Nematode control in 'green' ruminant production systems

Collectively, nematode parasites of domestic ruminants continue to pose the greatest disease problem in grazing livestock systems worldwide, despite the powerful and extensive chemotherapeutic arsenal available for their control. The widespread development of anthelmintic resistance, particularly in nematode parasites of small ruminants, and the trend towards nonchemical (ecological, organic, green) farming of livestock has provided an impetus for the research and development of alternative parasite control methods. This article provides a brief overview of the non-chemotherapeutic options for parasite control and how they might play a role either in organic farming or in other low-input farming systems

Anaplasma phagocytophilum in Danish sheep: confirmation by DNA sequencing

<p>Abstract</p> <p>Background</p> <p>The presence of <it>Anaplasma phagocytophilum</it>, an <it>Ixodes ricinus </it>transmitted bacterium, was investigated in two flocks of Danish grazing lambs. Direct PCR detection was performed on DNA extracted from blood and serum with subsequent confirmation by DNA sequencing.</p> <p>Methods</p> <p>31 samples obtained from clinically normal lambs in 2000 from Fussingø, Jutland and 12 samples from ten lambs and two ewes from a clinical outbreak at Feddet, Zealand in 2006 were included in the study. Some of the animals from Feddet had shown clinical signs of polyarthritis and general unthriftiness prior to sampling. DNA extraction was optimized from blood and serum and detection achieved by a 16S rRNA targeted PCR with verification of the product by DNA sequencing.</p> <p>Results</p> <p>Five DNA extracts were found positive by PCR, including two samples from 2000 and three from 2006. For both series of samples the product was verified as <it>A. phagocytophilum </it>by DNA sequencing.</p> <p>Conclusions</p> <p><it>A. phagocytophilum </it>was detected by molecular methods for the first time in Danish grazing lambs during the two seasons investigated (2000 and 2006).</p

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

The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR

BACKGROUND: The Internal Transcribed Spacer 2 (ITS2) is a candidate diagnostic marker of the pathogenic cattle nematode Ostertagia ostertagi. The aims of this study were: (i) to document and quantify how the development of O. ostertagi eggs affects ITS2 copies under different storage conditions, and (ii) to suggest optimal storage conditions for faecal samples in a diagnostic pipeline that involves detection and semi-quantification by real-time semi-quantitative polymerase chain reaction (qPCR). FINDINGS: Eggs of Ostertagia ostertagi were obtained from fresh faeces and stored at 4 °C or 25 °C under aerobic or anaerobic (vacuum packing) conditions. Development was monitored by microscopy for up to 336 h, and the ITS2 copies were determined by qPCR from a fixed number of parasites. Under aerobic conditions at 25 °C, embryonation and a significant increase of ITS2 copies (P < 0.0001) were observed after 12 h. At 4 °C, embryonation occurred after 168 h with a trend towards increased ITS2 copies. Anaerobic conditions inhibited egg development at both temperatures and no significant increase in ITS2 copies was noticed (P = 0.90). ITS2 copies were analysed for each parasite stage: first-stage larvae (L1) exhibited significantly higher copy numbers (20,353 ± 1,950) than unembryonated eggs (568 ± 168; P < 0.0001) with lower coefficient of variation (33 vs 266 %). CONCLUSIONS: Aerobic storage of O. ostertagi eggs at 25 °C led to a significant increase in ITS2 copies after 12 h due to embryonation and subsequent hatching. In contrast, anaerobic storage (vacuum packing) at 25 °C completely inhibited egg development and any undesirable semi-quantification bias for up to 336 h. Hence, vacuum packing is an optimal storage strategy prior to molecular diagnostic analyses. Alternatively, aerobic storage at 4 °C for up to 72 h can be used. Due to high copy numbers and lower genetic variation, the L1 stage may be considered for diagnostics and further molecular research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1657-4) contains supplementary material, which is available to authorized users

A quantitative assessment method for Ascaris eggs on hands.

The importance of hands in the transmission of soil transmitted helminths, especially Ascaris and Trichuris infections, is under-researched. This is partly because of the absence of a reliable method to quantify the number of eggs on hands. Therefore, the aim of this study was to develop a method to assess the number of Ascaris eggs on hands and determine the egg recovery rate of the method. Under laboratory conditions, hands were seeded with a known number of Ascaris eggs, air dried and washed in a plastic bag retaining the washing water, in order to determine recovery rates of eggs for four different detergents (cationic [benzethonium chloride 0.1% and cetylpyridinium chloride CPC 0.1%], anionic [7X 1% - quadrafos, glycol ether, and dioctyl sulfoccinate sodium salt] and non-ionic [Tween80 0.1% -polyethylene glycol sorbitan monooleate]) and two egg detection methods (McMaster technique and FLOTAC). A modified concentration McMaster technique showed the highest egg recovery rate from bags. Two of the four diluted detergents (benzethonium chloride 0.1% and 7X 1%) also showed a higher egg recovery rate and were then compared with de-ionized water for recovery of helminth eggs from hands. The highest recovery rate (95.6%) was achieved with a hand rinse performed with 7X 1%. Washing hands with de-ionized water resulted in an egg recovery rate of 82.7%. This washing method performed with a low concentration of detergent offers potential for quantitative investigation of contamination of hands with Ascaris eggs and of their role in human infection. Follow-up studies are needed that validate the hand washing method under field conditions, e.g. including people of different age, lower levels of contamination and various levels of hand cleanliness

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

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