Immunization of pigs with culture antigens of Taenia solium

An evaluation has been made of the protective effect of immunizing pigs with excretory-secretory homologous antigens on Taenia solium infections. This procedure reduced the number of cysticerci established from a challenge infection.     

Purification of larval Taenia solium antigens by gel filtration.

Crude larval Taenia solium extracts were fractionated by Sephacryl S-200 gel filtration into four fractions (W1-W4). The sensitivities of the fractions to rabbit and pig antiserum against Taenia solium were tested by double immunodiffusion, immunoelectrophoresis, and ELISA. Fraction W2 which was highly sensitive to antisera was shown by immunoblotting to contain antigen B (95 and 105 kDa). The four fractions were shown to contain antigenic determinants common with pig serum proteins and crude extracts of other Platyhelminthes (especially Taenia hydatigena). Fraction W2 has the potential to be used as a serodiagnostic antigen.     

Taenia solium oncosphere antigens induce immunity in pigs against experimental cysticercosis

Immunity to Taenia solium infection was investigated using an experimental intramuscular oncosphere infection assay (IMOA) model in pigs. Three naturally infected pigs with cysticercosis were treated with oxfendazole (OFZ), a drug demonstrated to kill cysts in porcine muscle. These animals were then challenged with oncospheres but did not develop any cysts while three uninfected pigs that were similarly challenged, did develop intramuscular cysts. In another study, two groups of three pigs each were immunized with crude T. solium oncosphere and metacestode antigens, respectively, and tested with the IMOA. Immunization with crude oncosphere antigens (OAs) induced 100% protection, while metacestode antigens provided only partial protection. Immunoblots showed that pigs with complete immune protection to oncosphere intramuscular challenge had antibodies to two OAs at 31.3 and 22.5 kDa, respectively. Antibody to these two antigens was absent in pigs immunized with metacestodes or in uninfected control pigs. This study demonstrated the presence of two antigens that are unique to the oncosphere. Although, antibody to these two antigens is consistently present in pigs that are protected from an oncosphere intramuscular challenge their role in preventing infection by T. solium larval cysts is still hypothetical.     

Kinetics of circulating antigens in pigs experimentally infected with Taenia solium eggs

Three groups of four piglets were experimentally infected with different doses (10(3), 10(4) and 10(5)) of Taenia solium eggs whereas a fourth group of two pigs received gravid proglottids. At autopsy 6 months post infection, the two latter pigs were heavily infected with more than 3000 living cysts per kg of muscle. Ten of the 12 other pigs harboured light infections, i.e. between 2 and 107 cysticerci, 42.4% of which were degenerated. The two remaining pigs had no detectable cysts at post mortem examination. Circulating antigens (CA) were detected in the sera of all pigs harbouring living cysticerci using a monoclonal antibody based ELISA. CA were first detected between 2 and 6 weeks post infection and remained present generally throughout the entire observation period even in pigs carrying only five to eight living cysts, although strong fluctuations of the level of CA were observed in some pigs. In animals without living cysts at post mortem CA were only detected for a short period and disappeared presumably when the cysticerci became degenerated. The minimum number of living cysts, which could be detected using this ELISA, was 1.     

猪带绦虫不同阶段及羊囊尾蚴蛋白质分析

利用浓度梯度SDS－聚丙烯酰胺凝胶电泳对猪囊尾蚴的囊液和虫体，猪带绦虫成虫的头颈节、成节、孕节，猪带绦虫的虫卵以及羊囊尾蚴的蛋白成分进行了对比分析。结果发现：囊液、虫体、头颈节、成虫、孕节、虫卵、羊囊尾蚴分别有35条（15．0－150．4kd）、44条（15．0－133．7kd)、22条（13．3－163．9kd)、19条（13．3－90．1kd)、19条（13．3－90．1kd)、30条（13．3－99．4）和38条（15．0－133．4kd)蛋白带；其中15．0kd和35．5kd两条共同抗原蛋白带，在猪带绦虫各阶段和羊囊尾蚴中含量最大的蛋白带同时也是阶段性的共同抗原蛋白带。这些结果为下一步的保护性抗原筛选奠定了基础。     

携带猪囊虫表位的嵌合蛋白疫苗的构建及其免疫学研究

用PCR法将猪囊虫抗原表位n1、n2插入乙肝病毒核心蛋白（HBc）序列的第78～79位之间，将猪囊虫抗原表位n3接在149位之后，再将该序列克隆入pET28a载体中，构建了重组表达质粒pET28a—△c-3n，在大肠杆菌中表达出融合蛋白。命名为PCCE。将PCCE纯化后免疫小鼠，以ELISA检测小鼠的体液免疫应答，Western blot检测抗体的特异性．Dot ELISA验证所选表位的保护性。另用绦虫卵攻击免疫小鼠，以观察疫苗的保护作用。测序结果表明，重组质粒构建成功；SDS—PAGE显示，融合蛋白表达正确，电镜证实有蛋白颗粒形成。对免疫小鼠应用ELISA检测到高滴度抗体；Western blot结果表明。免疫鼠体内诱导出了3种特异性抗体；Dot ELISA结果表明，所逸表位对宿主可能具有保护性。绦虫卵攻击免疫小鼠试验表明，疫苗PCCE的相对保护率为89％。结论：成功地表达和纯化了以HBc为载体的携带3个猪囊虫表位的融合蛋白（PCCE），以PCCE作为疫苗免疫小鼠，能诱导较强的特异性体液免疫反应，免疫小鼠对绦虫卵攻击具有较好的免疫保护作用。     

Cross-reactivity of anti-Taenia crassiceps cysticerci immune antibodies with Taenia solium antigens

A comparative study of antibody production was carried out using BALB/c mice immunized with 20 or 50microg vesicular fluid (VF)-Tcra (Taenia crassiceps) antigens, and gel of <30kD or eluate from <30kD peptides. Good IgM, IgA and IgG levels were detected by ELISA-Tcra and the antibodies presented reactivity with the <20kD peptides when tested by immunoblotting-Tcra. The antibodies from animals immunized with 20 and 50microg presented high anti-Tso cross-reactivity in ELISA (IgG>IgM and IgA). All groups presented IgG antibodies identifying the 12kD Tso-peptide.     

Performance of the ELISA test for swine cysticercosis using antigens of Taenia solium and Taenia crassiceps cysticerci

Studies were conducted to evaluate antigens of Taenia solium (Tso) and Taenia crassiceps (Tcra) cysticerci in the ELISA test for the diagnosis of swine cysticercosis. The samples analyzed were cysticercosis positive and negative control sera and heterologous sera. Four antigens were assayed: vesicular fluid (VF) and crude (T) Tcra and scolex (S) and crude (T) Tso. All antigens showed good performance, but VF-Tcra was the best followed by T-Tcra. Sensitivity rates of ELISA were respectively, in 2nd and 3rd standard deviation cut-offs, 96.0 and 80.0% for the VF antigen and specificity of 97.5 and 100.0%. Cross-reactivity was verified only for hidatidosis and ascaridiosis. Due to the high performance observed, the ELISA test using Tcra antigens should be recommended for the diagnosis of swine cysticercosis.     

猪囊尾蚴dUTPase的同源建模及分析

通过SWISS-MODEL服务器对猪囊尾蚴dUTPase（Cysticercus cellulosae dUTPase，CYdUT-Pase）进行同源建模，用SPDBV和DSViewer对模型进行修饰，模型经WHATIF服务器进行质量评估。对已知dUTPase的晶体结构分析后，推测CYdUTPase属于同源三聚体，通过SymmDock服务器进行CYdUTPase三聚物的分子对接，并与人dUTPase3D结构叠合。结果表明，建立的CYdUTPase3D模型是成功的，具有良好的真实性，CYdUTPase三级结构的预测为此酶的结构功能研究和新型抗囊虫药物的开发奠定了基础。     

Effect of Cysticercus cellulosae on neutrophil function and death

Neutrophils, eosinophils and macrophages interact with invading parasites and naive hosts. The initial reaction of leukocytes is the generation of reactive oxygen species (ROS). The cytotoxic effects of extracts derived from intact Cysticercus cellulosae and from the scolex or membrane fractions on neutrophils were examined. DNA fragmentation of neutrophils was observed when cells were incubated with an extract from the intact metacestode; however, the addition of antioxidant enzymes to the incubation medium had a protective effect. The scolex and membrane extracts did not affect DNA fragmentation of neutrophils. Hydrogen peroxide production of neutrophils incubated with metacestode fractions from C. cellulosae increased by 190% (total extract), 120% (scolex) or 44% (membrane). An increase in antioxidant catalase activity (28%) concomitant with the increased production of ROS was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. ROS production by neutrophils in the presence of the intact cysticerci extract did not alter phagocytosis. In contrast, the scolex and membrane fractions increased the phagocytic capacity of neutrophils by 44 and 28%, respectively. The results showed that the extract from intact C. cellulosae was toxic for neutrophils via ROS production, leading to DNA fragmentation and inhibition of phagocytic capacity, but neutrophils are able to protect themselves against oxidative stress by via catalase activity.     

Cross-reactions of bovine herpesvirus 1 antigens with those of other cattle herpesviruses

Bovine embryonic kidney cells were infected with bovine herpesviruses (BHV1, 2, or 3), suid herpesvirus 1 (SHV1), or were sham-inoculated. When cytopathic effect was apparent, the cells were solubilized using Triton X-100 detergent. Resulting antigen preparations were tested by 2-dimensional immunoelectrophoresis using bovine fetal serum and antisera directed against BHV1, BHV2, BHV3, SHV1 or a restricted spectrum of BHV1 antigens. Interaction of BHV1 antiserum with BHV1 antigen preparations resulted in 11 precipitation arcs. The same antiserum produced 3 arcs with BHV2, none with BHV3, and 5 with SHV1. The interaction of BHV1 antigen preparations with BHV2, BHV3, or SHV1 antisera failed to produce demonstrable arcs. However, when heterologous antigen or antibody preparations were added to BHV1 homologous 2-dimensional immunoelectrophoresis tests, all 11 BVH1 arcs were modified by BHV1, 2 by BHV2, 4 by BHV3 and 4 by SHV1 preparations. Two antigens were common to the 4 herpesviruses. Antigen preparations were tested for their ability to inhibit virus neutralization by BHV1 antiserum; only the BHV1 preparation was active. Sera were tested for BHV1 neutralizing activity; only BHV1 antiserum and a serum specific for a restricted spectrum of BHV1 antigens were active. A glycoprotein antigen associated with BHV1 neutralization was identified which may be important in the protection of animals against disease.     

Concurrent infection with Trichinella spiralis and other helminths in pigs

The aim of this study was to investigate possible influence of different helmintosis in the development of Trichinella spiralis in experimental infected pigs. Forty-two Iberian pigs were allocated to six groups. Three groups were single inoculated with Ascaris suum, Metastrongylus apri or T. spiralis, respectively. Two groups were co-infected with T. spiralis and A. suum or T. spiralis and M. apri, respectively, while the last group included uninfected control pigs. Clinical signs were only observed in pigs with single or concurrent M. apri infections, with more severe respiratory symptoms in pigs with mixed M. apri infection. The number of A. suum and M. apri lung larvae, intestinal larvae of A. suum and adult M. apri were reduced in pigs with mixed Trichinella infections compared to pigs with single infections. In contrast, the number of liver white spots was higher in pigs with mixed infections. While T. spiralis muscular larval burdens were increased in pigs concomitantly infected with M. apri, they were reduced in pigs concomitantly infected with A. suum, compared to pigs receiving single infections with either of these helminths. Pigs with single or mixed A. suum infections showed higher eosinophil levels compared to the remaining groups. IgGt, IgG1, IgG2 and IgM against T. spiralis antigen could not be detected in pigs with single Ascaris or Metastrongylus infections, indicating that no cross-antibodies were produced. IgGt, IgG1 and IgM antibodies were detected earlier and generally at higher levels in mixed T. spiralis infections compared to single T. spiralis infections. The results suggest that T. spiralis had a low synergistic interaction with M. apri in concomitantly infected pigs, and an antagonistic interaction in concurrent infection with A. suum.     

猪囊尾蚴T24基因的克隆与表达

采用RT—PCR方法扩增猪囊尾蚴T24免疫原基因，将扩增产物与pGEM—Teasy载体连接，重组质粒经PCR、酶切鉴定后进行测序；构建T24基因的pGEX-4T-1原核表达载体，经IPTG诱导表达后，进行SDS-PAGE、Western—blotting；用所表达的蛋白免疫小鼠，经ELISA检测血清抗体，验证其免疫原性。结果显示，所克隆的T24基因片段长716bp，含有1个678bp的开放阅读框，其编码226个氨基酸，与已报道的猪囊虫T24基因核苷酸序列同源性为100％；表达的融合蛋白大小为40ku，并能被猪囊虫阳性血清识别；免疫小鼠在免疫1周后即可检测到血清抗体，第30d达到较高水平，表明该融合蛋白具有较好的免疫原性。     

猪囊尾蚴及其培养细胞的超微结构观察

应用透射电镜和扫描电镜观察了采自猪体的猪囊尾蚴及其体外培养细胞的形态结构特征。猪囊尾蚴的基本结构包括皮层、间质层和实质区。皮层密布微绒毛;内部是实质区,含光面内质网,大量囊泡和圆形空泡。线粒体多分布在基质区的近基膜处。皮层以基膜与间质分开。间质层为网状纤维样,并形成分支深入实质区,起支架作用。肌组织位于间质层中,间质层向内为实质区,有实质细胞、皮层细胞、成肌细胞及成石灰颗粒细胞。排泄系统中除不同孔径的排泄管外,还有三五成群的焰细胞与毛细管相通。体外培养的细胞结构与体内自然生长的无明显差异,猪囊尾蚴培养细胞的内部细胞器较其体细胞清晰,与体细胞相比培养细胞有些细胞内部细胞器减少。猪囊尾蚴细胞的外部形态主要以圆形、椭圆形为主,多数细胞表面有凹槽、脊及绒毛。     

Changes in serum enzyme activities in pigs naturally infected with the metacestodes of Taenia solium

The serum enzymes of pigs naturally infected with the metacestodes of Taenia solium and of uninfected pigs were assayed. Aspartate aminotransferase, alanine aminotransferase, ornithine carbamyl transferase, sorbitol dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase, alkaline phosphatase and ceruloplasmin activities were significantly increased in the serum of the infected pigs.     

猪囊尾蚴特异性单克隆抗体的制备及其识别抗原成分的鉴定

通过SDS-PAGE方法对猪囊尾蚴囊液抗原成分进行了分离鉴定,并分别分离纯化了其中的16 000和10 000特异性蛋白质成分;将16 000和10 000纯化蛋白质成分分别做成佛氏佐剂苗,分组免疫BALB/c小鼠,超免后,无菌取脾脏制备脾细胞,分别与NS0骨髓瘤细胞进行融合,经阳性筛选和特异性鉴定获得2株分泌猪囊尾蚴特异性单抗的杂交瘤细胞,命名为TSCF-1611H12B8和TSCF-1012G5B5。免疫学鉴定结果表明,单抗TSCF-1611H12B8和TSCF-1012G5B5与猪细颈囊尾蚴、旋毛虫、住肉孢子虫和蛔虫抗原间不存在交叉反应;单抗TSCF-1611H12B8识别囊液中16 000和10 000蛋白质抗原条带,而单抗TSCF-1012G5B5识别囊液中的10 000蛋白质条带。     

Castration and pregnancy of rural pigs significantly increase the prevalence of naturally acquired Taenia solium cysticercosis

Cuentepec is a rural village of central Mexico, where 1300 pigs were bred at the time of the study in conditions that favor Taenia solium transmission. The tongues of 1087 (84%) of these pigs were visually examined and 33% were found to be cysticercotic. Castration of male pigs increased prevalence from 23 to 50% (P < 0.001) and pregnancy in sows also increased their prevalence from 28 to 59% (P < 0.001). Thus, endocrinological conditions characterized by low levels of androgens or high levels of female hormones probably influence the susceptibility of pigs to T. solium cysticercosis as observed in mice infected with Taenia crassiceps. Delaying castration of male pigs and confinement of sows during pregnancy might significantly decrease the prevalence of pig-cysticercosis and help curb transmission without much cost or difficulty.     

Efficacy of the immunoblot assay for cysticercosis in pigs and modulated expression of distinct IgM/IgG activities to Taenia solium antigens in experimental infections

A recently invented immunoblot assay for human cysticercosis was evaluated for efficacy in pigs. The test population consists of 45 pigs with parasitologically confirmed cysticercosis, 47 with heterologous infections, 45 SPF or concrete raised control animals. With this group of 137 animals the test performance was 100% sensitive and 100% specific. The antigen-specific responses of immunoglobulin A (IgA), IgG and IgM in four pigs infected with Taenia solium eggs derived from a human were quantified by immunoblot. Antigen-specific activities were observed as early as 1 week postinfection. The first antigen-specific isotypic response was IgM antibodies directed against a glycoprotein at 97 KD (GP97). This activity generally disappeared between the sixth and ninth week postinfection. Between Weeks 5 and 8, IgG activity rose as IgM activity fell. The IgG activity, however, was directed mostly towards GP50 and GP42 antigens. If the same response occurs in people with cysticercosis, identifying specific isotype activity may help to distinguish new infection from old.