Epitope specificities and antibody responses to the EG95 hydatid vaccine

Antibody isotype and epitope specificities were examined in sheep immunized with EG95, a protective recombinant vaccine against hydatid disease. All sheep immunized with EG95 as a fusion protein with glutathione S-transferase (GST) produced prominent IgG antibodies against the EG95 portion of the protein. Linear, antibody-binding epitope specificities of EG95 were mapped using a series of 25 overlapping synthetic peptides. Three immunodominant regions were identified which generated specific IgG₁ and IgG₂ antibodies in the majority of vaccinated sheep. These regions corresponded to the EG95-derived sequences SLKAVNPSDPLVYKRQTAKF, DIETPRAGKKESTVMTSGSA and SALTSAIAGFVFSC. An additional immunogenic region was identified which induced almost exclusively IgG₂ antibody. This epitope was located within the sequence TETPLRKHFNLTPV. The anti-parasitic, protective effects of the EG95 vaccine correlated with the detection of specific antibody to two or more of the four linear immunogenic regions. The identification of these immunogenic peptides of EG95 maybe useful in the development of a synthetic peptide vaccine as a derivative of the EG95 recombinant.     

Antigenic differences between the EG95‐related proteins from Echinococcus granulosus G1 and G6 genotypes: implications for vaccination

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.     

Vaccination against hydatidosis using a defined recombinant antigen

Echinococcus granulosus is the causative agent of hydatid disease in humans and animals. Natural transmission of the parasite occurs between dogs as definitive hosts and animal intermediate hosts. There is an urgent need for improved methods to control the parasite's transmission. Here we describe the development of a vaccine based on a cloned recombinant antigen from the parasite egg (oncosphere). Sheep vaccinated with the antigen, designated EG95, are protected (mean 96–98%) against hydatidosis developing from an experimental challenge infection with E. granulosus eggs. The vaccine will provide a valuable new tool to aid in control of transmission of this important human pathogen. It also has the potential to prevent hydatid disease directly through vaccination of humans.     

细粒棘球绦虫Eg95基因疫苗和重组抗原诱导小鼠免疫应答的比较研究

目的观察比较细粒棘球绦虫Eg95重组抗原和基因疫苗诱导小鼠的免疫应答状况。方法实验组和对照组小鼠分别注射Eg95重组抗原(rEg95)、费氏佐剂(FCA)、pcDNA3-Eg95基因疫苗、pcDNA3质粒和生理盐水,收集各组血清用酶联免疫吸附(ELISA法)检测抗体IgG和IgG2a亚类水平;采集脾细胞用四甲基偶氮唑盐试验(MTT法)检测免疫小鼠的脾淋巴细胞增殖反应。结果rEg95免疫组小鼠在第二次免疫后开始检测到抗Eg95抗原的IgG,并随着免疫次数的增多,血清抗体效价升高,在第1次免疫后第10周时,免疫抗体滴度可达到1∶25,600。Eg95基因疫苗免疫的小鼠产生抗体滴度随免疫次数的增加而升高,最高可达1∶3,200,但是低于Eg95重组蛋白免疫小鼠产生的抗体滴度水平。pcDNA3-Eg95免疫组产生IgG2a亚类抗体水平明显高于对照组和rEg95组。在第四次免疫后,进行淋巴细胞转化试验,MTT法检测证实rEg95和pcD-NA3-Eg95免疫的小鼠,其脾细胞均可在体外被特异性刺激增生。结论细粒棘球绦虫Eg95重组抗原和基因疫苗均可诱发小鼠产生特异性免疫应答。     

细粒棘球绦虫重组Bb-Eg95-EgA31疫苗诱导BALB/c小鼠的保护力观察

目的探讨细粒棘球绦虫(Eg)重组双歧杆菌(Bb)-Eg95-EgA31疫苗免疫和Eg原头节攻击后小鼠的囊重抑制率、抗体及其亚类的变化。方法将细粒棘球绦虫重组Bb-Eg95-EgA31疫苗分别采用皮下注射、肌肉注射、鼻腔内接种和口服灌胃4种途径免疫BALB/c小鼠,免疫后8周每鼠用50个Eg原头节攻击感染,感染后25周剖杀小鼠,分离细粒棘球蚴包囊并称重,计算囊重抑制率;收集血清,常规ELISA测定血清IgG及其亚类和IgE水平,试验设有空载体、Bb和MRS对照。结果皮下注射组、肌肉注射组、鼻腔内接种组和口服灌胃组免疫小鼠的囊重抑制率分别为45.33%、41.33%、70.67%和62.67%,血清IgGI、gG2aI、gG2b和IgG1水平显著升高,IgG3和IgE水平显著降低。结论接种Bb-Eg95-EgA31疫苗能诱导小鼠产生保护力,IgGI、gG2aI、gG2b和IgG1起重要作用。     

Serology and longevity of immunity against Echinococcus granulosus in sheep and llama induced by an oil‐based EG95 vaccine

An oil‐based formulation of the EG95 vaccine to protect grazing animals against infection with Echinococcus granulosus was formulated in Argentina. The efficacy of the vaccine was monitored by serology in sheep and llama (Lama glama) and was compared to the serology in sheep previously published using a QuilA‐adjuvanted vaccine. Long‐term efficacy was also tested in sheep by challenging with E. granulosus eggs of the G1 strain 4 years after the beginning of the trial. The serological results for both sheep and llama were similar to those described previously, except that there was a more rapid response after the first vaccination. A third vaccination given after 1 year resulted in a transient boost in serology that lasted for about 12 months, which was similar to results previously described. Sheep challenged after 4 years with three vaccinations presented 84·2% reduction of live cysts counts compared with control group, and after a fourth vaccination prior to challenge, this reduction was 94·7%. The oil‐based vaccine appeared to be bio‐equivalent to the QuilA vaccine.     

Spontaneous primary intrathoracic,extrapulmonary hydatid cyst in a broiler rabbit

Echinococcus granulosus, a zoonotic tapeworm with a dog-herbivore life cycle, is known to use ruminants, horses, pigs, etc., as intermediate hosts. Natural infections of hydatid cysts have not been documented in small animals like rabbits in India. This paper records spontaneous intrathoracic, extrapulmonary hydatid cysts of E. granulosus in a cage reared rabbit. The presence of non-invasive unilocular cyst with typical protoscolices containing rostellar hooks favoured the diagnosis of E. granulosus over E. multilocularis, the only other Echinococcus species found in India. The presence of fertile hydatid cyst points to the fact that rabbits can also act as natural intermediate hosts for E. granulosus. The significance of the findings in relation to public health importance is discussed.     

Identification and diagnostic value of a major antibody epitope on the 12 kDa antigen from Echinococcus granulosus (hydatid disease) cyst fluid

An IgG1 monoclonal antibody (MoAb), designated C9E7H8, has been produced against an epitope on the 12 kDa antigen of Echinococcus granulosus cyst fluid, believed to represent the smallest subunit of antigen B. This MoAb, raised against purified 12 kDa antigen eluted from a reducing SDS-PAGE gel, demonstrated strong binding to native sheep cyst fluid in ELISA and recognition of all three subunits of antigen B (at 12, 16, 23 kDa) by immunoblot under both reducing and non-reducing conditions. Immunoblot analysis also indicated that the complementary epitope is conserved amongst cyst fluids from different intermediate hosts of E. granulosus, including fluids from cysts of two distinct strains, and is present in cyst fluid from E. multilocularis. The monoclonal displays binding to a cDNA clone, EgPS-3, which we have previously shown expresses part of the 12 kDa molecule. EgPS-3, expressed as a glutathione-S-transferase fusion protein, was successful in positive detection of 74% of cystic hydatid patients, although cross-reactions were observed with 25% of sera from alveolar hydatid and 22% of sera from schistosomiasis japonica patients. Three peptides, based on the predicted amino acid sequence of EgPS-3. showed increased specificity but slightly reduced sensitivity in the detection of antibody from E. granulosus patients. The predominant epitope recognized by human antibody occurs in the Nterminal 27 amino acids (peptide 65) of EgPS-3 which also correlates with the location of the monoclonal antibody epitope.     

青海省达日县棘球蚴病流行病学调查

目的 分析青海省果洛藏族自治州达日县棘球蚴病的流行分布现状,为制定预防控制措施提供科学依据。 方法 于2007年8～9月对达日县6个乡各2～3个自然村的3周岁以上常驻牧民分别用B超、间接红细胞凝集试验（IHA）和间接ELISA法（重组Ag B和Em 18抗原）检查两型棘球蚴病患病和感染情况。并调查当地啮齿类动物、牦牛、绵羊和野犬的感染情况,对采集的棘球绦虫和棘球蚴用PCR-RFLP方法进行虫种鉴定,并确定其基因型。收集牧民的家犬粪便,用双抗体夹心法检测粪抗原阳性率。 结果 共调查牧民1 723人,B超查出棘球蚴病患者236例（占13.7%）,其中囊型和泡型棘球蚴病患病率分别为5.5%（95/1 723）和8.2%（141/1 723）。男、女性棘球蚴病患病率分别为11.6%和16.0%（χ ²=7.0,P＜0.05）。家犬粪抗原阳性率为11.3%（31/275）。剖检9只无主犬,其中5只棘球绦虫感染阳性,对检获的虫体经PCR-RFLP鉴定,1只犬感染细粒棘球绦虫,基因型为G1,4只犬感染多房棘球绦虫。牦牛、绵羊的细粒棘球蚴感染率分别为26.4%（14/53）和5/16,对从牦牛、绵羊检获的细粒棘球蚴经PCR-RFLP鉴定,基因型均为G1。捕获高原鼠兔239只,石渠棘球绦虫感染率为11.3%（27/239）。 结论 达日县存在细粒棘球绦虫、多房棘球绦虫和石渠棘球绦虫的分布,泡型和囊型棘球蚴病在人群中严重流行,犬是细粒棘球绦虫和多房棘球绦虫主要传染源。     

Short report: Echinococcus granulosus from Xinjiang,PR China: cDNAS encoding the EG95 vaccine antigen are expressed in different life cycle stages and are conserved in the oncosphere

The EG95-based vaccine protects sheep from infection with the dog tapeworm Echinococcus granulosus. The EG95 encoding gene is a member of a multigene family, several members of which are expressed in the oncosphere, believed to be the target of immunity induced by the vaccine. E. granulosus exhibits extensive intraspecific (strain) variation, and variability of the eg95 gene in different isolates of E. granulosus may directly impact the effectiveness of the EG95-based vaccine. We analyzed the eg95 gene from E. granulosus collected in Xinjiang, in northwest China, where hydatid disease is hyperendemic. The gene is expressed in oncospheres, protoscoleces, and immature and mature adult worms, and the eg95 gene family was shown to comprise two basic sequence types. Very limited sequence variation was evident in the EG95 protein from oncospheres. This high degree of sequence conservation predicts that the vaccine will continue to be effective in China and elsewhere.     

绵羊CTLA-4胞外区对细粒棘球蚴EG95抗原的免疫佐剂作用

目的探索绵羊细胞毒性T细胞相关抗原-4(cytotoxic Tlymphocyte-associated antigen4,CTLA-4)胞外区靶向提呈羊细粒棘球蚴(Echinococcus granulosus,Eg)EG95抗原的可行性。方法用RT-PCR扩增CTLA-4胞外区编码序列,插入原核表达载体pET-30a获得融合表达载体pET-IgV;根据蛋白质二级结构分析结果 ,用PCR扩增EG95亲水区编码序列,将串联的3拷贝EG95编码序列分别插入表达载体pGEX-6P-1和pET-IgV,诱导重组大肠杆菌表达并纯化重组蛋白;用相同剂量的GST-3EG95、IgV-3EG95包涵体或可溶性蛋白免疫小鼠,比较EG95抗体的应答水平。结果用RT-PCR扩增得绵羊CTLA-4胞外编码区,IPTG诱导的重组大肠杆菌表达预期的约63kDa GST-3EG95和60kDa IgV-3EG95融合蛋白,两者均能被EG95抗血清识别;经两次免疫后,IgV-3EG95免疫组的EG95抗体水平显著高于GST-3EG95免疫组,可溶性蛋白的免疫效果优于包涵体。结论绵羊CTLA-4胞外区可作为分子佐剂促进细粒棘球蚴EG95抗原的抗体应答水平。     

Muscular cystic hydatidosis: case report

Background  

Hydatidosis is a zoonosis caused by Echinococcus granulosus, and ingesting eggs released through the faeces from infected dogs infects humans. The location of the hydatid cysts is mostly hepatic and/or pulmonary, whereas musculoskeletal hydatidosis is very rare.     

Molecular characterization of human and animal Echinococcus granulosus isolates in Isfahan, Iran

Cystic hydatid disease (CHD) is one of the most important zoonotic diseases in different parts of Iran. While it causes major health problem, there is limited information about its transmission cycles and reservoirs of human infection. Therefore we aimed to characterize the existence Echinococcus granulosus cysts in humans and animals in the province of Isfahan, central region of Iran. We collected hydatid cysts from the liver and lungs of patients who underwent surgery procedure and also cysts were obtained from domestic animals at slaughterhouses. DNA was extracted from the protoscoleces and examined by polymerase chain reaction (PCR) of rDNA internal transcribed spacer1 (ITS1-PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). In addition, fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) were sequenced. Among the two different identified strains/genotypes (sheep and camel), the sheep strain was shown to be the most common genotype of E. granulosus affecting humans, sheep, cattle, goats and occasionally camels. Nine out of 26 camel samples were infected with sheep strain. However the camel genotype was observed in humans, camels and cattle. Seventeen out of 26 camel isolates, 6 out of 31 human and 5 out of 14 cattle samples were infected with the camel genotype. The camel genotypes had PCR and RFLP patterns which were different from the PCR and RFLP patterns of the rest of isolates (sheep strain). The results of this study showed that the ‘camel’ strain was actual source of infection to humans which circulates between intermediate hosts including camels and cattle, and it confirms the camel–dog transmission cycle in Isfahan.     

Pericyst: The outermost layer of hydatid cyst

Hydatid disease,caused by the parasite Echinococcus granulosus,mostly affects the liver and the lungs with hydatid cysts which consist of three layers:(1)the outer pericyst;(2)the middle laminated membrane;and(3)the inner germinal layer.Pericyst,as the outermost layer of the hydatid cyst,is made by host cells encasing the hydatid cyst.An extremely close interaction exists between this host tissue and the parasite,and any degenerative changes of the pericyst would result in hydatid cyst degeneration or rupture.The pericyst plays an undeniably important role in the development and survival of the hydatid cyst.     

Studies on stage-specific immunity against Taenia Taeniaeformis metacestodes in mice

The possible existence of stage-specific immune responses to Taenia taeniaeformis infection was investigated in C3H/He mice vaccinated with antigens prepared from either the oncosphere or metacestode stages. Mice were immunized twice, 2 weeks apart, with antigen in Freund's complete adjuvant. Two weeks after the second immunization they were challenged with 250 T. taeniaeformis eggs and killed day 0, 5, 10, 15, 20, 25, 30, 45 and 60 after infection. Gross examination of the livers revealed marked differences between oncosphere (TtO) and metacestode (TtM) vaccinated mice. Very few metacestodes were found in the first group but most of those that evaded the initial host attack developed like the cysts found in the control group. In contrast, many degenerating metacestodes were found in the TtM vaccinated group. In a subsequent experiment groups of mice were vaccinated with varying doses of either TtO or TtM to determine whether the qualitative differences observed above were due to antigen dose effects. However, varying antigen doses gave the same results. These data show that vaccination with oncospheres generates an immune response capable of killing invading larvae soon after infection whereas vaccination with TtM results in larvae being killed at a later stage, suggesting that there are stage-specific, host-protective antigens.     

复频高强度聚焦超声对离体细粒棘球蚴囊壁超微结构的影响

目的 分别用单频和多频聚焦超声体外照射细粒棘球蚴包囊, 观察对其囊壁的损伤情况。 方法 用羊肝细粒棘球蚴原头节接种小鼠, 1年后处死, 自腹腔取出包囊, 选择直径约2 cm的包囊40只随机均分4组。对照组剪破包囊壁, 用3%戊二醛固定并立即冷藏。实验A组包囊用2号单频换能器（4 W）照射, 实验B组用2及3号双频换能器（功率分别为4和5 W）同时照射, 实验C组用1、2及3号3频换能器（功率分别为4、4及5 W ）同时照射。各组均照射 1 min, 然后立即用3%戊二醛固定并冷藏。用肉眼、扫描电镜及透射电镜观察其变化。 结果 肉眼见实验组囊壁变厚, 颜色浑浊。透射电镜观察显示, 随超声频数增加囊壁结构被破坏程度加重, 角皮层纤维增粗、紊乱, 生发层细胞核肿胀破裂, 线粒体明显受损, 微绒毛变短, 断裂或消失。部分区域仅剩细胞碎片和破裂的细胞核。扫描电镜观察显示, 随超声频数增加, 囊壁内外表面被破坏程度加重, 最终正常结构被完全破坏。 结论 在体外, 复频高强度聚焦超声对小鼠细粒棘球蚴囊壁损伤明显, 其损伤程度随超声频数的增加而加重。     

Recent advancements and new perspectives in animal models for Neurocysticercosis immunopathogenesis

Neurocysticercosis (NCC), one of the most common parasitic diseases of the central nervous system, is caused by Taenia solium. This parasite involves two hosts, intermediate hosts (pig and human) and a definitive host (human) and has various stages in its complex life cycle (eggs, oncosphere, cysticerci and adult tapeworm). Hence, developing an animal model for T. solium that mimics its natural course of infection is quite challenging. We have reviewed here the animal models frequently used to study immunopathogenesis of cysticercosis and also discussed their usefulness for NCC studies. We found that researchers have used mice, rats, guinea pigs, dogs, cats and pigs as models for this disease with varying degrees of success. Mice and rats models have been utilized extensively for immunopathogenesis studies due to their relative ease of handling and abundance of commercially available reagents to study these small animal models. These models have provided some very exciting results for in‐depth understanding of the disease. Of late, the experimentally/naturally infected swine model is turning out to be the best animal model as the disease progression closely resembles human infection in pigs. However, handling large experimental animals has its own challenges and limitations.     

细粒棘球蚴EG95s重组蛋白串联表达免疫原性分析

目的 比较、分析EG95s重组蛋白的免疫原性。方法 本研究分别诱导表达含有1、2、3拷贝EG95s的pET-1EG95s、pET-2EG95s和pET-3EG95s重组质粒,并用His亲和纯化HIS-1EG95s、HIS-2EG95s和HIS-3EG95s3种重组蛋白。再以相同的免疫程序分别免疫8周龄BALB/c小鼠,通过间接ELISA方法检测小鼠体内抗体水平的变化分析其免疫原性。结果 经过改造的EG95s重组蛋白:HIS-1EG95s、HIS-2EG95s、HIS-3EG95s均保留了免疫原性,能诱导小鼠产生特异性抗体。3者抗体水平的比较结果显示HIS-1EG95s首次免疫后1周产生的抗体水平明显高于HIS-2EG95s、HIS-3EG95s,但免疫后2周开始,HIS-3EG95s抗体水平超过HIS-1EG95s组,并在二次免疫及三次免疫后持续高于HIS-1EG95s组。免疫后最终抗体效价显示HIS-1EG95s和HIS-2EG95s两组均为1∶819 200,而HIS-3EG95s组为1∶1 638 400。HIS-1EG95s、HIS-2EG95s、HIS-3EG95s均诱导机体产生IFN-γ,但差异不显著,与羊包虫阳性血清反应结果显示HIS-1EG95s的反应性均显著低于HIS-2EG95s和HIS-3EG95s。结论 HIS-1EG95s、HIS-2EG95s与HIS-3EG95s均具有很好的免疫原性,虽HIS-EG95s在免疫初期免疫效果好,但串联3拷贝的EG95s后使得重组蛋白免疫效果更持久,更有利于产生更长效的免疫保护作用。本研究为羊包虫病疫苗的研制及免疫预防奠定了科学基础。     

The effect of Echinococcus granulosus on spleen cells and TGF‐β expression in the peripheral blood of BALB/c mice

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus (E. granulosus) is a zoonotic parasitic disease. The effective immune evasion mechanisms of E. granulosus allow it to parasitize its hosts. However, the status of the innate and adaptive immune cells and their contributions to E. granulosus progression remain poorly understood. In this study, we aimed to determine the impact of E. granulosus infection on T cells, NK cell responses and TGF‐β expression during the early infection phase in BALB/c mice. In E. granulosus infections, there was an increasing tendency in the percentage of CD4⁺CD25⁺ T cells and CD4⁺Foxp3⁺ T cells and peripheral blood TGF‐β levels and relative expression of the Foxp3 gene. Moreover, there were a decreasing tendency in the percentage of NK cells and NK cell cytotoxicity and the expression of NKG2D on NK cells. The TGF‐β1/Smad pathway was activated by E. granulosus in mice. Above results can be reversed by the inhibitor SB‐525334 (potent activin receptor‐like kinase 5 inhibitor). These results suggest that the TGF‐β/Smad pathway plays an important role in changes of T‐cell or NK cell responses. These results may contribute to revealing the preliminary molecular mechanisms in establishing hydatid infection.     

Study of cystic echinococcosis in slaughtered animals in Al Baha region, Saudi Arabia: Interaction between some biotic and abiotic factors

The variation in cystic echinococcosis (CE) prevalence and mean intensity was studied in relation to site, season and host age and sex. A total of 12,911 slaughtered animals, 140 camels, 2668 cattle, 6525 sheep and 3578 goats were inspected for hydatid cysts in Al Baha region, Saudi Arabia, in three study areas during four seasons from June 2008 to May 2009. The prevalence of infection was 32.85%, 8.28%, 12.61% and 6.56% in camels, cattle, sheep and goats respectively. The prevalence of the parasite varied significantly in relation to site, season and host age classes and sex in most host species. Spring showed the highest prevalence in camels, cattle and sheep. A significant association was found among host age classes and likelihood of infection in all examined hosts and the oldest age class was significantly more likely to be infected. The main effects in parasite intensity were host sex and age in most examined host species. A positive correlation was found between intensity of CE and host age class in all animal species examined. The most commonly infected organs were liver and lungs which constituted 48.75% and 32.83% respectively, of the total infected organs. There was a significant difference among host species in fertile cysts (P < 0.0001). The higher percentages of fertile cysts were in sheep (47.67%) and goats (23.99%) indicating that sheep and goats are the most important intermediate hosts for Echinococcus granulosus. Examined hydatid cysts of the liver had a higher fertility rate (38.79%) than those of the lungs (25.13%). Cysts size ranged from 1 to 8 cm in diameter. The mean cyst diameter was found in the lungs higher than that in the liver in all hosts. The range in the number of cysts was 1-33 in infected animals. The mean number of cysts was higher in lungs than that in liver in all examined animals. The viability rate of protoscoleces of liver fertile cysts (62.20%) was significantly higher than that of lung cysts (52.73%). In conclusion, these findings of infection, mean abundance and fertility rates of CE in slaughtered animals, prompt plans for further epidemiological studies and control programmes.