Genetic polymorphisms of Echinococcus granulosus sensu stricto in the Middle East.

Echinococcus granulosus sensu stricto is a cosmopolitan parasite causing cystic echinococcosis in humans and livestock. Recent molecular phylogeographic studies suggested the rapid dispersal of the parasite by the anthropogenic movement of domestic animal hosts. In the present study, genetic polymorphism of E. granulosus s. s. in the Middle East, where the domestication started, was investigated to validate the dispersal history of the parasite. Thirty-five and 26 hydatid cysts were collected from Iran and Jordan, respectively, and mitochondrial cytochrome c oxidase subunit I (cox1) gene was sequenced. Chinese and Peruvian specimens were also analyzed for comparison. Haplotype network analysis demonstrated the existence of a common haplotype EG01 in all populations. Although EG01 and its one-step neighbors were the majority in all regions, most of the neighboring haplotypes were unique in each locality. Haplotype diversity was high but nucleotide diversity was low in Iran, Jordan and China. Both diversities were lowest and only a few haplotypes were found in Peru. Neutrality indices were significantly negative in Iran, Jordan and China, and positive but not significant in Peru. Pairwise fixation index was significant for all pairwise comparisons, indicating genetic differentiation among populations. These results suggest a evolutionary history of E. granulosus s. s. in which a genetic subgroup including EG01 was selected at the dawn of domestication, and then it was rapidly dispersed worldwide through the diffusion of stock raising. To approach the origin of the ancestral strain, extensive sampling is needed in many endemic regions. To evaluate the hypothetical evolutionary scenario, further study is needed to analyze specimens from diverse host species in wider regions.

Production of monoclonal antibodies against the 8 kDa subunit of Echinococcus granulosus Antigen B (EgAgB8/2) using DNA immunization.

Cystic echinococcosis (CE), an endemic cosmopolitan zoonotic helminthic disease caused by the larval stage of Echinococcus granulosus, lacks reliable diagnostic tools that fulfill the criteria of high sensitivity and specificity. Antigen B (AgB), a thermostable lipoprotein that constitutes a considerable fraction of the cystic hydatid fluid (HF), is being considered as a suitable source for vaccination and immunodiagnosis of CE due to its high specificity. Genetic immunization was used to immunize BALB/c mice with the second subunit of antigen B (EgAgB8/2) for the production of monoclonal antibodies (MAb). Fusion products between the spleen cells and myeloma cells produced six MAbs of the following isotypes: IgG2a (two clones), IgG2b (three clones), and IgM (one clone). The MAbs were tested for their specificity to crude sheep hydatid fluid (CSHF) versus other antigens prepared from other helminthic parasites including Toxocara canis, Acanthocheilonema viteae, Fasciola hepatica, Schistosoma mansoni, and Taenia. Five MAbs reacted with E. granulosus antigens, one showed cross reactivity with S. mansonia antigens, and one showed a high reactivity with E. granulosus but was cross reactive with all helminthic antigens tested. Using SDS-PAGE and immunoblotting under reducing conditions, all MAbs identified the four AgB subunits with molecular weights of 8, 16, 24, and 36 kDa. Further work on the specificity and sensitivity of these MAbs as well as their use in detecting circulating parasite antigens and in antigen purification will be assessed in future studies.

The induction of T helper type 1 response by cytokine gene transfection protects mice against secondary hydatidosis.

Infection of BALB/c mice with protoscoleces of Echinococcus granulosus constitutes a model for the study of secondary hydatidosis (SH) and the associated immune response in immunization and infection trials. This study aimed at testing the efficacy of the cytokine gene expression approach to modulate the immune response and the magnitude of cyst development in mice with secondary hydatidosis. At the time of cyst development (28 days post infection), mice were injected intramuscularly with an expression vector containing murine promoter and carrying the open reading frames of IFN-gamma, IL-12 (Th1 cytokines), or IL-4 (Th2 cytokine). Assessment of cyst load at 22 weeks of infection showed a significant reduction in cyst load in mice injected with IFN-gamma and IL-12 genes at 60% and 47%, respectively. In contrast, the IL-4-gene-injected mice displayed six times higher cyst load in comparison to control-infected mice (injected with empty plasmids). Parasite-specific IgG2a peaked in IL-12-gene-injected mice at week 7 of infection (3 weeks after gene transfection), whereas in IFN-gamma-gene-injected mice IgG2a started to elevate after week 9 and continued to increase steadily until the termination of the experiment (22 weeks post infection). In contrast, in IL-4-gene-transfected mice, the IgG1 elevation started after week 9 and continued steadily thereafter. In conclusion, a significant high protection rate against secondary hydatidosis in BALB/c mice was accompanied with the induction of Th1 response. Moreover, in vivo IL-12 gene expression induced earlier IgG2a in comparison to IFN-gamma.

Humoral and cytokine response during protection of mice against secondary hydatidosis caused by Echinococcus granulosus.

Infection of BALB/c mouse with protoscoleces of Echinococcus granulosus constitutes a model for the study of secondary hydatidosis and the associated immune response in immunization and infection trials. The aims of this study were to induce a protective immunity against secondary hydatidosis using conventional vaccination approaches and to analyse the immune responses that accompany this protection. Mice immunized with antigen B (AgB), a component of crude sheep hydatid fluid (CSHF), showed a significant level of protection as indicated by a 98.3% reduction in cyst load. This reduction in cyst development was accompanied by a high concentration of interferon gamma secreted by antigen-stimulated spleen cells, as compared with those secreted by cells of mice immunized with CSHF or protoscoleces homogenate (PSH) antigens. In contrast, interleukin-4 was significantly higher in the supernatants of cells stimulated with CSHF or PSH compared with AgB (191.5, 195.7 and 127.5 pg, respectively). Kinetic analysis of immunoglobulin subclasses showed persistently high levels of IgG1 and IgG2a subclasses in immunized infected animals until 6 months of infection, whereas IgG3 showed a significant decline after 1 month of infection. In infected non-immunized control mice, all IgG subclasses showed a gradual increase after the first month of infection until the experiment termination (8 months after infection).

Echinococcus granulosus in Jordan: assessment of various antigenic preparations for use in the serodiagnosis of surgically confirmed cases using enzyme immuno assays and the indirect haemagglutination test.

The Enzyme linked immunosorbent Assay (ELISA), indirect haemagglutination (IHA), and immunoblot techniques (IB) were used for the serodiagnosis of surgically confirmed cystic echinococcosis (CE) caused by the tapeworm Echinococcus granulosus. Antigens used for the detection of IgG or total antibodies included crude sheep hydatid fluid (CSHF), autoclaved antigen B (AAB), boiled antigen B (BAB), and homogenate protoscoleces antigen (HPA). The overall sensitivity of the ELISA and IHA tests used for the serodiagnosis of 57 surgically confirmed human cases was 91.2% and 68.4%, respectively. The sensitivity of both tests was comparable in groups whose sera were collected one week before surgery and up to one year after surgery at 95.8% and 87.5%, respectively. In contrast, the sensitivity of the ELISA was significantly higher than that of IHA for sera of patients collected after one year of surgery. There was a positive correlation (r = 0.61) between the titers of antibodies detected by the ELISA and IHA. Using the IB technique, antigen B fractions (8/12, 16, and 24 KDa) were detectable by sera of 68.4% using either CSHF or AAB, 49.1% using BAB and 22.8% using HPA as detecting antigens. The overall sensitivity of the three AgB fractions was identical or similar to that of the 8/12 KDa fraction alone, indicating that the detection of the latter fraction is sufficient for the serodiagnosis of CE infection in humans. In conclusion, the ELISA is the test of choice for the serodiagnosis of CE and the follow up of cases following surgery using CSHF as an antigen. The IB test is a confirmatory test when antigen B fractions of CSHF or AAB are detected.

Copro-diagnosis of Echinococcus granulosus infection in dogs by amplification of a newly identified repeated DNA sequence.

Diagnosis of Echinococcus granulosus infection in dogs by detecting adult worms recovered post mortem or purged from the intestines after treatment with arecoline is not suitable for mass screening. Large-scale diagnosis by detection of copro-antigens is useful but only with relatively high intensity infections, and only by genus. To provide a more sensitive and specific diagnosis, a polymerase chain reaction (PCR) assay was developed, that amplified a target repeated sequence (EgG1 Hae III) newly identified in the genome of the common sheep strain of E. granulosus. This repeated sequence consists of approximately 6,900 copies, arranged in tandem, in groups of 2-6 repeats. The corresponding primers used in the PCR easily detected a single egg with no cross-amplification of DNA from closely related cestodes, including E. multilocularis and Taenia spp. Fecal samples from naturally infected dogs, with 2-10,000 E. granulosus worms at necropsy, were all PCR positive, while E. multilocularis or Taenia spp. positive controls as well as non-endemic controls were all PCR negative. This copro-PCR assay was demonstrated to be 100% specific and also detected all necropsy-positive E. granulosus-infected dogs. It is suggested that this copro-PCR assay has the potential for pre-mortem diagnosis of E. granulosus infection even in areas where E. granulosus and E. multilocularis are co-endemic.

Retrospective surgical incidence and case distribution of cystic echinococcosis in Jordan between 1994 and 2000.

A retrospective follow-up study on the surgical incidence of cystic echinococcosis (CE) was carried out in major governmental, military and private hospitals throughout Jordan between 1994 and 2000. A total of 472 cases were recorded over the 7-year period and an overall mean annual surgical incidence (MASI) of 2.3 per 100,000 inhabitants was estimated. The highest number of surgical cases was recorded in hospitals of the middle region of the country. The highest MASI (3.6 per 100,000) was found in the southern region while the lowest (1.4 per 100,000) was in the northern region of the country. Taking into consideration the population size and the origin of surgically confirmed cases of each region, a relative surgical index (RSI) was calculated at which the highest (RSI=3.4) was among cases originated from the southern region and the lowest (RSI=0.7) was among those originated from the middle region of Jordan. The northern region and desert areas (badia) showed comparable RSI at 1.0 and 1.1, respectively. Males younger than 15 years of age showed significantly higher surgical incidence than females of comparable age at a ratio of 1.6:1, whereas the number of female cases of different age groups over 15 years of age was consistently higher than that of males at a ratio of 1.25-2.5:1.0. The liver was the primary site of cyst development in 69.4% of the cases and the lung involvement accounted for 13.3% of the cases. Diagnosis of CE in Jordan relies mostly on imaging methods with serological techniques being rarely used for diagnosis. The frequency of CE recurrence was 27.5% of the cases, which may be attributed to the low use of chemotherapeutic antihelminthics among surgically treated cases.

Canine echinococcosis in northern Jordan: increased prevalence and dominance of sheep/dog strain.

A total of 112 stray and semi-stray dogs (Canis familiaris) from four different geographical areas in northern and middle Jordan were necropsied to evaluate the prevalence and intensity of intestinal helminthiasis. Of these, 33 dogs (29.5%) were infected with Echinococcus granulosus and 61 (54.5%) with other Taenia species. Other cestodes found included Dipylidium caninum in 36 dogs (32.1%), Diplopylidium in 6 dogs (5.4%), Mesocestoides sp. in 3 dogs (2.7%) and Joyuexiella in 1 dog (0.9%). Toxocara nematodes were found in 10 dogs (9.2%) and only 1 dog was positive for acanthocephalans. Among the dogs infected with E. granulosus, 8 dogs (24.2%) had a worm load higher than 1,000 worms. The ratio of infected male to female dogs was 1.9:1.0. Strain analysis of E. granulosus using random primers revealed the dominance of the G1 strain (sheep/dog strain) in the region. Only one dog harbored another E. granulosus strain, which resembled the G4 strain pattern.