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This article was published in 1978
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Worm Resistance to Anthelmintics
L.F. Le Jambre, B.S., M.S., Ph.D., C.S.I.R.O., Division of Animal Health, Pastoral Research Laboratory, Armidale
INTRODUCTION
In 1914 Melander asked what now may be considered a naive question 'Can insects become resistant to sprays' (?) (Melander, 1914). Today with more than 364 species of arthropods resistant to insecticides (Georghiou and Taylor, 1977) the answer is a resounding yes. In fact since the introduction of potent synthetic insecticides following 1965, the main technical problem has been the development of resistance to them by insects they formerly controlled.
Since the introduction of phenothiazine in the late 1930's - chemicals have become the mainstay of roundworm control in sheep. However, parasitologists have been more fortunate than their colleagues in entomology as until recently, resistance in nematodes has been much slower to develop under field conditions. For example, although resistance to phenothiazine did develop in some H. contortus strains in the United States and the use of phenothiazine gradually decreased, this was not due to resistance; rather, it was replaced by newer, more efficient anthelmintics.
The most important of these new anthelmintics was thiabendazole, released in 1961. Thiabendazole revolutionised the control of helminth diseases as it was effective against a broad spectrum of nematodes and had a high safety margin. Unfortunately, as early as 1964 the forces of evolution began to strike, back when Drudge et al. (1964) reported finding a strain of H. contortus resistant to thiabendazole. Thus began an increasing interest amongst parasitologists in the development of anthelmintic resistance in nematodes.
From the outset, two terms that should be clearly distinguished from one another are tolerance and resistance. Tolerance should be reserved to describe the natural ability of a species population to withstand its first exposure to a toxicant. This ability may vary between populations of the same species and according to the chemical used and method of exposure but when these criteria are fixed, it becomes a basic measure of the tolerance of the population. Resistance can then be defined as a significant increase above the tolerance level in the ability of the population to withstand the toxicant. This increased ability is acquired by breeding from those individuals which survive exposures to the toxicant insufficient to destroy the entire population, i.e. resistance is inherited.
DETECTING RESISTANCE
Generally field observations cannot provide definite evidence of the early development of resistance because so many factors may be involved, such as faulty application or defective drench. It usually takes control failure with stock losses before anthelmintic failure is diagnosed in the field. Ideally, techniques and facilities would be available to provide a continuous surveillance for an increase in resistance of a worm population. Although this ideal state may be approached in monitoring some tick and insect pests, field observations of nematode parasite populations remain the main source of information on changes in susceptibility to anthelmintics.
At present, small quantitive changes in levels of resistance to anthelmintics usually can only be determined through slaughter trials. This involves dosing worn-free sheep with worm Larvae and allowing the infection to develop for 30-35 days. Then the sheep are injected intra-rumenally with the anthelmintic. Three to seven days later the sheep are killed and the number in the treated group compared with the number in untreated controls to give a percentage reduction due to treatment.
To differentiate between strain comparisons it is necessary to determine the percentage kill of worms over a range of anthelmintic dosage levels. Then the percentage kill is statistically rearranged to produce a straight line when plotted against an increasing dose of anthelmintics. From this line one can then determine the dose required to kill 50%, 95% and 99% of the worm population. These doses are called the LD50, LD95 and LD99, respectively. The resistance ratio (or resistance factor =Rf) is the ration of LD50 of the strain suspected of resistance to that of a known susceptible strain and is generally used in entomology as a measure of resistance. This concept of resistance has now been applied to nematodes by Le Jambre et al. (1976). Benzimidazole resistance can also be detected by hatching worm eggs in a series of concentrations of thiabendazole in a 0.1% NaC1 solution. Eggs from resistant strains hatch in higher concentrations than those from nonresistant strains. The percentage hatch can then be used to provide between strain comparisons in the same manner as percentage kill in slaughter trials. (Le Jambre, 1976).
GENETICS OF RESISTANCE
Evolutionary theory explains the appearance of a resistant strain by saying that the total population of worms contained in a few rare individuals that possessed the ability to survive the anthelmintic. They must have been rare, otherwise the drug wouldn't have worked. Since the drug kills all the susceptible worms, the next generation will consist of the offspring of the resistant minority. Many of these worms will have inherited their parents' ability to survive the drug. Therefore single worms are either non-resistant or resistant to a fixed by varying degree. Populations of worms and not individuals become resistant.
If the character that gives resistance is controlled by a single gene, then the resistant populations build up very rapidly. Often though, resistance is controlled by several genes that need to work together. This is known as polygenic resistance. In this sort of situation - where selection may have to operate over several generations to produce the best combination of genes, and so the most resistant individuals - the whole process is much slower. Thiabendazole resistance in Haemonchus contortus has been found to be an example of polygenic resistance (Le Jambre, et al. 1978)
ANTHELMINTIC RESISTANCE IN AUSTRALIA
In Australia, thiabendazole resistant H. contortus was first reported by Smeal et al. (1968) from three properties in northern New South Wales. A recent survey of 40 properties in the same area found that 42.5% of the properties contained resistant strains of H. contortus. The area surveyed covered three Pastures Protection districts and the incidence of properties carrying resistant strains in each district was 50% for Armidale, 20% for Glen Innes and 50% for Tenterfield (Webb et al. 1978). In a follow-up of this survey, even those strains listed as nonresistant were found to contain a high proportion of resistant individuals (Le Jambre et al. 1978a).
Following a single dose of thiabendazole and using the egg-hatch assay (Le Jambre, 1976), the resistance ration increased and remained high in the subsequent generation.
Thiabendazole resistance in Trichostrongylus colubriformis was first discovered in two strains in New South Wales, (Hotson et al. 1970). These strains occurred in flocks on research properties where thiabendazole had been used frequently. The LD of this strain was 42 mg/kg.
When tested, thiabendazole-resistant field strains of both H. contortus and T. colubriformis have been found to have cross resistance to other benzimidazole anthelmintics (parbendazole, cambendazole membendazole, oxibendazole, fenbendazole, oxfendazole and thiophanate).
The first published report of either levamisole or morantel tartrate resistance in a field strain of ovine parasites comes from a research property in New South Wales, where thiabendazole morantel tartrate and levamisole were used in a rotational drenching programme. On this property a strain of O. circumcincta has developed resistance to all three anthelmintics. The LF05's for thiabendazole, morantel tartrate and levamisole were reported as 88, 7.0 and 5 mg/kg respectively. This compares with corresponding values of 20, 3.0 and 2.0 mg/kg in non-resistant O. circumcincta (Le Jambre, et al. 1977).
DEVELOPMENT OF RESISTANCE IN THE LABORATORY
Recently, there has been an increase in work on selection for resistance to broad spectrum anthelmintics in laboratory populations of worms. It must be recognised, however, that the environment in the laboratory is different from natural selection in the field. Laboratory strains often fail to respond to selection as strongly as field populations since they have such limited genetic variance. Consequently, when selection in the laboratory leads to the development of resistance it can be concluded that resistance is likely to develop in parent strains in the field. Little can be concluded, however, when laboratory selection fails to develop resistance.
At the CSIRO'S Pastoral Research Laboratory strains from 2 genera of sheep parasitic nematodes (H. contortus and O. circumcincta) were selected for either single or multiple resistances to thiabendazole, morantel tartrate and levamisole. At the beginning of the selection programmes the H. contortus strain had a degree of thiabendazole resistance while the O. circumcincta did not. Originally none of the strains were resistant to either morantel tartrate or levamisole. The H. contortus strain was selected over six generations for resistance to 50 mg/kg thiabendazole. After this time, selection on one strain was continued at 50 mg/g and selection on a second line was extended to include 8.8 mg/kg morantel tartrate. In the fourth generation following the division into two lines, both strains together with the unselected parent strain were assayed for resistance to thiabendazole, morantel tartrate and levamisole. The LD50 of both selected strains was greater than 200 mg/kg thiabendazole compared with 64 mg/kg in the parent strain. Against morantel tartrate, the LD50 of the thiabendazole - morantel tartrate strain increased to 3.9 mg/kg while for the thiabendazole selected strain it remained the same as the parent strain at 1.8 mg/kg. This was the first indication that H. contortus could develop resistance to morantel tartrate and furthermore, it appeared that H. contortus had the necessary genetic variation to develop resistances to two anthelmintics at the same rate as to one.
To determine whether O. circumcincta had sufficient genetic variation to develop resistance, Le Jambre and co-workers divided a field isolate into five strains based on anthelmintic selection in the laboratory. The first strain was selected with 50 mg/kg thiabendazole, the second with 4 mg/kg morantel tartrate, the third with 3.2 mg/kg levamisole, the fourth was not selected and the fifth strain was selected with all three anthelmintics in each generation. This selection was continued for seven generations; in the eighth a drug assay was conducted on the five strains. In the adult O. circumcincta the LD95 for thiabendazole, morantel tartrate and levamisole was 172.0, 9.2 and 8.4 mg/kg respectively in the multi-selected strain, compared with corresponding values of greater than 200, 6.1 and 6.9 for the single selected strains and 14.5, 2.8 and less than 1.6 in the unselected parent strain. Thus, O. circumcincta, like H. contortus can apparently develop resistances to several different anthelmintics simultaneously at rates that are equal to or greater than those in single selected lines.
WHY ANTHELMINTIC RESISTANCE IS SLOW TO DEVELOP
Results from laboratory selection programmes demonstrate that nematodes have the necessary genetic variability to develop resistance to several unrelated anthelmintics. If resistance genes are as common as indicated by these studies, why has resistance not been reported more often? A likely reason is that most sheep are drenched in response to obvious parasitic disease, which is usually associated with maximum larval availability on pasture. During these periods, while there may be thousands of worms in the sheep there are thousands of millions on pasture. Consequently, anthelmintic treatment selects only a very small portion of the worm population.
The impact of a portion of the population missing exposure to the drug on delaying resistance is suggested by a simulation model of the evolution of insecticide resistance. In this model, when the number of individuals escaping treatment was zero, 50% of the population were resistant in one generation. However, when 0.1 of the population missed treatment, it took six generations to reach this level. Once 0.5 of the population were missed, the time required for 50% of the population to become resistant exceeded 20 generations. (Georgiou and Taylor, 1977).
RETURN TO SUSCEPTIBILITY
Since the genes causing anthelmintic resistance were at low frequency in the population before the anthelmintic began to be applied, it must ordinarily be true that they are to some extent disadvantageous; otherwise they would have been common. Therefore, the selection for resistance should ordinarily involve the replacement of the original genes with resistance factors that, in every respect, except resistance, are deleterious for survival. One should then expect that when the anthelmintic is removed from its environment the strain will return to susceptibility. This is known as reversion.
However, Le Jambre et al. (1978) reported that when isolated in 1975, a T. colubriformis strain had a high level of resistance to thiabordazole (LD 110 mg/kg) even though this anthelmintic had not been used against the strain since 1971. Thus, it is likely that selection in nature is accompanied by intense natural selection for general viability and resistance factors would not be selected if they were otherwise highly detrimental. Hence, reversion should be slow and does not offer much hope that a short period of absence of the anthelmintic will lead to a susceptible population.
ALTERNATING ANTHELMINTICS The use of several different anthelmintics in rotation has been suggested as a means of preventing or retarding the development of resistance (Belschner, 1976). However, there is no evidence that rotational use of anthelmintics would have this effect. In theory, there should be no difference in the rate of development of resistance whether one alternates the drenches frequently, or uses one for a long time before changing to the other as long as the alteration occurs between generations of worms. Thus, if resistance to anthelmintic X can develop in eight generations and the anthelmintic Y also in eight generations, then one could choose between having X resistant worms in eight generations and switching to y for a total of 16 generations or alternating anthelmintics between generations and having X and Y resistance develop simultaneously at generation 16.
DELAYING RESISTANCE
Present trends in parasite control are now leading towards decreasing the total worm population by reducing the numbers of infective larvae on pasture. When this is accomplished by drenching the host and moving it to a 'safe' pasture or by monthly drenching, selection for anthelmintic resistance is increased and it can be expected that after a few generations resistance of the population will be much greater.
It becomes important to use anthelmintics in a manner that is most likely to retard the development of resistant strains of nematodes. One method is to return to drenching only those sheep with obvious symptoms of parasitosis. Clearly, the cost in loss of production before treatment and deaths, due to late treatment is too high a price to pay for a possible delay in the onset of anthelmintic resistance.
It is possible that when an anthelmintic like thiabendazole is first used in the field, no individual worm carries the right combination of genes needed to survive it. This makes it feasible to aim at the complete elimination of the local population, thus avoiding the escape of survivors that carry resistant traits. Even if the population isn't completely wiped out, the likelihood of a very small number of survivors producing individuals with a strongly resistant gene combination will be very small. Geneticists say that selection does not operate effectively when the genetic variability of a population is very low.
For example, if there were 10 genes for resistance, each with a frequently of .01, the number of worms of maximum resistance would be one in 1020, or one followed by 20 zeros. If 20 recessive genes were required to produce maximum resistance, the number would be one in 1080 (the same as the estimated number of electrons [atoms] in the visible [known] universe!). Hence recommendations that include the use of a broad spectrum anthelmintic to control nematodes should stress the importance of using the full recommended dose (or perhaps more) to remove as many worms as possible from the host.
REFERENCES
Belschner, H.G. (1976) Sheep management and diseases. 10th ed. Angus & Robertson, Sydney>/p>
Crow, J.F. (1952), National Academy of Sciences - National Research Council Publication No. 219, p72
Drudge, J.H., Szanto, J., Wyant, Z.N. & Elam, G. (1964) American Journal of Veterinary Research 25, 1512
Georghiou, G.P. & Taylor, C.E. (1977) J. Econ. Entomol. 70, 319
Hotson, I.K., Campbell, N.J. & smeal, M.G. (1970) American Journal of Veterinary Research 46, 356
Le Jambre, L.F. (1976) Veterinary Parasit. 2, 785
Le Jambre, L.F., Martin, P.J. and Webb, R.F. (1978) Aust. Vet. J. (In preparation)
Le Jambre, L.F., Royal, W.M., & Martin, P.J. (1978) Parasit. (Submitted)
Le Jambre, LF., Southcott, W.H. & Dash, K.M. (1976) Int. J. Parasit. (In press)
Le Jambre, L.F., Southcott, W.H. & Dash, K.M. (1976) Int. J. Parasit. 6, 217
Le Jambre, L.F., Southcott, W.H. & Dash, K.M. (1978) Int. J. Parasit. (Submitted)
Le Jambre, L.F., Southcott, W.H. & Dash, K.M. (1978) Aust. Vet. J. (Submitted)
Melander, A.L. (1914) J. Econ. Entomol. 2, 167
Smeal, M.G., Gough, P.A., Jackson, A.R. & Hotson, I.K. (1968) Aust. Vet. J. 44, 108
Webb, R.F., McCully, C. , Clark, F.L., Greentree, P., and Honey, P. (1978) Aust. Vet. J. (In press)