捻转血矛线虫免疫调节型DNA疫苗的构建及在山羊体内表达研究

为构建免疫调节型DNA疫苗,将山羊IL-2基因分别与捻转血矛线虫H11-1、H11-2和H11-3基因串联构建融合表达载体pcDNA/IL-2-H11-1、pcDNA/IL-2-H11-2和pcDNA/IL-2-H11-3。为检测疫苗在山羊体内的表达情况,将纯化的疫苗质粒肌肉注射山羊后取注射部位肌肉组织,于首次免疫7 d后和二次免疫10 d后分别用RT-PCR和Western Blot等方法检测H11抗原的转录和翻译情况。结果发现H11基因和IL-2基因均能在注射部位肌肉获得转录和翻译。     

捻转血矛线虫Hc38基因DNA疫苗对绵羊免疫保护性效果评价

为了研究基因疫苗对绵羊的免疫保护效果,本研究构建了捻转血矛线虫(H.contortus)Hc38基因DNA疫苗.将H.contortus Hc38基因保守结构域克隆到真核表达载体pcDNA3.1中,免疫鼠8d后用RT-PCR检测到该疫苗在鼠肌肉组织中进行了转录.将纯化的DNA疫苗免疫绵羊后,用western blot和ELISA方法检测疫苗在绵羊体内的翻译和诱导IgG的产生.二免后2周用10 000条H.contortus第3期幼虫攻击实验动物,检测绵羊粪便虫卵排出、成虫数量等免疫保护性指标.该H.contortus Hc38 DNA疫苗与对照组比较,免疫组绵羊排出虫卵减少66.6%、成虫减少33.1%.特别值得注意的是免疫组的静脉注射方式产生抗体最高,相应羊的虫卵数和成虫数低.本实验证明Hc38基因DNA疫苗对绵羊虫卵及成虫发育具有明显的抑制作用.     

不同药物对放牧山羊捻转血矛线虫驱虫效果比较试验

为了筛选山羊捻转血矛线虫的理想驱虫药物,对8组检出有捻转血矛线虫感染的山羊群分别采用不同剂量的阿苯达唑、盐酸左旋咪唑、伊维菌素以及阿苯达唑-伊维菌素预混剂进行驱虫效果对比试验。结果盐酸左旋咪唑、伊维菌素以及阿苯达唑-伊维菌素预混剂对山羊捻转血矛线虫驱虫后,虫卵减少率均达95%以上,而阿苯达唑驱虫后的虫卵减少率低于95%。表明左旋咪唑、伊维菌素以及阿苯达唑-伊维菌素预混剂对山羊捻转血矛线虫的驱虫效果较好,而阿苯达唑有一定的抗药性。     

感染两种蠕虫病的山羊血液生理生化指标比较

[目的]了解山羊自然感染肝片吸虫及自然感染捻转血矛线虫后血液生理生化指标的差异性.[方法]分别对10只自然感染肝片吸虫山羊、10只自然感染捻转血矛线虫山羊以及10只粪检蠕虫卵阴性的健康对照组山羊进行血液生理生化指标测定.[结果]2个试验组与健康对照组相比,白细胞总数(WBC)、粒细胞数(GRAN)、尿酸(UA)的指标均明显增高,差异极显著(P<0.01);白蛋白(ALB)、红细胞总数(RBC)、血红蛋白(HCT)的指标降低,差异显著(P<0.05).肝片吸虫感染组的谷草转氨酶(AST)、碱性磷酸酶(ALP)、谷氨酰胺氨基转移酶(GGT)、总胆红素(TBIL)、肌酸激酶(CK)、乳酸脱氢酶(LDH)、α-羟丁酸脱氢酶(HBDH)等指标均明显高于捻转血矛线虫感染组,差异极显著(P<0.01);捻转血矛线虫感染组的红细胞总数和血红蛋白等指标低于肝片吸虫感染组,两组间差异显著(P<0.05).[结论]自然感染不同寄生蠕虫对山羊血液生理生化指标影响程度存在一些异同点,通过对比山羊肝片吸虫病和捻转血矛线虫病的血液生理生化指标,可为这两种寄生蠕虫病的间接诊断积累一些参考数据.     

捻转血矛线虫纯净3期幼虫制备方法的改进

从自然感染羊体内获得捻转血矛线虫雌虫后,收集其子宫中的虫卵进行培养,再将纯净的第3期幼虫感染保种山羊,于虫卵排出高峰期大量收集粪便,进行幼虫培养,可获得大量纯净的捻转血矛线虫第3期幼虫。     

绵阳地区山羊粪便中捻转血矛线虫的分子鉴定

为鉴定绵阳地区山羊粪便中疑似血矛属线虫虫卵的虫种,本研究提取绵阳地区三个县山羊粪便中疑似血矛属线虫的虫卵DNA 60份,采用PCR技术扩增其核糖体第二内转录间隔区(rDNA ITS-2)基因序列,并与GenBank上公布的捻转血矛线虫同源序列进行比较分析。结果显示:60份疑似血矛属线虫的虫卵DNA样品均得到191bp的ITS-2片段;60条ITS-2序列共形成15条不同的序列,含有17个单变异位点,与GenBank中多株捻转血矛线虫的同源性达95.8%~100%;ML系统发育树显示,本研究得到的15种类型ITS-2序列与已报道的捻转血矛线虫(Haemonchus contortus)聚类形成一个分支。综上可得,60份来自山羊的血矛属线虫均为捻转血矛线虫。     

福建省放牧山羊主要蠕虫粪检虫卵数与荷虫量的相关性研究

【目的】为了了解山羊主要蠕虫克粪便虫卵数(EPG)与其荷虫量的相关性。【方法】通过对15例肝片吸虫病例、13例捻转血矛线虫病例、12例莫尼茨绦虫病例、9例阔盘吸虫病例以及11例同盘吸虫病例的克粪便虫卵数与其荷虫量计数,计算出两者之间的相关系数。【结果】肝片吸虫、捻转血矛线虫、莫尼茨绦虫、阔盘吸虫和同盘吸虫的EPG值和荷虫量的相关系数(r)分别为0.803、0.643、-0.176、0.985、0.926,表明肝片吸虫病、阔盘吸虫病、同盘吸虫病的EPG与其荷虫量的相关性都极显著(P0.01);捻转血矛线虫病的EPG与其荷虫量的相关性较显著(P0.05);莫尼茨绦虫病的的EPG与其荷虫量没有直接相关。【结论】山羊主要蠕虫的EPG与其荷虫量的相关系数大小可为临床上判断蠕虫感染强度以及制定合理的驱虫方案提供依据。     

崇明白山羊寄生虫病调查和消长动态的研究

对上海市崇明地区白山羊的寄生虫病进行了调查,危害崇明白山羊的寄生虫主要是捻转血矛线虫。哨羊全年粪便中捻转血矛线虫虫卵数形成的３ 个高峰与月平均最高温度和月降水量曲线形成的３ 个高峰相吻合,证实每年７、８ 月份崇明地区白山羊群中大批羊只消瘦、死亡是由于捻转血矛线虫严重感染引起的,并提出了防治措施的建议。     

崇明白山羊寄生虫病调查与消长动态的研究

对上海市崇明地区白山羊的寄生虫病进行了调查,危害崇明白山羊的寄生虫主要是捻转血矛线虫,哨羊全年粪便中捻转血矛线虫虫卵数形成的３个高峰与月平均最高温度和月降水量曲线形成的３个高峰相吻合,证实每年７,８月份崇明地区白山羊群中大批羊只消瘦,死亡是由于捻转血矛线虫严重感染引起的,并提出了防治措施的建议。     

羊消化道4种易混淆线虫虫卵的形态学观察与比较

为了方便临床上鉴定羊消化道常见且易混淆线虫虫卵并丰富相关资料,试验从羊消化道中采集疑似捻转血矛线虫、夏伯特线虫、食道口线虫和奥斯特线虫共4种线虫,采用形态学和分子生物学方法鉴定虫种,将4种线虫于体外短暂培养后得到纯种虫卵,通过观察虫卵不同发育阶段的胚胎形态变化获取每种线虫虫卵形态特征,并对虫卵的长径、宽径和长宽比进行测定。结果表明：经鉴定4种线虫分别为捻转血矛线虫、绵羊夏伯特线虫、粗纹食道口线虫和普通奥斯特线虫。4种线虫虫卵初期胚胎多呈收缩状态且形似桑葚,在外观形态上具有相似性,均为双层卵壳,多数对称,少数不对称。捻转血矛线虫虫卵呈短椭圆形,颜色较浅;绵羊夏伯特线虫虫卵呈椭圆形,颜色较捻转血矛线虫虫卵深;粗纹食道口线虫虫卵呈长椭圆形,颜色较捻转血矛线虫虫卵深,与绵羊夏伯特线虫虫卵相近;普通奥斯特线虫虫卵亦呈长椭圆形,颜色与捻转血矛线虫相近,较绵羊夏伯特线虫和粗纹食道口线虫虫卵浅。虫卵在生理盐水中培养1～6 d后,捻转血矛线虫、绵羊夏伯特线虫和普通奥斯特线虫依次进入蝌蚪期和幼虫期,但仅凭肉眼依然难以区分虫卵种类;绵羊食道口线虫未观察到含蝌蚪或含幼虫的虫卵,仅在桑葚期观察到胚胎收缩成1团及...     

Evaluation of immune response to artificial infections of Haemonchus contortus in Gulf Coast Native compared with Suffolk lambs

Due to their critical functions in worm physiology, glutathione peroxidases in Haemonchus contortus are potential candidates for vaccine to control haemonchosis. However, information on the protection potential of these molecules is lacking. In this study, recombinant HC29 antigen was tested for protection against experimental H. contortus infections in goats. Fifteen animals were allocated into three trial groups. The animals of HC29 group was vaccinated with a recombinant HC29 DNA vaccine twice at day 0 and day 14, and then challenged with 5000 infective H. contortus L3 (third larval stage) on the 28th day. An unvaccinated positive control group was challenged with L3 at the same time. An unvaccinated negative control group was not challenged with L3. The results indicated HC29 DNA vaccine was transcribed at local injection sites and expressed in vivo post immunizations, respectively. Following L3 challenge, the mean eggs per gram feces and worm burdens of HC29 group were reduced by 36.1% and 35.6% respectively compared to the positive control group. After immunization with the DNA vaccine, significantly high levels of serum IgG, serum IgA, mucosal IgA and CD4(+) T lymphocytes were detected. These results suggest that recombinant H. contortus HC29 glutathione peroxidase DNA vaccine induced a partial immune response and has protective potentials against caprine haemonchosis.     

Expression of acquired immunity to a local isolate of Haemonchus contortus by the Nigerian West African Dwarf goat.

The capacity of young Nigerian West African Dwarf (WAD) goats to express good acquired immunity to their native geographic strain of Haemonchus contortus and the correlates of this responsiveness were studied in a laboratory experiment involving forty 7-8 month old kids. A primary immunising infection with 2000 L3 (equivalent to 260-450 L3/kg body weight) with or without challenge on D42 with 2000 L3 resulted in a mild chronic infection with a pre-patent period of 18-20 days and little or no reduction in worm burden between D14 and D56. In contrast, another group (D) of kids, whose immunising infection had been truncated with fenbendazole on D35 and later received similar challenge infection, developed good protection against challenge. Thus, worm burdens were largest in group E (challenge control), larger in group C (primary+challenge) and least in group D. Of the measures of infection used, namely faecal worm egg counts (FECs), circulating eosinophil (EOS) responses, packed cell volume (PCV) and body weight, FEC and EOS responses exhibited marked individual variability, but only FEC (geometric mean of transformed counts) and PCV showed strong correlation with worm burden. There was also a significant negative correlation between FEC and PCV. The size of inoculum used was well tolerated by the kids, as it induced only mild changes in PCV in some goats and no effect at all on body weights. This suggests that the WAD goat may possess a good measure of resistance to the pathogenic effects of its native strain of H. contortus. The wide individual variability in FEC and its strong relationships to worm burden and PCV are pointers to its likely genetic basis. There are, therefore, good prospects for further studies to identify H. contortus resistant genotypes among the WAD goat population.     

Effect of feeding sericea lespedeza leaf meal in goats experimentally infected with Haemonchus contortus

Effect of sericea lespedeza [SL; Lespedeza cuneata (Dum-Cours.) G. Don.] leaf meal feeding was evaluated in two experiments in indoor reared goats with experimental infection of Haemonchus contortus larvae. In the first experiment, ten 8-10 month old male Spanish and Alpine cross kids pair matched for body weight and age were fed SL or bermudagrass [BG; Cynodon dactylon (L.) Pers.] hay one week before infection and were infected with 5000 H. contortus L(3). The animals were maintained on the same diet for the remaining period and were slaughtered 28 days post-infection (DPI) to determine the establishment of incoming infective larvae. Goats fed SL had lower establishment (P<0.05) of H. contortus larvae than that of the control goats fed BG hay. In the second experiment, twenty-five 8-10 months old male Alpine cross, Saanen, Nubian×Saanen and Spanish kids reared in confinement on BG were experimentally infected with 5000 H. contortus L(3). On 35 DPI, the animals were allocated to two groups after blocking by fecal egg count (FEC), and one group was fed SL leaf meal (n=13), and another control group remained on BG (n=12). Four goats/group were slaughtered successively on days 7, 14, and 28 days post SL feeding, except on day 7, when five SL fed goats were slaughtered. Fecal egg counts and blood packed cell volume (PCV) were measured at weekly intervals and worm count, female worm fecundity, worm length and mucosal eosinophils, mast cells and globule leucocytes were measured after slaughter. Goats fed SL had a lower FEC (P<0.05) one week after feeding, as compared to those fed on BG, and the values remained at low level thereafter. Similarly, PCV was also significantly affected by feeding (P<0.01), and feeding and time interaction (P<0.05). However, worm burden, female worm fecundity, parasite length, and mucosal inflammatory cell count were similar between the groups. Feeding SL reduced the establishment of infective larvae and FEC of H. contortus in experimental studies and this plant could be used for biological control of parasite infection under field conditions to limit the harmful effects of the parasites in goats.     

Lack of effects of quebracho and sainfoin hay on incoming third-stage larvae of Haemonchus contortus in goats

The effects of tannins on adult populations of Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta in goats are characterised mainly by a decrease in egg excretion without any significant changes in worm number. In contrast, the impact of tannins on T. colubriformis or T. circumcincta third-stage larvae (L3) is associated with a significant reduction in worm establishment. The objective of the present study was to examine the effects of quebracho extract tannins on H. contortus L3. The consequences of consumption of sainfoin hay were also examined. Twenty-one na?ve kids were divided into three experimental groups. Group Q received quebracho extract and group S received sainfoin hay from days D3 to D5. Group C remained as an infected control group. All kids received 1500 L3 H. contortus on D0, D1 and D2. On D18, post-infection, the kids were slaughtered and the worm populations compared in the different groups. Compared to the control values, the worm counts decreased, respectively, by 33% and 38% in groups Q and S but the differences were not significant. No differences were found in pathophysiological measurements between the three groups. The results confirm differences in tannin effect according to nematode species but not parasitic stage.     

Effect of dietary supplementation on resistance to experimental infection with Haemonchus contortus in Creole kids

The aim of the present study was to test the effect of dietary supplementation on resistance to experimental infection with Haemonchus contortus in Creole kids. One trial with three replicates involved a total of 154 female kids that were chosen from three successive cohorts of the Creole flock of INRA-Gardel in 2007. The kids were placed into four treatments according to the amount of concentrate they received: G0 (no concentrate and a quality Dichantium spp. hay ad libitum, HAY), G1 (HAY+100g commercial concentrate d(-1)), G2 (HAY+200 g commercial concentrate d(-1)), G3 (HAY+300 g commercial concentrate d(-1)). The G0-G3 groups were infected with a single dose of 10,000 H. contortus third stage larvae (L(3)) at Day 0 (D0). Each infected group was comprised of one half resistant and one half susceptible genetically indexed kids. The average breeding values on egg excretion at 11 months of age were distant of 0.70, 0.65, 0.61 and 0.61 genetic standard deviations in G0, G1, G2 and G3, respectively. The faecal egg count (FEC), packed cell volume (PCV), eosinophilia (EOSI) and dry matter intake (DMI) indices were monitored weekly until 42 days post-infection. Enzyme-linked immunosorbent assay was carried out on serum samples to determine the level of IgA anti-H. contortus L(3) crude extracts and adult excretion/secretion products (ESP). The 10,000 L(3) dose received by the kids induced a severe infection: 8000 eggs per gram at the FEC peak, a PCV less than 15% and mortality. Interestingly, the supplemented animals in G3 showed a higher level of EOSI but a lower level of IgA anti-L3 and IgA anti-ESP than non-supplemented animals (G0). Resistant and susceptible kids had significantly different FEC variations within the groups. Susceptible kids had a 1.6 times higher egg output than resistant kids in G0. This difference was not found in the supplemented groups. The results of this study showed that supplementary feeding improved resistance of Creole kids to H. contortus experimental infection.     

Serum antibody responses in Creole kids experimentally infected with Haemonchus contortus

The objective of this study was to evaluate the relationship of parasite-specific serum antibodies with the resistance status of Creole kids. The average breeding values on egg output predicted in a context of natural infection at 11 months of age were distant of 1.07 genetic standard deviation between resistant and susceptible animals. After drenching the animals were maintained worm-free during 1 month until experimental infection with 10,000 Haemonchus contortus infective larvae (L3). Enzyme-linked immunosorbent assay was carried out in serum samples to determine the level of IgG, IgA and IgE anti-H. contortus L3 crude extracts and adult excretion/secretion products (ESP). Parasitological and blood immunological parameters were measured on the 2 extreme groups. Despite the absence of any typical signs of haemonchosis, susceptible kids had more than 11 times higher faecal egg counts (FEC) at 35 days post-infection (d.p.i.) than resistant kids had. Levels of immunoglobulin against H. contortus L3 and ESP increased significantly after infection in both groups. However, no difference in the host immune response mediated by immunoglobulin against H. contortus was evidenced between groups. This finding suggests that, in goats previously infected by H. contortus, a degree of protection occurred and the phenotypic and genetic segregation in resistant and susceptible animals were not related to the humoral immune response. The correlation coefficients between FEC and IgE anti-ESP (r=0.593; P<0.05 was significant in both resistant and susceptible animals. Such correlation suggesting a hypersensitivity reaction dependent on worm prolificacy has never been described. This result needs further studies to understand the mechanisms underlying this observation.     

Combining the effects of supplementary feeding and copper oxide needles for the control of gastrointestinal nematodes in browsing goats

The aim was to assess the benefits obtained from combining supplementary feeding and copper needles (COWP), compared to the use of both approaches independently, for the control of gastrointestinal nematode (GIN) infections in browsing kids. Forty-four nematode free Criollo kids were exposed to natural parasite infection. The kids were divided into six experimental groups: not treated, supplemented (NT-S), not treated, not supplemented (NT-NS), moxidectin treated, supplemented (M-S), moxidectin treated not supplemented (M-NS), copper treated, supplemented (COWP-S) and copper treated, non-supplemented (COWP-NS). Copper treated groups received Copinox (2 g capsules) on day 0 and on day 60 of the trial. Moxidectin treated groups received Cydectin (0.2 mg/kg of body weight s.c.) every 28 days. Three of the groups received individual supplementation (100 g of feed/day fresh basis; 74% sorghum: 26% soybean meal; NT-S, M-S and COWP-S) and the other three groups were not supplemented (NT-NS, M-NS and COWP-NS). Animals browsed native vegetation (6.5 h/day) during the wet season (154 days). Kids were weighed every 14 days to determine live weight gain (LWG) and blood and faecal samples were obtained to determine packed cell volume (PCV), haemoglobin (Hb), peripheral eosinophil counts (PEC) and faecal egg counts (FEC). At the end of the trial, four kids of each group were euthanatized (six kids in each COWP treated group). Worm burdens, female worm lengths and prolificacy were determined. Liver samples were used to determine copper concentration and were stained with haematoxylin-eosin to determine microscopic lesions. Animals receiving the combination of supplementary feeding and COWP improved their LWG, PCV and Hb to similar levels of animals with suppressive AH treatment. This was not the case when COWP was used without supplementation. Liver copper concentration in COWP treated groups increased significantly especially in the COWP-NS kids but this was not associated with liver lesions or clinical signs. Post-mortem Haemonchus contortus and Trichostrongylus colubriformis worm counts had a tendency to be reduced in the different groups (66-35% reduction) compared to NT-NS group at the end of the trial (P>0.05). Also, COWP treatment and/or supplementation reduced female worm length of T. colubriformis and prolificacy of H. contortus and T. colubriformis. This study, confirmed the value of nutritional supplementation in the control of GIN in growing kids. The use of COWP in addition to supplementation had a limited contribution on the kids' resilience against GIN. This may be due to the reduced infection of H. contortus during this trial.