Screening for Indian Isolates of Predacious Fungi for Use in Biological Control against Nematode Parasites of Ruminants

Four isolates of predacious fungi, two each of Arthrobotrys oligospora isolated from a sheep and a male crossbred calf and of Duddingtonia flagrans isolated from a sheep and a female buffalo in western India, were studied for their suitability as biocontrol agents against parasitic nematodes of ruminants, using growth assay, predatory activity, germination potential and ability to survive passing through the ruminants gut as criteria. The study showed that isolates of D. flagrans grew well in artificial media, had encouraging predatory activity, produced profuse chlamydospores that germinated easily at 25°C and could survive passage through the ruminant gut. The ovine isolate of D. flagrans was superior in all respects to the isolate from buffalo and was the most promising candidate for biological control of nematode parasites of ruminants.     

Implications of Fungicidal Effects of Benzimidazole Compounds on Duddingtonia flagrans in Integrated Nematode Parasite Management in Livestock

An in vitro trial with carbendazim fungicide on the growth profile of the predatory fungus Duddingtonia flagrans was undertaken and in vivo trials in sheep and buffaloes, fed on chlamydospores of D. flagrans and administered albendazole anthelmintic, were conducted. Although no growth inhibition was detected at a carbendazim concentration of 0.05 ppm, growth inhibition was recorded of 50% and above at concentrations of 0.25 and 1.00 ppm (p < 0.001) and of around 90% at concentrations of 2.00 to 5.00 ppm (p <0.0001). Scanty recovery of the fungus was made from faecal culture 48 h following a single dose of albendazole both in sheep and buffaloes. However, profuse fungal recovery was made from 96 h post dosing onwards. When the drug was used as an intraruminal slow-release capsule, no faecal fungal recovery could be made from day 3 after administration of the capsule, when the albendazole sulphoxide concentration was around 1.0 microg/ml. However, profuse and scanty fungal recovery could be made on days 1 and 2, respectively, after administration of the capsule, when the plasma albendazole sulphoxide concentration was around 0.4 and 0.9 microg/ml, respectively. The implications for use of a combination of anthelmintics and biological control in sustainable parasite control programmes are discussed.     

Assessing the efficacy of Duddingtonia flagrans chlamydospores per gram of faeces to control Haemonchus contortus larvae

The aims were (a) to quantify the number of Duddingtonia flagrans chlamydospores per gram of faeces (CPG) recovered from sheep administered with different oral doses and, (b) to describe the relationship between CPG and eggs per gram of faeces (EPG) on the efficacy to reduce Haemonchus contortus infective larvae. Three doses of chlamydospores per kg BW were orally administered during seven days: (T1) non treated control group, (T2) 1 × 10⁶, (T3) 2.5 × 10⁶ and (T4) 5 × 10⁶. Three lambs, infected with H. contortus, were used per group. Faeces were obtained from the rectum of each lamb during the fungal administration period (days 0–6) and for six days after that period. Four coproculture replicates were made from each animal in days 2, 4, 6, 8 and 10. A higher chlamydospore dose produced higher CPG in faeces (p < 0.05), but a clear dose dependent effect was not found either in the larvae reduction or in the CPG:EPG ratio. When ratios were re-analyzed, independently of the treatment groups of origin, a better efficacy was obtained with a ratio from 5 to 10 CPG:EPG and a higher ratio (>10 per egg) showed a lower reduction efficacy (p < 0.05). The binomial analysis showed that for each unit of increment in CPG:EPG ratio there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test indicated that the number of larvae was reduced by CPG until possible saturation. The highest CPG:EPG ratios did not necessarily improve efficacy of D. flagrans.     

Cholinesterase and Phosphatase Activities in Adults and Infective-stage Larvae of Levamisole-resistant and Levamisole-susceptible Isolates of Haemonchus contortus

Cholinesterase (ChE) and acid phosphatase (AP) activities, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of adult and infective-stage larvae of levamisole-resistant and levamisole-susceptible Haemonchus contortus. In contrast to other gastrointestinal nematodes, the ChE activity was higher in L₃ than in adults and, in both cases, was mainly associated with membranes. ChE activity was inhibited by Triton X-100 and was only detected in membrane-bound fractions when the detergent was removed. Differences between resistant and susceptible L₃ were observed in the response to inhibitors (cytosolic fraction) and in the enzymatic content (membrane-bound fraction). Phosphatase activity was detected at acidic pH in all fractions, being higher in the adult than in the L₃ stage. In the former, most of the enzyme was localized in the membrane-bound fractions, whereas in the latter it was mainly in cytosolic fractions. This difference could be correlated with the activity in the gut. In inhibition assays, a difference between cytosolic fractions from resistant and susceptible adults was observed in their response to 1 mmol/L tartaric acid.     

Development and survival of Haemonchus contortus larvae on pasture at Vom,Plateau State,Nigeria

The development and survival of the eggs of Haemonchus contortus on pasture at Vom were studied by depositing faecal pellets on grass plots over a period of 12 months. Development and survival to the infective larvae occurred throughout the study except during the dry season months of December to April. More infective larvae were recovered from the herbage in June, July and August than in other months. The survival time of the infective larvae ranged from 2 weeks in October to 10 weeks in June, July and August. Rainfall was the most important epizootiological factor influencing the development and survival of the infective larvae. Temperature was not a limiting factor.     

Efficiency of feeding <Emphasis Type="Italic">Duddingtonia flagrans</Emphasis> chlamydospores to control nematode parasites of first-season grazing goats in France

A field trial, conducted over two consecutive years, was aimed at assessing the efficacy of the administration of spores of the nematophagous fungus Duddingtonia flagrans to young goats for the control of nematode parasite infections on a French commercial dairy goat flock. For both years, the first-year grazing kids were divided into two similarly managed groups (fungus and control groups): in 2003 a daily dose rate of 5 × 10⁵ spores/kg body weight was given to the fungus-group animals, while in 2004 a daily dose rate of 10⁶ spores/kg body weight was used; the other half of the kids, acting as control, did not receive the spores. Parameters measured every 3 weeks included nematode egg excretion, larval development in faecal cultures and pasture larval counts. Additionally, at the beginning, the middle and the end of each grazing season, the goats were weighed and blood samples for pepsinogen determination were collected. In 2003, similar results were recorded for all the measured parameters in the control and fungus groups. In contrast, in 2004, the kids receiving the spores showed lower faecal egg counts and pepsinogen levels at the end of the season and higher growth rate compared to kids of the control group.     

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.     

Response of Corriedale and Crioula Lanada Sheep to Artificial Primary Infection with Haemonchus Contortus

Clinical, parasitological and biochemical parameters were evaluated in Corriedale and Crioula Lanada sheep after a single experimental infection with Haemonchus contortus. Ten 4-month-old worm-free lambs, of each breed, were infected with 200 L₃ H. contortus per kg live weight and four uninfected animals of each breed were used as controls. Every week, the animals were weighed and blood and faecal samples were collected for measurement of packed cell volume (PCV), total serum protein (TSP) and albumin (ALB), and the number of eggs per gram of faeces (EPG), respectively. Twelve weeks after infection, the animals were slaughtered. The worm burden was determined and samples of the abomasal mucosa were processed for determination of the number of eosinophils, mast cells and globule leukocytes. No significant differences in PCV, TSP, ALB, parasite burden or the cell populations of the abomasal mucosa were observed between breeds, but Crioula lambs had a lower EPG count. The comparison of the infected groups with their respective controls revealed significant alterations in PCV, TSP and ALB in the Corriedale lambs and in PCV, TSP, ALB and the density of eosinophils and mast cells in the Crioula lambs.     

Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil

The viability of a fungal formulation using the nematode-trapping fungus Duddingtonia flagrans was assessed for the biological control of horse cyathostomin. Two groups (fungus-treated and control without fungus treatment), consisting of eight crossbred mares (3–18 years of age) were fed on Cynodon sp. pasture naturally infected with equine cyathostome larvae. Each animal of the treated group received oral doses of sodium alginate mycelial pellets (1 g/(10 kg live weight week)), during 6 months. Significant reduction (p < 0.01) in the number of eggs per gram of feces and coprocultures was found for animals of the fungus-treated group compared with the control group. There was difference (p < 0.01) of 78.5% reduction in herbage samples collected up to (0–20 cm) between the fungus-treated group and the control group, during the experimental period (May–October). Difference of 82.5% (p < 0.01) was found between the fungus-treated group and the control group in the sampling distance (20–40 cm) from fecal pats. During the last 3 months of the experimental period (August, September and October), fungus-treated mares had significant weight gain (p < 0.01) compared with the control group, an increment of 38 kg. The treatment with sodium alginate pellets containing the nematode-trapping fungus D. flagrans reduced cyathostomin in tropical southeastern Brazil and could be an effective tool for biological control of this parasitic nematode in horses.     

Screening for the presence of nematophagous fungi collected from Irish sheep pastures

With worldwide development of anthelmintic resistance, alternative approaches to the chemotherapeutic dominant approach for the control of parasitic nematodes in sheep are urgently required. As natural enemies of nematodes, nematophagous fungi offer the exciting possibility of an alternative to the dominant anthelmintic approach for parasite control in ruminants. Permanent sheep pasture harbor a promising array of nematophagous fungi and merits further investigation. One hundred and fifty samples of soil, old and fresh faeces were collected from 10 Irish sheep pastures. The three methods employed for the isolation of nematophagous fungi include the Baermann technique, flotation method and the sprinkling–baiting technique. Twenty-nine nematophagous fungi were observed of which 12 were predacious and 17 were endoparasitic. The most prevalent fungi were Cystopage lateralis, Stylopage hadra, Drechmeria coniospora and Meristacrum asterosperum. Permanent sheep pasture is a good source of nematophagous fungi and hence may harbor potential biological control agents. Monacrosporium cionopaga, Duddingtonia flagrans, D. coniospora and Hirsutella rhoissilensis were detected in fresh faecal samples indicating they may have survived the gastrointestinal tract and therefore a viable option as a biological control agent.     

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.     

Efficacy of Copper Oxide Needles for the Control of Nematode Parasites in Dairy Goats

The spread of benzimidazole-resistant nematodes in dairy goat farms is of a great concern as probably more than 70% of the flocks are involved. As there are very few other anthelmintic options during the lactating period, we have evaluated the efficacy of copper oxide needles (CON, Copinox, Bayer, UK) in both experimental and natural infections in goats. The curative effect of CON (2–4 g) on existing worm burdens was assessed in goats experimentally infected with Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis, compared to controls. The preventive effect of CON (4 g) on worm establishment was monitored for 2 months in animals experimentally infected with H. contortus and for 3 months in naturally infected animals on a farm exhibiting predominant infections with T. circumcincta and Oesophagostomum venulosum. In both experimental and natural conditions, the efficacy of CON was nil against Teladorsagia, Trichostrongylus and Oesophagostomum infections. In contrast, the efficacy of CON against Haemonchus was clearly established in reducing the worm burden (75%) as well as in lowering the egg output (37–95%) in relation to the establishment of new infections over several weeks. Copper oxide needles may represent an alternative to conventional anthelmintics in the control of Haemonchus infection in some goat farms.     

Resistance to Ivermectin and the Effect of Topical Eprinomectin on Faecal Egg Counts in St Croix White Hair Sheep

Veterinary Research Communications -     

Ovine Metabolism of Saponins: Evaluation of a Method for Estimating the Ovine Uptake of Steroidal Saponins from Narthecium ossifragum

A sheep was dosed three times per day over six consecutive days with 70 g Narthecium ossifragum, and once on the seventh day with 70 g N. ossifragum. Additionally, it was dosed once on days 1–7 with 20 mg of [20,23,23-²H₃]sarsasapogenin. After 7 days, the sheep was killed and GC-MS analysis of the free and conjugated sapogenin content in bile, urine, rumen, duodenum, jejunum, colon and rectum samples collected from the sheep, faecal samples collected on days 4–7, and dosed plant material was performed. The N. ossifragum contained mainly sarsasapogenin and smilagenin. Only negligible levels of deuterium-labelled sarsasapogenins were detected in the samples from the animal. Ingested saponins were quickly hydrolysed in the rumen to free sapogenins and, in part, epimerized at C-3 to afford episapogenins. The absorption of free sapogenins appeared to occur in the jejunum. The concentration of sapogenins in faeces reached a plateau 108 h after dosing started.