寄生虫抗肿瘤研究进展

虽然旋毛虫抗肿瘤的分子机制仍不清楚,但旋毛虫具有抗肿瘤的效应却已被许多实验所证实。小鼠感染旋毛虫后对肿瘤细胞在体内的增殖或对移植进体内的肿瘤生长的抑制作用,其可能的机理是旋毛虫感染机体后激发宿主免疫网络系统促使机体产生多种免疫活性细胞因子,发挥特异性和非特异性抗肿瘤免疫功能;或者是由于寄生虫本身就具有能够分泌排泄一些抗肿瘤活性物质而发挥抗肿瘤效应;或者是由于寄生虫感染诱导肿瘤的基因表达发生变化而产生了抗肿瘤的效应。     

Larval viability and serological response in horses with long-term Trichinella spiralis infection

The horse is considered an aberrant host for the nematode parasite Trichinella spiralis, and many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood. It has been reported that experimentally-infected horses produce a transient serological response to infection and that muscle larvae are cleared more rapidly than in parasite-adapted hosts such as the pig and humans. However, limited numbers of animals have been studied, and both the longevity of larvae in horse musculature and the immune response to Trichinella larvae remain unclear. In this study, we infected 35 horses with 1000, 5000, or 10,000 T. spiralis muscle larvae and followed the course of infection for 1 year, assessing larval burdens in selected muscles, the condition and infectivity of recovered larvae, and the serological response of infected horses. The results demonstrated that T. spiralis establishes infection in horses in a dose dependent manner. Anti-Trichinella IgG antibodies peaked between weeks 6-10 post-inoculation. Viable, infective larvae persisted in horse musculature for the duration of the study (12 months), and exhibited no apparent reduction in muscle burdens over this period. Encapsulated larvae showed no obvious signs of degeneration in histological sections. Larval capsules were surrounded by infiltrates consisting of mature plasma cells and eosinophils. Macrophages were notably absent. Given the lack of a detectable serological response by 26 weeks p.i. and the persistence of infective muscle larvae for at least 1 year, parasite recovery methods are currently the only suitable detection assays for both meat inspection and epidemiological studies of Trichinella infection in the horse.     

Differential detection of Trichinella papuae, T. spiralis and T. pseudospiralis by real-time fluorescence resonance energy transfer PCR and melting curve analysis

Trichinellosis caused by nematodes of Trichinella spp. is a zoonotic foodborne disease. Three Trichinella species of the parasite including Trichinella spiralis, Trichinella papuae and Trichinella pseudospiralis, have been etiologic agents of human trichinellosis in Thailand. Definite diagnosis of this helminthiasis is based on a finding of the Trichinella larva (e) in a muscle biopsy. The parasite species or genotype can be determined using molecular methods, e.g., polymerase chain reaction (PCR). This study has utilized real-time fluorescence resonance energy transfer PCR (real-time FRET PCR) and a melting curve analysis for the differential diagnosis of trichinellosis. Three common Trichinella species in Thailand were studied using one set of primers and fluorophore-labeled hybridization probes specific for the small subunit of the mitochondrial ribosomal RNA gene. Using fewer than 35 cycles as the cut-off for positivity and using different melting temperatures (T(m)), this assay detected T. spiralis, T. papuae and T. pseudospiralis in muscle tissue and found the mean T(m) ± SD values to be 51.79 ± 0.06, 66.09 ± 0.46 and 51.46 ± 0.09, respectively. The analytical sensitivity of the technique enabled the detection of a single Trichinella larva of each species, and the detection limit for the target DNA sequence was 16 copies of positive control plasmid. A test of the technique's analytical specificity showed no fluorescence signal for a panel of 19 non-Trichinella parasites or for human and mouse genomic DNA. Due to the sensitivity and specificity of the detection of these Trichinella species, as well as the fast and high-throughput nature of these tools, this method has application potential in differentiating non-encapsulated larvae of T. papuae from T. spiralis and T. pseudospiralis in tissues of infected humans and animals.     

Historical perspectives and current global challenges of Trichinella and trichinellosis

Trichinella spiralis and related species of Trichinella have had a long history of causing human disease, and as a foodborne pathogen have had a major impact on international commerce of pork and other meat animal species which are known to transmit the parasite. Our knowledge of Trichinella has increased substantially over the past few years particularly in the areas of phylogeny, host diversity, epidemiology and control. In this paper, we provide a brief overview of our understanding of Trichinella from its discovery to present time. Past and current challenges of the control of Trichinella and trichinellosis are summarized. As editors of this special issue of Veterinary Parasitology, we introduce a series of invited review articles prepared by experts from around the world, summarizing recent knowledge in Trichinella and trichinellosis.     

Trichinella spiralis infection induces angiogenic factor thymosin β4 expression

Trichinella spiralis (T. spiralis) has been reported to up-regulate the expression of the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation. In order to analyze the induction of angiogenesis by T. spiralis, the expression patterns of angiogenesis-related proteins were investigated by immunohistochemical analysis. VEGF expression was induced in the infected muscles at an early stage of infection (10 days after infection) and diminished after 3 weeks. Thymosin β4, a major factor which induces VEGF, showed increased expression in muscle fibers 10 days after infection, and the expression remained high in the nurse cells for 6 weeks, when the formation of the nurse cell complex was completed. The hypoxia inducible factor (HIF)-1α showed a diffuse expression pattern around the infected muscle fibers and was strongly expressed in inflammatory cells but was not related to the hypoxic condition caused by nurse cell formation. Localization of the hypoxic regions by the hypoxia marker, pimonidazole showed T. spiralis infection does not induce a hypoxic condition in nurse cells. These results suggest that the expression of VEGF and thymosin β4 induce angiogenesis and the expression of thymosin β4 remained elevated to potentially regulate nurse cell formation and maintenance.     

Peripheral blood mononuclear cell subsets during Trichinella spiralis infection in pigs

The immune response of 'Yugoslav meat breed' pigs inoculated with low doses of Trichinella spiralis muscle larvae was followed over two to nine weeks of primary infection, by analysing changes in peripheral blood mononuclear cell subsets, the development of a humoral antibody response and muscle larvae burden. During the course of the infection, infected animals showed a persistent elevation of both CD4+ and CD8+ T cell subsets from days 15 to 60 after the parasite exposure. During this time, the number of peripheral blood mononuclear cells expressing major histocompatibility complex class II antigens was also increased, while no significant differences were found in the number of circulating monocytes/macrophages and B cells over time. Humoral antibody responses to muscle larvae excretory-secretory products were evident as early as 41 days after infection, while the muscle larvae were recovered as early as 27 days after infection. The increased levels of CD4+ and CD8+ T cell subsets, as well as cells expressing major histocompatibility complex class II antigens in pigs exposed to T spiralis, may be indicative of some considerable alterations in cell subsets that are involved in the regulation of the swine immune response to this parasite.     

旋毛虫G10-7基因在大肠杆菌中的高效表达

旋毛虫新生幼虫期特异性 G10 - 7基因从 PBK- CMV- G10 - 7重组质粒中亚克隆到 p ET- 2 8b( )原核表达载体上 ,成功地构建了重组表达质粒 p ET- 2 8b- G10 - 7。将其转化到大肠杆菌 DE3感受态细胞中 ,经 IPTG诱导表达 ,其细菌裂解物经 SDS- PAGE电泳可检测到相对分子质量约为 34 0 0 0的目的蛋白带 ,且随诱导时间的延长蛋白表达量逐渐增加 ,诱导 4h达到峰值 ,薄层扫描结果显示 ,目的蛋白占菌体总蛋白的 6 1.7%。Western- blotting检测显示 ,此蛋白可被感染旋毛虫家猪阳性血清所识别 ,表明该蛋白具有良好的抗原性。     

抗旋毛虫单克隆抗体细胞林的建立及其特性鉴定

为筛选旋毛虫保护性抗原,建立了4株分泌抗旋毛虫McAb的细胞株。经连续培养30余代,仍稳定地分泌抗体。其中,2G3和2C10的靶抗原为旋毛虫幼虫表膜。免疫球蛋白 型及亚 鉴定表明,2G3和2C10为IgG_1,1D5为IgG_3,1C9为IgM。除2C10和IC9对伊氏锥虫可溶性抗原呈现阳性反应外,未见对猪圆线虫、猪囊尾蚴、伊氏锥虫表膜抗原和正常猪肉反应。经ELISA测定,2G3、2C10、1D5、1C9均可与施毛虫幼虫可溶性抗原作用,其腹水效价分别为1:50000、1:10000、1:1000、1:800。各McAb均与旋毛虫幼虫排泄分泌物抗原(ES抗原)作用,经ELISA测定表明,2G3腹水效价达1:320000。     

Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife

Only a few studies have compared the muscle distribution of the different Trichinella genotypes. In this study, data were obtained from a series of experimental infections in pigs, wild boars, foxes and horses, with the aim of evaluating the predilection sites of nine well-defined genotypes of Trichinella. Necropsy was performed at 5, 10, 20 and 40 weeks post inoculation. From all host species, corresponding muscles/muscle groups were examined by artificial digestion. In foxes where all Trichinella species established in high numbers, the encapsulating species were found primarily in the tongue, extremities and diaphragm, whereas the non-encapsulating species were found primarily in the diaphragm. In pigs and wild boars, only Trichinella spiralis, Trichinella pseudospiralis and Trichinella nelsoni showed extended persistency of muscle larvae (ML), but for all genotypes the tongue and the diaphragm were found to be predilection sites. This tendency was most obvious in light infections. In the horses, T. spiralis, Trichinella britovi, and T. pseudospiralis all established at high levels with predilection sites in the tongue, the masseter and the diaphragm. For all host species, high ML burdens appeared to be more evenly distributed with less obvious predilection than in light infections; predilection site muscles harbored a relatively higher percent of the larval burden in light infections than in heavy infections. This probably reflects increasing occupation of available muscle fibers as larger numbers of worms accumulate. Predilection sites appear to be influenced primarily by host species and secondarily by the age and level of infection.     

Factors affecting the flow among domestic, synanthropic and sylvatic cycles of Trichinella

Nematodes of the genus Trichinella are maintained in nature by sylvatic or domestic cycles. The sylvatic cycle is widespread on all continents, from frigid to torrid zones, and it is maintained by cannibalism and scavenging behavior of carnivores. Trichinella is primarily a parasite of carnivorous mammals, although one non-encapsulated species, Trichinella pseudospiralis, has also been detected in birds. The anaerobic metabolism of larvae in nurse cells allows their survival in extremely decayed meat. Encapsulated larvae in the decomposing carcass function similarly to the species-dispersing population of eggs or larvae of other nematodes, suggesting that the natural cycle of Trichinella includes a free-living stage when the parasite is no longer protected by the homeothermy of the host. Consequently, environmental temperature and humidity play an important role in the transmission of Trichinella among wildlife. Of the 10 recognized genotypes of Trichinella, only Trichinella spiralis is transmitted and maintained in a domestic cycle, although it can be present also in wildlife. All other genotypes (Trichinella nativa, Trichinella britovi, T. pseudospiralis, Trichinella murrelli, Trichinella nelsoni and Trichinella papuae, Trichinella T6, T8, and T9) are transmitted and maintained only in a sylvatic cycle. This generalization does not preclude sylvatic species of Trichinella from invading the domestic habitat, and T. spiralis may return to this habitat when humans fail in the management of wildlife and domestic animals. However, the presence of sylvatic genotypes of Trichinella in the domestic habitat represents a "dead-end" for the sylvatic cycle. Synanthropic animals (rats, foxes, mustelids, cats, dogs, etc.) contribute to the flow of sylvatic Trichinella genotypes from wildlife to domestic animals and of T. spiralis from domestic to sylvatic animals. Furthermore, human behavior not only influences the transmission patterns of Trichinella genotypes in the domestic habitat, but also it can contribute to the transmission and spread of this infection among wildlife, for example by improper hunting practices.     

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.     

Specific serum antibody responses following a Toxoplasma gondii and Trichinella spiralis co-infection in swine

The aim of this study was to examine the dynamics of parasite specific antibody development in Trichinella spiralis and Toxoplasma gondii co-infections in pigs and to compare these with antibody dynamics in T. spiralis and T. gondii single infections. In this experiment, fifty-four pigs were divided into five inoculated groups of ten animals, and one control group of four animals. Two groups were inoculated with a single dose of either T. gondii tissue cysts or T. spiralis muscle larvae, one group was inoculated simultaneously with both parasites and two groups were successively inoculated at an interval of four weeks. Specific IgG responses to the parasites were measured by ELISA. T. gondii burden was determined by MC-PCR carried out on heart muscle and T. spiralis burden by artificial digestion of diaphragm samples. Specific IgG responses to T. gondii and T. spiralis in single and simultaneously inoculated animals showed a respective T. gondii and T. spiralis inoculation effect but no significant interaction of these parasites to the development of specific antibodies with the serum dilutions used. Moreover, our data showed that the specific IgG response levels in groups of animals successively or simultaneously co-infected were independent of a respective previous or simultaneous infection with the other parasite. Additionally, no differences in parasite burden were found within groups inoculated with T. gondii and within groups inoculated with T. spiralis. Conclusively, for the infection doses tested in this experiment, the dynamics of specific antibody development does not differ between single and simultaneous or successive infection with T. gondii and T. spiralis. However, lower parasitic doses and other ratios of doses, like low-low, low-high and high-low of T. gondii and T. spiralis in co-infection, in combination with other time intervals between successive infections may have different outcomes and should therefore be studied in further detail.     

Host diversity and biological characteristics of the Trichinella genotypes and their effect on transmission

The host spectra and biological diversity of the Trichinella genotypes are reviewed. While all genotypes appear to reproduce equally well in carnivore hosts, their infectivity and persistence in omnivores and herbivores show remarkable differences. Most of the genotypes found in wildlife have low infectivity for pigs and some persist only for a few weeks; in herbivores this tendency is even more profound, but malnourished, environmentally stressed, or otherwise immuno-suppressed hosts are likely to be more susceptible to Trichinella genotypes that would otherwise cause no, or only low level infection in that particular host species. In the domestic habitat (e.g. domestic pig farms), Trichinella spiralis is found almost exclusively, but in the sylvatic habitat the other Trichinella genotypes have found individual ecological niches. Thus, when environmental stress is limited in the domestic habitat, the high reproductive capacity of T. spiralis has a selective advantage, but in nature, the tolerance of other (sylvatic) genotypes to high and low temperatures and decomposition of host tissue might be more important. Parasite distribution according to muscle appear to be independent of the genotype of Trichinella and predilection sites are primarily determined by host species and secondarily by the age and level of infection. The biological diversity of the Trichinella genotypes should definitely be considered when planning experimental studies, as the uniform high infectivity of all genotypes in carnivores probably make them more suited for comparative studies than rodents.     

不同发育时期旋毛虫脂肪酸组成的研究

为探讨旋毛虫生长发育过程中脂肪酸组分的变化及外界环境对其影响,本研究采用贝尔曼氏装置分别收集旋毛虫肌幼虫和成虫,同时,将少量骨骼肌和肠黏膜进行酯化处理,通过气相色谱质谱联用技术进行分析。结果显示:肌幼虫和成虫的脂肪酸组分涵盖了C12～C22的22种,主要为16、18和20碳脂肪酸,但旋毛虫肌幼虫与成虫的各脂肪酸相对含量有明显差异(p0.05)。分别对旋毛虫肌幼虫和成虫及其各自寄生部位骨骼肌和肠黏膜中的脂肪酸成份进行比较分析,结果显示:各脂肪酸组成相似,但在相对含量上却有显著差异(p0.05)。表明不同发育时期旋毛虫的脂肪酸组成在种类和相对含量上均存在明显差异,这可能与其所处的不同宿主环境和不同发育时期虫体的特殊生理结构有关。     

Trichinella: differing effects of antigens from encapsulated and non-encapsulated species on in vitro nitric oxide production

Trichinellosis is a cosmopolitan zoonotic disease affecting a wide variety of animals, including man. Non-encapsulated and encapsulated species diverge with respect to their developmental strategies. Little is known at the molecular level about parasite-derived mediators responsible for host muscle cell transformation occurring during trichinellosis. In this context, host-parasite relationships in Trichinella-infected animals could be related to different host-immune and cell mediators, e.g. nitric oxide (NO). Here, we investigate the stimulatory/inhibitory role of L1 antigens from four encapsulated (T. spiralis, T. britovi, T. nelsoni and T. nativa) and one non-encapsulated (T. pseudospiralis) Trichinella species on NO production from rat macrophages in vitro. Our results demonstrate that encapsulated and non-encapsulated Trichinella species differ in their capacity to stimulate the secretion of NO from host macrophages. Biological significance of these differences should be further assessed in the available experimental models.     

Predilection muscles and physical condition of raccoon dogs (Nyctereutes procyonoides) experimentally infected with Trichinella spiralis and Trichinella nativa

The predilection muscles of Trichinella spiralis and T. nativa were studied in 2 experimental groups of 6 raccoon dogs (Nyctereutes procyonoides), the third group serving as a control for clinical signs. The infection dose for both parasites was 1 larva/g body weight. After 12 weeks, the animals were euthanized and 13 sampling sites were analysed by the digestion method. Larvae were found in all sampled skeleton muscles of the infected animals, but not in the specimens from the heart or intestinal musculature. Both parasite species reproduced equally well in the raccoon dog. The median density of infection in positive tissues was 353 larvae per gram (lpg) with T. spiralis and 343 lpg with T. nativa. All the infected animals had the highest larvae numbers in the carpal flexors (M. flexor carpi ulnaris). Also tongue and eye muscles had high infection levels. There were no significant differences in the predilection sites between these 2 parasite species. Trichinellosis increased the relative amount of fat, but not the body weight in the captive raccoon dogs. Thus, Trichinella as a muscle parasite might have catabolic effect on these animals.     

Influence of adjuvant formulation on the induced protection of mice immunized with total soluble antigen of Trichinella spiralis

Vaccination of pigs against the helminth nematode Trichinella could be a good alternative to prevent the risk of human infection. In order to develop an efficient and safe vaccine, the choice of the adjuvant is an important issue. In this study, two adjuvants were selected to prepare vaccines based on total soluble Trichinella spiralis muscle larvae (ML) antigen: Montanide ISA 70 water in oil emulsion and Montanide IMS nanoparticles. Aluminium hydroxide was used as a reference adjuvant. The immune response was checked by ELISA of parasite antigen specific IgG1 and IgE. Finally, protection induced in vaccinated mice was measured after a T. spiralis challenge by counting ML burdens. The results clearly showed an impact of adjuvants on the specific IgG1 and IgE antibody responses against T. spiralis. Differences were observed between the rates of protection induced according to the type of formulation, although the three adjuvants tested were able to enhance the humoral immune response. This work demonstrated the need to use an adjuvant to obtain a specific IgG1 and IgE responses directed against the total soluble extract of T. spiralis.     

旋毛虫调节宿主免疫反应的机制

旋毛虫释放的排泄分泌产物（excretory-secretory,ES）对于宿主免疫系统产生了一系列复杂的作用,发挥激活、活化以及抑制等功能影响机体的免疫反应,使其能够逃避宿主主动免疫,从而成功完成寄生过程。在此过程中,旋毛虫诱导的免疫调节机制还可以减轻免疫性疾病、过敏和癌症等疾病的症状。     

旋毛虫肌幼虫p53cDNA的克隆及鉴定

根据GenBank中旋毛虫53000抗原基因的序列设计引物，用PCR技术直接从旋毛虫肌幼虫cDNA文库中扩增靶基因p53cDNA，将其克隆到pMD-18T载体，转化至大肠埃希氏菌NovaBlue后测序分析，结果表明，克隆到1176bp的p53cDNA，共编码391个氨基酸。序列分析表明，p53cDNA序列与GenBank中的旋毛虫p53基因序列相比共有12个核苷酸发生改变，二者的同源性为98％，所编码蛋白质氨基酸序列的同源性为98％。将其克隆到原核表达载体pET28a并转化至表达菌BL21star（DE3），经IPTG诱导表达后获得约48000的重组蛋白。Western blotting检测证实，p53重组蛋白可以被旋毛虫感染猪血清识别，具有很好的抗原性。