World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.): Third edition of the guideline for evaluating efficacy of anthelmintics in ruminants (bovine, ovine, caprine).

This guideline is aimed at those who are involved in the assessment of anthelmintic efficacy in ruminant livestock species (bovine, ovine and caprine). The intent is to provide a framework that can be adopted worldwide for the testing of anthelmintics in ruminants, such that studies carried out in different countries can be compared and thereby unnecessary duplication can be reduced. Recommendations are made for the selection, housing and feeding of study animals, the type of studies required, the method used to conduct those studies, the assessment of results and the standards for defining anthelmintic efficacy.

Production trials involving use of the FAMACHA system for haemonchosis in sheep: preliminary results.

In three trials conducted on two separate farms the production of sheep treated for naturally acquired haemonchosis using the FAMACHA system of targeted selective treatment (TST) (i.e. to treat only those animals unable to manage unaided in the face of heavy Haemonchus challenge) was compared to that of suppressively drenched sheep in the same flock. As expected by the research team who developed and evaluated the FAMACHA system, TST did result in some loss in production. However, despite high levels of worm challenge in two of the trials and the fact that the comparison was with suppressive drenching which is not sustainable, the total effect was relatively small in relation to the important advantage of using the TST as regards reduced selection for anthelmintic resistance (AR). Concerning the sustainability of worm control, it is concluded that the development of drug resistance to anthelmintics leaves sheep and goat farmers in South Africa no choice but to use methods of TST such as FAMACHA. The FAMACHA system can also be a useful clinical aid for early on-farm detection of AR by farmers; the degree of improvement in the colour of the ocular mucous membrane from pale to red in individually drenched anaemic animals over a period of 7-14 days can give a good indication of the efficacy of the compound(s) used.

Targeted selective treatment for worm management--how do we sell rational programs to farmers?

Seriously escalating global anthelmintic resistance in gastrointestinal nematodes of small ruminants has spawned a variety of alternatives to anthelmintics for worm management, based on the need for sustainable Integrated Parasite Management (sIPM). Pivotal to the sIPM approach is the concept of refugia, the proportion of a given parasite population that escapes exposure to control measures. By balancing drug applications with the maintenance of refugia, the accumulation of anthelmintic resistance alleles in worm populations can be considerably delayed, while still providing good levels of control. The over-dispersed nature of parasitic infections provides an opportunity to achieve this balance, by targeting treatments to the members of a flock or herd that are least tolerant to nematode infection. However, implementation of this strategy has only recently become feasible, with the development of the FAMACHA((c)) system for clinical evaluation of anaemia due to haemonchosis. Subsequently, the use of milk yields has proven an effective indicator in dairy goats infected predominantly with nematodes other than Haemonchus contortus. In addition, short-term weight changes and perhaps also body condition scoring may provide indices of parasitism, permitting the rapid identification of animals likely to benefit from treatment. However, sIPM and refugia-based approaches are more complex than whole-flock treatments in conventional programs, and adoption by farmers is most likely where the theoretical basis is understood. As close communication with informed advisors is generally limited, there is a danger that sIPM will remain a theoretical concept without alternative modes of communication. The development of computer-based decision support programs, which use epidemiological, seasonal and clinical information to provide recommendations for specific situations, should be accorded high priority in the future development of worm management systems.

The Pathophysiology, Ecology and Epidemiology of Haemonchus contortus Infection in Small Ruminants.

The parasitic nematode Haemonchus contortus occurs commonly in small ruminants, and it is an especially significant threat to the health and production of sheep and goats in tropical and warm temperate zones. The main signs of disease (haemonchosis) relate to its blood-feeding activity, leading to anaemia, weakness and frequently to deaths, unless treatment is provided. Due to the high biotic potential, large burdens of H. contortus may develop rapidly when environmental conditions favour the free-living stages, and deaths may occur with little prior warning. More chronic forms of haemonchosis, resulting in reduced animal production and eventually deaths, occur with smaller persistent infections, especially in situations of prolonged, poor nutrition. The global distribution of the main haemonchosis-endemic zones is consistent with the critical requirements of the egg and larval stages of H. contortus for moisture and moderate to relatively warm temperatures, but the seasonal propensity for hypobiosis (inhibition of the fourth-stage larvae within the host) largely explains the common, though sporadic, outbreaks of haemonchosis in arid and colder environments. The wide climatic distribution may also reflect the adaptation of local isolates to less favourable ecological conditions, while an apparent increase in the prevalence of outbreaks in environments not previously considered endemic for haemonchosis - especially cold, temperate zones - may be attributable to climatic changes. Although the risk of haemonchosis varies considerably on a local level, even where H. contortus is endemic, the extensive range of ecological investigations provides a sound basis for predictions of the relative geographical and seasonal risk in relation to climatic conditions.

Diagnosis, Treatment and Management of Haemonchus contortus in Small Ruminants.

Haemonchus contortus is a highly pathogenic, blood-feeding nematode of small ruminants, and a significant cause of mortalities worldwide. Haemonchosis is a particularly significant threat in tropical, subtropical and warm temperate regions, where warm and moist conditions favour the free-living stages, but periodic outbreaks occur more widely during periods of transient environmental favourability. The clinical diagnosis of haemonchosis is based mostly on the detection of anaemia in association with a characteristic epidemiological picture, and confirmed at postmortem by the finding of large numbers of H. contortus in the abomasum. The detection of impending haemonchosis relies chiefly on periodic monitoring for anaemia, including through the 'FAMACHA' conjunctival-colour index, or through faecal worm egg counts and other laboratory procedures. A range of anthelmintics for use against H. contortus is available, but in most endemic situations anthelmintic resistance significantly limits the available treatment options. Effective preventative programmes vary depending on environments and enterprise types, and according to the scale of the haemonchosis risk and the local epidemiology of infections, but should aim to prevent disease outbreaks while maintaining anthelmintic efficacy. Appropriate strategies include animal management programmes to avoid excessive H. contortus challenge, genetic and nutritional approaches to enhance resistance and resilience to infection, and the monitoring of H. contortus infection on an individual animal or flock basis. Specific strategies to manage anthelmintic resistance centre on the appropriate use of effective anthelmintics, and refugia-based treatment schedules. Alternative approaches, such as biological control, may also prove useful, and vaccination against H. contortus appears to have significant potential in control programmes.

Detection of resistance to ivermectin in Haemonchus contortus.

Infective, third-stage (L3) larvae of Haemonchus contortus isolates resistant to ivermectin (IVM) show a decreased sensitivity to IVM-induced paralysis in vitro. The inhibition of larval motility by IVM can be detected in L3 larvae incubated in the dark on an agar matrix containing IVM, by the failure of affected larvae to move when stimulated by exposure to light. Optimally, avermectin (AVM) potency is quantified after three cycles, each involving storage in the dark for 24 h followed by a brief exposure to light. For IVM-susceptible isolates, a 50% inhibition of motility (LP50) was achieved with IVM concentrations between 0.30 and 0.49 microM, while LP50 values in IVM-resistant isolates ranged from 0.8 to 2.6 microM depending on the in vivo resistance status of the isolate. A limited study of structure-activity relationships within the AVM class indicated that in vitro inhibition of L3 motility was consistent with the known in vivo efficacy of each analogue. Resistance factors for IVM-resistant isolates were dependent on AVM structure with the more polar AVM B2 analogue being a particularly sensitive probe of IVM-resistance status.

Avermectin inhibition of larval development in Haemonchus contortus--effects of ivermectin resistance.

Avermectin (AVM) inhibition of the development of the free-living stages of Haemonchus contortus has been quantified in an assay in which nematode eggs are placed on an agar matrix containing serial dilutions of a drug in the wells of a microtitre plate. Development is allowed to proceed for 6 days by which time larvae in control wells (no drug) have reached the infective, third (L3) stage. At high concentrations (> 30 nM) ivermectin (IVM) paralyses L1 larvae soon after hatching, however, much lower concentrations (approximately 1 nM) are sufficient to inhibit development to the L3 stage which suggests that effects of the drug other than those relating to gross motor activity are responsible for the latter effect. The larval stages of IVM-susceptible H. contortus isolates from both Australia and South Africa, including isolates known to be resistant to levamisole or rafoxanide and/or the benzimidazoles, were equally sensitive to inhibition by AVMs. In contrast, 6 isolates of H. contortus resistant to IVM in vivo showed a reduced sensitivity to AVM inhibition of development. The order of potency of a limited range of AVMs as inhibitors of larval development was consistent with in vivo efficacy. Resistance ratios for IVM-resistant isolates were dependent on AVM structure, with AVM B2 the most sensitive probe for IVM resistance in the isolates tested.

Hydroxyl radical production in the presence of fibres by a Fenton-type reaction.

Glass fibres are considered to be inert and therefore thought to present no real hazard to the health of people who inhale them. Results in the present study however indicate that these fibres are able to produce hydroxyl radicals in the presence of hydrogen peroxide by a Fenton-type reaction. Since hydroxyl radical is implicated in lipid peroxidation, single-strand DNA breaks and carcinogenesis, care should be exercised when dealing with glass fibres.

A novel approach to the control of anthelmintic-resistant Haemonchus contortus in sheep.

This paper reports attempts to control a resistant strain of Haemonchus contortus on pasture by replacing it with a susceptible strain. By making use of artificially infected donor sheep, six camps (paddocks) were seeded with a resistant field strain of H. contortus until it was confirmed by means of worm-free tracer lambs that the grazing had become infective. Thereafter, using donor sheep infected with a susceptible laboratory strain of H. contortus for seeding the pasture, attempts were made at various times of the year to replace the resistant strain on the pasture with the susceptible strain in five of the camps. The sixth remained as a control camp, in which no attempt was made to replace the resistant strain. In two of the five test camps, the susceptible strain was introduced in the autumn after 8-10 weeks of nil grazing; in the remaining three camps the introduction was made in spring (two camps) or summer without having a period of nil grazing. The susceptibility of the worm strains introduced initially, as well as of those that developed in the various camps, was gauged both by controlled non-parametric anthelmintic slaughter trials at the beginning and at the conclusion of the trial, and by an in vitro egg hatch test. A reversion to susceptibility occurred in three of the five camps. These included both of the camps infested with the susceptible strain in the spring and one of the two infested in the autumn. Possible reasons for the failure to replace the resistant strain in the remaining two test camps are discussed.

Effects of time of day, season and stratum on Haemonchus contortus and Haemonchus placei third-stage larvae on irrigated pasture.

The effect of time of day, season and stratum of herbage and soil on the availability of Haemonchus contortus and Haemonchus placei third-stage larvae (L3) on pasture was assessed. Feces from infected calves and lambs were placed on pasture plots and samples of upper herbage, lower herbage, mat and soil were collected at five intervals per day throughout the daylight hours on 18 sample days over 12 months. Using recovery rate factors derived from a preliminary investigation on the efficacy of larval recovery from each stratum, the data on larval recoveries were analyzed for the effect of season, time and stratum, and their interactions. Significant (P less than 0.05) differences were found for season, stratum and the season-with-stratum interaction for both parasites. No significant differences were detected for larval counts at different times of the day. Larval recoveries of H. contortus were larger throughout the study than those of H. placei. Most H. contortus L3 were recovered in the summer and autumn, and H. placei in the spring and summer. For both parasites, the recoveries of larvae from the upper and lower herbage were larger than those from the mat and soil. The implications of these findings are discussed in terms of control strategies.

Morphological identification of nematode larvae of small ruminants and cattle simplified.

A simplified system has been developed to facilitate identification of nematode larvae of the common nematodes of cattle, sheep and goats. Firstly, in addition to the characteristics conventionally used for identification (such as the shape of the cranial extremity and numbers of intestinal cells), the lengths of the infective sheath tails of infective larvae of each genus/species are related to that of Trichostrongylus spp. instead of using measurements for differentiation. For instance, if the mean length of the sheath tail (the distance the sheath extends caudad beyond the caudal tip of the larva) of Trichostrongylus spp. is assumed to be "X", then that of Haemonchus contortus is 2-2.7"X", and that of Oesophagostomum spp. from sheep is 4-7"X", etc. Secondly, by estimating the proportion of the sheath tail of a larva comprised of a terminal thin whip-like filament, identification is aided, particularly in those L3 of species that resemble one another closely, such as Chabertia ovina and Oesophagostomum venulosum or Oesophagostomum columbianum. After some practice with the system it is usually necessary to measure only one or two sheath tails of L3 in a mixed population, whereupon the identity of most of the remaining L3 can be estimated in relation to those measured, without a need for further measurements. The keys were found to facilitate differential larval identification and are particularly useful for training.

How long before resistance makes it impossible to control some field strains of Haemonchus contortus in South Africa with any of the modern anthelmintics?

This paper describes an exceptional spectrum of multiple anthelmintic resistance in two strains of Haemonchus contortus in South Africa, one from Howick in KwaZulu-Natal, and the other from Badplaas, in Mpumalanga. Apparently for the first time, a helminth strain is described with resistance to compounds from all five of the modern anthelmintic groups used for nematode control in sheep; also, two strains of H. contortus show resistance to the two substituted phenols, dinitrophenol and nitroxynil. Only closantel at 5 mg kg-1 of nine compounds tested appears to have undiminished efficacy against the Howick strain, but even in the case of closantel, the residual activity and minimal effective level need to be tested before it can be concluded that its efficiency is unaffected. The exceptional resistance of the Howick strain is demonstrated by the fact that sequential daily drenching of sheep infected with the strain, with levamisole at 18 mg kg-1, oxfendazole at 20 mg kg-1, levamisole at 20 mg kg-1 and a mixture of fenbendazole at 10 mg kg-1 plus trichlorfon at 132 mg kg-1 on the fourth day, failed to clear sheep of the infection. There are strong indications that side-resistance occurs between dinitrophenol and nitroxynil, on the one hand, and the salicylanilides, on the other, and it is suggested that, before long, strains of H. contortus will be found with high levels of resistance to all the currently available anthelmintics.

Parenterally administered gastrointestinal nematode infective larvae viable after more than 15 years in liquid nitrogen.

After cryopreservation for 13.3-15.8 years, the viability of the infective larvae (L3) of Trichostrongylus axei, T. colubriformis, Oesophagostomum columbianum, Haemonchus contortus, Ostertagia circumcincta, T. falculatus, Nematodirus spathiger, Chabertia ovina and Dictyocaulus filaria was assessed in sheep, by being deposited at their predilection sites. D. filaria was, however, an exception, in that the L3 were injected into the jugular vein. The mean development of all the species was 22.8%, but if three species (O. columbianum, C. ovina and D. filaria), that developed poorly are disregarded, then the mean development was 33.4%, similar to previous tests after shorter periods of cryopreservation. The L3 of some of the species appeared sluggish when examined 10-15 min after being thawed, and in the case of H. contortus practically all the larvae of the original batch tested in the previous trials of the series appeared dead when thawed for use in the present trial, and were replaced by another batch of L3 of the same species. When re-examined after about 8 h, however, a high percentage of the L3 of the original batch appeared to have become revitalised, and their viability was tested in a trial reported elsewhere. The intestinal cells of the majority of the L3 of N. spathiger, O. circumcincta and C. ovina were vesiculated when they were thawed. Nevertheless, the degree of development of the former two species was of the highest in the trial, and it can be concluded that this phenomenon does not necessarily impede the viability of larvae.

Preliminary observations on the potential of gut membrane proteins of Haemonchus contortus as candidate vaccine antigens in sheep on naturally infected pasture.

In a trial lasting 11 months in South Africa, faecal egg counts and haematocrits of sheep vaccinated with gut membrane proteins of adult Haemonchus contortus were compared with unvaccinated controls grazing pasture contaminated with the parasite. Vaccination reduced egg output by >82% on average during one 4 month period of the trial and simultaneously significantly reduced the degree of anaemia and deaths due to haemonchosis. Although vaccine immunity was not sufficiently long lasting to prevent a surge in egg output which occurred after the onset of a period of irrigation, re-vaccinating the sheep at this point cleared their newly acquired infection and rapidly restored protection to approximately the level observed beforehand. It was clear that a vaccine based on parasite gut membrane proteins could offer substantial benefits in the control of natural haemonchosis.

Wildlife parasites: lessons for parasite control in livestock.

For sustainable livestock production it is suggested that the parasitologist take a leaf out of Nature's book in the search for solutions to the mounting problems concerning parasite control. While the farmer has come to regard all parasites affecting livestock as entirely without benefit, indigenous parasites and diseases are normal and play an essential role as interacting components of a natural environment in an ecosystem such as the 19,000 km2-sized Kruger National Park, Republic of South Africa. The parasites help to select their hosts for fitness and are assisted by predators and intra-species territorial aggression which continually eliminate the weak individuals from the system. It is essential to guard against the introduction of foreign parasites or infectious agents which have no real ecological niche or role in an established ecosystem, however, as they cause untoward interactions, sometimes of a violent nature. The policy must be to block off or, failing that, to control or eliminate these foreign parasites and diseases as far as possible. Often, when Man intervenes in an ecosystem, it leads to stress, overcrowding and stagnation and predisposes to disease and death. Intensification of the system, as in farming units, denies Nature the chance to manage on its own, because of clashing interests with Man. Frank parasitism and disease should almost invariably be seen as indicators of an imbalance in the ecosystem and should be rectified. Chemicals and vaccines should be used to produce sufficient food for all, but without exploiting Nature, or else Nature will be unable to continue catering for Man's needs.

Quality control in generic anthelmintics: is it adequate?

We became increasingly concerned about indications of possible substandard efficacy of some generic anthelmintics, particularly after P.C. van Schalkwyk (personal communication, 1990) had found some batches of imported generic products obtained from international brokers to be poorly active, despite apparently normal physical characteristics. Therefore, considering the serious consequences this would have for sheep farming, it was decided to test the efficacy of some of the generic rafoxanide products available on the South African market. One of the three commercial formulations (of highly reputable companies) tested against a known susceptible strain of Haemonchus contortus in sheep was markedly substandard, with an arithmetic mean efficacy of 66.2% (Class B, Reinecke, 1973), compared to Class A efficacy of the other two, which also differed significantly from one another (Mann-Whitney; P = 0.01). Larger differences were found between the three products against a natural infection with a partially resistant strain of H. contortus than against the susceptible strain, with corresponding arithmetic mean efficacies of 28.7% (Class X, or ineffective), 71.3% (Class B) and 87.7% (also Class B). It is concluded that the most likely reason for the observed differences is that international brokers do not disclose the sources of supply of different batches of active ingredient (with the result that the companies buying anthelmintics from them have no way of telling when a source of supply is changed); that the efficacy of such batches differs; and that efficacy testing of individual batches in some cases is inadequate. It is suggested that registering authorities should consider simplified efficacy testing of each new batch of active ingredient before it may be marketed.

A rapid method for differentiating between the infective larvae of Oesophagostomum columbianum and Chabertia ovina.

When freshly-collected, exsheathed infective larvae of Oesophagostomum columbianum and Chabertia ovina were frozen in liquid nitrogen and subsequently thawed, conspicuous vesicles appeared in the intestinal cells of the larvae. The mean number of vesicles, which differed in size according to the species was 20 for O. columbianum and 32 for C. ovina.

An apparatus for the restraint of sheep during infestation with schistosomes.

With this adjustable apparatus sheep aged from five months to five years and varying in mass from 16-95 kg could be restrained effectively. Cercarial suspension was split by only 2/44 sheep during infestation with Schistosoma mattheei. Using a battery of seven cages, it was possible to infest 23 sheep, each for 30 minutes, in one day. No cercariae were more than 4-1/2 hours old at the end of exposure.

A comparison of the infectivity of cryopreserved versus unfrozen infective larvae of Haemonchus contortus, Trichostrongylus colubriformis and Trichostrongylus axei. Results of the Onderstepoort Veterinary Institute and collaborators from 1977 to the present.

The infectivity for sheep of cryopreserved infective larvae (L3) of various strains of Haemonchus contortus, Trichostrongylus colubriformis and Trichostrongylus axei is compared using previously published results of trials conducted at the Onderstepoort Veterinary Institute laboratories, and of collaborators. The means and ranges of development were similar for both frozen and unfrozen larvae of two of the three worm species reviewed. A mean of 33.4% (range, 12.7-63.0%) of cryopreserved H. contortus L3 developed, compared to a mean of 43.7% (range 2.4-78.7%) of unfrozen L3 of this worm species. The corresponding values for T. colubriformis were 33.0% (range 10.3-62.7%), and 33.5% (range 8.3-52.2%), respectively. In the case of T. axei, the development of the cryopreserved L3 (tested in only three trials) was markedly lower than that of unfrozen L3 in the single trial in which the latter was evaluated. It is concluded that development of cryopreserved L3 is probably similar to that of unfrozen L3 and that, for several reasons, maintaining nematode larvae in the frozen state in liquid nitrogen is a much superior method to that of one which entails cycling worm strains continually in their final hosts.

Refugia--overlooked as perhaps the most potent factor concerning the development of anthelmintic resistance.

Anthelmintic resistance involving particularly the gastrointestinal nematodes of small ruminants is escalating globally, to the extent that in certain countries, such as South Africa, it has already reached alarming proportions, and is affecting practically all the anthelmintics. In this paper it is argued that the high levels of resistance in nematodes of veterinary importance indicate that the drugs have been used incorrectly. It is suggested that the phenomenon of refugia plays a much more important role in the selection of anthelmintic resistance than other phenomena that are more frequently investigated and recommended for counteracting it, such as reduced drenching frequency and avoiding under-dosing. While refugia is commonly mentioned in passing in most papers on anthelmintic resistance, it is, almost without exception, not incorporated in the final control/ management proposals. On the strength of the conclusions arrived at in the present paper strategies such as the drench-and-move system in which all the animals in a flock are drenched before they are moved to pastures containing few or no worms in refugia, and the system of strategic drenching on safe pastures should be condemned and never recommended. If such strategies are indeed unavoidable, the farmer should be warned that the farming system would probably not be sustainable even in the short term, in view of the generally high levels of resistance already present in most of the important sheep-producing regions. Farmers should be educated to consider refugia above all else when designing worm management programmes. Finally there seems to be too much complacency concerning the possibility that anthelmintic resistance may also escalate in cattle, eventually to reach the proportions that it has in sheep.