Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia

A commercially available ELISA for detecting antibodies to liver fluke was evaluated for use in Australia. Milk and serum samples from cattle and sheep in which infection with Fasciola hepatica was confirmed by detection of eggs in faeces were used to estimate sensitivity. Similar samples collected from cattle and sheep outside the F. hepatica-endemic area were used to estimate specificity. The ELISA was also evaluated for detecting antibodies to F. hepatica in milk from sheep and antibodies to Fasciola gigantica in sera from cattle and buffaloes, but with small numbers of samples. In cattle, the sensitivity and specificity of the ELISA were 98.2% and 98.3% using serum and 97.7% and 99.3% using milk. In infected herds, 41.4% and 41.5% of animals were positive in the serum and milk ELISAs, respectively, whereas F. hepatica eggs were found in faecal samples from 26.5% of animals. In sheep, the sensitivity of the ELISA was 96.9% and the specificity was 99.4%. In infected flocks, 60.2% of animals were positive in the serum ELISA and F. hepatica eggs were found in faecal samples 52.2% of animals. There was perfect agreement in the ELISA between paired serum and milk samples collected from ewes. The assay detected antibodies in sera from cattle and buffaloes with natural and experimental F. gigantica infections. In the experimentally infected animals, antibodies were detected 2 weeks post-infection. We conclude that the ELISA will be a valuable tool for diagnosing F. hepatica infections in cattle and sheep. The assay may also be useful for diagnosing F. gigantica infections but further studies are required to establish sensitivity and specificity.     

国产三氯苯咪唑驱除山羊肝片形吸虫效果试验

以国产三氯苯咪唑对自然感染肝片形吸虫的山羊进行了驱虫试验，并以硝氯酚为药物对照。结果表明，三氯本咪唑5mg/kg体重的剂量对肝片形吸虫成虫和童虫的驱虫率分别为99.3%和92.9%，驱净率为75％；三氯苯咪唑10mg/kg体重的剂量对肝片形吸虫成虫和童虫的驱虫率均为100％，驱净率为100％；硝氯酚5mg/kg体重剂量对肝片形吸虫成虫驱虫率为100％，但对童虫的仅为74.3%，而驱净率仅为60％。在整个试验期间试验羊均无不良反应。     

三氯苯咪唑混悬剂对牦牛肝片吸虫的驱虫试验

应用三氯苯咪唑混悬剂,选择自然感染肝片吸虫的牦牛60头进行了驱虫试验。结果表明:①三氯苯咪唑混悬剂5mg/kg体重剂量对肝片吸虫的虫卵转阴率、减少率分别为83.3%、90.0%,对成虫的驱净率和驱虫率分别为91.7%、93.8%,对童虫的驱净率和驱虫率分别为83.3%、90.1%;10、15mg/kg剂量对肝片吸虫虫卵转阴率、减少率及成虫、童虫的驱净率和驱虫率均达100%;②三氯苯咪唑混悬剂10mg/kg剂量与同剂量三氯苯咪唑片剂的驱虫效果无明显差异;③三氯苯咪唑混悬剂3个剂量组试验牛未见异常反应。试验证明三氯苯咪唑混悬剂10、15mg/kg剂量驱除牦牛肝片吸虫高效安全,临床使用以10mg/kg剂量为宜。     

Determining the Prevalence and Seasonality of Fasciola hepatica in Pasture-based Dairy herds in Ireland using a Bulk Tank Milk ELISA

Background

Fasciola hepatica is a helminth parasite of global importance in livestock, with major economic impact. However information on F. hepatica infections in Irish pasture-based dairy herds is limited. Therefore this study was conducted in order to determine the prevalence, seasonality and management factors associated with F. hepatica. A total of 319 Irish dairy herds were selected for this study. Bulk tank milk (BTM) samples were collected from 290 dairy farms on a quarter year basis, while from a further 29 dairy farms BTM samples were collected on a monthly basis to provide a more detailed pattern of F. hepatica exposure in Irish herds. BTM samples were analysed using a commercially available F. hepatica antibody detection ELISA. Furthermore, within-herd prevalence of F. hepatica was assessed in a subset of these 29 herds (n = 17); both individual serum samples and bulk tank milk samples were collected.

Results

A within-herd prevalence of ≤ 50 % was found for herds with negative bulk tank milk samples. The mean prevalence of the 290 study herds was 75.4 % (Range 52 %–75.1 %), with the highest prevalence being observed in November (75.1 %). The seasonal pattern of F. hepatica shows elevated antibodies as the grazing season progressed, reaching a peak in January. A significant association was found between F. hepatica and age at first calving.

Conclusion

This study demonstrates that F. hepatica is present in a large proportion of Irish dairy herds and provides a basis on which control practices, particularly in adult dairy cows, can be reviewed.     

肝片吸虫对奶牛生理生化水平的影响及分析

[目的]试验选择济南地区3个奶牛场中5头感染肝片吸虫的奶牛和5头健康奶牛为试验动物,对感染肝片吸虫的奶牛生理生化指标进行了探讨。[方法]通过对奶牛体温、呼吸、心率和血液中红细胞、白细胞、血红蛋白和红细胞压积的测定来观察奶牛机体生理指标的影响;通过对血清中血糖、谷丙转氨酶和总胆红素的测定来观察肝片吸虫对奶牛血液生化指标的影响。[结果]感染肝片吸虫的奶牛体温、呼吸和心跳与对照组相比显著升高,血液红细胞总数、红细胞压积、血红蛋白降低,白细胞总数升高;血清中血糖、谷丙转氨酶和总胆红素的含量与健康组相比显著升高。[结论]肝片吸虫对奶牛的生理和生化水平都有一定程度的影响。     

实验肝片吸虫感染对大鼠机体自由基代谢影响的研究

通过大鼠感染肝片吸虫复制感染模型 ,研究肝片吸虫感染后血清抗氧化功能的变化。 60只大鼠随机分成感染组和对照组 ,每组 30只。感染组大鼠一次口服 2 5个囊蚴 ,对照组不感染。于感染前 (0周 )和感染后 (1 ,3,5,7,9周 )采集血样 ,检测感染后谷胱甘肽过氧化物酶(GSH Px)、超氧化物歧化酶 (SOD)、过氧化物酶 (CAT)活性和丙二醛 (MDA)含量的变化。结果表明大鼠感染肝片吸虫后 ,感染组血清中GSH Px先升高后下降 ,SOD活性无明显变化 ,CAT活性在感染后下降 ,MDA含量在前 5周变化不明显 ;说明自由基参与了肝片吸虫病的发病过程     

肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

肝片吸虫免疫显性抗原基因的筛选及鉴定

为筛选出特异的肝片吸虫诊断候选抗原,拓展诊断靶标,利用肝片吸虫阳性血清筛选肝片吸虫cDNA表达文库,获得肝片吸虫特异性抗原基因,采用RT-PCR技术扩增目的基因,连接表达载体pET-32a(+),构建重组质粒,转化入感受态细胞BL21(DE3)中,利用IPTG对重组蛋白进行诱导表达,通过SDS-PAGE及Western blot技术对蛋白表达情况进行鉴定。结果显示:经筛选获得了17个肝片吸虫免疫显性抗原基因,其中假定蛋白Fh010935为肝片吸虫特异性抗原基因;扩增得到的Fh010935核苷酸序列大小为144 bp,编码48个氨基酸,A+T含量为55.56%;Fh010935蛋白由1个α-螺旋,2个β-折叠和3个β-转角构成,具有3个抗原表位,推测该蛋白可能具有较好的抗原性;重组蛋白主要以包涵体形式表达,分子量大小约24 ku,可以被肝片吸虫感染阳性血清特异性识别,具有较好的反应原性。提示:假定蛋白Fh010935可作为肝片吸虫病诊断候选抗原,为疾病诊断制剂的开发提供前期基础。     

实验感染肝片吸虫山羊的病理生理学——Ⅲ.细胞因子水平的变化

试验选择6只健康白山羊（经粪便学检查和Dot-ELISA检测，确认无肝片吸虫感染）随机分成Ⅰ、Ⅱ、Ⅲ组，其中Ⅰ、Ⅱ组山羊每只分别1次口服接种200、500个肝片吸虫囊蚴，Ⅲ组作为不感染对照组，每周颈静脉采血1次，连续11周，检测肝片吸虫感染后山羊血清中IL-2、TNF-α水平的变化和外周血淋巴细胞分泌IL-2的能力。结果表明，山羊试验感染肝片吸虫后，Ⅰ组山羊血清IL-2水平在感染后有所下降，而Ⅱ组山羊血清IL-2水平在感染后有所升高；感染不同剂量肝片吸虫的山羊血清TNF-α水平和淋巴细胞IL-2分泌水平均有不同程度的升高，在感染后第1周淋巴细胞就有反应，并且对非特异性刺激原和特异性刺激原的应答都有显著升高。这一结果提示，TNF-α可能参与肝片吸虫对肝的损伤过程，肝片吸虫感染剂量对宿主的IL-2含量影响较大，同时IL-2在宿主防御免疫中有重要作用。     

绵羊肝片吸虫谷胱甘肽硫转移酶基因的克隆与序列分析

本研究扩增肝片吸虫(Fasciola hepatica,Fh)谷胱甘肽硫转移酶(GST)基因,并进行同源性分析。根据GenBank发表的部分Fh GST基因序列设计并合成一对特异性引物,利用RT-PCR方法扩增出Fh GST基因完整的开放阅读框(open reading frame,ORF),测定序列,使用分子生物学软件进行同源性分析。获得Fh GST基因全长682 bp,编码218个氨基酸,与澳大利亚分离的肝片吸虫GST同源性较高,与大片吸虫和卫氏并殖吸虫的GST也有较高的同源性。不同虫株GST基因具有较高的同源性,因此Fh GST蛋白不适合用作诊断抗原,但由于其存在交叉反应,GST基因作为分子疫苗的候选基因具有重要意义。肝片吸虫GST基因的克隆,为进一步研究GST蛋白的功能和作用奠定了基础。     

Fasciola hepatica infection in farmed emus (Dromaius novaehollandiae)

Objective To describe two cases of infection with Fasciola hepatica i n young farmed emus, subacute and chronic fasci-olosis and a response to treatment of the flock with albenda-zole.
Procedure Gross lesions were found at necropsy and hepatic lesions in microscopic examination. The parasite recovered from one emu was identified by its morphological characteristics and an egg count reduction test was carried out after treatment of the flock with albendazole.
Results Hepatic lesions resembling subacute and chronic fasciolosis of ruminants were identified. An adult fluke was recovered from the liver of one of the birds and was identified as F hepatica . The eggs of the fluke were irregular in shape and size. No fluke eggs were identifiable in faeces of live emus 10 days after treatment of the flock with albendazole at a dose of 10 mg/kg.
Conclusions This is the first reported case of infection with F hepatica i n farmed emus and the first report of the occurrence of Fasciola infection is the class Aves. The irregular shape and size of the eggs may be attributable to infection of an aberrant host. Treatment with albendazole eliminated eggs from the faeces of the flock.     

一氧化氮（NO）在山羊感染肝片吸虫过程中作用

8～12月龄雄性山羊(n=5)人工感染肝片吸虫后,每周定时颈静脉采血1次,用硝酸还原酶法检测血清中一氧化氮(NO)的动态变化,并观察了NO与IgG抗体、嗜酸性粒细胞(EOS)之间的相关性.结果,山羊在感染后第1周,NO含量明显低于对照组,第2～6周或高或低呈波动趋势,自第6周后明显高于对照组,其中第7～8周达高峰,即(93.73±17.233)μmol/L,第7周与对照组相比P<0.01,第8、10周P<0.05;IgG抗体用间接ELISA最早在感染后第2周就可检出,并从第4周起保持高水平,第8～9周达到最高峰,血清中NO含量与IgG水平的相关系数为r＝0.511(P<0.05),而对照组的NO与IgG相关系数为r＝0.25(P>0.05);感染山羊在整个试验期间的EOS明显升高,其中第8周达峰值(P<0.01).本试验结果表明,NO、IgG和EOS在山羊感染肝片吸虫过程中的变化趋势基本一致并呈正相关,且与虫体在宿主体内的移行、发育密切关联,显示NO、IgG和EOS共同参与宿主抗肝片吸虫感染的作用.     

Closantel缓释剂驱除山羊实验感染肝片吸虫的效果

选用 5只实验感染肝片吸虫山羊进行 Closantel(氯氰碘柳胺 )缓释剂驱虫试验。试验用山羊每只一次性口服肝片吸虫囊蚴 15 0个 ,感染后第 15周随机将山羊分成对照组 (n=2 )和驱虫组 (n=3) ,驱虫组每只羊口服 2丸 Closantel缓释剂 (含 15 .5 % Closantel,每丸 7.6 g) ,每周定时采集颈静脉血和进行粪便检查 ,每月称重 ,试验结束时剖检测量肝重和计数肝内虫体数 ,以驱虫前后山羊的红细胞、白细胞、嗜酸性粒细胞、血清抗体和一氧化氮的变化、体重变化、剖检时肝重 /体重之比、虫卵的变化以及虫体减少率作为衡量驱虫效果的指标。结果表明 ,投药后嗜酸性粒细胞在第 1、2、3周与对照组相比有显著下降 ,其他各项指标与对照组相比虽有一定差异但不显著。虫体减少率为 4 1.2 2 %。提示Closantel缓释剂对驱除实验感染肝片吸虫有一定效果 ,在虫体感染前投药会起到预防作用。     

苏皖水牛肝片吸虫和前后盘吸虫感染情况的调查

苏皖两省１０县（市）共调查３０２农户的３０７头水牛,粪便学检查肝片吸虫阳性４３头,阳性率１４．０％,其中安徽的阳性率为１１．６１％（１８／１５５）,江苏阳性率为１６．４８％（２５／１５２）;母牛阳性率为１１．８％,公牛为１１．４％;前后盘吸虫阳性１７０头,阳性率为５５．４％（１７０／３０７）,其中安徽的阳性率为５８．１％（９０／１５５）;江苏的阳性为５２．６％（８０／１５２）,母牛的阳性率为６０．１％（１３７／２２８）,公牛的阳必国４１．８％（３３／７９）,两种吸虫的阳性率均呈现随年龄增加而提高的趋势,经统计学分析,两种吸虫的感染与地形无显著差异（Ｐ〉０．０５）,肝片吸虫的感染与性别、年龄亦无显著差异（Ｐ〉０．０５）,但对前后盘吸虫来说,感染与性别、年龄的差异显著（Ｐ〈０．０５）。而各县间两种吸虫感染则有显著关     

肝片形明虫 长菲策吸虫及日本血吸虫DNA的多态性检测

本试验用随机扩增DNA多态性技术对肝片形吸虫、长菲策吸虫和日本血吸虫虫体总DNA的多态性进行检测，结果筛选出三条具有种鉴别意义的RAPD谱带。这些特征性条带可望标记成探针用于粪便学检查中虫卵的鉴定。     

两种片形吸虫感染家兔的试验比较

用大片形吸虫和肝片形吸虫感染家兔以便选择大片形吸虫对动物的最佳感染量,及明确肝片形吸虫和大片形吸虫的生物学和对动物宿主的致病力的差别。结果显示肝片形吸虫虫体在兔体内发育成熟的时间早于大片形吸虫,感染成活率更高,对动物的病理损害明显比感染大片形吸虫兔的病变要轻。本试验证实这两种片形吸虫除了形态学的差异外,在对动物致病力、病理损害等方面确实存在差别。     

实验感染肝片吸虫山羊的病理生理学Ⅱ.血清NO和花生四烯酸代谢物的变化

选择 9只 6～ 10月龄健康雄性白山羊 ,经粪便检查和 Dot- EL ISA检测 ,确认无肝片吸虫感染 ,随机分成感染组(n=5 )和对照组 (n=4 ) ,试验组每只一次性口服接种 15 0个肝片吸虫囊蚴 ,感染前 (0周 )至感染后 2 3周每周从颈静脉采集血液 1次 ,分离血清 ,测定一氧化氮含量和花生四烯酸代谢物 6 -酮 -前列腺素 F1α(6 - keto- PGF1α)和血栓素 B2(TXB2 )的变化。结果表明 ,肝片吸虫感染后第 6～ 2 1周试验组血清中的 NO一直高于对照组 ,并在第 7、10周时显著地高于对照组 ;血浆中 TXB2 水平变化较为明显 ,在 1～ 3周与对照组相比无显著差异 ,从第 5周开始到试验结束 ,试验组血浆中 TXB2 水平均显著或极显著地高于对照组 ;在整个试验期间 ,血浆中 6 - keto- PGF1α水平变化不显著。这一结果提示 ,NO及花生四烯酸代谢物可能参与山羊肝片吸虫病的发生发展过程。     

Serum-gamma-glutamyltranspeptidase (gamma-GT) and aspartate-aminotransferase (AspAT) activities in adult cattle with chronic Fasciola hepatica infection

Sera from adult cattle with fluke-infected and normal livers were analyzed for γ-GT and AspAT activities. The analyses revealed no significant increases in either γ-GT or AspAT values of the animals with fascioliasis as compared with the non-infected animals. The advanced chronic stage of the infection characterized by hepatic fibrosis, limited injury of parenchyma and low fluke burdens may explain the lack of enzymatic increase.     

Wild fallow deer (Dama dama) as definitive hosts of Fasciola hepatica (liver fluke) in alpine New South Wales

To determine the extent to which wild deer are contributing in the transmission of Fasciola hepatica (liver fluke) livers from deer shot by hunters, farmers undertaking population control on their farms and vertebrate pest controllers were collected and frozen. The livers were later thawed, sliced and examined for the presence of adult flukes or evidence of past infection. Livers from 19 deer were examined (18 fallow [Dama dama] and one sambar [Rusa unicolor]). Seventeen of the fallow deer were animals collected on farms near Jindabyne, New South Wales. The remaining fallow deer was collected in the Australian Capital Territory and one sambar deer was collected in north-eastern Victoria. Nine of the 17 deer (53%) from the Jindabyne area were either infected with Fasciola hepatica (liver fluke) or had thickened bile ducts indicating past infection. Infection levels in the infected animals varied widely from 3 liver fluke to over 50 per liver. No sign of infection was present in the deer from the Australian Capital Territory or Victoria. Fallow deer are wide-spread in the Jindabyne area and their population is increasing. It is likely their contribution to the maintenance and distribution of F. hepatica to livestock in the Jindabyne area, and in other livestock rearing areas of south-eastern Australia, is important and increasing.