Field efficacy of four anthelmintics and confirmation of drug-resistant nematodes by controlled efficacy test and pyrosequencing on a sheep and goat farm in Denmark

AbstractWe describe a case of anthelmintic resistance on one of the largest organic small ruminant farms in Denmark. The flock was established in 2007 by purchase of animals from other Danish farms and had history of clinical parasitism, high mortality of young stock and anthelmintic treatment failure. In October 2011, 40 lambs and 40 kids were selected for a faecal egg count reduction test (FECRT) with fenbendazole (FBZ), ivermectin (IVM), moxidectin (MOX) and levamisole (LEV). Lambs were treated with the recommended sheep dose of each product while kids received the sheep dose of IVM, 1.5× sheep dose of MOX and 2× sheep dose of FBZ and LEV. Untreated lambs and kids were also included and three methods for calculating faecal egg count (FEC) reduction were compared. In a subsequent investigation, a controlled efficacy test (CET) with FBZ and IVM was performed in lambs infected with Haemonchus contortus and Trichostrongylus colubriformis isolated from adult goats on the farm. Recovered specimens of H. contortus were subjected to pyrosequencing for detection of single nucleotide polymorphisms (SNPs) related to benzimidazole (BZ) resistance. During the FECRT, FECs in untreated lambs dropped significantly by 47%. No FEC reduction was detected in untreated kids. After FBZ treatments, FEC reductions in lambs and kids ranged from 15 to 54% and 49–56%, respectively, according to the different calculation methods. Post IVM treatments, FEC reductions in lambs and kids varied between 71–90% and 81–83%, correspondingly. LEV and MOX reduced FECs by 98–100% in both species. In the CET, FBZ reduced H. contortus worm counts by 52–56% and no reduction in T. colubriformis counts were detected after treatment. IVM eliminated 100% of H. contortus and reduced T. colubriformis counts by 84–92%, according to different calculation methods. Pyrosequencing of isolated H. contortus revealed increased frequencies of the BZ resistance-related SNP in codon 200 of the β-tubulin isotype 1 gene. Frequency of BZ resistance-related SNPs in codons 167 and 198 were very low and did not exceed levels as obtained in the susceptible reference isolate. Anthelmintic resistance was confirmed in this recently established organic farm and low field efficacy of FBZ was verified by CET and pyrosequencing. BZ-resistant populations of H. contortus and T. colubriformis were isolated for the first time in Denmark. Problems with correct dosing of goats, the observed FEC reduction in untreated lambs and the relevance of including a control group in the FECRT are discussed

Sesquiterpene lactone-containing extracts from two chicory cultivars show different anthelmintic activity in vitro against Ostertagia ostertagi

Mechanisms behind reported in vivo anthelmintic effects of chicory (Cichorium intybus) in ruminants are poorly understood but it is likely that plant compounds, like sesquiterpene lactones (SL), play a role. Objectives: The aim was to test the inhibitory activity of SL-containing extracts from two chicory cultivars on free-living and parasitic stages of Ostertagia ostertagi. Methods: Leaves from chicory cv. Spadona and cv. Puna II were freeze-dried and SL extracted with methanol/water. Resulting extracts were incubated with cellulase enzyme and SL were purified from other plant compounds by normal solid-phase extraction. Purified extracts were dissolved in DMSO. O. ostertagi eggs from a mono-infected calf were hatched and first-stage larvae (L1) were used in a larval feeding inhibition assay (LFIA), while L3 cultured from faeces were used in a larval exsheathment inhibition assay (LEIA). O. ostertagi adult worms recovered post-mortem were used for motility inhibition assays (AMIA) and worm motility was evaluated after 6, 24 and 48 h of incubation (37oC). In all in vitro assays, decreasing concentrations of chicory extracts in PBS (1% DMSO) were tested in triplicates with 1% DMSO in PBS as negative control. Chemical profile of the extracts was analysed by liquid chromatography (LC). Results: In the LFIA Spadona-extract inhibited larval feeding at significantly lower concentrations than Puna II-extract (EC50=31.5 [CI=25.9-38.3] g Spadona-extract/mL vs. EC50=121.1 [CI=95.2-153.8] g Puna II-extract/mL; p<0.0001). In the LEIA extracts from neither of the two cultivars interfered with the exsheathment of L3 at any of the tested concentrations. In the AMIA, Spadona-extract showed a significantly higher potency and exerted faster worm paralysis than Puna II-extract at all time points when tested at equal concentrations (p<0.0001). Preliminary LC analyses revealed different SL profiles of the extracts and further chemical characterization is undergoing. Discussion: The observed anthelmintic effects of SL-containing extracts from chicory seem to be stage-specific as L1 and adult O. ostertagi but not L3 were affected. Different anthelmintic potency of SL-containing extracts from different chicory cultivars may help the identification of the most active(s) compound(s) and of cultivars with higher antiparasitic potential

Feeding chicory ( Cichorium intybus ) selectively reduces Ostertagia ostertagi infection levels in cattle

Objectives: Studies were conducted to test the potential use of chicory against gastrointestinal nematode infections in cattle. Methods: In study 1, fifteen 2-4 months-old dairy calves were allocated into a chicory (CHI, n=9) or control (CTL, n=6) group. CHI and CTL were stabled and fed with chicory silage or hay, resp., ad lib for 56 days. Protein/energy intakes were equalized between groups throughout the study. After 14 days on the diet all calves were infected with 10,000 Ostertagia ostertagi and 66,000 Cooperia oncophora third-stage (L3) larvae. In study 2, twenty 4-6 months-old dairy calves grazed a second-year, pure chicory sward (CHI, n=10) or a ryegrass/white clover pasture (CTL, n=10) for 43 days. After 7 days on the diet all calves were infected with 20,000 O. ostertagi L3. In both studies, individual live weights were recorded and faecal egg counts were calculated as number of eggs per g of dried feces (FECDM). At day 56 (study 1) calves were killed for worm recovery. Live weights and log-transformed FECDM were analysed by ANOVA using repeated measurements. Log-transformed worm counts were analysed by t-test. Results: In study 1 daily live weight gains were 500 and 329 g/day in CHI and CTL animals, resp. (p=0.02). Mean FECDM were not significantly different between groups (p=0.19). O. ostertagi geo mean worm counts were 1599 (± 296) and 3752 (± 258) in CHI and CTL groups, resp. (p0.05). From this point, egg excretion in CHI calves was significantly reduced and by day 36 post-infection FECDM was decreased by 48-65% compared to CTL (P<0.05). Discussion: Feeding on a chicory diet demonstrated a marked anthelmintic effect against O. ostertagi in both trials, whereas C. oncophora in study 1 was unaffected. Apparently, chicory does not interfere with worm establishment of O. ostertagi but significantly reduces egg excretion and adult worm counts. The lower weight gains in study 2 probably reflect lower energy consumption in this group and suggest that duration of grazing of pure chicory should be limited to selectively target established O. ostertagi adult populations

Anthelmintic effects of forage chicory (Cichorium intybus) against gastrointestinal nematode parasites in experimentally infected cattle

Two experiments studied the effects of dietary chicory against gastrointestinal nematodes in cattle. In Experiment (Exp.) 1, stabled calves were fed chicory silage (CHI1; n = 9) or ryegrass/clover hay (CTL1; n = 6) with balanced protein/energy intakes between groups. After 16 days, all calves received 10 000 Ostertagia ostertagi and 66 000 Cooperia oncophora third-stage larvae (L3) [day (D) 0 post-infection (p.i.)]. In Exp. 2, calves were assigned to pure chicory (CHI2; n=10) or ryegrass/clover (CTL2; n = 10) pastures. After 7 days, animals received 20 000 O. ostertagi L3/calf (D0 p.i.) and were moved regularly preventing pasture-borne infections. Due to poor regrowth of the chicory pasture, CHI2 was supplemented with chicory silage. At D40 p.i. (Exp. 1) and D35 p.i. (Exp. 2) calves were slaughtered for worm recovery. In Exp.1, fecal egg counts (FEC) were similar between groups. However, O. ostertagi counts were significantly reduced in CHI1 by 60% (geometric mean; P &lt; 0·01), whereas C. oncophora burdens were unaffected (P = 0·12). In Exp. 2, FEC were markedly lowered in CHI2 from D22 p.i onwards (P &lt; 0·01). Ostertagia ostertagi adult burdens were significantly reduced in CHI2 by 66% (P &lt; 0·001). Sesquiterpene lactones were identified only in chicory (fresh/silage). Chicory shows promise as an anti-Ostertagia feed for cattle and further studies should investigate its on-farm use

Anti-parasitic activity of pelleted sainfoin (Onobrychis viciifolia) against Ostertagia ostertagi and Cooperia oncophora in calves

BACKGROUND: Increasing anthelmintic-resistance in nematodes of ruminants emphasises the need for sustainable parasite control. Condensed tannin-containing legume forages such as sainfoin (Onobrychis viciifolia) have shown promising anthelmintic properties in small ruminants but this has never been explored in cattle. Therefore, our aim was to examine the efficacy of sainfoin against cattle nematodes in vivo. METHODS: Fifteen Jersey male calves (2–4 month-old) were allocated into two groups and fed isoproteic and isoenergetic diets mainly composed of sainfoin pellets (Group SF; n = 9, three pens) or concentrate and grass-clover hay (Group CO; n = 6, two pens). After 16 days of adaptation, all animals were experimentally infected with 10,000 and 66,000 third-stage larvae of Ostertagia ostertagi and Cooperia oncophora, respectively. Egg excretion, blood parameters and bodyweights were recorded throughout the study. Worms were harvested by sieving for quantification and scanning electron microscopy (SEM) 42 days post-infection (dpi) when the calves were necropsied. RESULTS: The number of O. ostertagi adults in the abomasum was reduced by 50 % in Group SF compared with Group CO (P < 0.05). This was further reflected in higher albumin (P < 0.1) and lower pepsinogen levels (P < 0.05) in Group SF at 21 dpi, and structural damage of the worm cuticle could be visualised by SEM. Yet, the nematode egg excretion in Group SF was not significantly different from that of the controls (P > 0.05). Likewise, no statistical difference in total worm burdens of C. oncophora was found between the groups. Weight gains were lower for Group SF (P < 0.05), which may reflect lower digestibility and phosphorus levels in the SF diet, despite similar feed intake at pen-level. CONCLUSIONS: Overall, the effect of sainfoin on abomasal nematodes corroborates results from studies with small ruminants and encourages further investigations of the use of this crop for control of cattle nematodes