In vitro larval migration and kinetics of exsheathment of Haemonchus contortus larvae exposed to four tropical tanniniferous plant extracts

As for some temperate forage, some tropical tanniniferous plants (TTP) from browsing might represent an alternative to chemical anthelmintic. The anthelmintic effect of four TTP (Acacia pennatula, Lysiloma latisiliquum, Piscidia piscipula, Leucaena leucocephala) on Haemonchus contortus was measured using two in vitro assays. First, the effects of increasing concentrations of lyophilized extracts (150, 300, 600, 1200 microg/ml PBS) were tested on H. contortus larvae (L(3)) using the larval migration inhibition (LMI) test. An inhibitor of tannin, polyvinyl polypyrrolidone (PVPP), was used to verify whether tannins were responsible for the AH effect. Secondly, the effects of extracts on larval exsheathment were examined. Larvae (L(3)) were in contact with extracts (1200 microg/ml) for 3h, and then were exposed to an artificial exsheathment procedure with observations of the process at 10 min intervals. A general lineal model (GLM) test was used to determine the dose effect in the LMI test and the difference of the percentage of exsheathed larvae between the control and the treatment groups. A Kruskal Wallis test was used to determine the effect of PVPP on LMI results. The LMI test showed a dose-dependent anthelmintic effect for A. pennatula, L. latisiliquum and L. leucocephala (P<0.01), which disappeared after PVPP addition, confirming the role of tannins. No effect was found for P. piscipula on H. contortus in the LMI test. However, all four plant extracts interfered with the process of L(3) exsheathment which might be involved as a mechanism of action of tannins on H. contortus larvae. A. pennatula, L. latisiliquum and L. leucocephala could be used as an anthelmintic for the control of H. contortus after confirmation based on in vivo studies.     

A modified larval migration assay for detection of resistance to macrocyclic lactones in Haemonchus contortus, and drug screening with Trichostrongylidae parasites

We have developed a modified migration assay system in 96-well plate format which is able to detect resistance to the macrocyclic lactone group of drugs in Haemonchus contortus. The assay involves exposure of infective stage larvae to drug for a 24 h period, then counting the numbers of larvae that are able to migrate through an agar and filter mesh system over a further 48 h. The agar barrier greatly increased the sensitivity of the assay for resistance detection compared to use of filter mesh alone. The assay was able to detect the presence of 10% resistant worms in an otherwise susceptible background. However, the assay was ineffective with Trichostrongylus colubriformis and Ostertagia circumcincta indicating that its usefulness for field monitoring will be restricted to situations where H. contortus is of most significance. A small-scale drug screening exercise showed that the assay identifies some anthelmintic activities distinct from those identified by larval development assays. The assay therefore also has a potential role in drug discovery programmes in screening for new anthelmintics.     

Effect of molecular weight and concentration of legume condensed tannins on in vitro larval migration inhibition of Haemonchus contortus

The effect of molecular weight of condensed tannins (CT) from a variety of warm-season perennial legumes commonly consumed by sheep and goats on anthelmintic activity has not been previously explored. The objectives of this study were to determine if molecular weight of CT from warm-season perennial legumes could predict the biological activity of CT relative to anthelmintic activity against ivermectin resistant L3 stage Haemonchus contortus (HC) using a larval migration inhibition (LMI) assay. A second objective was to determine if CT from warm-season perennial legumes possess anthelmintic properties against L3 stage (HC). Lespedeza stuevei had the greatest concentration of total condensed tannin (TCT; 11.7%), whereas, with the exception of Arachis glabrata, a CT-free negative control, Leucaena retusa had the least TCT (3.3%). Weight-average molecular weight of CT ranged from 552 Da for L. stuevei to 1483 Da for Lespedeza cuneata. The treatments demonstrating the greatest percent LMI were L. retusa, L. stuevei and Acacia angustissima var. hirta (65.4%, 63.1% and 42.2%, respectively). The ivermectin treatment had the smallest percent LMI (12.5%) against ivermectin resistant L3 HC. There was a weak correlation (R² = 0.34; P = 0.05) between CT M_W and percent LMI, suggesting that molecular weight of CT is a weak contributing factor to CT biological activity as it relates to LMI of L3 stage HC. L. stuevei, L. retusa and A. angustissima var. hirta STP5 warrant further evaluation of anthelmintic properties in vivo.     

The requirement for early exposure of Haemonchus contortus larvae to Bacillus thuringiensis for effective inhibition of larval development

The potential for a nematocidal Bacillus thuringiensis (Bt) to target the free-living larval stages of Haemonchus contortus was examined using in vitro larval development and migration assays. Bt toxicity in larval development assays decreased as the time period between egg hatch and initial exposure to the Bt was increased; a time lag of 48 h resulted in a 350-fold increase in the IC(50) (from 2.6 ng/ml to 910 ng/ml). The effects on larval migration largely paralleled the effects on larval development, indicating that the larvae which reached the infective stage after exposure to Bt were generally as 'fit' as control worms in terms of migration ability. However, a comparison of the two assays also showed the presence of a level of Bt exposure which showed significantly more toxicity in migration assays than development assays, indicating that, in some cases, fully developed Bt-exposed larvae were less able to migrate than controls, and hence may be compromised in their ability to infect sheep. The rapid decrease in toxicity when exposure to the Bt is delayed highlights a significant issue concerning the use of Bt for control of the free-living larval stages of animal-parasitic nematodes. Targeting the larvae by delivering bacterial spores to the faeces through the host animal's digestive tract would require the spores to germinate upon defecation, grow through a vegetative phase, to then produce crystal toxin protein upon subsequent sporulation. This period of bacterial development will introduce a time lag between worm egg hatching and initial exposure of the larvae to the Bt, which, as demonstrated in the present study, may allow the worm larvae to develop to late larval stages which are relatively insensitive to the toxin.     

Critical controlled test to evaluate resistance of field strains of Haemonchus contortus to thiophanate

Following reports of suspected gastrointestinal strongylid nematode resistance to thiophanate in sheep and goats at Sokoine University of Agriculture (SUA), Tanzania, field strains of Haemonchus contortus, one from SUA and another from Kimamba, Tanzania, were selected for a critical controlled test to evaluate the efficacy of thiophanate. The SUA H. contortus strain in goats showed a marked resistance to thiophanate at an oral dose of 50 mg kg-1 liveweight (efficacy = 24.1%), while the Kimamba H. contortus strain showed very marked susceptibility (efficacy = 99.7%) to thiophanate at the same oral dose. This is the first report from East Africa of a field strain of H. contortus resistant to thiophanate. The incidence of resistant strains of strongyloid parasites to benzimidazole anthelmintics in sheep and goats in East Africa is on the increase. The magnitude of this problem should be evaluated and remedial solutions sought.     

A strain of Haemonchus contortus resistant to thiophanate

The occurrence of a field strain of Haemonchus contortus resistant to thiophanate is reported for the first time in New Zealand. In a controlled anthelmintic trial with experimentally infected animals, thiophanate at 50-100 mg/kg had no appreciable effect on the Haemonchus burden. Albendazole reduced faecal strongylate egg counts by 95% in animals with naturally acquired 59 infections.     

Susceptibility assay of Haemonchus contortus to commonly used anthelmintics in Jimma, southwest Ethiopia

An experimental study to investigate the susceptibility pattern of Haemonchus contortus to commonly used anthelmintics (albendazole, tetramisole, ivermectin, and triclobendazole) was conducted between June and September 2009. Adult H. contortus parasites were collected from a total of 30 sheep slaughtered at Jimma municipal abattoir. The anthelmintics were assessed for egg hatch inhibition ability against H. contortus eggs. The eggs (500 eggs/ml) were incubated with different concentrations (0.25, 0.125, 0.0625, 0.03125, and 0.015625?μg/ml) at 26°C for 48?h. Egg hatching inhibition of different anthelmintics at different concentrations was recorded. The overall mean percent inhibition showed that there was a significant difference (p?相似文献   

Interaction between the nematode-destroying fungus Arthrobotrys robusta (Hyphomycetales) and Haemonchus contortus infective larvae in vitro.

In an in vitro trial, the effect of the nematode-destroying fungus Arthrobotrys robusta on Haemonchus contortus infective larvae was evaluated in petri dishes containing corn meal agar. After seven days incubation at 25 degrees C, 92.33% (+/- 4.1) predation was recorded.     

Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus

The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 μg/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P<0.001) and A. gaumeri (P<0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L(3) larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage.     

Detection of benzimidazole resistance in Haemonchus contortus using RFLP-PCR technique

Benzimidazole (BZ) resistance in Haemonchus contortus is linked primarily with the mutation in the beta-tubulin isotype 1 gene that substitute phenylalanine (Phe) to tyrosine (Tyr) at 200 codon of the gene. In the present study, a new restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique has been developed for detection of BZ resistance in the beta-tubulin isotype 1 gene of H. contortus. The technique utilizes two primers viz. AvikaF and AvikaR to amplify the region containing mutation in the beta-tubulin gene followed by restriction digestion. After digestion, the 'rr' individuals (homozygous resistant) revealed 257 and 48 bp bands, the 'rS' individuals (heterozygous) showed 305, 257 and 48 bp bands, while 'SS' individuals (homozygous susceptible) revealed uncut 305 bp band. A total of 162 adult male H. contortus collected from Avikanagar, Jaipur and Bikaner regions (54 from each region) were genotyped for analyzing BZ resistance in the beta-tubulin gene. Out of which, 130 adults were 'rr' types, 20 'rS' types and 12 'SS' types. The results showed that genotypic frequencies of different genotypes (rr, rS and SS) were highly significant difference among the three regions (P<0.001). The 'rr' individuals were higher (98%) in Jaipur followed by Avikanagar (93%) and Bikaner (50%) regions. Overall, the prevalence of BZ resistant allele (r) was higher (86%) as compared to BZ susceptible allele (S) (14%). The technique was also found suitable for genotyping of larvae of H. contortus and yielded reproducible results. The study indicated that RFLP-PCR is an easy, reproducible and less expensive than allele specific PCR. This technique will be helpful in establishing the prevalence rate of BZ resistance in H. contortus and can also be utilized for existing worm control programme.     

The anthelmintic efficacy of thiabendazole and levamisole against inhibited Haemonchus contortus larvae in sheep

Fifty-five dogs, 6 to 18 months old, from 11 areas of New Zealand were examined for gastro-intestinal helminths. Thirty-eight (69.1%) were infected. Twenty-one (38.2%) had Toxocara canis, 20 (36.4%) Trichuris vulpis, 20 (36.4%) Uncinaria stenocephala, 3 (5.5%) Toxuscaris leonina, 3 (5.5%) Dipylidium caninum and 1 (1.8%) Taenia sp. Helminth infections were less frequent and populations were smaller in females than in males.     

捻转血矛线虫感染性三期幼虫gst基因的筛选及抗干燥功能分析

为研究干燥作用对捻转血矛线虫L3期幼虫mRNA表达的影响,筛选并分析干燥相关表达基因,本试验利用mRNA差异显示技术得到了捻转血矛线虫干燥相关基因,通过相对荧光定量PCR验证所获基因的表达情况,并在秀丽隐杆线虫中通过RNA干扰抑制相关同源基因的表达检测干燥后复苏虫体生存率以研究基因抗干燥功能。结果显示,差异表达序列GH-U02A与已知捻转血矛线虫谷胱甘肽-S-转移酶(GST)氨基酸同源性为94%。对秀丽隐杆线虫Ce-gst-7基因进行RNA干扰后,虫体在干燥后的生存率降低,证明gst基因在线虫中有抗干燥功能。     

Comparison of three in vitro techniques to estimate benzimidazole resistance in Haemonchus contortus of sheep

Three in vitro assays to detect benzimidazole resistance, namely, the egg-hatch assay, tubulin-binding assay, and a larval-development assay, were evaluated by estimating the level of benzimidazole resistance in three field isolates of Haemonchus contortus compared with a susceptible reference strain. Comparisons were also made with estimates of benzimidazole resistance of the three field strains obtained from an in vivo controlled anthelmintic efficacy test. All three in vitro tests showed similar, consistent results which also suggested greater sensitivity than the in vivo assay. These results indicate that selection of an in vitro technique to determine benzimidazole resistance should therefore be based on considerations other than precision, such as technical expertise, availability of equipment, cost and speed in which diagnosis is required.     

Changes in LC50 in an in vitro egg development assay during the patent period of Haemonchus contortus in sheep

Sera of various species inhibit the activity of haemolysin produced by Treponema hyodysenteriae. Low density and high density lipoprotein fractions from swine and human sera suppressed the haemolysin, with no single class of lipoproteins in swine serum showing predominant activity. Albumin, immunoglobulin G or blood sugars in swine serum showed no inhibitory activities.