P.J. Waller

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance

Methods have been described to assist in the detection of anthelmintic resistance in strongylid nematodes of ruminants, horses and pigs. Two tests are recommended, an in vivo test, the faecal egg count reduction test for use in infected animals, and an in vitro test, the egg hatch test for detection of benzimidazole resistance in nematodes that hatch shortly after embryonation. Anaerobic storage for submission of faecal samples from the field for use in the in vitro test is of value and the procedure is described. The tests should enable comparable data to be obtained in surveys in all parts of the world.

The prevalence of anthelmintic resistance in nematode parasites of sheep in Southern Latin America: Argentina

Sixty-five sheep farms in the northern provinces of Buenos Aires, Entre Rios, Corrientes, Cordoba and Sante Fe were used in this survey on anthelmintic resistance. Anthelmintic groups tested were the benzimidazoles, levamisole, the combination levamisole + benzimidazole product and the avermectins. The overall level of resistance was 46% of properties, with resistance to the individual drug groups being 40%, 22%, 11% and 6%, respectively. On a large proportion of farms the resistance recorded was to two, or more, anthelmintic groups. Haemonchus contortus, Ostertagia spp. and Trichostrongylus spp. were the most abundant parasite species recorded. Resistance was greatest in the province of Corrientes where the frequency of treatment is generally high due to the endemic nature of H. contortus. Management practices were also important with resistance greatest on farms where frequent drenching is carried out and on farms carrying only sheep.

The prevalence of anthelmintic resistance in nematode parasites of sheep in southern Latin America: general overview.

South America has a large population of small ruminants. Currents estimates are approximately 100 million sheep and 23 million goats. A large percentage of these flocks are raised in the humid tropics/sub-tropics. Nematode parasitism is singly the most important disease of these animals and typically farmers resort to frequent anthelmintic treatment in attempts to control this problem. Because of this reliance on drugs, price consideration is an important determinant in a farmers choice of anthelmintic. In some circumstances, this has led to unethical practices of drug adulteration and substitution, or the offering to farmers of cheap, sub-standard generic products. The prevalence of anthelmintic resistance has not been investigated in any widespread sense in South America, although some of the first reports of resistance were made in southern Brazil almost 30 years ago. The following series of papers outline surveys conducted in the humid topics/sub-tropics of southern Latin America to assess the significance of resistance to the broad-spectrum anthelmintic groups in nematode parasites of sheep flocks.

THE POTENTIAL OF NEMATOPHAGOUS FUNGI TO CONTROL THE FREE-LIVING STAGES OF NEMATODE PARASITES OF SHEEP : PASTURE PLOT STUDY WITH DUDDINGTONIA FLAGRANS

The nematophagous fungus, Duddingtonia flagrans, isolated from a fresh sheep faecal sample obtained from a farm in northern New South Wales, Australia, was subjected to a number of in vivo investigations in both surgically modified and normal sheep to determine its capacity to survive passage through the gastrointestinal tract. Single and sustained dosing experiments established that between 5 x 10(5) and 10(6) chlamydospores/day resulted in a substantial (> 80%) reduction in the number of infective larvae derived from nematode eggs in faeces. This effect can be maintained if dosing continues. These results demonstrate for the first time the potential of nematophagous fungi to be deployed by means of sustained release technology in the biological control of nematode parasites of livestock.

Population dynamics of Trichostrongylus colubriformis in sheep: The effect of infection rate on the establishment of infective larvae and parasite fecundity

The establishment of Trichostrongylus colubriformis was estimated in helminthologically naive 20-week-old Merino sheep given third stage infective larvae (L3) at rates of 2000, 632 or 200 L3 per day, 5 days per week. After varying periods of continuous L3 intake, a levamisole-susceptible strain of T. colubriformis was replaced with a highly resistant strain for 1 week. The animals were then treated with levamisole to remove the susceptible population, and establishment of the cohort of resistant worms was estimated. In previously uninfected sheep, approximately 65% of the L3 given in the first week became established as adults. This fell to low levels (less than 5%) after 7, 10 and 14 weeks of continuous L3 intake for the high, medium and low infection rates, respectively. At the low infection rate, establishment remained at maximum levels for the first 4 weeks, but then fell at a rate similar to that observed for the higher infection rates. This implied that a threshold of worm exposure was required before resistance to establishment developed. Parasite egg production, expressed as eggs per gram of faeces, was proportional to infection rate and is explained by higher worm burdens occurring at high infection rates. However, estimates of fecundity in eggs per female per day showed the opposite relationship with rate of infection. Fecundity stayed high (approximately 600) for 5 weeks at the low infection rate but only maintained this level for 3 weeks and 1 week at the medium and high rates, respectively. This suggests that fecundity, like establishment, was similarly affected at threshold levels of immunological recognition.

The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: survey for the presence of fungi in fresh faeces of grazing livestock in Australia

In the course of 12 months, 1742 fresh faecal samples from grazing livestock, principally ruminants, from various States of Australia were examined for the presence of nematophagous fungi. In total, 48 separate isolations were made representing various species from the genus Arthrobotrys and also 16 isolates of the single Duddingtonia species, Duddingtonia flagrans. These isolates will be used in continuing studies to select the most suitable species, on the basis of nematophagous capability and ruminant gut survival capacity, for development of a biological means of controlling nematode parasites of livestock.

Drug Resistance in Nematodes

Drug resistance in nematodes is almost solely confined to the strongylid parasites of the alimentary tract of grazing livestock. Few reports have been made on the resistance in plant-parasitic nematodes (Foot, 1980), and none for nematode parasites of man. Unlike drug resistance in bacteria, protozoa, and arthropods, resistance in nematodes to anthelmintics has been slow to develop and is patchy in worldwide distribution. However, this does not constitute grounds for complacency. It is not necessary for anthelmintic resistance to have reached a high level to be a serious problem. Evidence that it has increased from negligibly small levels and that it exists in two of the three widely used broad-spectrum drug groups means it would be foolhardy to assume that its prevalence and thus importance will not escalate, unless effective countermeasures are undertaken.

An egg-hatch assay for resistance to levamisole in trichostrongyloid nematode parasites

An in vitro technique is described for detecting resistance of nematodes to the anthelmintic levamisole hydrochloride. Samples of eggs are developed under controlled temperature conditions until just prior to the commencement of hatching. They are then exposed to different concentrations of the drug and, when hatching is almost complete, the test samples are killed and preserved. The proportion of unhatched eggs at each drug concentration can then be counted at leisure. Provided a suitable range of drug concentrations is chosen for each test isolate, this assay provides results which may be satisfactorily fitted to a log-concentration-probit regression model. Comparisons with in vivo anthelmintic assays have shown that the technique provides an accurate reflection of the resistance status of parasite populations.

Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep

Abstract Waller P. J. , Dobson R. J. , Donald A. D. , Griffiths D. A. and Smith E.F. 1985. Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep. International Journal for Parasitology15: 669–676. A T. colubriformis population (BCK), formerly resistant to benzimidazole anthelmintics, but now highly resistant to levamisole after 6 years exposure to this drug alone in the field, was passed through 12 generations in the laboratory in three separate lines exposed either to selection with thiabendazole or levamisole, or to no selection. Another population (McM) not previously exposed to these anthelmintics was treated similarly in two lines, selected with thiabendazole or not selected. Selection with thiabendazole resulted in a return of benzimidazole resistance in the BCK line which occurred faster than in the McM line, but a similar level of resistance was reached in each by the twelfth generation. Resistance ratios in both selected lines compared with the unselected McM line were less than 20: 1, and only 1.5 times the recommended dose rate of thiabendazole was required to remove more than half of the resistant population. This suggests that a polygenic vigour tolerance rather than a specific resistance had been selected. In the case of levamisole resistance, the BCK population was found to contain two distinct subpopulations, one susceptible and the other highly resistant. Resistance ratios for the highly resistant subpopulation were greater than 4000: 1, implying a specific resistance controlled by a major gene. During the 12 generations of levamisole selection, the proportion of resistant phenotypes fluctuated about an average level of 70%, suggesting that susceptibility alleles were being maintained in the population through superior heterozygote fitness. This conclusion is supported by a significant decline in levamisole resistance in the absence of levamisole selection. Moreover, thiabendazole selection hastened the reversion to levamisole suceptibility. The results provide support for the reintroduction of a benzimidazole anthelmintic to control this helminth population, and for a slow rotation in the use of drugs with different modes of action.

The potential of nematophagus fungi to control the free-living stages of nematode parasites of sheep: in vitro and in vivo studies

Following in vitro screening investigations on approximately 100 nematophagous fungi reported previously, eight species were selected for further investigation. Fungal elements (mycelium and conidia) were subjected to in vitro stress selection designed to simulate rumen and abomasal conditions. From these studies, three species, namely, Arthrobotrys oligospora, Arthrobotrys oviformis and Geniculifera eudermata, were selected for in vivo survival studies in sheep surgically fitted with abomasal and ileal cannulae. Doses of fungal conidia were administered orally or via the abomasal cannulae and samples of digesta were taken from the abomasum, the terminal ileum and faeces. The viability of the three fungal species at these sites was demonstrated. The abundance of fungi throughout the gut was dose-dependent but in all cases only very small volumes of fungal suspension containing unprotected conidia were used. These results demonstrate that a practical means of orally administering nematophagous fungi to control free-living stages of nematodes in faeces may become an achievable objective.