External Parasites

Summary

Pesticide resistance has been found in three species of arthropod parasites of Australian livestock. Strains of Lucilia cuprina, the Australian sheep blowfly, are resistant to organophosphate insecticides and strains of Damalinia ovis, the sheep body louse, are resistant to synthetic pyrethroids. Resistance to organophosphates and synthetic pyrethroids has been found in strains of Boophilus microplus (cattle tick). Some mechanisms of resistance and methods of controlling resistant strains are discussed.

Organosphosphate (OP) Resistance In The Sheep Blowfly (Lucilia cuprina)

History

Organophosphates (OPs) were introduced around 1950, as a replacement for the cyclodiene compounds (chlorinated hydrocarbons) to which significant resistance had occurred (Shanahan and Hart, 1966). The OP insecticide most used has been diazinon, with fenthion ethyl, chlorfenvinphos, dichlofenthion and coumaphos also being used to a lesser extent (Hughes, 1981). A more recent introduction is propetamphos (Gruss et al. 1988).

The compounds act by blocking the activity of the enzyme cholinesterase, which is required for removing the neurotransmitter acetylcholine from the post-synaptic membrane after the passage of a nerve impulse. These insecticides are frequently referred to as anticholinesterase compounds.

OPs are applied to sheep by either dipping or jetting. There is also a diazinon and cypermethrin combination which is applied as a long wool backline treatment.

Emergence of resistance

OP tolerance in L. cuprina was first reported in Australia by Shanahan and Hart (1966). They found that a field isolate from Dubbo in central western New South Wales had a 3.lx tolerance to diazinon and a 3.5x tolerance to ronnel, indicating a non-specific tolerance to both diethoxy and dimethoxy OP esters. Further investigation found some level of OP tolerance in 25 out of 29 field strains. Shanahan (1967) also reported tolerance to butacarb (a carbamate insecticide with a similar mode of action to OPs) in a field strain of L. cuprina. By 1974, strains tolerant to chlorfenvinphos, diazinon, fenchlorphos and fenthion ethyl had been found (Shanahan and Roxburgh, 1974). The protection period afforded against resistant strains tested in the laboratory was between 7 and 11 weeks (a reduction of 4 to 8 weeks) depending on whici~ OP was used. The protection period against carbamate resistant strains was reduced from 14 weeks to 4 weeks (Shanahan and Roxburgh, 1974). The situation was not the same as when cyclodiene resistance developed. In that case, resistances of several hundred times normal were recorded and resistant strains were generally unaffected by the insecticide. A genetic basis for this phenomenon was proposed by McKenzie and Whitten (1984). The emergence of tolerance to the OPs nevertheless demonstrated the ability of L. cuprina to adapt itself to new insecticides (Shanahan, 1967).

Resistance in the field

The field resistance situation was surveyed in New South Wales, South Australia and Western Australia in 1974, where resistance factors were found to be higher than in Queensland, which was surveyed in 1977-78, although all of the 19 strains collected in Queensland showed resistance to diazinon, dichiofenthion, fenthion ethyl and chlorfenvinphos. Protective periods against resistant strains were reduced to 2 to 3 weeks when blowflies were very active. The combination of resistance, poor insecticide penetration of the fleece and the presence of organic matter contaminating the fleece were considered by O’Flynn and Green (1980) to be collectively responsible for the poor performance of the OP compounds against resistant strains.

Genetics and mechanisms of resistance

The resistance status of an insect population is usually a function of the level of insecticide use, with the resistant phenotypes losing their advantage in the absence of the insecticide. In L. cuprina, however, McKenzie et al. (1980) found that resistance was present at a high level in all of the populations that they surveyed and when selection pressure was relaxed, the level of resistance generated a stable plateau, with little evidence of regression to susceptibility.

Hughes (1981) found that the OP resistance gene was present at a high frequency in field populations of L. cuprina and suggested that the diazinon resistance gene may confer cross-resistance to other OPs used for blowfly control. Some strains, however, were resistant to diazinon but susceptible to coumaphos. There also appeared to be a separate mechanism for carbamate resistance in the field.

The selective advantage of resistant over susceptible larvae is greatest in the period between 10 and 30 weeks after treatment, when the insecticide concentration on the sheep does not afford any further protection from fly strike, so, although treatment directed specifically at blowflies is sporadic, under commercial management conditions selection for resistance is continuous and is compounded by the use of OPs at lower concentrations against other ectoparasites (McKenzie and Whitten, 1984).

The resistance mechanisms developed by L. cuprina may involve one or a combination of reduced penetration of the insecticide across the cuticle and degradation and excretion of the insecticide (McKenzie, 1984).

The discovery of specific resistance to malathion (Hughes et al. 1984) and its presence in 11% of flies collected from the field was unusual, since malathion was not used for the control of L. cuprina except in limited experimental applications, which proved unsatisfactory, because the compound was not repellent to adults and the larvicidal activity was less than 7 weeks (Riches and O'Sullivan, 1957). The use of the malathion synergist, triphenyl phosphate, together with malathion abolished the resistance, thus indicating that the mechanism of malathion resistance was probably enhanced carboxyl esterase activity in the blowfly (Hughes et al. 1984). The resistance was subsequently found to be due to a single incompletely dominant gene on the same chromosome as the major OP resistance gene which confers resistance to the wider range of OP insecticides. The mechanism of this wider resistance was also found to be an esterase (Hughes et al. 1984; Raftos and Hughes, 1986).

Control of resistant strains

Strategic treatment to disrupt the blowfly lifecycle

If the spring emergence of overwintering blowflies coincides with warm, wet, humid conditions and the presence of large numbers of susceptible sheep, then blowfly numbers increase rapidly and remain high for the rest of the season. If the number of larvae entering the soil in the autumn is reduced, blowfly numbers in the following spring are depressed for a long period and catastrophic strikes are avoided.

Early autumn treatment to prevent susceptibility to strike during the autumn reduces the bank of larvae entering the soil to overwinter and therefore reduces the number of spring emergents. Long-term knowledge of blowfly annual cycles and strike patterns characteristic of particular areas is needed to determine other opportune times when early treatment would be beneficial (Anderson, 1989).

An alternative insecticide to the OPs can be used for this type of strategic blowfly treatment. Cyromazine is a triazine insecticide developed from existing herbicides, which interferes with moulting in L. cuprina larvae. The compound is primarily absorbed by the larvae from the stomach after it has been ingested, but also has some contact effect (Hart et al. 1979). Shanahan and Hughes (1980) found that the compound was effective against first instar larvae of both susceptible and OP resistant strains of L. cuprina. There was no indication that OP resistant stains had any cross-resistance to cyromazine. A concentration of 1 g/L gave treated sheep effective protection for up to 14 weeks (Hart et al. 1979) with nearly complete protection from fly strike for 9 weeks in the majority of trials (Hart et al. 1982). Levot and Ship (1983) found that cyromazine at doses of up to 4 μg per adult female fly did not affect egg hatching but did reduce the number of larvae reaching development, indicating that oral uptake by larvae was not essential to the action of the compound and that sufficient amounts could be transported to the eggs of gravid females to have a delayed toxic effect. The compound is applied by jetting, plunge or shower dipping.

Protection against blowfly strike is also provided by the use of synthetic pyrethroids as oviposition suppressants. There are currently two synthetic pyrethroid plus diazinon formulations registered for blowfly and lice control. One of these is applied through a manual applicator and the other using an automatic race. The insecticide is applied to the tip of the fleece where it acts as an oviposition suppressant. The products must be applied along the backline and around the crutch in order to achieve efficacy against blowflies. The backline method of application is considered to give less efficient control of flystrike than thorough hand jetting (Arundel and Sutherland, 1988).

Reducing sheep susceptibility

Crutching, mulesing, shearing and insecticide treatments reduce the feeding and breeding sites for blowflies. The development of OP resistance means that this group of insecticides, while no longer providing reliable long-term protection against blowfly strike, still has a use for the treatment of struck sheep where an immediate kill of larvae is necessary.

Direct reduction of blowfly numbers

A bait bin has been developed which, when placed in the preferred blowfly habitats such as creek beds, permanent sheep watering points, camps and yards where sheep are regularly mustered, attracts and kills large numbers of blowflies. Excluder wire is used on the bins to reduce the kill of native blowflies which are the main competitors with L. cuprina on carcases (Anderson, 1989).

Selection of sheep for reduced susceptibility to blowfly strike

Breeding sheep for reduced susceptibility to flystrike should focus on body strike, as other types of strike can be controlled by management. Fleece rot renders sheep highly susceptible to body strike (Merit and Watts, 1987 a, b) and fleece rot resistance is the best known breeding indicator for body strike resistance.

Considerable genetic variation exists in resistance of sheep to body strike and fleece rot and in merinos this has been identified between strains, between blood lines and between individuals within a flock. Raadsma (1987) suggested that the utilisation of differences between strains and bloodlines offers the best potential for rapid improvement in resistance to these diseases. Reliable identification of the more resistant flocks is a limiting factor.

Direct selection can be undertaken based on the expression of fleece rot, water stain and faults such as malformed withers in individual sheep.

Indirect selection on the basis of fleece and skin characters is not as successful as direct selection. The usefulness of indirect selection traits depends on their heritability, correlation with fleece rot and body strike, cost and ease of measurement and degree of expression. The best indications so far are greasy wool colour, fibre diameter variability and possibly wax content, but Raadsma (1987) cautions that they should not be incorporated into sheep breeding programs until their effect on resistance to fly strike and other production traits is known.

Alternatives to insecticides

Bacteria, protozoa and viruses

Several biological alternatives to the use of insecticides for the control of L. cuprina have been investigated. Of these, the use of bacterial pathogens which act as larvicides would seem to offer the most promise. Cooper and Pinnock (1983) reported that first instar L. cuprina larvae are highly susceptible to two strains of Bacillus thuringiensis. Other organisms offering potential biological control are the microsporidean protozoa Octosporea muscaedomestricae which affects both adult and larval flies (Cooper and Pinnock, 1983) and some iridoviruses.Thomson and Bushell (1983) reported the infectivity of Chilo, Tipula and Sericesthis iridescent viruses in L. cuprina, but oral transmission of the viruses does not occur naturally in the field or experimental populations of flies. Modification of the virus so that it is protected from destruction in the foregut of the fly and can then reach the hindgut, which is most susceptible to infection, may make natural oral transmission possible (Thomson and Bushell, 1983).

Parasitic nematodes

The control of blowfly larvae using parasitic steinemematid and heterorhabditid nematodes was investigated by Bededing (1983). He suggested that strains of nematodes selected for a high level of infectivity in L. cuprina larvae, could be distributed in parasitised larvae in the spring and autumn and could act as a source of infection in quiescent field populations of larvae which remain dormant and susceptible in the soil for an extended period. Bedding (1983) suggested that distribution within sheep campsites where blowfly pupariation may be concentrated, could provide useful control. Trials were also conducted to determine whether nematodes would infect larvae feeding on sheep, but while some nematodes survived on the fleece, there was negligible infection of the L. cuprina larvae (Bedding,1983).

Genetic control

Irradiation or chemosterilisation of males (SIRM) - considered biologically feasible but uneconomical for the control of the sheep blowfly.

Exploitation of unique genetic phenomena - naturally occurring lethal or deleterious genes.

Cytogenic techniques - also called chromosome mechanics, this technique is being evaluated and tried in the field.

Genetic engineering - this is also being evaluated (Whitten and Maddern, 1983). One of the main problems with genetic control is the lowered competitiveness of laboratory-reared strains compared with field strains. Mahon (1983) concluded that an improvement in competitiveness would at best be desirable and under some conditions would be essential for the successful implementation of a genetic control program.

Vaccination

There are two potential sources from which vaccines may be produced.

Vaccination against 1arvae - Field observations by Watts (1979) indicated that sheep may acquire resistance to strike through repeated infestation and Sandeman et al. (1986) demonstrated protective resistance in some sheep in a group exposed to successive larval challenges. O’Donnell et al. (1981) found that while antigens extracted from third instar larvae induced high levels of circulating antibody in immunised sheep, the titres produced were not protective against first instar larvae. Montague et al. (1986) reported that a vaccine based on culture liquors of L. sericata achieved no-strike rates of 80-90% compared with 40-50% in controls. However, such vaccines were not protective against L. cuprina.

They also found that homogenates of whole larvae were unsuccessful as vaccines as were extracts of dissected organs. They concluded that cloning of blowfly genes to allow production of pure immunogens may lead to production of useful vaccines (Montague et al. 1986).

Vaccination against fleece rot - Work by Burrell (1985) indicated the feasibility of indirect control of flystrike through the vaccination of sheep against Pseudomonas aeruginosa. More extensive field trials are required for a full evaluation of this method (Arundel and Sutherland, 1988).

In the immediate future, insecticides will probably continue to be the main method of controlling L. cuprina, with the possibility of significant assistance from biological and/or genetic control or vaccination in the longer term.

Synthethic Pyrethroid Resistance In The Sheep Body Louse (Damalinia ovis)

History

The first synthetic pyrethroid (SP) compound (allethrin) was produced for commercial use in 1949, but the early compounds had a pronounced knockdown effect without necessarily killing the target insect. They also broke down rapidly in ultra-violet light and therefore had little persistency after application. The second generation of photostable SPs appeared in the 1970s (Elliott et al. 1973). Subsequently the pour-on application of SPs, deltamethrin (Bayvel et al. 1981; Kettle et al. 1983), cypermethrin (Henderson and McPhee, 1983; MacQuillin et al. 1983) and alphamethrin (Sherwood and Page, 1988) became widely used for the control of D. ovis in Australia. In this technique a high concentration of the insecticide is deposited in a band down the sheep’s back. The insecticide then moves to the rest of the fleece travelling in the grease on the wool and skin surface. The concentration of the insecticide decreases with the distance from the backline (Kettle et al. 1983; Johnson et al. unpublished data).

Using autoradiography, Jenkinson et al. (1986) found that cypermethrin in a pour-on formulation spread rapidly to a distance of 8 cm from a spot application and also penetrated the stratum corneum, particularly at the site of application. Using a similar technique, Johson and Dixon (unpublished data) found labelled insecticide on wool fibres both above the surface and in the follicles in the epidermis and dermis. Work by Darwish et al. (unpublished data) indicates that the amount of synthetic pyrethroid on the wool fibres may be greater than that on the skin surface. Johnson and Boray (unpublished data) used a liquid scintillation counting technique on full thickness skin biopsies to demonstrate the movement of deltamethrin around the bodies of lice and itchmite infested sheep and an uninfested sheep. The insecticide moved from the backline within 12-24 hours after application and the results indicated that movement was more rapid on the ectoparasite infested sheep. The results also suggested that the concentration of insecticide varied considerably over the body of the sheep and this is currently under investigation in our laboratory.

SP compounds are also efficient against lice when applied in plunge and shower dips. Hall (1978) found that cypermethrin was efficient at concentrations down to I ppm and that a concentration of 10 ppm would eradicate lice from infested sheep and prevent reinfestation for up to 19 weeks. Cypermethrin, cyhalothrin and alphamethrin dips are currently registered (July 1990).

The insecticidal activity of pyrethroids is due to their neurotoxic effect. They act at the nerve membrane to modify the sodium channels, probably impeding protein conformational changes at the lipid-protein interface. The resulting neurophysiological changes depend on the nerve element, temperature, and the structure of the applied compound, but typically include repetitive firing, blockade of impulse conduction or neuromuscular transmission and spontaneous depolarisation of the resting potential. Sensory neurones, neurosecretory cells and nerve endings seem particularly sensitive to these effects. The knockdown effect, which is a characteristic of the pyrethroids, is probably produced by peripheral intoxication, while the lethal activity of the compounds involves both peripheral and central neurons (Zerba, 1988).

The relative toxicities of the currently registered synthetic pyrethroids are, in order of increasing toxicity:

1. cypermethrin

2. alpha-cypermethrin

3. deltamethrin

4. lambdacyhalothrin

Emergence of resistance

Resistance to SP insecticides has developed in a number of arthropod species of agronomic or veterinary importance, including members of the following Orders:

Diptera (flies), Lepidoptera (butterflies and moths), Coleoptera (beetles), Hemiptera (bugs and leafhoppers) and Acarmna (mites) (Georghiou, 1986). Order Phthiraptera can also be added to this list, with the development of resistance in D. ovis (Boray et al. 1988, 1989; Johnson et al. 1989; de Chaneet et al. 1989; Levot and Hughes 1989, in press).

Synthetic pyrethroid resistance in ectoparasistes of animals was first reported in populations of horn flies (Haematobia irritans) affecting cattle in the United States (Sheppard and Hinkle, 1985; Sparks et al. 1985), where it was associated with the use of fenvalerate and flucythrinate impregnated ear tags. The purpose of these tags was to provide a sustalned release of the insecticide over a number of weeks. The pyrethroid was released in a fine powder which was spread over the body of the animal by normal grooming and contact with other animals. Between 1979 and 1984, control of horn fly with pyrethroid impregnated ear tags was reduced from 100% to 28%. Synthetic pyrethroid sprays were still effective against resistant strains. Taylor et al. (1987) found that the concentration of cypermethrin released from similar ear tags was highest around the head and back of the cattle and declined markedly down the flanks and towards the udder. In reviewing the development of SP resistance in horn flies, Sparks et al. (1985) discussed the operational and biological conditions

necessary for the development of resistance and concluded that most of the predisposing factors for the rapid development of resistance were present in some control methods using slow release devices. Some of the operational conditions may also be applicable to the pour-on technology for lice control, such as:

• prolonged exposure to a single insecticide group with a persistent action;

• multiple generations of the insect being selected;

• high selection pressure, no refuge for the exposed population;

• widespread use of the insecticide; and

• a low population threshold for the application of the control measures.

In the case of pour-on louse treatments, the insecticide takes time to diffuse around the body and to reach a concentration lethal to lice. If movement of the insecticide is inefficient for any reason, then sub-lethal concentrations of insecticide may occur on some areas of the skin.

Under these circumstances, aspects of louse biology also favour selection for resistance:

• limited migration between populations;

• the monophagous diet of the parasite;

• a comparatively short generation time;

• relatively large numbers of offspring per generation.

Offsetting these factors is the tendency for treatments against lice to be restricted to one per year. This may facilitate the survival of non-selected strains.

Field SP pour-on failures caused by resistance

Boray (personal communication) predicted that the use of pour-on formulations was likely to select for synthetic pyrethroid resistance in the D. ovis population in the field. Since 1986, resistance to synthetic pyrethroids applied as pour-on treatments has emerged as a serious problem and poses a threat to the usefulness of synthetic pyrethroids for lice control (Boray et al. 1988).

In 1986, a number of field failures of pour-on SP treatments which could not be explained by poor application technique or inappropriate use, were received from different areas in NSW. Given the propensity for the development of resistance associated with pour-on treatments, it was decided to obtain infested sheep from some of these flocks for further examination.

In-vitro testing

To date, 35 strains of lice have been examined using an in-vitro contact test for susceptibility to synthetic pyrethroids (Levot and Hughes, 1989, in press). Two fully susceptible strains were obtained from the field to provide a reference for the evaluation of unknown strains using probit regression analysis. The results of this work indicate that some strains of lice have developed an increased tolerance to all of the SP compounds registered for lice control. The resistance factors obtained for the various synthetic pyrethroids are shown in Table I.

A field lice resistance detection test has been developed to assist veterinarians, livestock officers and Pastures Protection Board staff to identify resistant strains of D. ovis (Boray et al. 1989).

Table 1. Synthetic pyrethroid resistance factors* in strains of sheep body lice (Damalinia ovis).

STRAIN	SP: cyper-methrin	delta-methrin	alpha-methrin	cyhal-othrin
PeakHill**	0	0	0	0
Graben Gullen	0.8	0.5	3.2	1.1
Hillston Selected	1.8	0.9	2.4	0.8
Glencoc	1.9	1.9	3.2	2.7
Western Australia	2.2	0.9	4.3	2.1
Manildra	2.6	1.2	3.3	1.3
Singapore	3.0	4.7	7.5	7.4
Wee Jasper	3.9	3.3	6.8	4.8
Merrygoen	4.2	3.1	5.2	4.5
Junee	4.2	3.7	6.2	3.2
Gundagai	4.2	3.9	5.9	4.2
Adelong	4.2	3.9	6.9	5.7
Yarrow	4.4	3.3	4.2	4.7
Gravesend	4.9	2.5	4.1	3.8
Holbrook Selected	5.1	3.5	10.4	6.7
Temora	5.6	8.2	9.8	8.1
Holbrook	5.7	4.8	13.3	7.7
Armidale	6.0	5.4	7.7	7.1
Carcoar	6.6	5.4	6.2	8.3
Dewrang	11.8	5.3	14.6	5.8
Adams Scrub	12.7	5.9	8.8	2.9
Wallangra	14.9	10.3	13.5	26.7
Claremont	19.4	13.5	26.2	18.1

** Susceptible reference strain

* Resistance Factor is determined by LD50 Unknown Strain LD 50 Susceptible Strain

LD 50 is calculated using probit analysis of in-vitro test data.

Dipping and pour-on trials

Dipping titrations

Confirmatory trials were carried Out by individually dipping sheep infested with resistant strains of lice in concentrations of cypermethrin ranging from 1 ppm (the discriminating dose for fully susceptible lice) to 4 ppm (Figure 1). Agreement was obtained between the in-vitro bioassays and in-vivo results (Boray et a!.1988, 1989; Johnson et al. 1989).

Figure 1 Efficacy of Cypermethrin Against Resistant and Susceptible Strains of Damalinia avis.

% Reduction after 8 weeks

Resistance Factors:

Manil. 1.9-2.6 Holb. (R) 5.1-9.1

W.Jasp. (R) 3.9-6.7 War. (R) 8.6

Sheep carrying a strain with a resistance factor of 11.8 were dipped at concentrations of cyperniethrin ranging from 2 ppm to 12 ppm. The mean lice count in the group dipped at 2 ppm was reduced by 7.6% at 8 weeks after treatment and live lice persisted in the group dipped at 6 ppm up to 7 weeks after treatment. Live lice were found on the sheep dipped at 12 ppm up to 3 weeks after treatment (Figure 2).

Figure 2 Efficacy of Cypermethrin Against Resistant Dewrang Strain of Damalinia ovis.

% Reduction after 8 weeks

Resistance Factor: 11.8

Titrations with two synthetic pyrethroid dips

A study comparing titrations of cypermethrin, the reference SP used in dipping titration studies and cyhalothrin, another SP registered for lice control, was carried out using a strain of lice with SP resistance factors to these insecticides of 4.2 and 3.2, respectively. Groups of 3 sheep were dipped at concentrations of each insecticide ranging from 0.5 to 6 ppm.

Live lice were found up to 8 weeks after treatment on both the cyhalothrin and cypermethrin treated groups dipped at 2 ppm. The percentage reductions were 90.5% and 94.3%, respectively. Small numbers of lice survived on both groups up to 6 weeks after dipping at 4 ppm.

Live lice were found up to 3 weeks after treatment on both the cypermethrin and cyhalothrin treated groups, dipped at a concentration of 6 ppm, but no live lice were found after week 5 in either group (Figure 3).

Figure 3 Efficacy of Cypermethrin and Cyhalothrin Against the SP Resistant Junee Strain of Damalinia avis

Percent reduction

Weeks 1 to 3 after treatment

Efficacy of Cypermethrin and Cyhalothrin against the SP resistant Junee strain of Damalinia avis

Percent reduction

Off-shears and long wool pour-on treatments

In field and laboratory trials, sheep in tested with resistant strains were also treated with commercial off-shears and long wool pour - on SP products, all of which failed to give adequate control (Tables 2 and 3). Confirmatory trials are being undertaken in the laboratory.

Table 2. Efficacy of off-shears backline treatments against Damalinia ovis (lice counts on groups of 6 sheep at 9 weeks after treatment)

Strain	Chemical	Dose Ml	Mean Lice Pre-Treat	Mean Percent Lice Reduction Post-Treat
Holbrook	Deltamethrin	8-15	191.5	4.3 97.7
Res.
	Cypermethrin	10-15	141.5	18.0 87.3
Wee Jasper	Alphamethrin	10	205.2	36.8 82.1
Res.
Peak Hill	Cypermethrin	10	360.5	0 100
Susc.
Manildra	Deltamethrin	8	105.8	0 100
Susc.
	Alphamethrin	10	107.5	0 100
Mixed Susc.	Cypermethrin	10-15	244.0	11.7 95.2

Res. = Resistant; Susc. = Susceptible

Holbrook strain resistance factors: deltramethrin 4.8
cypermethrin 5.7

Wee Jasper strain resistance factor alphamethrin 6.8

Peak Hill strain resistance factor cypermethrin 0 (reference susceptible strain)

Manildra strain resistance factors cypermethrin 2.6
deltamethrin 1 .2

alphamethrin 3.3

All sheep were treated according to label directions with doses given according to the bodyweight of each individual in each group.

Table 3. Efficacy of deltamethrin against the SP resistant Dewrang strain of Damalinia ovis

	MEAN LICE COUNTS
Pre-Treatment	Week 2	Week 4	Week 6	Week 8
336.3	40.2	8.5	5.7	2.2
+/- 33.3	+/- 18.4	+/-3.7	+/- 3.4	+/-1.3

Dewrang strain: LC 50 deltramethrin 0.80

Resistance factor deltamethrin 5.3

Deltramethrin fornmlation: Clout-S Coopers Animal Health North Ryde, NSW Australia

Dose: 8-12 ml (according to bodyweight, range 31.0-57.4 kg)

A group of 6 sheep was used in the trial.

Dipping with commercial strength dips

Resistant strains were controlled with plunge or shower dips containing cypermethrin at a concentration of 19 ppm (Boray et al. 1989; Johnson et al. 1989). The results of dipping treatments against resistant strains are shown in Table 4.

Currently, the problem of SP resistance in D. avis is confined to the pour-on formulations of these insecticides, despite the fact that pour-on treatments deposit 5-15 times more insecticide on the sheep than a plunge or shower dip. This suggests that the rate and degree of movement of the insecticide from the backline on some sheep may be a significant factor in the failure of pour-ons to control lice infestations and in the selection of lice for resistance. The amount of insecticide deposited on a 50 kg sheep, using different treatment techniques, is shown in Table 5.

Although strains of lice which have high SP resistance factors are controlled using SPs in plunge or shower dips, the dipping experiments carried out on them suggest that they survive for longer after treatment than fully susceptible strains. It may therefore be desirable to isolate dipped sheep from other sheep for a minimum of 3 weeks after treatment in order to minimise any risk of transmission of SP resistant lice.

Table 4. Efficacy of cypermethrin applied by dipping 4 weeks after shearing against the resistant Wee Jasper strain of Damalinia ovis.

	MEAN NUMBER OF LICE
Dipping Method	Pre-Treat	3 Weeks Post-Treat	6 And 9 Weeks Post-Treat	Percentage Reduction
Plunge dip 19 ppm	106.5	0.7	0	100
	+/- 19.3	+/- 0.2
Shower dip 19 ppm	108.2	0	0	100
*	±23.1

The experiment was carried out using groups of 6 sheep.

The dips were charged with cypermethrin according to label directions.

Wee Jasper strain: resistance factor to cypermethrin 3.9.

Cypermethrin: Robust

Youngs Animal Health, Blackburn, Vie. Australia

* Buzacott power spray dip

Buzacott Rural Machinery, Tamworth, NSW, Australia

Table 5. Insecticide deposited on a 50 kg sheep after treatment for sheep body louse (Damalinia ovis) infestation.

Treatment Type: Insecticide	Amount Of Active Ingredient applied (Mg)
Off-Shears Backline:
Cypermethrin	100 - 312
Dehamethrin	100
Alphamethrin	200
Plunge or Shower Dip:
Cypermethrin	20
Cyhalothrin	20
Coumaphos	375
Diazinon	150
Long Wool Backline:
Cypermethrin	756
+ Diazinon	504
Alphamethrin	250 - 1000
Jetting:
Cyhalothrin	75 - 100
Diazinon	600 - 800

Prevalence of SP resistant strains

Resistant strains have been obtained from all of the sheep producing areas of New South Wales, but the overall prevalence of resistant strains is unknown. Resistant strains have also been identified in Western Australia (de Chaneet et al. 1989), South Australia (James, 1989, personal communication), Queensland (O’Sullivan, 1989, personal communication) and Victoria (Campbell and Presidente, 1989, personal communication). What can be said is that pour-on formulations are still being widely used and are giving satisfactory results in the majority of cases. The emergence of resistance has, however, caused a problem for a significant number of graziers and has created the need for an assessment of lice control strategies on the management and the past performance of insecticides applied by different treatment methods.

Mechanisms of resistance

Nothing is known about the mechanisms of pyrethroid resistance in D. ovis. SP resistance mechanisms in other insect species were reviewed by Miller (1988) and can be grouped within four categories:

(i) Behavioural resistance, where the insect’s behaviour becomes modified so it no longer comes in contact with the insecticide.

(ii) Penetration resistance, where the composition of the insect’s exoskeleton becomes modified so as to prevent insecticide penetration.

(iii) Site insensitivity, where the chemical site of action for the insecticide becomes modified to have reduced sensitivity to the active form of the insecticide.

(iv) Metabolic resistance, where the metabolic pathways of the insect become modified in ways that detoxify the insecticide or disallow metabolism of the applied compound into its toxic forms. The most important forms of metabolic resistance involve multifunction oxidases, gluthathione-S-transferase and, in the case of pyrethroids (which are almost all esters), esterases.

In general, site insensitivity or metabolic detoxification are the main resistance mechanisms in insects and physiological resistance is the result of the interaction between these factors. In the case of pyrethroid insecticides, resistance to knockdown and to lethal effects does not necessarily involve the same mechanisms. Knockdown resistance usually involves site-insensitivity and generally confers resistance to killing, but the converse is not always true. Insects may develop kill resistance but remain susceptible to knockdown. Resistance based on multifunction oxidase activity can be demonstrated by the use of an insecticide synergist such as piperonyl butoxide, which retards detoxification of the insecticide. Resistant strains are susceptible to the insecticide plus synergist mixture.

In the case of D. ovis the mechanism of resistance has yet to be determined. There are two broad possibilities for the nature of the resistance. The first is that susceptibility to SPs is lower, due to innate genetic variants in vigorous strains of lice and these have been selected by prolonged use of the chemicals in the field. The second possibility is that some strains of lice have developed a physiological resistance due to a specific genetic mutation.

Strategies for the control of lice

Preferred treatment

Shower or plunge dip with registered synthetic pyrethroid or organophosphate insecticides. Preferred because OP and SP insecticides are efficient when applied in a plunge or shower dip, against both SP resistant and susceptible strains of lice.

Pour-on off-shears SP treatments

Are acceptable provided:

(1) no split shearing;

(2) not used within 8 weeks of lambing;

(3) not used when there are lambs at foot;

(4) all sheep in the mob are treated at the same time: no stragglers. (This is also important for dips and long wool treatments);

(5) if sheep are bought: shear, treat and isolate from other sheep for 8 weeks;

(6) previous acceptable results with pour-ons: no evidence of SP resistance. If one pour-on formulation has failed there is no point in using other off-shears or long wool pour-ons;

(7) the mob is dosed according to the heaviest individuals. (The same principle applies as when sheep are being drenched for internal parasites. When you treat sheep with pour-ons according to bodyweight, you are using bodyweight as an estimator of surface area).

Long wool treatments

SP susceptible lice

• Thorough hand jetting with cyhalothrin or diazinon.

• Pour-on treatment with alpha-cypermethrin or cypermethrin/diazinon.

• Level of control achieved depends on wool length and thoroughness of treatment.

• Check sheep thoroughly at next shearing and apply off-shears treatment if necessary.

• The need for long wool treatment indicates a problem with the control of lice in the flock. The reason for the infestation should be investigated.

SP resistant lice

• Thorough hand jetting with diazinon.

• If SP resistance is suspected, the use of long wool treatments will cause further selection of the resistant strain and SP based long wool treatments should not be used.

Acaricide Resistance In The Cattle Tick (Boophilus microplus)

Organophosphate Resistance

History

Cattle tick (Boophilus microplus) is an important pest of beef and dairy cattle in tropical areas of Australia. European (Bos taurus) breeds are more severely affected than Bos indicus breeds. The parasite is relatively immobile, achieving significant dispersal only on cattle and it is highly seasonal, particularly in south-eastern Queensland. There are four parasitic generations per year, progressively increasing in size from spring to autumn-winter (Sutherst and Comins, 1979).

Resistance to OPs was first recognised in 1963 on a property at Ridgeland, near Rockhampton, when dioxathion failed to control ticks. This strain showed cross-resistance to carbophenothion, diazinon and carbaryl, a carbamate insecticide, and was recovered from 8 properties surrounding the initial outbreak and 6 other properties. By 1967, it had been found on 72 properties between Townsville and Brisbane (Seddon, 1967; Wharton, 1967).

In 1966, a strain of ticks appeared at Biarra in the Brisbane valley, which was found to be resistant not only to the four insecticides resisted by the Ridgelands strain, but also to ethion and coumaphos. By 1967 the strain had been found on 34 properties (Seddon, 1967). Roulston et al. (1971) reported that the emergence of these two strains together with the Mackay, Mount Alford and Gracemere strains (the latter two having increased resistance to both Dursban and diazinon), necessitated the development of an alternative chemical group for the control of OP resistant strains.

In the mid- 1970s there were 8 OP resistant strains recorded in Queensland and the OP compounds, while still registered, were rarely used as the sole chemical in the dip (Arundel and Sutherland, 1988). Three new strains wet-c reported by Roulston et al. (1977), one from a property which experienced a failure with chlorpyrifos, one from a property on which ethion failed and one from a property on which the owner experienced difficulties with coum aphos, clenpyri n and chlorpyrifos.

Mechanisms of Resistance

Stone (1972) reported that increased detoxication of coumaphos in the body was the mechanism of resistance in the Mackay strain of 13. microplus, but insensitivity to OPs in the enzyme acetylcholinesterase was found to be the mechanism in some other strains (Nolan et al. 1972; Schuntner and Smallman, 1972; Riddles and Nolan, 1986).

A single biochemical mechanism and one genetic factor for resistance was reported in the Ridgelands, Biarra and Mackay strains by Stone (1972). Subsequently, Riddles and Nolan (1986) concluded that the most incisive mechanism that the cattle tick uses in resistance to the OP compounds is the changing of the target enzyme acetylcholinesterase. Observations on different strains of B. microplus indicate that resistance may arise from at least two different types of mutations, possibly affecting the same gene (Riddles and Nolan, 1986).

Stone (1972) found little reversion to susceptibility in OP resistant strains reared in the laboratory, when selection pressure was relaxed, leading to the conclusion that the prospects for reuse of acaricides once resistance had occurred were very poor. Balancing this, resistance to the OPs was found to be genetically incompletely dominant (Sutherst and Comins, 1979) and, previously, Stone (1972) had concluded that the likelihood of all known resistance genes appearing in all populations at the same time was remote and the system of early detection and characterisation of resistance, based on biochemical testing of strains, allowed for intelligent application of the available chemicals. Subsequently, alternative acaricides were developed.

Alternatives to OPs

Amitraz

One of the amidine group of compounds, amitraz is currently registered for cattle tick control and is widely used. Sonic resistant strains have been identified (Arundel and Sutherland, 1988).

Synthetic Pyrethroids

The SP compounds control OP resistant strains but show some cross-resistance to DDT resistant strains, some of which have persisted in the field since the withdrawal from use in 1962 of the chlorinated hydrocarbon compounds, because of residue problems. Nolan et al. (1979) found that cyperinethrin and decamethrin (deltamethrin) were effective against OP susceptible and resistant strains and were effective at higher concentrations against DDT resistant strains. As a result of these studies, Nolan et al. (1979) also confirmed that SP compounds were potentiated by OPs. Combinations of OP and SP compounds were suggested (Nolan et al. 1979) to exploit this potentiation. Such compounds are now registered (cyperinethrin-chlorfenvinphos, deltamethrin-ethion) for the control of all strains and are extensively used (Arundel and Sutherland, 1988).

Flumethrin is registered alone in plunge dipping and pour-on formulations for the control of OP. amidine and DDT resistant strains. Cyhalothrin is also registered alone in a plunge dip formulation for the control of multi-resistant strains.

Ivermectin

Ivermectin is one of a group of avermitilin compounds derived from the actinoinycete Streptomyces avermitilis (Benz, 1985). The drug has efficacy against a range of internal and blood sucking external parasites, including B. microplus (Drummond,1985). A dose rate of 0.2 mg/kg gives control of cattle tick for 21 days following an initial lag period of 2 days (Nolan et al. 1981). In circumstances where cattle need to be cleansed of ticks before moving to tick free areas, two treatments with ivermectin using a dose rate of 0.2 mg/kg administered at an interval of 4 days has been shown to be as effective as the currently accepted practice of applying three treatments of an SP-OP acaricide mixture (Arundel and Sutherland, 1988).

Management of cattle tick

Use of Acaricides

Strategies for the control of resistant strains of B. microplus were reviewed by Sutherst and Comins (1979), who suggested that the profligate use of chemicals may result in the squandering of the supply of acaricide susceptible strains of the parasite, which should be seen as a depletable natural resource. They concluded that the use of frequent acaricide treatments instead of, rather than in support of stock and pasture management is an extremely expensive policy in the long term. Their suggestions for the management of resistant strains are summarised in the following points.

(1) Resistance problems make dipping costlier and indicate the need for a reduction in the extent of control. To reduce these problems it may be necessary to ignore light infestations or use alternative management practices such as pasture spelling or switch to Bos indicus type cattle.

(2) Acaricide use can be effectively reduced by increasing the control thresholds for commencement of dipping of the spring to autumn generations of the parasite, particularly the first two generations which tend to be small because of the unfavourable conditions for reproduction in the preceding autumn-winter.

(3) High concentrations of non-residual acaricides may delay the occurence of resistance by killing the heterozygotes of strains with low resistance. The use of mixtures of acaricides with different modes of action reduce the risk of multiple resistance alleles, but should be used in conjunction with cattle and pasture management.

(4) Avoid unnecessary dipping, the long term costs of which are high, although the short term cost may be inapparent.

(5) Awareness amongst graziers of the ease with which resistant strains can be spread on infested cattle and awareness on the part of buyers that cattle purchased should be quarantined until they have been thoroughly examined and treated if necessary.

Pasture Spelling

This has been summarised by Arundel and Sutherland (1988) and is based on the reproduction and survival of the parasite in different climates. Moving Bos taurus type cattle to a spelled paddock in May, when engorged ticks produce few or no progeny, and subsequently alternating them between two paddocks at 4-monthly intervals, enables the tick population to be controlled without the need for treatment or with fewer treatments than are required if pasture spelling is not practised.

An alternative system utilises a small disinfection paddock, in which cattle are placed until all ticks have matured and dropped off. The cattle are then moved to the main grazing paddocks before the progeny of the dropped ticks are available to reinfest them. In this system the use of the disinfection paddock replaces the usual dipping treatment applied when cattle change paddocks. The system requires increased fencing and some graziers believe it results in under-utilisation and loss of pasture productivity because of prolonged spelling. Epidemiological data shows that paddocks should be spelled for 2-3 months in the summer and 3-4 months in the autumn and winter in order to achieve a substantial reduction in tick numbers (Arundel and Sutherland, 1988).

Vaccination

Resistance to B. microplus is acquired after exposure to the parasite. Using extracts derived from adult female ticks, Johnston et al. (1986) showed that partial immunity to the parasite was induced in both Bos taurus and Bos taurus x Bos indicus breeds. The immunity induced persisted after 14 weeks of daily challenge with 1000 larvae and tick populations on vaccinated cattle being reduced by 70% compared with control animals. In a second experiment, cattle were challenged with 20,000 larvae on two occasions in the field and tick populations in the vaccinated cattle were reduced by 93% compared with unvaccinated controls. Immunity was variable between individual animals, with no significant breed differences being observed (Johnston et al. 1986).

Histological examination by Agbede and Kemp (1986) of the ticks that had fed on vaccinated cattle showed that the primary site of damage was the gut. Within 24-48 hours of attachment to the host, digest cells lining the gut were either sloughed off into the lumen or were completely destroyed leaving only the basal lamina and muscle layer. Subsequent rupture of the gut allowed host leucocytes to enter the haemocoel and attack other tissues. Damage to muscle, Malphigian tubules and the accessory gland of the reproductive organ in males was observed (Agbede and Kemp. 1986).

The commercial development of the vaccine is proceeding.

Acknowledgement

The work carried out in our laboratories on resistance to synthetic pyrethroids in the sheep body louse is supported by a research grant from the Wool Research and Development Fund of The Australian Wool Corporation.

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