Internal Parasites Resistance of Haemonchus contort us to Broad and Narrow Spectrum Anthelmintics

Resistance to anti-parasitic agents in sheep has emerged as the most important limitation for successful production of wool and sheepmeat in Australia. Intestinal parasitism, and specifically parasitism caused by Haemonchus contortus, has been identified as an important cause of production loss in sheep which has largely been controlled by anthelmintics. In response to the rapid and widespread emergence of resistance to broad spectrum anthelmintics, strategic control programs have been developed (Dash, 1986; Wailer, 1987). These aim to reduce the frequency of treatment with anthelmintics and to maximise the potential benefit by considering environmental and management factors.

H. contortus has developed resistance before other intestinal parasites in sheep. This is related in part to the large biotic potential, principally fecundity, of this species and the frequency of treatments required to control the parasite. At present in Australia, resistance to benzimidazole by H. contortus is widespread (up to 90% of farms). However, levamisole, ivermectin and naphthalophos remain effective. Resistance to salicylanilides is emerging as a significant problem.

Development of Resistance to Various Classes of Anthelmintics

1. Phenothiazine

Resistance was reported by Drudge et al. (1957), Leland et al. (1957), Drudge (1959) and Enzie (1960) which was subsequently found to be related to either fine (Levine and Garrigus, 1962) or coarse (Hasche and Todd, 1963) particle size. Resistance has emerged as a result of long term, low dose, prophylactic control of H. contortus.

2. Benzimidazoles

Resistance in H. contortus to thiabendazole (TBZ) was first reported by Drudge et al. (1964) and later defined by Partosoedjono et al. (1969). Resistance emerged after minimal exposure to the anthelmintic which indicates the likely presence of resistant genotypes prior to the selection by the anthelmintic. Smeal (1968) reported resistance to TBZ on three farms in the New England region of New South Wales and it was later confirmed in surveys (Welsh et al. 1979; Green et al. 1982). High frequency of treatment was closely related to the development of resistance (Barton, 1980). In New Zealand, benzimidazole resistance was reported by Vlassoff and Kettle (1980) and confirmed by surveys that showed some resistance on 20% of sheep farms in 1982 (Kemp and Smith, 1982) but a much more serious situation on goat farms (Kettle et al. 1983). In Europe, the development of resistance in H. contortus has been much slower and less significant (Jordi,1980; Boersema et al. 1982; Cawthorne and Cheong,1984; Kerboent and Hubert, 1985) and confined to benzimidazoles. The reasons for this are unclear. However, the large percentage of parasites not selected in the free living refugia during the growing season has been cited as one reason (Wailer, 1985).

3. Ivermectin

Since the release of ivermectin as an anti-parasitic agent, it has been used on all continents to control intestinal nematodes (Campbell and Benz, 1984) and arthropods (Jackson, 1989). Recent reports have confirmed the emergence of resistance in H. contortus before any other species. Carmichael et al. (1987) advised of a preliminary finding of resistance in South Africa after regular treatment at 3 to 5 week intervals. More recently, Van Wyk and Malan (1988); Echevarria et al. (1989) and Van Wyk et al. (1989) described resistance to ivermectin in South America and South Africa; the ulimate result in one case was to abandon livestock production where no anthelmintic classes were effective (Van Wyk et al. 1989). This report indicates that resistance has emerged in quite different situations; with intensive treatment on irrigated pastures, in locations where H. contortus was not considered important and, of more concern, where only three treatments were used before resistance was apparent. The development of resistance in this way reinforces the view that major resistant genes are present in normal susceptible sub-populations and are rapidly selected after minimal treatments with ivermectin. Alternately, regular and prolonged treatment will select populations that are “more fit”, but with lower gene(s) frequency for the relevant traits.

4. Salicylanilides

Salicylanilides have been used extensively to control H. contortus where benzimidazole resistance was extreme and there was a need to use an effective anthelmintic in rotation with levarnisole and ivermectin. These compounds have a number of advantages. Firstly, they are narrow spectrum anthelmintics with activity agalnst intestinal parasites that ingest blood or have a close association with blood. Secondly, they have little activity against the other major intestinal parasites affecting sheep, therefore no selection is applied to these parasites with the regular use required for the control of H. contortus.

The most recently released salicylanilide, closantel, has an added advantage that treatment removes existing burdens and will protect against ingested larvae so that faecal egg output is effectively eliminated for six weeks (Hall et al. 1981). This attribute has led to the development of strategic treatment programs to control haemonchosis using closantel and broad spectrum anthelmintics to control other trichostrongylid infections (Dash, 1986). Resistance to salicylanilides has recently emerged in South Africa (Van Wyk and Gerber, 1980; Van Wyk et al. 1982, 1987; Van Wyk and Malan, 1988; Van Wyk et al. 1989) and Australia (Rolfe and Boray, 1990).

The development of resistance to closantel is not unexpected given the suboptimal doses that ingested larvae receive after the initial peak in plasma concentrations in a treated animal and the widespread use of rafoxanide for control of either H. contortus or Fascio!a hepati Ca. However, in Australia the previous use of salicylanilides has not predisposed to resistance in isolates where histories of treatment were available and in other cases resistance has emerged after only minimal use of closantel and no history of use of rafoxanide (Rolfe et al. 1990). Closantel resistance has been the subject of recent intense investigation in Australia (Rolfe and Boray, 1989; Rolfe et al. 1989; Rolfe and Boray, 1990).

Field isolates of H. contortus have been obtained from farms in various locations including the northern tablelands, north western slopes and north coast of New South Wales, South Australia and Western Australia. Most of the properties had either fair control of H. contortus (presence of H. contortus on larval differentiation sufficient to be of concern in the Wormkill program) or poor control of H. contortus (actual haemonchosis occurred in lambs/weaners). Most had used salicylanilides at some stage either for control of nematodes or trematodes. No salicylanilides had been used on the farms where four isolates were recovered.

Efficacy Against Existing Worm Burdens

Resistance to closantel can be expressed as decreased efficacy against pre-existing burdens or by a decreased period of protection against ingested larvae.The efficacy of closantel (2.5 mg/kg) was assessed against isolates of H. contortus in slaughter trials (n = 4). The dose rate was used because of the exceptional high efficacy at 1/3 the recommended dose rate from studies completed in susceptible isolates. Resistance will be expressed, initially at least, at lower dose rates.

Twenty isolates of forty-nine assessed were determined to be resistant, that is, defined as a greater frequency of individuals within a population able to tolerate doses of a compound than in a normal population of the same species. Some isolates had been transferred from other farms, but at least six different and separate loci of resistance have been found. It is important to note that resistance has been found on properties where there has been extensive use of salicylanilides (especially the northern tablelands) and also where this group of chemicals has not been used or used minimally (north west slopes). The Warialda S strain had no apparent previous exposure to any salicylanilides either on the property or through introductions of sheep. Similarly, many isolates were susceptible to closantel that had extensive previous use of salicylanilides. Resistance has emerged in two distinct situations: firstly, in the expected manner after repeated exposures to closantel perhaps with a “background” resistance caused by use of rafoxanide (northern tablelands) or, alternatively, by slow selection for a “more fit” genotype; secondly, with minimal or no exposure to closantel (north west slopes). The latter circumstance raises the possibililty of rare gene(s) in the resistant population of H. contortus that could be expressed rapidly after some exposure to closantel.

The first confirmed resistance in goat flocks was observed from the north coast of NSW (G. Fraser, personal communication, 1989). This isolate was exposed to regular repeated closantel treatments (four times a year for three to four years). Resistance was expressed initially as increased percentages of H. contortus in routine cultures and rapidly progressed to control failure and clinical haemonchosis.

In our studies, selected resistant and susceptible isolates were examined for susceptibility to closantel at increasing dose rates to assess the relative susceptibility and development of resistance between strains and to determine the dose rate required to gain maximum efficacy. Also, susceptible isolates were examined to ascertain the range of efficacies in normal populations. The three susceptible isolates were highly susceptible to closantel at 2.5 mg/kg and higher dose rates (Table 1). Armidale 2 and South Australia 1 strains had extreme and no previous exposure to salicylanilides respectively. Four resistant isolates were examined. The first field isolates with suspected resistance (Wallangra 1 and 2) were the least resistant with high efficacy of closantel at 5.0 mg/kg. The Armidale 4 was of intermediate resistance; suboptimal efficacy was present at 7.5 mg/kg indicating that pre-existing burdens will not be completely removed by the recommended dose rate. The Manning 1 strain (titration studies courtesy of Dr G. Fraser, Wollongbar Regional Veterinary Laboratory) was the most resistant with inadequate efficacy even at 10 mg/kg. The latter two isolates represent those that have developed resistance in the predicted manner; the Wallangra 1 strain and possibly Armidale 4 represent the unique emergence of resistance after minimal selection.

Table 1. Efficacy of closantel against Hoemonchus contortusin sheep

STRAIN	EFFICACY % Dose Rate (mg/kg)
A. Susceptible	0.625	1.25	2.5	5.0	75	10.0	15.0
McMaster	-	70	99.5	99.5	99.9	-	-
Armidale	-	99.3	99.9	99.6	-	-	-
South Australia	31.7	12.6	98.1	99.9	99.8	-	-
B. Resistant
Wallangra 1	0	43.2	85.3	98.4	99,7	100	-
Manning 1	-	-	48.9	8.9	53,2	72.9	99.9
Armidale 4	0	39.9	17.4	62.6	93,9	99.5	-
Wallangra 2	-	-	68.2	96	98.7	100	l00

Protection Period Studies

Two isolates were examined to determine the period of protection given by a single treatment with closantel compared to a susceptible strain using prolonged trickle infection.

Wallangra 1

The period of protection given by closantel at 7.5 mg/kg was reduced from 43 days (McMaster susceptible) to 23 days when lambs were regularly infected (3 x week, n = 15 per group). Untreated lambs infected with either strain began passing eggs in faeces at 14-17 days. Total worm counts (n = 5) at various stages after treatment reflected the trends observed in egg counts.

These results indicate that even at a low level resistance, the period of protection was greatly reduced and practically eliminated despite removing pre-existing burdens. Therefore, programs that rely on the period of protection given by closantel will be jeopardised.

Manning 1

A similar pattern emerged with this isolate as occured with Wallangra 1. The protection period was reduced from 48 days (McMaster susceptible) to 19 days (Manning). The lambs used in this trial were worm free but not naive resulting in a delay in onset of egg output in faeces in treated and untreated lambs. Effectively the period of protection was eliminated in treated lambs. Lambs treated with nitroxynil, a related monophenolic compound, were also assessed for efficacy against closantel resistant isolates and the period of protection it gave after treatment. A short period of protection was given against a susceptible strain, although side resistance to closantel was present.

Efficacy Against Larval Stages

A comparison was made between the efficacy of closantel against larvae and early adults of susceptible and mildly resistant strains. Four and 11 day old larvae were very resistant to closantel (28.7 and 29.5% reduction, respectively) compared to susceptible larvae of the same age (96.9 and 95.1%, respectively) and to adult (21 days old) resistant strains. Enhanced resistance at this stage helps to explain the elimination of the protection period even against mildly resistant isolates.

5. Levamisole

Resistance to this anthelmintic has not emerged in H. contortus despite its widespread use over many years. Green et al. (1981, 1982) reported resistance in goats. However, levamisole has been found to have inadequate efficacy in goats compared to sheep (Gillham and Obendorf, 1985). The continued reliability of levamisole against H. contortus should be the subject of investigation. Why has this anthelmintic remained effective when treatments have not been applied with any greater discipline than other anthelmintic classes? Research effort into the specific mode of action would be very beneficial and provide useful insights into why levamisole remains effective.

6. Organophosphates

Organophosphates have been widely used in Australia as a short acting anthelmintic, principally to control H. contortus where resistance to broad spectrum anthelmintics was present. A narrow spectrum anthelmintic from a separate chemical class has distinct advantages where regular treatment is necessary to control H. contortus, but less frequent strategic treatments are needed to control other intestinal nematodes. Naphthalophos has been withdrawn from the Australian market by the manufacturer; no resistance has been detected to this anthelmintic.

Detection of Resistance

A wide range of tests has been developed to detect resistance for research and diagnostic purposes (Presidente, 1985). In-vitro assays have been a useful tool for research but with limited application for routine testing of isolates from farms especially where mixed populations of parasites are present. The tests for detecting benzimidazole and levarnisole resistance include the egg hatch assay (Le Jambre, 1976), tubulin binding assay (Lacey, 1985) and more recently the larval development assay (E. Lacey, personal communication, 1989). However, they do not successfully detect ivermectin and closantel resistance (Lacey, unpublished data; Rolfe, unpublished data). In the case of closantel, it appears that eggs and larvae up till the ex-sheathed L₃ stage are not affected by closantel in-vitro or that differences between resistant and susceptible strains are not manifest at this stage. The effect of benzimidazoles on polymerisation of tubulin to microtubules in nematodes and the avoidance of this effect in resistant worms has led to the development of the tubulin binding assay as a method of detection of resistance (for review see Lacey, 1988). This test is the only biochemical test routinely available that is based on a primary mechanism of resistance. Further studies on a specific enzyme activity responsible for drug tolerance are being researched in Australia at this time which will lead to a diagnostic assay.

The most consistently reliable and practical method for detecting resistance in the field in sheep has been the faecal egg count reduction trial. It has been used as a survey tool in many countries and has been recently standardised and used extensively for routine diagnosis in all Australian states. The results correlate well with in-vitro techniques (Lacey, 1985; Presidente, 1985). A modification of the technique to test the efficacy of closantel against H. contortus takes advantage of the high efficacy of closantel at 2.5 mg/kg against pre-existing burdens. Post-treatment samples are taken at 10-14 days after treatment with an expected 95% reduction in the treated group compared to untreated controls. The technique requires that accurate differentiations of larvae are performed on treated and control groups. Alternatively, the normal recommended dose can be given and the faecal egg output monitored up to 45 days after treatment. In sheep infected with a susceptible strain, faecal egg production should not occur before 40-45 days if the correct dose is given. Both procedures require that a reasonable burden of H. contortus is present, the latter method is dependent on continuous exposure to infective H. contortus larvae.

Egg count reduction percentages and reduction in worm counts in critical slaughter studies, 7 days after treatment are very comparable in those strains examined so far particularly when a 5% confidence limit is considered (Rolfe, unpublished data). Strains that are resistant may have a percentage reduction in egg count close to 95% but have a 5% confidence limit less than 90%. Ivermectin resistance in H. contortus should be detectable in the faecal egg count reduction test based on the history of the White River and Stellenbosch strain (Van Wyk et al. 1985). However, a recent report by Mason (1990) indicates that one manifestation of resistance is the suppression of egg production in resistant Ostertagia circumcincta. Based on this report eggs in faeces after treatment cannot be used as an indicator of the efficacy of ivermectin against this parasite species at least.

A priority for closantel resistance research is for a good in-Vitro diagnostic method. Currently a number of alternatives are being assessed. Two procedures are promising. The first is the in-vitro treatment and assessment of the motility of 21 day old adult worms in-vitro 8-12 hours after treatment. The second is the use of guinea pigs as a model for H. contortus infection in sheep. Previous studies have concentrated on the guinea pig as a model for immunological expulsion (B.M. Wagland, personal communication, 1989). Differences have been observed between limited studies on resistant and susceptible strains when treated 3 days after infection at varying dose rates (Rolfe, unpublished data).

Detection tests based on the mode of action of closantel have not been developed. Its action is still unclear except that as a group the salicylanilides act as uncouplers of oxidative phosphorylation at the mitochondrial membrane by interruption of the reduction of fumarate to succinate. This process does not discount other possible mechanisms of action. H. contortus resistant to closantel presumably avoid the disruption of aerobic metabolism by some competitive mechanism, although the mechanism is poorly understood.

Combinations of levamisole and benzimidazoles have been shown to delay the development of resistance to both anthelmintic classes (Martin, 1990). The maximum benefit from combinations will occur when the frequency of resistant genes in a parasite population is at a low level. This is not the case in Australia. Combination anthelmintics have been found to have acceptable efficacy in 30% of farms where, if given individually, the components fail. While combinations will be useful for control of T. colubriformis and O. circumcincta, where good control can be gained in strategic treatments of short acting anthelmintics, the control of H. contortus will require regular treatment with short acting anthelmintics and therefore is undesirable. Combinations will at least be a short term measure to extend the life of benzimidaxole and levamisole compounds.

There is also an urgent need for a reliable method for diagnosis of ivermectin resistance. Currently available methods involving molecular genetics will be the most rewarding area of research. However, they need to be in place before resistance is established and widespread.

Ivermectins apparently act by potentiation of the action of gamma-aminobutyric acid (GABA) at the interneuronal junctions of nerves which use GABA as a neurotransmitter (reviewed by Campbell, 1985; Jackson, 1989). The potentiation of GABA is thought to result in an influx of chloride ions and hyperpolarisation of motor neurons leading to paralysis, although other neuromuscular effects may be present. Resistance to ivermectin will necessarily circumvent the effects of ivermectin at the neuronal synapse.

Management of Resistance

The inevitable outcome of resistance is the need to implement strategies to delay the onset and severity of the problem. However, too often strategies have only been considered after the appearance of resistance when the frequency of resistant genotypes in a parasite population is at a high level. Unfortunately the marketing of anthelmintics to farmers has not taken into account the best options in formulation, duration of effect and dose rate to slow the development of resistance, but rather to maximise chemical sales in an environment where resistance and demise of chemical classes is considered inevitable.

A number of strategies have been developed in Australia to slow the development of resistance to all anthelmintic classes in the three main parasite genera affecting sheep. They are based on treatments timed for maximum benefit based on epidemiological studies, annual rotation of effective anthelmintics, combinations of anthelmintics, provision of ‘low worm’ pastures and adequate nutrition for susceptible sheep. In summer rainfall regions the strategic use of a combination of effective broad spectrum anthelmintics against O. circumcincta and Trichostrongylus co!ubriformis and prolonged protection with closantel against H. contortus has been successful (Dash, 1986). Whilst alternatives usually exist for broad spectrum control of resistant T. colubriformis and O. circumcincta no obvious options exist for control of H. contortus resistant to closantel.

Several strategies are being examined on experimental sites and farms where closantel resistance is present. Levamisole is still effective against H. contortus although resistance is widespread in the other major parasites. Treatment at strategic times with levamisole or naphthalophos (August, November, February) and movement to pastures with low numbers of infective larvae will control H. contortus (Barger, 1978). However, it is incompatible with intensive and economic wool production. Four weekly treatment in lambs has been the preferred option before the introduction of closantel; the disadvantages are the rapid selection for resistance in non-target species, the high labour inputs required for treatment and maintenance of a reservoir of infection on pasture. Treatment at 2 weekly intervals at the onset of the rise in available infective larvae on pasture is required for a period likely to outlast the life expectancy of eggs and larvae on pasture. The effect of such intense treatment on selection for resistance in target and non-target species is being assessed. Another option was to use closantel at higher dose rates and more frequent intervals more like a conventional short activity anthelmintic. The danger is that resistance will be selected at a greater rate and exacerbate the problem.

Slow release dissolution devices have been recently introduced onto the Australian market (reviewed by Anderson, 1985). Albendazole is released at a rate of 32.5 mg/day for approximately 100 days by an intraruminal device. Despite widespread benzimidazole resistance in H. contortus, effective control is being achieved on some properties and plot studies when the device is administered in late winter (E. Hall, personal cummunication,1990; P. Rolfe, unpublished data). The opportunities would appear to exist to incorporate levamisole or closantel in these devices to provide sustained protection with minimal or less selection for resistance.

Ivermectin has not been promoted as a means of controlling H. contortus because of the frequency of treatment required for H. contortus and concurrent selection for resistance in the three major parasite species. Large scale promotion of programs to slow the development of resistance has greatly increased the awareness of fanners of the need to use effective anthelmintics sparingly, although many farmers are faced with the need to use ivermectin every year and at frequent intervals if resistance to levamisole, benzimidazoles and closantel is present. Clearly the economic imperative to change management systems will not be present until the failure of more economic but resistance plagued solutions. The advisory messages to farmers on non-chemotherapeutic solutions have largely been ignored. A greater awareness of interaction between parasites, stocking rates, nutrition and safe, low worm pastures will be necessary in the short term. Long term solutions are not available; other anthelmintic classes may be found, but a considerable time is required for development and registration. The development of vaccines is a long and difficult process and at best will realistically provide a useful adjunct to other control measures.

Acknowledgement

The work carried out in our laboratory on salicylanilide resistance has been supported by a research grant from the Wool Research and Development Council of the Australian Wool Corporation.

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