characteristics of livestock and human hydatid cyst isolates from Hamadan area, western Iran. Methods: Sampling was conducted in Hamadan industrial slaughterhouse and Beast Hospital of Hamadan City, western Iran, from 2015 to 2016. Overall, 74 livestock isolates including 69 sheep, 3 cattle and 2 goats and 9 human hydatid cysts were genotyped by PCR amplification of the rDNA ITS1 region and followed restriction fragment length polymorphism (RFLP) analysis with four restriction endonuclease enzymes, RsaI, HpaII, AluI, and TaqΙ , and sequencing. Results: The PCR amplicon size of each isolate was approximately 1 kb which was the same with that of sheep strain. According to the RFLP patterns, the isolates belonged to a single species, E. GRANULOSUS sensu stricto (G1– G3 complex). Furthermore, sequencing of representative amplicons confirmed that the RFLP-genotyped isolates corresponded to E. GRANULOSUS sensu stricto. Conclusion: E. GRANULOSUS sensu stricto is the prevailing species of E. GRANULOSUS sensu lato in the region and pointed out the importance of sheep/dog cycle in human transmission.

Definitive hosts of the ECHINOCOCCUS GRANULOSUS (E. GRANULOSUS) parasite are carnivores such as dogs, wolves and foxes. Detection of this parasite through faecal examination is not possible. In this study, dotblotting test for E. GRANULOSUS-specific coproantigens has been evaluated in dog. Three 2–3-month-old puppies were treated with piperazine and then faecal samples were collected as pre-infection samples. Seven days later, hydatid cysts from livers and lungs of sheep were fed to the puppies. Faecal samples were collected weekly for five weeks as post-infection samples. Soluble protein of pre- and post-infection faecal samples was prepared and dot-blotting test was conducted. In parallel experiments, the presence of E. GRANULOSUS eggs and also dot-blotting test were evaluated in 15 faecal samples of dogs collected from Razi Veterinary Hospital in Mashhad. For the detection of protein bands in pre-infection and fifth-week postinfection samples, polypeptide profile was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that incremental spot colours was observed in samples of experimentally infected dogs collected from the first to fifth post-infection weeks. In dot-blot analysis of faecal samples in 15 dogs, 4 samples were positive, and also these four samples were positive for E. GRANULOSUS eggs. In SDS-PAGE, one band in pre-infection and four bands in fifth-week post-infection samples were observed. The molecular weight of pre-infection sample of experimentally infected dogs was 16 kDa and the molecular weights of the samples collected five weeks post-infection were 14, 22, 36 and 45 kDa, respectively. In conclusion, the results of this experiment showed that the dot-blotting method does not produce a reliable outcome. For evaluation of the specific coproantigens of E. GRANULOSUS in dogs, coproantigen-ELISA test is needed.

Cystic hydatid disease is caused by cystic larval (metacestode) stages of ECHINOCOCCUS GRANULOSUS. This parasite can potentially occur all over the world especially in Mediterranean and Middle East countries and some parts of Africa, Latin America and China which have major foci of human infections. The cyst wall of metacestodes consists of inner, middle and external layers. To date, little attention has been paid to the immunological studies of the laminated (middle) layer. Because of the presence of diagnostic carbohydrate antigens, this layer is important. In this report, the characterisation of the laminated layer carbohydrate of the E. GRANULOSUS was investigated by peroxidase-labelled lectin conjugate blots. The comparison of SDS-PAGE and Western blot analysis confirms that many laminated layer bands are glycoproteins, especially 50-66 kDa and 25-29 kDa bands. These glycoproteins contain a-Methyl-D-mannoside, N-Acetyl-b-D-glucosamine, a-L-Fucose and N-Acetyl-b-D-galactosamine

Background: Despite advances toward an improved understanding of the evasive mechanisms leading to the establishment of cystic echinococcosis, the discovery of specific immunosuppressive mechanisms and related factors are of great interest in the development of an immunotherapeutic approach. Objective: To elucidate immunosuppressive effects of bioactive factors contained in chromatographic fractions from hydatid cystic fluid (HCF) of ECHINOCOCCUS GRANULOSUS. Methods: Hydatid cystic fluid was fractionated by reverse phase chromatography. Non-specific Concanavalin Adriven proliferation of spleen cells was used to determine specific inhibitory fractions. Trypan blue exclusion test and flowcytometry analysis were performed to check whether highly inhibitory fractions of HCF have apoptotic effect on peritoneal macrophages. Western blot analysis was used to determine proteolytic effects of parasitic antigens on major histocompatibility complex (MHC) class II (I-a) contained in membrane proteins extract from macrophages. Results: High concentrations of HCF and few of chromatographic fractions suppressed spleen cells proliferation. Fractions 7 and 35 were the highest inhibitory fractions. Specifically fraction 35 and to a lesser extent HCF induced apoptosis in peritoneal naive macrophages. However, HCF and the fraction 7 proteolytically altered the expression of MHC class II molecules on peritoneal macrophages. The proteolytic molecule was identified to be a serine protease. Macrophages taken at the chronic and end phase from cystic echinococcosis-infected mice were able to uptake and process C-Ovalbumine-FITC. These cells expressed a drastically reduced level of (I-a) molecules. Conclusion: Our study present new aspects of immune suppression function of E. GRANULOSUS. Further molecular characterization of apoptotic and proteolytic factors might be useful to develop immunotherapeutic procedure to break down their inhibitory effects.