An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.     

捻转血矛线虫HLJ株15 ES抗原基因的原核表达

为了对羊源捻转血矛线虫(H.contortus)15 ES抗原基因进行原核表达,本研究利用RT-PCR技术从黑龙江省羊源H.contortus成虫总RNA中扩增得到相对分子质量15Ku ES蛋白基因(H15 ES),序列分析表明,其核苷酸序列与国外已发表的15 ku排泄分泌抗原基因的同源性为99.78%.将H15 ES克隆至pET-32a(+)中,构建重组表达质粒pET-32a-H15 ES,转化大肠杆菌,IPTG诱导表达,经SDS-PAGE电泳和western blot分析表明,表达蛋白相对分子质量约为35.2 ku.本研究为H.contortus病诊断抗原和保护性抗原的大量制备及H.contortus核酸疫苗的研究奠定了基础.     

Excretory/secretory products of Haemonchus contortus inhibit aminopyrine accumulation by rabbit gastric glands in vitro.

Abomasal parasites inhibit gastric acid secretion and reduce the number of acid secreting parietal cells either through physical contact with gastric tissue, the release of inhibitory excretory/secretory (ES) products or by initiating the host inflammatory response. To examine the role of parasite ES products, adult Haemonchus contortus were incubated in a medium based on Hank's balanced salt solution and three incubates were tested for the presence of inhibitors of acid secretion by dispersed rabbit gastric glands in vitro, using the intracellular accumulation of 14C-aminopyrine (AP) as an indirect measure of acid secretion. Acceptably sensitive rabbit preparations (80%) for testing ES products showed increased 14C-AP accumulation with either 10(-5) or 10(-4)M histamine. Sheep glands proved unsuitable as a test system as only two of six preparations responded weakly to histamine. Inhibitory activity was demonstrated in all the three parasite incubates, although responses varied quantitatively between tests, even with the same batch of incubate. In single tests, 16% ES products significantly reduced the 14C-AP ratio (P<0.05) of 10(-4)M histamine-stimulated glands (15 of 19 tests with Batch 1 and two of six with Batch 3). Even at 1 and 5%, ES products (Batch 2) were inhibitory for 10(-5)M histamine-stimulated glands: at 1% the mean reduction was 26.0% (range 7.8-54.8%, n=10), four of which were statistically significant and at 5% was 17.6% (range -15.3-53.8%, n=17), four of which were statistically significant. ES products were overall inhibitory (-12%, P<0.05), when tested on glands exposed to increasing histamine concentrations from 10(-6)M to 10(-3)M (which increased the 14C-AP ratio (P<0.001)). Responses by individual gland preparations differed (P<0.001). The active component(s) of the parasite ES products were less than 5000 molecular weight. Ammonium chloride, tested over the range of concentrations of ammonia present in worm incubates (0.2-1 mM, final concentrations in the gland incubations of 0.005-0.1 mM), had variable, but overall inhibitory effects on 10(-5)M histamine-stimulated glands (n=3). When tested with a range of histamine concentrations, 0.01 mM NH4Cl overall reduced the response by 8.6% (P<0.05)(n=4). The similarity of responses of rabbit glands to parasite ES products and to NH4Cl suggests that ammonia may be the small molecular weight ES product of adult H. contortus, which inhibited acid secretion in vitro.     

猪囊尾蚴循环抗原和排泄分泌抗原研究进展

猪囊尾蚴病是由带科(Taeniidae)带属(Taenia)的猪带绦虫(有钩绦虫)(Taenia solium)的幼虫-猪囊尾蚴(Cysticercus cellulosae)寄生于猪的肌肉和其他器官中而引起的一种人畜共患寄生虫病,俗称囊虫病。该病主     

Motility response of levamisole/benzimidazole-resistant Haemonchus contortus larvae

Electronic measurements of the motility of third stage larvae of a susceptible and a levamisole/benzimidazole-resistant strain of Haemonchus contortus were made after incubation in solutions of anthelmintics for 24 h. Results confirmed the resistance to benzimidazoles, but failed to show differences in response to levamisole. Visual observations of paralysis of first and third stage larvae in solutions of levamisole also failed to show significant differences between the two strains. The tests as conducted failed to demonstrate levamisole resistance, suggesting that nematode strains may vary in their mechanisms of resistance to levamisole.     

伊维菌素耐药及敏感捻转血矛线虫国内分离株的鉴定

为解决国内开展驱虫药耐药性相关研究中虫种资源缺乏问题,该研究采用国际通用方法开展了华北地区捻转血矛线虫耐伊维菌素虫株的分离工作,并采用传统形态学方法对其进行鉴定。对来源不同的耐伊维菌素捻转血矛线虫虫株进行分离,结果显示：华北地区分离到了4株对伊维菌素6倍耐药及1株对其敏感的捻转血矛线虫。该研究将为国内开展捻转血矛线虫耐伊维菌素研究及新型驱虫药物的开发提供虫种资源。     

Effect of Fasciola gigantica excretory secretory antigen on rat hematological indices

The present study was undertaken to investigate the effect of Fasciola gigantica excretory secretory antigen (Fg-ESA) on rat hematological indices. Fg-ESA was prepared by keeping thoroughly washed 40 F. gigantica flukes in 100 ml phosphate buffer saline (PBS) for 2 h at 37℃, and centrifuging the supernatant at 12,000 g at 4℃ for 30 min. The protein content of Fg-ESA was adjusted to 1.8 mg/ml. The rats were randomly divided into two groups of six rats each. Rats in group A received 0.5 ml of Fg-ESA intraperitoneally (i.p.) for 7 days, whereas control rats in group B received 0.5 ml of PBS i.p. for 7 days. Hemograms of both groups were studied initially and on days 0, 2, 4, 14 and 21 after the final injection of Fg-ESA or PBS. Progressive and significant (p < 0.01) declines in the values of hemoglobin, hematocrit, and total erythrocyte count were observed without significant (p > 0.05) changes in the values of mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, or mean corpuscular volume in group A. Thus, we conclude that Fg-ESA induces normocytic normochromic anemia in rats.     

Effects of dietary molybdenum on nematode and host during Haemonchus contortus infection in lambs.

The addition of molybdenum (0.05 mmol kg-1 dry-matter) to the diet of lambs given a trickle infection of Haemonchus contortus larvae (500 third stage larvae d-1 over six weeks) reduced mean faecal egg counts (epg) from 3952 to 2312 +/- 402 by 32 days (P less than 0.02) and greatly reduced the mean number of worms recovered from the abomasum 14 days after infection ceased (907 compared with 4167: P less than 0.01). Infection reduced haemoglobin concentrations less in lambs given molybdenum although the difference was small relative to the reduction in worm burden. Lambs not given molybdenum had low intraepithelial mast cell counts in the abomasal mucosa and less abomasal hypertrophy than expected from abomasal parasitism. Molybdenum did not consistently reduce the copper status of the host or the parasite. Previous exposure to molybdenum greatly reduced protein but not proteinase activity in, or secreted by, adult worms cultured for eight hours. It is suggested that molybdenum either increased the inflammatory response which preceded worm rejection or that it indirectly enhanced that reaction by reducing the effectiveness of copper-dependent, anti-inflammatory enzymes in the gastrointestinal mucosa.     

Recombinant galectins of male and female Haemonchus contortus do not hemagglutinate erythrocytes of their natural host

Recombinant galectins of female and male adult worms of Haemonchus contortus were expressed in Escherichia coli and their hemagglutinating activities to human and different animal erythrocytes were analyzed. The results showed that female and male galectins could be highly expressed in E. coli using a temperature-sensitive plasmid, with the recombinant protein being mainly appeared in inclusion bodies. Hemagglutinating activity assays showed that both of the galectins hemagglutinated human A, B, O type, dog, rabbit, chicken and mouse erythrocytes at the high concentration of 40 microg/well, but did not hemagglutinate erythrocytes of the natural host of H. contortus, the goat. Sugar inhibition assays confirmed that, out of eight sugars tested, only lactose was effective to inhibit agglutination of human type B erythrocytes by the recombinant galectins.     

Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil

Haemonchus contortus is the most prevalent nematode in Brazil. The objective of this study was to select 6 populations of H. contortus of known or suspected benzimidazole resistance status and characterize these using quantitative real-time polymerase chain reaction (qPCR) for single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the β-tubulin isotype 1 gene. qPCR was performed using DNA from a pool of 10 adult male H. contortus from a single animal per farm. Faecal egg count reduction test (FECRT) and egg hatch test (EHT) were used to determine the resistance status. Samples were obtained from 6 farms located in 5 counties in the Ceará State: Tauá, Boa Viagem, Quixadá, Santa Quitéria and Solonópole. The inbred-susceptible-Edinburgh (ISE) isolate was used as reference for comparative purposes in the qPCR. Benzimidazole resistance was detected by FECRT on all farms with efficacy values ranging from 0 to 51%. EC50 values as determined by EHT were all above 1.49 μg/ml. High frequencies of the resistant SNPs F200Y and F167Y alleles were detected but no resistance was detected at SNP E198A. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations.     

浏阳黑山羊捻转血矛线虫感染情况调查及pcox1基因分析

为调查浏阳黑山羊捻转血矛线虫的感染情况,为养殖者提供科学数据,本研究通过洗涤法对浏阳黑山羊捻转血矛线虫进行了感染情况调查,结果显示不同季度捻转血矛线虫感染情况不同,第一季度的感染率为39.1%,第二季度的感染率为46.7%,第三季度的感染率为81.3%,第四季度的感染率为53.3%;通过线粒体DNA pcox1基因对捻转血矛线虫虫种进行鉴定,目的基因片段大小约为480bp。     

Functional investigation of a QTL affecting resistance to Haemonchus contortus in sheep

This study reports a functional characterization of a limited segment (QTL) of sheep chromosome 12 associated with resistance to the abomasal nematode Haemonchus contortus. The first objective was to validate the identified QTL through the comparison of genetically susceptible (N) and resistant (R) sheep produced from Martinik × Romane back-cross sheep. The R and N genotype groups were then experimentally infected with 10 000 H. contortus larvae and measured for FEC (every three days from 18 to 30 days post-challenge), haematocrit, worm burden and fertility. Significant differences in FEC and haematocrit drop were found between R and N sheep. In addition, the female worms recovered from R sheep were less fecund. The second step of the characterization was to investigate functional mechanisms associated with the QTL, thanks to a gene expression analysis performed on the abomasal mucosa and the abomasal lymph node. The gene expression level of a candidate gene lying within the QTL region (PAPP-A2) was measured. In addition, putative interactions between the chromosome segment under study and the top ten differentially expressed genes between resistant MBB and susceptible RMN sheep highlighted in a previous microarray experiment were investigated. We found an induction of Th-2 related cytokine genes expression in the abomasal mucosa of R sheep. Down-regulation of the PAPP-A2 gene expression was observed between naïve and challenged sheep although no differential expression was recorded between challenged R and N sheep. The genotyping of this limited region should contribute to the ability to predict the intrinsic resistance level of sheep.     

Control of Haemonchus contortus in goats with a sustained-release multi-trace element/vitamin ruminal bolus containing copper

The objective of this study was to determine the effectiveness of a sustained-release multi-trace element/vitamin ruminal bolus (TEB) containing copper administered to mature does for control of gastrointestinal nematodes (GIN) during summer months and during late gestation. Haemonchus contortus was the predominant nematode during these trials. In Experiment 1, yearling Spanish does were untreated or administered TEB (n=11/group) on Day 0 (August 2005) prior to breeding. In Experiment 2, Spanish and Boer does were untreated or administered TEB 6 weeks before kidding. Fecal egg counts (FEC) and blood packed cell volume (PCV) were determined weekly between Days 0 and 42. In both experiments, FEC were reduced within 7 days in TEB-treated compared with untreated does. PCV was similar between treatment groups (Experiment 1) or tended to be lower in the TEB group (Experiment 2). GIN control did not persist more than 28 days. These studies suggest that TEB may be an effective means of GIN control in mature goats, but additional control measures may be necessary.     

Protection of lambs with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae

Helminth-free lambs (approx 4 months old) were inoculated SC with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae. The metabolite was obtained from in vitro cultivation and was identified as XL3FA1. After 3 periodic XL3FA1 inoculations, lambs were challenge exposed with H contortus. Multiple vaccinations with XL3FA1 did not inhibit the development of worm populations in the lambs; however, worm egg production seemed to be inhibited.     

The effects of transfer factor, parasitic antigen and precipitated immunoglobulin treatments in lambs infected with Haemonchus contortus

Significant reductions in adult worm burdens in lambs less than six months old infected with 5000 third stage Haemonchus contortus larvae were achieved following the combination of a single transfer factor treatment with multiple parenteral treatments of adult parasitic antigen and precipitated immunoglobulin. Specific cellular responses in the abomasa associated with individual treatments were discernible in all the experimental groups and quantitative differences in these responses reflected the varying treatment levels and treatment combinations. The reactions associated with transfer factor were similar to immediate type hypersensitivity responses and their role as possible effector mechanisms in correcting partial immune incompetency in lambs is discussed.     

Immune exclusion, a mechanism of protection against the ovine nematode Haemonchus contortus

The fate of exsheathed Haemonchus contortus larvae injected directly into the abomasal lumen was studied in groups of naive and hyperimmune sheep. Within 48 hours of challenge, more than 90 per cent of the larvae were lost from hyperimmune sheep. The larvae had a propensity to associate with the superficial mucus in both groups and, in naive sheep, approximately 50 per cent of the larvae entered glandular tissue within four hours of challenge. By contrast, although initially associated with the mucosal surface, larvae in immune sheep were excluded from the glandular tissues. It is postulated that the rapid loss of worms from the hyperimmune abomasum is a consequence of 'immune exclusion' and the possible mechanisms underlying this process are discussed.     

Evaluation of a laboratory test to detect resistance to closantel in Haemonchus contortus

OBJECTIVE: To evaluate a laboratory test for closantel resistance in Haemonchus contortus. PROCEDURE: Field isolates of H contortus, known to be resistant to closantel, were tested in the assay. In addition, mixtures of closantel-susceptible and closantel-resistant laboratory reference strains were tested to develop a method of predicting the proportion of resistant worms in a sample from the field. RESULTS: The assay correctly identified as resistant all of the closantel-resistant field isolates of H contortus. It also identified one isolate with an in vivo efficacy of 98% as having emerging resistance. Testing of the mixtures of laboratory reference strains revealed that an isolate would be classified as resistant when it consists of about 25% or more resistant worms. Test samples that are not fully susceptible yet contain less than 25% resistant worms may be classified as emerging resistance. CONCLUSION: The in vitro migration assay is a sensitive method of detecting closantel resistance in H contortus.