Temperature and metal ions-dependent activity of the family I inorganic pyrophosphatase from the swine roundworm Ascaris suum

Temperature dependence, heat stability and metal ions-dependent activity were examined on the Family I inorganic pyrophosphatase (PPase) recently identified from Ascaris suum. Recombinant A. suum PPase (rAsPPase) showed an optimal activity at 55 degrees C. The rAsPPase was heat stable at 40 degrees C in the absence of added Mg(2+) and at 50 degrees C in its presence. The enzyme required divalent metal ions for its activity. The preferences for the metal ions (5 mM concentration) were in the order: Mg(2+)> Co(2+)> Cu(2+)> Fe(2+)> Zn(2+)> Mn(2+). On the contrary, enzyme activity was inhibited by Ca(2+). These findings suggest that catalytic features of AsPPase are consistent with the Family I PPases reported from a wide range of organisms.     

Screen for anthelmintics, using larvae of Ascaris suum

A multiwell culture system was used to assay the effects of 12 known anthelmintic compounds on Ascaris suum larval development from 2nd-stage (L2; hatched from eggs) to early 3rd-stage (L3) and from in vivo-derived late L3 to early 4th-stage (L4). Larval survival, development, and motility were monitored for drug effects. Development of L2 to L3 was sensitive to thiabendazole, albendazole (ABZ), ABZ/sulfoxide, ABZ/sulfone (SO), mebendazole, L-tetramisole, D-tetramisole, piperazine, or closantel at a concentration of 0.01 microgram/ml; however, the effects of these drugs on larval development did not correlate well with known effects in vivo. The development of L3 to L4 was blocked by ABZ or mebendazole at 0.01 microgram/ml, by thiabendazole or ABZ/sulfoxide at 0.1 microgram/ml, and by ABZ/SO at 1.0 microgram/ml; however, except for ABZ/SO, most larvae were viable at these concentrations. In contrast, L-tetramisole or morantel appeared to inhibit development of L3 to L4 and to reduce survival at concentrations of greater than or equal to 1 microgram/ml; however, D-tetramisole was at least 10 times less effective. Haloxon, ivermectin, and closantel blocked development of L3 to L4 at 0.1, 1, and 10 micrograms/ml, respectively, in the absence of serum, but their activity was reduced by the presence of serum. Seemingly, in vitro development of A suum larvae was a convenient and sensitive bioassay for anthelmintic activity and could serve as a screen for anthelmintic residues in edible tissues.     

Ascaris suum: seasonal egg development rates in a Saskatchewan pig barn.

This study investigated the effects of seasonal temperature variations on the rate of development to infectivity of Ascaris suum eggs under western Canadian climatic conditions. Flasks containing a suspension of A. suum eggs were placed inside a pig barn located near Saskatoon, Saskatchewan, Canada, at monthly intervals from July 1997 to July 1998. Eggs from each flask were monitored weekly for development to the infective larval stage. Infectivity of eggs was confirmed using a mouse bioassay. Development to the infective stage took three to four weeks in summer when in-barn temperatures were similar to the external ambient temperature. Fall, winter and early spring egg development took longer as in-barn temperatures were cooler. Mid-winter egg development took as long as 11-12 weeks. The in-barn mean temperature during this period was approximately 17 C, close to the development threshold of A. suum eggs. These data are discussed in relation to similar studies from northern Europe and to potential control measures.     

Establishment of Ascaris suum in the pig: development of immunity following a single primary infection

Development of immunity after a single primary infection of Ascaris suum in pigs was investigated with regard to the worm population dynamics of a superimposed A. suum infection, host immune response and gross liver pathological changes. Group A was given a primary infection of 60,000 infective A. suum eggs and group B was left uninfected. Four weeks later both groups A and B were inoculated with 1,000 A. suum eggs, and subgroups were slaughtered 7, 14 and 21 days post challenge infection (p.c.i.). An uninfected control group C was slaughtered on day 21 p.c.i. The challenge worm recovery in group A was reduced compared to group B by 12%, 50% and 75% on day 7, 14 and 21 days p.c.i., respectively. In both groups was the expulsion of worms initiated between day 14 and 21 p.c.i. However, in group A the worms were recovered more posteriorly in the small intestine and 21 days p.c.i. the mean worm length was significantly shorter than in group B (p = 0.01). The results above were associated with significantly higher (p < 0.05) antibody response and higher eosinophil counts in group A compared to group B. The present results suggest that the larval growth and survival of a challenge infection are decreased, probably due to higher antibody and eosinophil attack during the migratory phase.     

The protective effect of developmental stages in an Ascaris suum infection in the guinea pig

The capacity of various developmental stages of Ascaris suum to induce a protective immune response in the guinea pig model was evaluated. Larvated eggs, second, third or fourth stage larvae were injected into guinea pigs by either the suncutaneous, intramuscular, ear vein or mesenteric vein route. Animals were challenged with a mesenteric vein injection of artificially hatched infective larvae of A. suum. Second stage larvae produced the highest degree of immunity and fourth stage larvae produced the least protection comparing all routes of administration. The most effective route of immunization for all the developmental stages was the mesenteric vein. Antibody titer as assessed by indirect hemagglutination was not correlated with the degree of protection.     

Bayesian estimation of diagnostic accuracy of a new bead-based antibody detection test to reveal Toxoplasma gondii infections in pig populations

The success of a Toxoplasma gondii surveillance program in European pig production systems depends partly on the quality of the test to detect infection in the population. The test accuracy of a recently developed serological bead-based assay (BBA) was investigated earlier using sera from experimentally infected animals. In this study, the accuracy of the BBA was determined by the use of sera from animals from two field subpopulations. As no T. gondii infection information of these animals was available, test accuracy was determined through a Bayesian approach allowing for conditional dependency between BBA and an ELISA test. The priors for prevalence were based on available information from literature, whereas for specificity vague non-informative priors were used. Priors for sensitivity were based either on available information or specified as non-informative. Posterior estimates for BBA sensitivity and specificity were (mode) 0.855 (Bayesian 95% credibility interval (bCI) 0.702–0.960) and 0.913 (bCI 0.893–0.931), respectively. Comparing the results of BBA and ELISA, sensitivity was higher for the BBA while specificity was higher for ELISA. Alternative priors for the sensitivity affected posterior estimates for sensitivity of both BBA and ELISA, but not for specificity. Because the difference in prevalence between the two subpopulations is small, and the number of infected animals is small as well, the precision of the posterior estimates for sensitivity may be less accurate in comparison to the estimates for specificity. The estimated value for specificity of BBA is at least optimally defined for testing pigs from conventional and organic Dutch farms.     

Susceptibility of fourth-stage Ascaris suum larvae to fenbendazole and to host response in the pig

Fenbendazole given at the rate of 2.5 g/kg of feed for 3 days had 100% efficacy against 4th-stage Ascaris suum larvae in 8 pigs. Eight control pigs had a total of 108 A suum. In 6 pigs infected 3 times with 3rd-stage A suum larvae and treated with fenbendazole after the larvae molted to the 4th stage, the challenge exposure-derived population was decreased by 64%. Similar sequential infections in 6 pigs similarly infected, but not treated with fenbendazole, decreased the challenge exposure-derived population by 98%; however, developing and/or adult worms from the vaccinating infections were present.     

Identification of a collectin-like protein in pig serum that binds a component in perienteric fluid from Ascaris suum

A collectin-like protein (CLP) of the acute phase protein family that binds the polysaccharides mannan and alpha-1-6 dextran was isolated from the serum of pigs infected with Ascaris suum. A monoclonal antibody generated against this protein and used to characterize the CLP revealed on SDS-PAGE and western blot analysis that the protein had a molecular weight of approximately 48 kDa under reducing conditions and greater than 100 kDa under nonreducing conditions. Enzyme-linked immunosorbent assay (ELISA) showed that the CLP bound to substances in the perienteric fluid of Ascaris suum (APF). Molecular weight fractionation of APF demonstrated that CLP binds primarily to APF substances of greater than 100 kDa. Binding of CLP to APF was partially blocked by phosphatidylinositol. This is the first report of a porcine CLP and the binding of a CLP to components of the common nematode Ascaris suum.     

猪蛔虫幼虫琼脂胶移行法的建立

本实验首次在国内建立起了线虫的琼脂胶移行法 (Agar- gel Migration Assay,AMA)。通过对影响幼虫活力的某些理化因素 ,如 :琼脂胶的温度、每孔加入的琼脂胶和幼虫混合液的体积、琼脂胶的浓度及幼虫在琼脂胶内移行的时间等研究发现 :加胶的温度和加胶的量对幼虫的移出率有较大的影响。幼虫的移出率随加胶温度的升高而降低 ,当温度达到 70℃时 ,则几乎无虫体从琼脂胶内移出 (移出率仅为 0 .5 % ) ;幼虫的移出率随加入琼脂胶量的增多而减小 ,当每孔加入 10 0 0μl时 ,幼虫的移出率为 16 .6 8% ,当加入 4 0 0μl时幼虫的移出率则能达到2 5 .17%。琼脂胶的浓度对幼虫移出的影响并不明显 ,采用 SAS软件 (for Windows V6 .12 )分析发现 1.2 %、1.0 %、0 .8%、0 .4 %的结果间无显著差异 (P>0 .0 5 ) ,但琼脂胶的浓度能影响胶液凝固的时间和凝固后的韧性。实验表明 :最适宜的加胶量为 4 0 0μl /孔 ,最适宜的加胶温度为 5 3℃ (Agar- gel,sigm a) ,在琼脂胶中最佳培养移行时间为 2 4 h,琼脂胶液的最适实验浓度为 1.5 %琼脂胶     

A comparison of modifications of the McMaster method for the enumeration of Ascaris suum eggs in pig faecal samples

The comparative efficacies of seven published McMaster method modifications for faecal egg counting were evaluated on pig faecal samples containing Ascaris suum eggs. Comparisons were made as to the number of samples found to be positive by each of the methods, the total egg counts per gram (EPG) of faeces, the variations in EPG obtained in the samples examined, and the ease of use of each of the methods. Each method was evaluated after the examination of 30 samples of faeces. The positive samples were identified by counting A. suum eggs in one, two and three sections of newly designed McMaster chamber. In the present study compared methods were reported by: I-Henriksen and Aagaard [Henriksen, S.A., Aagaard, K.A., 1976. A simple flotation and McMaster method. Nord. Vet. Med. 28, 392-397]; II-Kassai [Kassai, T., 1999. Veterinary Helminthology. Butterworth-Heinemann, Oxford, 260 pp.]; III and IV-Urquhart et al. [Urquhart, G.M., Armour, J., Duncan, J.L., Dunn, A.M., Jennings, F.W., 1996. Veterinary Parasitology, 2nd ed. Blackwell Science Ltd., Oxford, UK, 307 pp.] (centrifugation and non-centrifugation methods); V and VI-Gr?nvold [Gr?nvold, J., 1991. Laboratory diagnoses of helminths common routine methods used in Denmark. In: Nansen, P., Gr?nvold, J., Bj?rn, H. (Eds.), Seminars on Parasitic Problems in Farm Animals Related to Fodder Production and Management. The Estonian Academy of Sciences, Tartu, Estonia, pp. 47-48] (salt solution, and salt and glucose solution); VII-Thienpont et al. [Thienpont, D., Rochette, F., Vanparijs, O.F.J., 1986. Diagnosing Helminthiasis by Coprological Examination. Coprological Examination, 2nd ed. Janssen Research Foundation, Beerse, Belgium, 205 pp.]. The number of positive samples by examining single section ranged from 98.9% (method I), to 51.1% (method VII). Only with methods I and II, there was a 100% positivity in two out of three of the chambers examined, and FEC obtained using these methods were significantly (p<0.01) higher comparing to remaining methods. Mean FEC varied between 243 EPG (method I) and 82 EPG (method IV). Examination of all three chambers resulted in four methods (I, II, V and VI) having 100% sensitivity, while method VII had the lowest 83.3% sensitivity. Mean FEC in this case varied between 239 EPG (method I) and 81 EPG (method IV). Based on the mean FEC for two chambers, an efficiency coefficient (EF) was calculated and equated to 1 for the highest egg count (method I) and 0.87, 0.57, 0.34, 0.53, 0.49 and 0.50 for remaining methods (II-VII), respectively. Efficiency coefficients make it possible not only to recalculate and unify results of faeces examination obtained by any method but also to interpret coproscopical examinations by other authors. Method VII was the easiest and quickest but least sensitive, and method I the most complex but most sensitive. Examining two or three sections of the McMaster chamber resulted in increased sensitivity for all methods.     

Effects of multiple dose infections with Ascaris suum on blood gastrointestinal hormone levels in pigs

Ten consecutive daily doses of infective Ascaris suum eggs were administered to pigs in two experiments and the levels of gastrointestinal hormones in their blood were measured. The piglets in each experiment were divided into low-dose (LDI) and high-dose (HDI) infections and control groups. Infected pigs had lower feed consumption, lower weight gains, and lower feed efficiency than control pigs. Serum gastrin levels in infected pigs were significantly lower than the controls from Days 7 to 17 post first inoculation (PFI), and so were their serum glucagon levels from Days 12 to 24 PFI. Serum insulin levels in infected animals were sometimes lower than those in controls. These differences were usually more intense in the LDI pigs than in HDI pigs. The plasma cholecystokinin (CCK) levels in the LDI group were significantly higher than those in controls from Day 10 PFI to the end of the experiment, while the CCK levels in the HDI group did not differ significantly from the controls. Increased plasma CCK levels could be a satiety factor in A. suum infection since the time of occurrence of high levels of CCK matched the period of reduced feed consumption.     

Evaluation of the indirect hemagglutination assay as a practical serodiagnostic test for mycoplasmal pneumonia of swine

Sera from swine experimentally or naturally infected with Mycoplasma hyopneumoniae (the etiological agent of mycoplasmal pneumonia of swine, MPS) were tested by the indirect hemagglutination assay (IHA), the enzyme-linked immunosorbent assay (ELISA) and the complement fixation (CF) test. The IHA detected antibody at comparable times and levels to the other 2 serological tests following experimentally-induced infection. In the late antibody response (greater than or equal to 86 days post-infection), the ELISA titres were higher than either the IHA or the CF test. The IHA appeared least satisfactory when it was used to test sera from commercial swine herds. When 1000 sera were tested, the IHA was positive for only 30 (22%) of 135 sera which were positive by the ELISA and the CF test. The IHA titres were low; 20 of the 30 sera had a titre of only 10. The end-points for the IHA were difficult to read for sera of this low titre. The relationship between positive IHA results for the herd sera obtained at necropsy, and the occurrence of gross or microscopic lesions typical of MPS was poor (41 and 50% agreement, respectively). An agreement of 39% was noted between positive IHA results and the localization of mycoplasmal antigens by an indirect immunofluorescence (IIF) test. However, IHA results correlated significantly (P less than 0.05) with gross and microscopic lesions, but not with the IIF test. No significant correlation was noted between the IHA (or the other 2 serologic tests) and the cultural isolation of M. hyopneumoniae or M. flocculare. On the basis of these results, the IHA appears to have limited promise as a practical test for the diagnosis of MPS in commercial swine herds because of the low titres observed, poor correlation of the IHA and other indicators of MPS, the necessarily subjective determination of end-points, and other inherent technical limitations of the test.     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

苦楝皮乙醇提取物驱除猪蛔虫效果研究及对猪主要免疫指标的影响

进一步考察苦楝皮的驱虫效果和毒副作用。用云南苦楝皮乙醇提取物,对人工感染猪蛔虫的猪只进行不同药物浓度组与空白组的药效对比试验,并在用药前后对猪只进行免疫指标的检测。结果表明,苦楝皮乙醇提取物的高、中浓度对治疗猪蛔虫病的效果较好,驱虫率分别达到87．2％和79．5％,虫卵减少率分别为87．3％和80．2％,与对照组相比差异极显著;对各项指标的检测表明,苦楝皮提取物能提高猪的非特异性免疫机能,在治疗剂量内使用安全。     

猪蛔虫雄虫cDNA文库的构建

采用Tripure Isolation Reagent抽提猪蛔虫雄虫成虫的总RNA,用poly(A)PuristTM纯化试剂盒分离mR-NA。分离mRNA后,用Clontech公司的CreatorTMSMARTTMcDNA文库构建试剂盒构建了猪蛔虫雄虫成虫的cDNA文库。结果获得了7.26×105独立克隆,重组率达96.7%,插入片段的平均长度约为1 kb。猪蛔虫雄虫cDNA文库的成功构建为利用文库筛选雄虫差异表达基因提供了材料来源,为研究猪蛔虫性别发育的分子机制奠定了基础。     

96孔板-PCR排除法筛选猪蛔虫雌虫卵黄蛋白原基因

为了从猪蛔虫雌虫cDNA文库中筛选出猪蛔虫雌虫卵黄蛋白原基因,本研究根据猪蛔虫雌虫卵黄蛋白原基因的EST序列为模板设计引物,采用96孔板-PCR排除法对猪蛔虫雌虫cDNA文库进行筛选,并筛选出阳性克隆F993-G10-A10。测序结果表明,该基因序列长621bp,有完整的3'端。经BLAST分析,其推导的氨基酸序列与秀丽隐杆线虫(C.elegans)的卵黄蛋白原基因(Vit1、Vit2、Vit3、Vit4和Vit5)的氨基酸序列的一致性分别为35%、35%、34%、34%和35%,核苷酸序列相似性分别为55%、57%、54%、54%和53%。该阳性克隆的获得为该基因的深入研究奠定了基础。同时也证明了96孔-PCR排除法是一种高效、简便、低成本的筛选文库方法。     

应用RNA干扰技术对猪蛔虫性别相关基因功能的初步研究

针对编码猪蛔虫雌虫卵巢蛋白基因的EST序列设计了1对特异引物及连接T7启动子的引物，经PCR扩增，获得5’端连有T7启动子的双链DNA，在RNA聚合酶的作用下，转录合成dsRNA。通过浸泡的方式将dsRNA导入秀丽新杆线虫中，在浸泡后的5个不同的时间点，采用RT—PCR检测虫体浸泡后靶基因被干扰的效果。试验结果表明，在浸泡后的8～29h能检测到同源靶标的存在；而在浸泡后的43～57h不能检测到靶标RNA。另外，试验组没有观测到虫体明显的表型变化，而对照组在浸泡28h后，发现部分线虫体内有发育成形的虫卵。初步认为导入虫体的dsRNA发生了干扰效应。     

A novel technique for identification of Ascaris suum cohorts in pigs

The objective of the present study was to develop a fast, cheap and reliable technique for identifying different cohorts of the swine parasite, Ascaris suum. A polymerase chain reaction linked restriction fragment length polymorphism (PCR-RFLP) technique on mt-DNA was used to identify unique haplotypes of four gravid A. suum females on agarose gels after eggs were recovered from each of the worms. Each of four pigs was inoculated with 2000 embryonated eggs originating from one of the four identified Ascaris haplotypes, respectively. Ascaris larvae were isolated from the small intestine at day 14 post-infection using an agar technique. Single larvae from each pig were transferred to 96-well PCR plates and a simple DNA extraction using a worm lysis buffer was carried out and followed by the PCR-RFLP analysis. More than 100 larvae from each of the four pigs were analysed and all were found to have the same haplotype as the parental female. We conclude that unique haplotypes of female A. suum and offspring can be identified by means of PCR-RFLP on mt-DNA and suggest that this method can be used in future research on Ascaris population biology using cohorts with distinct mt-DNA profile.