Variation in allele frequencies in benzimidazole resistant and susceptible isolates of Haemonchus contortus during patent infection in lambs

We evaluated the variation in the frequency of benzimidazole (BZ) resistance-associated alleles at codons 200, 167 and 368 (F200Y, F167Y, V368L) of the β-tubulin isotype 1 gene during the patent period in isolates of Haemonchus contortus susceptible and resistant to BZ using pyrosequencing. Four lambs 5–6 months old were infected with 5000–6000 infective third-stage larvae (L3) of the susceptible MHco1 and the multi-resistant MHco4 isolates, respectively. Faecal samples were collected 28-times during 20–90 days post-infection (dpi). Coprocultures were subsequently prepared to produce L3 for genotyping. The frequency of the resistant allele (TAC) at codon 200 in MHco1 was lowest at 43 and 76 dpi with at each time point 0% and highest at 36 dpi with 10.85%, with a mean of 6.47% ± 2.39 and a coefficient of variation of 37.01%. The frequency of the TAC at codon 200 in MHco4 was lowest at 76 dpi with 25.6% and highest at 90 dpi with 49.25%, with a mean of 35.7% ± 4.42 and a coefficient of variation of 12.39%. No resistance alleles were detected in MHco1 at either codon 167 or 368. For MHco4 isolate, resistance alleles were detected only on codon 167 with a mean of 8.00% ± 4.83 and a mean coefficient of variation of 60.40%. Our results demonstrate the considerable variation in the frequency of resistant alleles in the susceptible and resistant isolates during the patent period. This variation should be considered when testing for the presence of BZ resistance in populations of gastrointestinal parasites, especially those with a low frequency of TAC

Multidrug resistance in Haemonchus contortus in sheep - can it be overcome?

Gastrointestinal nematodes pose a threat to animal health and affect farmers by negatively impacting farm management

Assessing the Efficacy of Albendazole against Fasciola hepatica in Naturally Infected Cattle by In Vivo and In Vitro Methods

This study was performed on a cattle farm with a long-term use of albendazole (ABZ) and a permanent history of fasciolosis for comparing in vivo and in vitro methods for the detection of anthelmintic resistance and drug efficacy. A selected group of 10 Charolais cows was treated in autumn 2020 with ABZ at a dose of 7.5 mg/kg body weight. Another group of 10 cows remained untreated as a control. The faecal egg count reduction test was used to determine in vivo efficacy. The percentage reduction of eggs on day 14 after treatment ranged from 77 to 81.8%, depending on the formula used for calculation. The in vitro egg hatch test (EHT) was used as a second diagnostic method. F. hepatica eggs for the EHT were isolated from faecal samples. The test was performed in two versions differing in the length of incubation with ABZ (12 h and 15 d). The percentage of eggs with inhibited development at a concentration of 0.5 μM in both versions of the EHT agreed with the in vivo results. Ovicidal activity at a concentration of 0.5 μM in the 12-h version suggested a reduced efficacy of ABZ (65.40%). An EHT prepared using pooled faecal samples was a prospective method for the detection of efficacy and ABZ resistance in F. hepatica

Seasonal Pattern of Prevalence and Excretion of Eggs of Baylisascaris transfuga in the Brown Bear (Ursus arctos)

The seasonal dynamics of the prevalence, abundance, and mean intensity of egg excretion by the nematode parasite Baylisascaris transfuga in the European brown bear (Ursus arctos) were monitored relative to environmental factors (mean temperature, humidity, and temperature) over three years. The prevalence, abundance, and mean intensity of egg excretion tended to increase from spring to autumn throughout the monitoring period. The seasonal prevalence (84.2–90.6%), abundance (470.2–545.3 eggs per gramme (EPG) of faeces), mean intensity of excretion (558.3–602.1 EPG), and number of eggs (1150 EPG) were highest in autumn. The prevalence of eggs (up to 48.5%), abundance (37.8–60.6 EPG), and mean intensity of excretion (94.4–125.0 EPG) were high in winter, despite the period of hibernation. The seasonal dynamics of B. transfuga abundance in bears, the mean temperature between spring and autumn, and the seasonal trend of increase in intensity of egg excretion with temperature from winter to summer were interrelated. Abundance differed significantly between winter and autumn, spring and autumn, and summer and autumn (p < 0.0001) in all years and between spring and summer in 2016 (p < 0.005), 2017 (p < 0.05). B. transfuga abundance differed significantly between the seasons over the three years only in spring (p ≤ 0.0001)

Assessing the Efficacy of Albendazole against <em>Fasciola hepatica</em> in Naturally Infected Cattle by In Vivo and In Vitro Methods

This study was performed on a cattle farm with a long-term use of albendazole (ABZ) and a permanent history of fasciolosis for comparing in vivo and in vitro methods for the detection of anthelmintic resistance and drug efficacy. A selected group of 10 Charolais cows was treated in autumn 2020 with ABZ at a dose of 7.5 mg/kg body weight. Another group of 10 cows remained untreated as a control. The faecal egg count reduction test was used to determine in vivo efficacy. The percentage reduction of eggs on day 14 after treatment ranged from 77 to 81.8%, depending on the formula used for calculation. The in vitro egg hatch test (EHT) was used as a second diagnostic method. F. hepatica eggs for the EHT were isolated from faecal samples. The test was performed in two versions differing in the length of incubation with ABZ (12 h and 15 d). The percentage of eggs with inhibited development at a concentration of 0.5 μM in both versions of the EHT agreed with the in vivo results. Ovicidal activity at a concentration of 0.5 μM in the 12-h version suggested a reduced efficacy of ABZ (65.40%). An EHT prepared using pooled faecal samples was a prospective method for the detection of efficacy and ABZ resistance in F. hepatica

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Considerable research has been directed towards optimising in vitro tests that can diagnose resistance in pre-parasitic stages of parasites. The objective of this study was to compare the in vivo faecal egg count reduction test (FECRT), the in vitro egg hatch test (EHT), and the molecular determination of the frequency of a codon 200 allele of β-tubulin isotype 1 associated with benzimidazole resistance in larval stages of Haemonchus contortus obtained from infected goats. Animals were infected with composite infective doses representing 10, 20, 30, 40, 60, and 80% resistant alleles. Faecal samples for the EHT were collected on 28, 33, and 35 days post-infection. The results of the in vivo FECRT indicated that albendazole treatment reduced infections consisting of composite doses of 10, 20, 30, 40, 60, and 80% larvae of the resistant isolate by 91.3, 78.0, 63.3, 48.4, 36.5, and 41.4%, respectively. The drug concentration at which 50% of the eggs were prevented from developing hatching larvae (ED50) in the in vitro EHT varied from 0.09 ± 0.01 to 15.63 ± 12.10 μg/mL thiabendazole. The results of the in vitro EHT indicated that the test could estimate in vivo resistance well. The EHT could thus accurately estimate the in vivo efficacy of the drug and percentage of the resistance allele in the population using hatching parameters in delineation doses. This finding was also supported by comparing the FECRT data to the hatching percentages in the EHT on 30 goat farms in Slovakia with natural mixed infections of gastrointestinal parasites

Anthelmintic Activity of Wormwood (Artemisia absinthium L.) and Mallow (Malva sylvestris L.) against Haemonchus contortus in Sheep

The objective of this study is to evaluate the effect of dry wormwood and mallow on the gastrointestinal parasite of small ruminants Haemonchus contortus. Twenty-four experimentally infected lambs were randomly divided into four groups of six animals each: unsupplemented lambs, lambs supplemented with wormwood, lambs supplemented with mallow and animals supplemented with a mix of both plants. Faecal samples from the lambs were collected on day 23, 29, 36, 43, 50, 57, 64 and 75 post-infection for quantification of the number of eggs per gram (EPG). The mix of both plants contained phenolic acids (10.7 g/kg DM) and flavonoids (5.51 g/kg DM). The nematode eggs were collected and in vitro egg hatch test was performed. The aqueous extracts of both plants exhibited strong ovicidal effect on H. contortus, with ED50 and ED99 values of 1.40 and 3.76 mg/mL and 2.17 and 5.89 mg/mL, respectively, in the in vitro tests. Despite the great individual differences between the treated lambs in eggs reduction, the mean EPG of the untreated and treated groups did not differ (p &gt; 0.05). Our results indicate that using wormwood and mallow as dietary supplements do not have a sufficient effect on lambs infected with H. contortus

Impact of Sainfoin (Onobrychis viciifolia) Pellets on Parasitological Status, Antibody Responses, and Antioxidant Parameters in Lambs Infected with Haemonchus contortus

Our study analyzed the parasitological status, antibody responses, and antioxidant parameters of lambs experimentally infected with a gastrointestinal nematode during the consumption of sainfoin pellets (SFPs) for 14 d. Twenty-four lambs infected with Haemonchus contortus were separated into two groups: untreated animals (control) and animals treated with SFPs (600 g dry matter/d). SFP treatment began on day (D) 30 post-infection. The number of eggs per gram (EPG) of feces was quantified on D18, D23, D26, D30, D33, D37, D40, and D44. The mean reductions in EPG on D40 and D44 were 33.6 and 36.7%, respectively. The number of abomasal worms was lower for the SFP than the control group (p &lt; 0.05). SFP treatment did not significantly affect either the total or the local antibody response (p &gt; 0.05). The blood activity of glutathione peroxidase was affected by the treatment (p &lt; 0.022). Adult worms were selected for scanning electron microscopy after necropsy, but surface structures of adult H. contortus females did not differ between the groups. The treatment of lambs with SFPs directly affected the dynamics of infection, probably indirectly by mobilizing the antioxidant defensive system and antibody response thus improving animal resistance

Effects of Medicinal Plants and Organic Selenium against Ovine Haemonchosis

The objective of this study was to determine the effect of traditional medicinal plants typical to Central Europe as well as organic selenium on increasing the resistance of lambs to gastrointestinal nematode (GIN) infection with Haemonchus contortus. 21 female lambs were infected with third-stage larvae of H. contortus on the day (D) 0 and re-infected on D49 and D77. The animals were divided into three groups based on a treatment diet: a basal diet (control), a diet enriched with dry plants (Herbmix), and a diet enriched with selenized yeast (Selplex). The number of eggs per gram (EPG) of feces was quantified on D21, D28, D35, D42, D49, D56, D63, D70, D77, D84, D91, D98, D105, D112, and D119. The mean reductions in EPG on D28 were 43.4 and 28.6% for Selplex and Herbmix, respectively. The reduction in egg output was nearly uniform throughout the experiment for Selplex. However, for Herbmix the mean reduction was only 19.8% up to D91 and 46.1% after D91. Glutathione peroxidase activity in the blood from D35 to D98 was two to three-fold higher for Selplex than the other groups. Both supplements slowed the dynamics of GIN infection and gradually increased the resistance of lambs against ovine haemonchosis

Effects of herbal nutraceuticals and/or zinc against Haemonchus contortus in lambs experimentally infected

Abstract Background The gastrointestinal parasitic nematode Haemonchus contortus is a pathogenic organism resistant to several anthelmintics. This study assessed the efficacy of a medicinal herbal mixture (Herbmix) and organic zinc, as an essential trace element for the proper functioning of both unspecific and specific immune defensive mechanisms, against experimental infections with H. contortus in lambs. All lambs were infected orally with approximately 5000 third-stage larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Twenty-four female lambs 3–4 months of age were divided into four groups: unsupplemented animals (control), animals supplemented with Herbmix (Hmix), animals supplemented with organic zinc (Zn) and animals supplemented with Herbmix and organic zinc (Hmix+Zn). Eggs per gram (EPG) of faeces were quantified 20, 28, 35, 42, 49, 56, 62 and 70 d post-infection and mean abomasal worm counts were assessed 70 d post-infection. Samples of blood were collected from each animal 7, 35, 49 and 70 d post-infection. Results Quantitative analyses of the bioactive compounds in Herbmix identified three main groups: flavonoids (9964.7 μg/g), diterpenes (4886.1 μg/g) and phenolic acids (3549.2 μg/g). Egg counts in the lambs treated with Hmix, Zn and Hmix+Zn decreased after 49 d. The EPGs in the Zn and Hmix+Zn groups were significantly lower on day 56 (P < 0.05 and P < 0.01, respectively), and the EPGs and mean worm counts were significantly lower on day 70 in all supplemented groups (P < 0.05 and P < 0.01). Hemograms of complete red blood cells of each animal identified clinical signs of haemonchosis after day 35. Serum calprotectin concentrations and IgA levels were significantly affected by treatment. The treatment influenced serum malondialdehyde concentrations (P < 0.05) and sulfhydryl groups (P < 0.01) of antioxidant status. The mineral status was unaltered in all lambs. Conclusion A direct anthelmintic impact on the viability of nematodes was not fully demonstrated, but the treatments with herbal nutraceuticals and zinc likely indirectly contributed to the increase in the resistance of the lambs to nematode infection