Interaction between the effects of evaporation rate and amount of simulated rainfall on development of the free-living stages of Haemonchus contortus

A factorial experiment (3 x 4 x 2 x 3) was conducted in programmable incubators to investigate interaction between the effects of rainfall amount, rainfall distribution and evaporation rate on development of Haemonchus contortus to L3. Sheep faeces containing H. contortus eggs were incubated on sterilised soil under variable temperatures typical of summer in the Northern Tablelands of NSW, Australia. Simulated rainfall was applied in 1 of 3 amounts (12, 24 or 32 mm) and 4 distributions (a single event on the day after deposition, or the same total amount split in 2, 3 or 4 equal events over 2, 3 or 4 days, respectively). Samples were incubated at either a Low or High rate of evaporation (Low: 2.1-3.4 mm/day and High: 3.8-6.1 mm/day), and faeces and soil were destructively sampled at 4, 7 and 14 days post-deposition. Recovery of L3 from the soil (extra-pellet L3) increased over time (up to 0.52% at day 14) and with each increment of rainfall (12 mm: <0.01%; 24 mm: 0.10%; 32 mm: 0.45%) but was reduced under the High evaporation rate (0.01%) compared with the Low evaporation rate (0.31%). All rainfall amounts yielded significantly different recoveries of L3 under Low evaporation rates but there was no difference between the 12 and 24 mm treatments under the High evaporation rate. The distribution of simulated rainfall did not significantly affect recovery of infective larvae. Faecal moisture content was positively associated with L3 recovery, as was the ratio of cumulative precipitation and cumulative evaporation (P/E), particularly when measured in the first 4 days post-deposition. The results show that evaporation rate plays a significant role in regulating the influence of rainfall amount on the success of L3 transmission.     

Haemonchus contortus with low inhibited development in sheep from the highveld of Zimbabwe

The abomasa of sheep grazing on natural pastures in the highveld of Zimbabwe were examined for Haemonchus contortus. Of 304 abomasa, 213 (70%) harboured H. contortus. The worm burden increased during the rains to reach the peak in February-March. This was followed by a decline with low worm loads throughout the dry season. The fourth stage larvae (L4) accounted for 1-7% of the total H. contortus population, except during July and August when they comprised 22 and 54% of the worm burden, respectively. It appears that the inhibition of H. contortus is not common in the commercial farming sector where sheep are treated very frequently.     

The establishment and development of Haemonchus contortus in goats

Twelve goats were inoculated with 40,000 third-stage Haemonchus contortus larvae and two were killed on each of Days 4, 7, 11, 14, 18 and 21 after inoculation (DAI). The number of worms that established, and the site of development were recorded. More worms established in the fundic, than in the middle or pyloric thirds of the abomasum. Early development occurred within the mucosa; emergence into the lumen started between 7 and 11 days after infection. By 4 DAI, all worms had completed the third moult to the L4 stage. At 11 DAI the majority of the worms were adults. A mean of 13.2% of the female worms had eggs in their uteri at 18 DAI; by 21 DAI more than half of the female worms had eggs in their uteri. The development of H. contortus was essentially similar to that described in sheep.     

Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans

With the problem of parasitic nematode drug resistance increasing, vaccine development offers an alternative sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly protective. However, recombinant forms of these proteins have failed to replicate this protection. This is thought to be due to differences in glycosylation and/or conformation between native and recombinant proteins. We have exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus aminopeptidase H11 glycoprotein, which is enriched in a gut membrane fraction capable of inducing significant protection against this important ovine gastrointestinal nematode. We show that H. contortus H11 expressed in C. elegans is enzymatically active and MALDI mass spectrometry identifies similar di- and tri-fucosylated structures to those on native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans-expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on rH11 do not contribute to the protective effect of H11 and that additional components present in native H11-enriched extract are likely required for enhancing the antibody response necessary for protection.     

The effects of ivermectin and moxidectin on egg viability and larval development of ivermectin-resistant Haemonchus contortus

The in vivo effects of ivermectin and moxidectin on egg viability and larval development of ivermectin-resistant Haemonchus contortus were examined over time after anthelmintic treatment of sheep. Twenty merino sheep, (12 months old) were allocated to five treatment groups and infected with ivermectin-resistant H. contortus. Thirty one days later, the sheep were treated with intraruminal ivermectin capsules, oral ivermectin, oral moxidectin or injectable moxidectin at the manufacturer's recommended dosages, or left untreated. At various times up to 112 days after treatment, faecal egg counts (FEC) were determined and development rates of infective larvae (L3) cultured in faeces or on agar were measured. Eggs in faecal cultures from ivermectin capsule treated sheep showed reduced L3 development percentages in comparison to faecal cultures from untreated sheep. Eggs from ivermectin capsule treated sheep, isolated from faeces, and cultured on agar showed similar L3 development to eggs from control sheep. These results demonstrate an inhibitory effect of excreted ivermectin in faeces on larval development of ivermectin-resistant H. contortus. L3 development in faecal culture from animals receiving oral ivermectin were reduced for only 3 days after treatment. Faecal egg counts and development of L3 larvae in both culture systems from moxidectin treated sheep were low, due to the high efficacy of the drug. Egg counts in moxidectin treated sheep were reduced by approximately 90% 24h after treatment, before decreasing to almost 100% at 48h, suggesting that the current quarantine recommendation of holding sheep off pasture for 24h after treatment may still lead to some subsequent pasture contamination with worm eggs.     

The effects of stage-specific selection on the development of benzimidazole resistance in Haemonchus contortus in sheep

Resistance to the benzimidazole (BDZ) class of anthelmintics in nematodes of sheep has become a common and global phenomenon. The rate at which the selection process and development of resistance occurs is influenced by a number of factors. Of these, the effects of stage-specific exposures to anthelmintic were investigated with a BDZ-resistant strain of Haemonchus contortus (HCR) over five parasite generations. Sheep were infected at each generation with the HCR strain and were treated with thiabendazole (TBZ), either 5 days post-infection (p.i.) (larval line), 21 days p.i. (adult line), or left untreated (no selection line). Additionally eggs from each generation were exposed to TBZ (egg line). Geometric worm burdens were calculated from post-mortem worm counts, both at the start of the study, and after the final selection studies for each of the selection lines. Egg hatch assays (EHAs) were also conducted throughout the study. All data relating to worm burdens and EHAs for each generation were analysed by linear regression to produce dose titration curves and lethal dose(50) (LD(50)) values for each of the selection lines. Over the five generations, LD(50) values on dose-response were increased and worm survival occurred at higher dose rates of TBZ irrespective of the parasite stage exposed to treatment. A similar picture was seen with ED(50) values, which showed a fluctuating but generally upward trend for each of the three selection lines. In contrast, LD(50) and ED(50) values were decreased in the no selection line, indicating some degree of reversion albeit to levels still considered to be BDZ-resistant.     

Histoenzymic effects of thiophenate and fenbendazole on the absorptive surfaces of Haemonchus contortus.

In vitro alterations induced by a 10 micrograms/ml and 50 micrograms/ml dose each of thiophenate and fenbendazole on the absorptive surfaces of Haemonchus contortus (Nematoda: Trichostrongylidae) were studied. The most significant changes were induced in the gut epithelium. Alkaline phosphatase and adenosine triphosphatase activities were decreased, succinic dehydrogenase activity was increased, while acid phosphatase and glucose-6-phosphatase were completely lost from the intestinal epithelium after treatment with either of the drugs. A stimulatory effect of these two anthelmintics was observe on lactic dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase distribution. Thiophenate caused an increase in the activities of glutamate dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G-6-PD) and nonspecific esterases and a decrease in reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-D) activity. Fenbendazole treatment led to the inhibition of GDH, while G-6-PD, NADPH-D, cytochrome oxidase, monoamine oxidase and nonspecific esterase activity remained unaltered in the epithelium.     

Development and survival of Haemonchus contortus infective larvae derived from sheep faeces under sub-tropical conditions in the Potohar region of Pakistan

Assessment on the development and survival of Haemonchus contortus larvae was made to evaluate the influence of microclimatic variables viz., relative humidity (%), temperature (°C), rainfall (mm) and intensity of sunlight (hrs). Pasture plots in a sub-tropical area, Pakistan were contaminated with H. contortus eggs at the start of every month. The plots were sampled on fortnightly basis after contamination for three consecutive months. The overall pattern indicated the most favorable conditions for survival, development and translation to herbage was during the wet season from July to October and the least favorable was during the dry season from April to June. Peak infective larvae (L₃) recovery was during the 15–45 day interval for each plot and the lowest number was during the 75–90 day interval. Herbage was collected in the morning, afternoon and evening and greatest recovery of L₃ was in the morning time and least in the afternoon. The number of L₃ on pasture was directly related to the pattern of rainfall and relative humidity with a significant (P < 0.05) positive correlation and temperature and intensity of sunshine were negatively correlated (P < 0.05). The results suggest that pastures can remain infective for up to 2 months and become relatively clean by 90 days after contamination. Thus, long pasture rest periods, especially during the high risk wet season, may be a helpful strategy to reduce the infection levels.     

Effects of the chitin synthesis inhibitor diflubenzuron on development of Ascaris suum and Haemonchus contortus

The potential of the chitin synthesis inhibitor diflubenzuron (DFB) to alter the development of the parasitic nematodes (Ascaris suum and Haemonchus contortus was investigated. DFB given orally (10 mg kg-1 per day for 30 days) to sheep inoculated with H. contortus infective larvae did not prevent the establishment of adults or affect fecal egg output. However, there was a significant (greater than 90%) decrease in the number of infective larvae recovered from fecal cultures derived from lambs harboring H. contortus adults that were treated with DFB. DFB did not affect egg hatching. Oral administration (10 mg kg-1 per day for 20 days) of DFB to swine harboring adult A. suum adults had no effect on the adult worm burden or on egg morphology, but eggs removed from worms obtained from DFB-treated swine contained less chitin than eggs removed from untreated control swine. DFB also inhibited chitin synthesis in vitro in the isolated reproductive tract of A. suum adults. These results indicate that DFB at high doses can inhibit the subsequent development of H. contortus larvae in the feces. Since H. contortus larvae lack chitin, DFB may act on these larvae by a mechanism independent of a direct effect on chitin synthesis.     

The effects of amount, timing and distribution of simulated rainfall on the development of Haemonchus contortus to the infective larval stage

Three studies were undertaken to determine the effects of amount, timing and distribution of simulated rainfall on the developmental success of Haemonchus contortus. Faeces containing H. contortus eggs were deposited onto pasture plots under a rainfall-activated retractable roof which eliminated incident rainfall. In October (spring) 2004 and January (summer) 2005, the effects of amount (6, 12, 18 or 24 mm) and timing (1, 4, 8 or 15 days post-faecal deposition) of a single simulated rainfall event was investigated via manual application of water to plots. More H. contortus pre-infective larvae (L1 and L2) developed under the d 1 simulated rainfall treatment than later treatments. There was no effect of rainfall amount on development in either experiment, and negligible development to infective larvae (L3). In February (summer) 2006, the effects of amount (12, 24 or 32 mm) and distribution (single event or three smaller but equal split events over 32 h) of simulated rainfall events was investigated with water applied via sprinkler. In this experiment L3 were recovered from the herbage in one-third of the plots harvested, however recovery was low (0.08% of eggs deposited) and there were no treatment effects. Recovery of L1 and L2 from faeces increased with simulated rainfall amount at d 4, and more L1 and L2 were recovered from the split distribution treatment at d 4. The results indicate that moisture conditions soon after faecal deposition are key determinants of H. contortus development success, with significant penalties on development when simulated rainfall was applied 7 days or more post-deposition, and when the duration of simulated rainfall was short. High rates of evaporation during both summer experiments resulted in rapid drying of the micro-environment and this appears to have limited development to L3.     

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.     

The effect of level of nutrition on the development of resistance to Haemonchus contortus in sheep

Merino wether weaners, 5-months old at the start of the experiment and fed lucerne-based pellets at 600 g per head per day, developed resistance to Haemonchus contortus given as a continuous or "trickle" infection at the rate of 500 larvae 3 times per week over a period of 17 weeks. By contrast, the development of resistance was impaired in weaners fed pellets at 400 g per head per day. Low food intake and protein-energy deprivation, therefore, have a major effect in impairing the development and expression of host-protective immunity against haemonchosis. The point at which low food intake causes outbreaks of haemonchosis in sheep at pasture is critical in determining procedures for prevention and control of the disease.     

The effect of oxfendazole terminated infections with Haemonchus contortus on the development of immunity in sheep

The relative contribution of third (L3), fourth (L4) or adult stages of Haemonchus contortus to the development of immunity was evaluated in three groups of sheep subjected to infections terminated by oxfendazole treatments at the L3, L4 or adult stage. A control group did not receive immunising infections. All the groups were challenged with 5000 L3, to evaluate the protection provided by the different protocols. All sheep were necropsied at the end of the experiment to count the abomasal worm burdens. A marked reduction in egg counts after challenge infection was only observed in sheep in which the infection was terminated in the adult stage (Group 4). A significant reduction in worm burden was also observed in Group 4. The immunising infections and/or the challenge infection resulted in moderately elevated IgG antibody levels against L3, L4 and adult somatic antigens in all the groups. In contrast, a strong IgG response against H. contortus excretory/secretory (ES) antigens was observed in the groups in which the immunising infection was terminated in the L4 and the adult stage. An elevated lymphocyte proliferation response against Haemonchus ES antigens was found only in the group that had their immunising infection terminated at the adult stage. The combined data suggest that exposure to and elicited immunological responses to ES antigens are important for the development of immunity against H. contortus.     

Effect of development of resistance to benzimidazoles, salicylanilides and ivermectin on the pathogenicity and survival of Haemonchus contortus

Two groups of six lambs were infected either with an anthelmintic-susceptible strain of Haemonchus contortus or with a strain resistant to benzimidazoles, ivermectin and salicylanilides. The pathogenicity of the two strains of parasite was compared by monitoring the development of anaemia, changes in plasma proteins and abomasal damage in the two groups of lambs. There were no significant differences between the groups, suggesting that the development of resistance to several anthelmintics did not correlate with an increase in the pathogenicity of the parasites. Furthermore, the establishment rates and egg production of the susceptible and resistant parasites were similar. However, fewer of the eggs of the resistant parasites survived and developed at a variety of temperatures.     

Lack of effects of quebracho and sainfoin hay on incoming third-stage larvae of Haemonchus contortus in goats

The effects of tannins on adult populations of Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta in goats are characterised mainly by a decrease in egg excretion without any significant changes in worm number. In contrast, the impact of tannins on T. colubriformis or T. circumcincta third-stage larvae (L3) is associated with a significant reduction in worm establishment. The objective of the present study was to examine the effects of quebracho extract tannins on H. contortus L3. The consequences of consumption of sainfoin hay were also examined. Twenty-one na?ve kids were divided into three experimental groups. Group Q received quebracho extract and group S received sainfoin hay from days D3 to D5. Group C remained as an infected control group. All kids received 1500 L3 H. contortus on D0, D1 and D2. On D18, post-infection, the kids were slaughtered and the worm populations compared in the different groups. Compared to the control values, the worm counts decreased, respectively, by 33% and 38% in groups Q and S but the differences were not significant. No differences were found in pathophysiological measurements between the three groups. The results confirm differences in tannin effect according to nematode species but not parasitic stage.     

Evaluation of multiple low doses of copper oxide wire particles compared with levamisole for control of Haemonchus contortus in lambs

High levels of anthelmintic resistance in gastrointestinal nematodes (GIN) of small ruminants have created the need for alternative approaches to parasite control. Copper oxide wire particles (COWP; 2g) have proven effective in decreasing GIN infection in lambs. However, the risk of copper toxicity has limited the usefulness of this approach. Recently, smaller doses (0.5 and 1g) have proven effective in GIN control, reducing the risk of toxicity. The objective of this study was to examine the effectiveness and risk of toxicity using multiple small doses of COWP for GIN control in lambs between weaning and market weight. Dorper crossbred ram lambs were orally administered levamisole (Levasol, 8.0mg/kg; n=8), 0.5g (n=9), or 1g COWP (n=9) at weaning (Day 0; 118+/-2 days of age; late May 2005) and again at 6-week intervals for a total of four treatments. A pooled fecal culture determined that Haemonchus contortus was the predominant gastrointestinal parasite at weaning. Lambs grazed bermudagrass pastures and were supplemented with up to 500g corn/soybean meal and free choice trace mineralized salt. Fecal egg counts (FEC), packed cell volume (PCV), and plasma aspartate aminotransferase (AST) activity were determined every 14 days and lambs weighed every 28 days. GIN infection reached a peak at Day 42 (high FEC, low PCV). COWP effectively reduced FEC on Days 0 and 42 compared with the previous week, but did not reduce FEC on Days 84 and 126 (treatment by time interaction, P<0.005). Plasma AST activity and weight gains were similar among treatment groups throughout the study period. Concentrations of copper in the liver on Day 155 were greater in COWP-treated lambs (P<0.001), but all concentrations were normal. Multiple doses of COWP were as effective as levamisole for control of H. contortus without risk of copper toxicity.