Low-cost molecular methods to characterise gastrointestinal nematode co-infections of goats in Africa.

BACKGROUND: Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. METHODS: Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and 'nemabiome' internal transcribed spacer 2 (ITS-2) amplicon sequencing. RESULTS: Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. CONCLUSIONS: These data represent the first 'nemabiome' sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas.

Assessment of anthelmintic potentials of Myrtus communis against Haemonchus contortus and Heligmosomoides polygyrus.

Anthelmintic resistance in grazing livestock systems has been spreading worldwide in prevalence and severity. Therefore, alternative measures including the use of herbal anthelmintic is considered as one of the successful approaches for the control of anthelmintic resistance. In the present report, we describe the chemical constituents of Myrtus communis essential oil, its in vitro anthelmintic effect against the most pathogenic gastrointestinal parasite of sheep; Haemonchus contortus and its in vivo anthelmintic potential using an in vivo gastrointestinal parasite model of rodents; i.e. Heligmosomoides polygyrus. Chromatographic analyzes of the essential oil (EO) extracted from the leaves of M. communis have shown that this oil was composed mainly of a α-pinene (33.59%), eucalyptol (23.85%) and limonene (14.70%). Regarding the in vitro anthelmintic potential, the ovicidal effect was confirmed in an egg hatch inhibition assay at IC50 = 0.7 mg/mL and with 95.83% of immobility of adult worm's after 8 h of exposure to 2 mg/mL of M. communis EO. The anthelmintic capacity of M. communis EO was also confirmed by in vivo assays conducted against the murine parasite H. polygyrus. In fact, at 1200 mg/kg bw of M. communis EO, a reduction of 99.70% in faecal egg counts was observed after 7 days of oral treatment, together with a 71.12% reduction in total worm counts. Based on the obtained results, M. communis EO showed relevant in vitro and in vivo anthelmintic effects against gastro-intestinal parasites.

The production costs of Haemonchus contortus and other nematode parasites in pre-weaned beef calves in New Zealand.

Haemonchus contortus can frequently be found infecting pre-weaned beef calves on sheep and beef farms around the North Island of New Zealand. The purpose of this study was to consider whether the presence of this parasite alone, or as part of a mixed infection, could be impacting growth rates of young animals, on three commercial farms in the North Island of New Zealand. Trials were conducted on commercial sheep and beef farms in each of the Northland, King Country and Gisborne regions, in late summer/autumn (February to April) of 2016 to measure the effect of treatment with narrow and broad spectrum anthelmintics on liveweight gain of spring-born calves pre-weaning. Each farm was chosen based on the presence of Haemonchus and that it was a beef cow/calf system with the cows and calves grazing the same pastures as sheep at some stage. Three sampling visits were made to each farm with the animals being weighed, faecal sampled and treated with one of two anthelmintics (Closantel alone to remove only Haemonchus or a triple combination containing moxidectin, levamisole and oxfendazole to remove all nematodes) or left untreated, on each of the first two visits. There was no significant difference in liveweight gain between any of the treatment groups, hence there was no evidence for an impact of Haemonchus alone, or a mixed nematode infection, on pre-weaned calf growth rates on these farms. It remains unclear whether there may be a justification to consider treatment of calves should they constitute a significant source of pasture larval infestation with H. contortus, in an integrated cattle-sheep system.

Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples.

BACKGROUND: Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR. RESULTS: DNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €). CONCLUSIONS: The Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL-1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

Combination of synthetic anthelmintics and monoterpenes: Assessment of efficacy, and ultrastructural and biophysical properties of Haemonchus contortus using atomic force microscopy.

The resistance of Haemonchus contortus to synthetic anthelmintics is of increasing concern; and different strategies are being evaluated to improve parasite control. The present study investigated the in vitro effects of combinations of synthetic compounds and monoterpenes. Additionally, the chemical association of the best combinations and their impact on the ultrastructural and biophysical properties of H. contortus eggs was evaluated. We assessed the efficacy of the monoterpenes, carvacrol, thymol, r-carvone, s-carvone, citral, and p-cymene and the anthelmintics, albendazole and levamisole using the egg hatch test (EHT) and the larval migration inhibition test (LMIT), respectively. The minimum effective concentrations of the monoterpenes, according to the EHT (efficacy ranging from 4.4%-11.8%) and LMIT (efficacy ranging from 5.6%-7.4%), were used in combination with different concentrations of synthetic compounds, and the IC50 and synergism rate (SR) were calculated. Fourier-transform infrared spectroscopy (FTIR) was used to analyze the chemical association between the best combinations as revealed by the in vitro tests (albendazole and levamisole with r-carvone or s-carvone). Atomic force microscopy (AFM) was used to assess the ultrastructural and biophysical properties of H. contortus eggs treated with the albendazole and r-carvone combination. Among the monoterpenes, the highest efficacies were exhibited by carvacrol (IC50 = 185.9 µg/mL) and thymol (IC50 = 187.0 µg/mL), according to the EHT, and s-carvone and carvacrol (IC50 = 1526.0 and 1785.3 µg/mL, respectively), according to the LMIT. According to the EHT, albendazole showed a slight statistically significant synergism in combination with r-carvone (SR = 3.8) and s-carvone (SR = 3.0). According to the LMIT, among the monoterpenes, r-carvone (SR = 1.7) and s-carvone (SR = 1.7) showed an increase in efficacy with levamisole; however, this was not statistically significant. The FTIR spectra of albendazole and levamisole, in association with r-carvone and s-carvone, indicated the presence of chemical interactions between the synthetic and natural molecules, contributing to the possible synergistic effects of these associations. Eggs treated with albendazole and r-carvone showed an increase in roughness and a decrease in height, suggesting that the treatment induced damage to the egg surface and an overflow of its internal contents. Overall, the combination of albendazole with r-carvone and s-carvone was efficacious against H. contortus, demonstrating a chemical association between the compounds; the significant changes in the egg ultrastructure justify this efficacy.

Assessment of the F200Y mutation frequency in the ß tubulin gene of Haemonchus contortus following the exposure to a discriminating concentration of thiabendazole in the egg hatch test.

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.

Pharmacokinetic-pharmacodynamic assessment of the ivermectin and abamectin nematodicidal interaction in cattle.

In a context of nematodicidal resistance, anthelmintic combinations have emerged as a reliable pharmacological strategy to control gastrointestinal nematodes in grazing systems of livestock production. The current work evaluated the potential drug-drug interactions following the coadministration of two macrocyclic lactones (ML) ivermectin (IVM) and abamectin (ABM) to parasitized cattle using a pharmacokinetic/pharmacodynamic (PK/PD) approach. The kinetic behavior of both compounds administered either separately or coadministered was assessed and the therapeutic response of the combination was evaluated under different resistance scenarios. In the pharmacological trial, calves received a single subcutaneous (s.c.) injection of IVM (100 µg/Kg); a single s.c. injection of ABM (100 µg/Kg) or IVM + ABM (50 µg/Kg each) administered in different injection sites to reach a final ML dose of 100 µg/Kg (Farm 1). Plasma samples were taken from those animals up to 20 days post-treatment. IVM and ABM plasma concentrations were quantified by HPLC. A parasitological trial was carried out in three farms with different status of nematodes resistance to IVM. Experimental animals received IVM (200 µg/Kg), ABM (200 µg/Kg) or IVM + ABM (100 µg/Kg each) in Farm 2, and IVM + ABM (200 µg/Kg each) in Farms 3 and 4. The anthelmintic efficacy was determined by fecal egg count reduction test (FECRT). PK analysis showed similar trends for IVM kinetic behavior after coadministration with ABM. Conversely, the ABM elimination half-life was prolonged and the systemic exposure during the elimination phase was increased in the presence of IVM. Although IVM alone failed to control Cooperia spp., the combination IVM + ABM was the only treatment that achieved an efficacy higher than 95% against resistant Cooperia spp. in all farms. In fact, when Cooperia spp. was the main genus within the nematode population and Haemonchus spp. was susceptible or slightly resistant to ML (Farms 2 and 4), the total FECR for the combination IVM + ABM was higher than 90%. Instead, when the predominant nematode genus was a highly resistant Haemonchus spp. (Farm 3), the total FECR after the combined treatment was as low as the single treatments. Therefore, the rational use of these pharmacological tools should be mainly based on the knowledge of the epidemiology and the nematode susceptibility status in each cattle farm.

Assessment of biophysical properties of Haemonchus contortus from different life cycle stages with atomic force microscopy.

Atomic force microscopy (AFM) was used in this work to investigate the ultrastructural and mechanical characteristics of Haemonchus contortus, the major gastrointestinal nematode that infects small ruminants worldwide. The biophysical characterization of this species is extremely important in order to reveal mechanisms of action of drugs and to classify its ultrastructure and biomechanical properties. High-resolution topographic images by AFM as well as data on biomechanical properties of cuticles were obtained at different stages of H. contortus. The results reveal details of the mechanical and structural properties of this nematode never observed before for nematodes parasite with other microscope techniques. Qualitative and quantitative reductions in the elasticity of the larvae stage egg were compared with those of the morulae stage, and the increased adhesion of unsheathed L3 were compared with the same stage of sheathed larvae. The results presented here open possibilities for understanding the mechanisms of drug and biomolecular actions that can be used to control infections caused by H. contortus.

Genetic parameter estimates for gastrointestinal nematode parasite resistance and maternal efficiency indicator traits in Santa Inês breed.

Infection with Haemonchus contortus is the most economically important gastrointestinal nematode parasitosis and the most important cause of mortality in sheep production. The aim of this study was to estimate variance components of gastrointestinal parasite resistance traits, maternal efficiency (ME) and ewe adult weight (AW) in Santa Inês breed in tropical conditions. The phenotypic records were collected from 700 animals, belonging to four flocks located south-east and north-east in Brazil. The evaluated traits were as follows: degree of anaemia assessed using the FAMACHA chart (FMC), haematocrit (HCT), white blood cell (WBC), red blood cell (RBC), haemoglobin (HGB), platelets (PLT), faecal egg count (EPGlog ), ME, metabolic maternal efficiency (MME), AW and metabolic ewe adult weight (MAW). From the 700 animals, 576 (82% of the evaluated population) were genotyped with the Ovine SNP12k BeadChip (Illumina, Inc.). Markers with unknown genomic position, located on sex chromosomes, monomorphic, with minor allele frequency <0.05, call rate <90% and with excess heterozygosity were excluded. The variance components were estimated using a single-trait animal model with ssGBLUP procedure. The correlation between the parasite's resistance indicators and the ME suggested that selecting animals with both higher adult weight and ME will also favour the selection of animals with better resistance to gastrointestinal nematodes parasites, specially H. contortus. Therefore, since there are few or no studies with Santa Inês breed in this area, it is important to study those traits to better manage selection programs.

Assessment of P-glycoprotein gene expression in adult stage of Haemonchus contortus in vivo exposed to ivermectin.

The efflux transporter P-glycoprotein (P-gp) has been implicated in multidrug resistance of different nematode parasites affecting livestock species. Increased expression of P-gp in nematodes after their in vitro as well as in vivo exposure to anthelmintics suggests a role of P-gp in drug resistance. The current study evaluated the P-gp gene expression in a highly-resistant isolate of the sheep nematode Haemonchus contortus, selected after exposure to ivermectin (IVM) treatments at 10-fold the therapeutic dose. Four lambs were artificially infected with L3 (7000 L3/animal) of a previously selected IVM highly resistant H. contortus isolate. Forty five (45) days after infection, adult worms were collected at 0 (untreated), 6, 12 and 24 h post-oral IVM (2 mg/kg) administration. The relative transcription levels of different H. contortus P-gp genes were studied by quantitative real-time PCR (qPCR) and confirmed by RNA-seq. P-gp1 and P-gp11 gene expressions did not change throughout the experimental sampling period. P-gp3 and P-gp9.1 transcripts decreased significantly at both 12 and 24 h post IVM exposure. P-gp2 expression was progressively increased in a time-dependent manner at 1.81 (6 h), 2.08 (12 h) and 2.49 (24 h)-fold compared to adult worms not exposed (control 0 h) to IVM, although without reaching statistically significant differences (P > 0.05). P-gp12 was neither detected by qPCR nor by RNA-seq analysis. These relatively modest changes in the P-gp gene expression could not be enough to explain the high level of IVM resistance displayed by the H. contortus isolate under assessment. Overexpression of membrane drug transporters including P-gp has been associated with IVM resistance in different nematode parasites. However, some evidences suggest that resistance to IVM and other macrocyclic lactones may develop by multiple mechanisms. Further studies are needed to improve the understanding of resistance mechanisms in adult stages of H. contortus.

Economic viability of anthelmintic treatment in naturally infected beef cattle under different nutritional strategies after weaning.

The aim of the present study was to evaluate the effects of treatment with different anthelmintic compounds on the productivity of naturally infected calves and the economic viability of these treatments within extensive breeding systems employing different nutritional strategies after weaning. For this purpose, 4 farms with 42-60 calves naturally infected with gastrointestinal nematodes were selected. The calves were distributed into 6 groups (7-10 animals each) per farm and treated with ivermectin 1%, ivermectin 3.15%, eprinomectin 5%, levamisole 7.5%, albendazole 15%, and control group (no treatment). These animals were evaluated over an experimental period of 150 days. Levamisole 7.5% presented the best capacity for the reduction of eggs per gram (EPG) of feces in all herds evaluated, followed by albendazole 15% and eprinomectin 5%. Parasite resistance to multiple drugs was found in all herds, especially those of Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. For farm 1, differences in weight gain and EPG reduction percentages led to a difference of US$285.06 between the levamisole and ivermectin 3.15% groups. Similar findings were noted for the levamisole and ivermectin 1% groups of farm 3, with a difference of US$399.37 because of the final weight gain in these groups. For farms 2 and 4, the ivermectin 3.15% and control groups, respectively, were the most profitable; these unexpected results were possibly influenced by variables not measured during the experimental period. This study suggested that anthelmintic treatments should always precede an efficacy test, once they are demonstrated to be most profitable under adequate breeding conditions, to ensure adequate control of gastrointestinal nematode infection.

Assessment of oxidative/nitrosative stress biomarkers and DNA damage in Haemonchus contortus, following exposure to zinc oxide nanoparticles.

Drug resistance in helminth parasites has incurred several difficulties to livestock industry and ranked among the top public health concerns. Therefore, seeking for new agents to control parasites is an urgent strategy. In the recent years, metallic nanoparticles have been considerably evaluated for anthelmintic effects. The current research was conducted to assess possible anthelmintic impacts of zinc oxide nanoparticles (ZnO-NPs) on a prevalent gastrointestinal nematode, H. contortus. Moreover, several biomarkers of oxidative/nitrosative stress and DNA damage were measured. Various concentrations of the nanoparticle were prepared and incubated with the worms for 24 hours. The parasite mobility, mortality rate, antioxidant enzymes activities (SOD, Catalase and GSH-Px), lipid peroxidation, total antioxidant status as well as nitric oxide (NO) contents and DNA damage were determined. ZnO-NPs exerted significant wormicidal effects via induction of oxidative/nitrosative stress and DNA damage. Conclusively, ZnO-NPs can be utilized as a novel and potential agent to control and treatment of helminth parasitic infections.

Assessment of anthelmintic activity and bio-guided chemical analysis of Persea americana seed extracts.

The aim of this study was to characterize the extracts and fractions of Persea americana Mill. (Avocado) seeds and to determine the composition and the in vitro anthelmintic activity against third-stage larvae (L3) of Haemonchus contortus. The fresh (F) and dried (H) avocado seeds (PA) were subjected to extraction with hot water (W-PAF, W-PAH), ethanol (E-PAF, E-PAH) or methanol 70% (v/v), and partition with solvents of increasing polarity [n-hexane (H-PAF, H-PAH), chloroform (C-PAF, C-PAH), ethyl acetate (Ea-PAF, Ea-PAH), and n-butanol (B-PAF, B-PAH)], yielding a total of 14 extracts/fractions. After considering the yield, water solubility, and the preliminary results of the larval migration test (LMT), the E-PAF, E-PAH, H-PAF, and H-PAH were selected for further experiments. E-PAH presented an efficiency concentration of 50% (EC50) of 36 µg/mL on the LMT. E-PAH showed the greatest efficiency when its EC50 was compared to the other fractions (E-PAF = 147 µg/mL; H-PAF = 801 µg/mL; H-PAH = 77 µg/mL). After that, the E-PAH was chemically characterized, considering its quantitative polyphenolic and flavonoid contents by colorimetric and chromatographic techniques. E-PAH presented 50, 38, and 24 mg/g of dry matter of total phenol, condensed tannins (CT), and flavonoid contents, respectively. Using high performance liquid chromatography (HPLC) analysis, E-PAH had shown to have epicatechin (4.7 µg/mL), rutin (2.8 µg/mL), and chlorogenic acid (1.4 µg/mL) as its main constituents besides quercetin. These isolated compounds were evaluated using the LMT in order to relate the composition to the anthelmintic activity observed for E-PAH. Quercetin (EC50 = 7.8 µg/mL) and epicatechin (EC50 = 10 µg/mL) presented a higher efficiency than rutin (EC50 = 30 µg/mL). Chlorogenic acid was also tested with the LMT but did not present a significant efficiency. According to the results, the phenolic composition of E-PAH and the EC50 values obtained for the isolated phenols, it can be suggested that, besides the CT content, the presence of epicatechin and rutin contributed to the larvicidal activity of E-PAH. In conclusion, avocado seeds may be used as a source of polyphenols with promising anthelmintic applications.

Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts.

The larval exsheathment inhibition assay (LEIA) of infective larvae (L3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia pennatula leaves was used to perform respective LEIA tests with H. contortus L3 of different ages (1-7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40µg/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraíso) strain. In order to identify changes in L3 vitality on different weeks (1-7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC50, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6µg/mL) compared to older larvae (P<0.05). The EC50 and EC90 for weeks 2-5 were similar (P>0.05), while older larvae tended to show higher EC50 and EC90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥-0.83; P <0.05) and EC50 (r≥-0.80; P<0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age.

An assessment of benzimidazole resistance against caprine nematodes in Central India.

Current status of resistance to benzimidazole (BZ) group of anthelmintic drugs against caprine nematodes in Central India at Amanala goat farm, Jabalpur, Madhya Pradesh (M. P.), was systematically investigated using faecal egg count reduction (FECR) test and egg hatch test (EHT). Besides, allele-specific PCR (AS-PCR) was deployed to ascertain the susceptible genotype (alleles) especially of the Haemonchus contortus. Randomly selected 30 goats, irrespective of age and sex, were divided into three groups of 10 each, to serve as treated and untreated controls. It was ensured that the animals were not administered with an anthelmintic drug for the past 3 months prior to undertaking the study, and faecal egg counts were estimated. FECR test evidenced fenbendazole resistance by partial elimination (24.90%) copro-egg counts in the treated group of animals vis-à-vis controls with a lower confidence interval of -26%. Further, EHT revealed ED-50 value of 0.335 µg of thiabendazole/ml, confirming benzimidazole resistance in the animals of that farm. AS-PCR showed that 62% of H. contortus larvae were homozygous resistant (rr), 24% heterozygous (rS) and 14% homozygous susceptible (SS). The genotypic frequencies of three genotypes (rr, rS and SS) were significantly (P < 0.01) different. The prevalence of benzimidazole resistance allele (r) was also significantly (P < 0.01) higher (74%) as compared to susceptible allele (S) (26%). The resistance to benzimidazole has been discussed while emphasizing improved managemental practices designed to reduce exposure of the goat population to parasites, minimize frequency of anthelmintic use at optimum dose and rotational use of different chemical groups of medicines with different mode of action, so as to overcome and combat the upcoming problem in the field.

In vitro assessment of Argemone mexicana, Taraxacum officinale, Ruta chalepensis and Tagetes filifolia against Haemonchus contortus nematode eggs and infective (L3) larvae.

Argemone mexicana, Taraxacum officinale, Ruta chalepensis and Tagetes filifolia are plants with deworming potential. The purpose of this study was to evaluate methanolic extracts of aerial parts of these plants against Haemonchus contortus eggs and infective larvae (L3) and identify compounds responsible for the anthelmintic activity. In vitro probes were performed to identify the anthelmintic activity of plant extracts: egg hatching inhibition (EHI) and larvae mortality. Open column Chromatography was used to bio-guided fractionation of the extract, which shows the best anthelmintic effect. The lethal concentration to inhibit 50% of H. contortus egg hatching or larvae mortality (LC50) was calculated using a Probit analysis. Bio-guided procedure led to the recognition of an active fraction (TF11) mainly composed by 1) quercetagitrin, 2) methyl chlorogenate and chlorogenic acid. Quercetagitrin (1) and methyl chlorogenate (2) did not show an important EHI activity (3-14%) (p < 0.05); however, chlorogenic acid (3) showed 100% of EHI (LC50 248 µg/mL) (p < 0.05). Chlorogenic acid is responsible of the ovicidal activity and it seems that, this compound is reported for the first time with anthelmintic activity against a parasite of importance in sheep industry.

Experimental infection of calves with Haemonchus placei or Haemonchus contortus: Assessment of clinical, hematological and biochemical parameters and histopathological characteristics of abomasums.

The present study had the primary objective of evaluating clinical, hematological and biochemical parameters, as well as observing anatomical and histopathological characteristics of abomasums, from calves prime-infected with Haemonchus contortus or H. placei. Ten male Holstein newborns were subdivided in three groups (GI placebo; GII infected with H. contortus; GIII inoculated with H. placei). Eye mucosa staining was evaluated. Hematological and biochemical tests were performed on animals. The euthanasia of all ten experimental calves was performed on the 42nd day post-inoculation. Fragments were collected from each of all 10 abomasums for histopathological analysis. Discrete submandibular edema was diagnosed in animals from both infected groups (H. contortus or H. placei). However, there were no significant changes (P > 0.05) in the color of the ocular mucosa of calves from all three experimental groups across the entire experimental period. Hematological and biochemical changes diagnosed on animals could not be linked to infections by species of Haemonchus spp. Regarding histopathological exams, it was possible to diagnose hypertrophy, hyperplasia, binucleated cells, inflammatory infiltrate, multifocal hemorrhage and edema in abomasums from calves of both groups infected with H. placei and H. contortus. It can, thus, be concluded that not only are calves susceptible to infections by both Haemonchus species, but they can also present clinical changes and similar anatomic histopathological lesions independent of being infected by Haemonchus placei or Haemonchus contortus. These results reflect a negative effect on helminth control by mixed grazing between sheep and cattle, especially when using calves.

Metabolic profiling and in vitro assessment of anthelmintic fractions of Picria fel-terrae Lour.

Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.

Correlation of polyphenolic content with radical-scavenging capacity and anthelmintic effects of Rubus ulmifolius (Rosaceae) against Haemonchus contortus.

Phenolic content, antioxidant and anthelmintic activities of herbal extracts are of particular interest to drug industry; plant extracts with significant anthelmintic activity have the potential to be used as alternatives to conventional chemical drugs. In the present study, Rubus ulmifolius fruit extracts obtained using solvents of increasing polarity (water, methanol, chloroform and hexane) were examined for their antioxidant and anthelmintic activities in correlation with their polyphenolic content. In vitro antioxidant activity of all extracts was carried out using free radical-scavenging activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethilenebenzotiazolin)-6-sulfonic acid (ABTS) radical cation. In vitro anthelmintic activities were investigated on the egg and adult worms of Haemonchus contortus from sheep in comparison to albendazole. Total polyphenol content of R. ulmifolius was higher in more polar extract, ranging from 64.5 in aqueous extract to 1.57 mg gallic acid equivalents per gram of dry weight (GAE/g DW) in hexanic extract. Likewise, highest amounts of flavonoids and condensed tannins were found in aqueous extract (28.06 mg QE/g and 7.42 mg CE/g DW, respectively) compared to hexanic extract (0.71 mg QE/g and 0.29 mg CE/g DW, respectively) (p<0.05). Both DPPH and ABTS antioxidant assays showed that all tested extracts possess free radical scavenging activity, while the inhibitory concentration 50% (IC50) range values were similar for both assays (2.13-45.54 µg/mL and 1.2-43.82 µg/mL, respectively). All plant extracts showed ovicidal activity at all tested concentrations. Fruit methanolic (IC50=2.76mg/mL) and aqueous (IC50=2.08 mg/mL) extracts showed higher inhibitory effects than chloroformic (IC50=7.62 mg/mL) and hexanic (IC50=12.93 mg/mL) extracts on egg hatching (p<0.05). There was a significant correlation of total polyphenol, flavonoids and tannins content with scavenging of either DPPH (r=0.722, 0.764 and 0.752, p<0.01, respectively) or ABTS radicals (r=0.893, 0.765 and 0.722, p<0.01, respectively) and with inhibition of egg hatching (r=0.874, 0.883 and 0.862, p<0.01, respectively). Highest inhibition of motility (100%) of worms was observed 8h post-exposure in aqueous and methanolic extract at 8 mg/mL. To our knowledge, these results depict for the first time that R. ulmifolius possesses in vitro anthelmintic properties.