猪囊尾蚴DNA疫苗pVAX1/TSOL18的安全性评估

目的探讨pVAX1/TSOL18重组表达质粒作为DNA疫苗在小鼠体内的组织分布和生物安全性。方法将pVAX1/TSOL18重组质粒经肌肉注射免疫昆明系小鼠,免疫后在不同时间剖杀小鼠,分别采集心、肝、脾等组织样品,抽提gDNA,利用PCR技术分析pVAX1/TSOL18在组织内的分布及残留时间。同时,以PCR技术检测免疫动物粪便,分析pVAX1/TSOL18重组表达质粒在外界环境中的释放情况。结果免疫pVAX1/TSOL18重组表达质粒后1 d和5 d,在小鼠组织gDNA均扩增出目的片段,不同个体小鼠,扩增出目的片段的组织也不完全相同,但在血液中均扩增出了目的片段,d5较d1扩增出的目的片段明显变暗。免疫后15 d,仅在一只小鼠的血液中检测到TSOL18基因;免疫后30 d在所有动物组织中均未检测到重组表达质粒。同时,免疫1 d、5 d小鼠的粪便中均未检测到TSOL18基因。结论pVAX1/TSOL18重组表达质粒作为疫苗对动物和环境是安全的。     

一种鸡球虫DNA疫苗的生物安全性评价

将一种鸡球虫DNA疫苗以剂量30μg/羽,经肌肉注射免疫1日龄雏鸡1次;以剂量30μg/羽,于7日龄、14日龄、21日龄分别经肌肉注射免疫雏鸡3次;以剂量80μg/羽,将不同时间制备地3批疫苗,分别经肌肉注射于14日龄雏鸡。分5次于免疫后每个月采取血液、心、肝、脾、肾和注射部位组织,用PCR技术检测质粒DNA在各个组织的残留情况;同时制备石蜡切片,观察上述组织有无病理变化。结果表明,疫苗免疫后的第1个月在被检组织中都能检测到质粒DNA的存在,随着时间的推移,所检测到质粒的浓度逐渐减少,免疫4个月之后,所有组织都检测不到质粒DNA存在,可推断质粒DNA并未整合入动物细胞基因组;同时组织也未发现病理学变化。认为该DNA疫苗对靶动物是安全的。     

猪干扰素-γ哺乳动物细胞重组表达质粒作为疫苗佐剂在小鼠体内的组织分布及环境安全性研究

重组表达质粒作为免疫佐剂从实验室走向临床必须解决其对机体和生物环境释放的安全性问题。本研究以pcDNA3．1／IFN-γ重组表达质粒作为免疫佐剂进行试验,探讨了其在小鼠体内组织的分布和生物安全性。将pcDNA3．1／IFN-γ重组表达质粒经肌肉途径免疫小鼠,免疫后不同时间迫杀,并取各种组织抽提基因组DNA：一是利用PCR技术检测其在组织内的分布及与细胞基因组发生整合的可能性;二是以PCR技术检测免疫动物现场环境样品,监测pcDNA3．1／IFN-γ重组表达质粒中的猪IFN-γ基因、CMV启动子基因和抗性基因是否转移和扩散到环境细菌中。结果表明,免疫24h后在小鼠的各组织中仅注射部位肌肉存在重组表达质粒,免疫5d后仅有1只小鼠注射部位肌肉和血液中存在重组表达质粒,免疫15d后所有动物的各组织中无重组表达质粒存在;同时未发现pcDNA3．1／IFN-γ重组表达质粒整合到宿主细胞基因组和转化到环境其他细菌中。因此,认为pcDNA3．1／IFN-γ重组表达质粒对动物和环境是安全的。     

猪白介素-4重组表达质粒在小鼠体内的组织分布及环境释放安全性

为了探讨pcDNA3.1/IL-4重组表达质粒作为疫苗佐剂进入临床应用的生物安全性,本试验就pcDNA3.1/IL-4重组表达质粒在小鼠体内的组织分布和环境释放安全性进行了研究.将pcDNA3.1/IL-4重组表达质粒经肌肉途径免疫BALB/c小鼠,免疫后不同时间剖杀动物并摘取各种组织,抽提基因组DNA.利用PCR技术分析其在组织内的分布及与细胞基因组发生整合的可能性,同时检测了免疫动物现场环境,以监测pcDNA3.1/IL-4重组表达质粒上的猪IL-4基因、CMV启动子基因和抗性基因是否在环境细菌中发生转移和扩散.结果表明,免疫后1 d在3/3小鼠各组织中仅注射部位肌肉存在重组表达质粒,免疫后5 d仅有1/3小鼠于注射部位肌肉中存在重组表达质粒,免疫后15 d所有小鼠的各组织中无重组表达质粒存在;未发现pcDNA3.1/IL-4重组表达质粒整合到宿主细胞基因组和转化到环境其他细菌中.因此,认为pcDNA3.1/IL-4重组表达质粒对动物和环境是安全的.     

PCR test for detecting Taenia solium cysticercosis in pig carcasses

Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.     

Taenia solium cysticercosis: immunity in pigs induced by primary infection

The present study demonstrates that pigs experimentally infected with Taenia solium eggs develop resistance to reinfection that lasts at least five months. Thirteen 2-month-old piglets were infected with eggs of Taenia solium. After 5 months, two pigs were euthanized and five were challenged with eggs from a second tapeworm. Nine months after the first infection, six pigs were challenged with a third tapeworm. All 11 challenged pigs were euthanized 2 months after reinfection. In order to confirm the infectivity of the eggs, several piglets were inoculated with each taenia. Two of the five pigs reinfected after 5 months did not develop metacestodes, two showed few caseous non-infective forms and in the fifth pig, 14% of the metacestodes were vesicular and 86% colloidal and caseous. In the six animals challenged 9 months after the first infection, three were heavily infected with vesicular metacestodes and the other three showed only colloid and caseous forms in muscles. All parasites found in brains were vesicular. We conclude that immunity due to primary infection lasts at least 5 months. At 2 months of infection antigens of 24 and 39-42 kDa were the most frequently recognised. In those pigs with only a few caseous cysts in muscles and/or vesicular ones in brains no antibodies were detected.     

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.     

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.     

Lymphocyte apoptosis in the inflammatory reaction around Taenia solium metacestodes in porcine cysticercosis

In the current research, we report apoptosis of lymphocytes in the inflammatory reaction around metacestodes in muscle tissue from cysticercotic pigs. Two events, high metacestode viability (100%) and high cysteine protease activity were found to be closely related to a high phosphatydilserine expression by inflammatory lymphocytes (56%). Testing the RPMI medium used for washing away inflammatory cells from metacestodes with 100% viability, with the fluorescent substrate Z-Phe-Ala-AFC for measuring cysteine protease activity, significant fluorescent values were found. In contrast, tests performed with RPMI medium used for washing away inflammatory cells from metacestodes with 90% viability or less, showed low fluorescence values. Flow cytometry analyses of inflammatory cells obtained from four naturally cysticercotic pigs, and stained with Annexin-V/PI, showed lymphocytes expressing phosphatidylserine with values of 0, 6, 41 and 56% on their outer surfaces. Electron microscopy studies of inflammatory cells from metacestodes with 100% viability, showed lymphocytes with strangled and fragmented nuclei, and heterochromatin displaced to the nuclear periphery. In addition, DNA from these cells showed fragmentation in electrophoresis assays. Apoptosis of lymphocytes in the inflammatory reaction around Taenia solium metacestodes, might have been induced by the parasite cysteine protease, and may be involved in impairing cell-mediated immune responses in human and porcine cysticercosis.     

Impact of naturally acquired Taenia solium cysticercosis on the hormonal levels of free ranging boars

In chronically infected BALBc/AnN male mice, Taenia crassiceps cysticercosis induces changes in the host's sex steroids hormone that lead to their estrogenization and deandrogenization, with possible repercussions on their susceptibility to infections. Here reported are the serum steroid levels in free range cysticercotic male boars. Therefore, the possible effects of Taenia solium cysticerci over the pig steroid levels were evaluated. Herein are described the sex steroids and cortisol levels of non-cysticercotic (n=25) and cysticercotic (n=22) adult boars, as diagnosed by tongue inspection, all free-ranging in a typical village of an endemic rural area in Mexico. A significant reduction of testosterone (P=0.022) and a likely one of 17beta-estradiol (P=0.08) levels were found in the cysticercotic boars in comparison with those non-cysticercotic, whilst no significant differences in the cortisol and DHEA levels were detected. Serum levels of specific antibodies did not correlate with infection nor with the levels of any of the hormones measured. Results suggest that T. solium cysticercosis significantly affects the hormonal status of its porcine host independently of their antibody response.     

Experimental infection model for Taenia solium cysticercosis in swine. Cysticercosis Working Group in Peru

A novel method for infecting pigs with Taenia solium using an intramuscular innoculum of oncospheres was investigated in a series of five experiments in 18 animals. The model is simple to perform, requires a minimal number of oncospheres, permits multiple infections per animal, and decreases the variation inherent in oral infection models. This intramuscular oncosphere assay (IMOA) may provide a valuable tool to evaluate therapeutic agents or potential vaccines for cysticercosis.     

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.     

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96. 4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.