A larval development test for the detection of anthelmintic resistance in nematodes of sheep

Research in Veterinary Science /990,49, /98-202

A larval development test for the detection of anthelmintic resistance in nematodes of sheep M. A. TAYLOR, Department 0/ Parasitology, Central Veterinary Laboratory, New Haw, Weybridge,

Surrey KT15 3NB

First stage larvae of a number of species of parasitic nematodes of sheep have been shown to develop to third stage larvae in the presence of a defined medium consisting of Earle's balanced salt solution and yeast extract. A larval development test, based on this culture technique,was used as a screen for detecting the presence of antbelmintic resistance in nematodes of sheep. It was.found to be sensitive and simple to use and also appeared capable of detecting resistance to any of the main anthelmintic groups. Available anthelmintic sensitive and resistant strains of Haemonchus contortus and Ostertagia circumcincta showed differences in development when incubated in the presence of either thiabendazole, levamisole and Ivermectln. These differences were expressed as the minimum inhibitory concentration required to prevent larval development over the incubation pe~od. . THE emergence of nematode parasites showing resistance to anthelmintic drugs is of increasing concernto all involved with grazing livestock. Anthelmintic resistance. has been detected most commonly among the gastrointestinal nematodes of sheep and goats (Kelly and Hall 1979, Donald 1983,. Waller 1985). On a worldwide basis, resistance has been commonly reported to the benzimidazole group of anthelmintics, also to levamisole and morantel, and more recently to ivermectin (Van Wyk and Malan 1988, Echevarria and Trindade 1989) and the salicylanilides, rafoxanide and closantel (Van Wyk et al 1987, Van Wyk and Malan 1988). A number of techniques have been described to detect the presence of resistance to anthelmintics. These techniques have been reviewed by Presidente (1985) and Taylor and Hunt (1989). Coles et al (1988) described a larval development test that could be used to detect benzimidazole and levamisole resistant strains of Haemonchus contortus. The test was further used for the detection of ivermectin resistance (Giordano et aI1988). This paper describes a modified larval development test for the detection of resistance to benzimidazole, levamisole and ivermectin anthelmintics.

Materials and methods

Nematode egg recovery technique Individual worm-free Dorset Horn sheep, reared indoors on concrete from birth, were orally infected with 5000 third stage larvae of each parasite strain used in the study. After 21 days, faecal samples were taken per rectum and examined for the presence of nematode eggs using a modified McMaster technique (MAFF 1986). To recover nematode eggs from the faeces, 10 to 20 g of faecal pellets, the amount depending on the egg count of the faeces under test, were added to approximately 200 ml of distilled water in a 1000 ml plastic graduated cylinder. The faeces were mixed using a perforated plunger, until the faecal material was in suspension. The suspension was then poured through a 100 mesh (150 pm) screen into a collecting bowl. The contents of the bowl were poured into eight Clayton Lane centrifuge tubes and centrifuged at 214 g for two minutes. The supernatant was poured off, and the residue resuspended in saturated salt solution. A positive meniscus was produced by carefully pipetting in further salt solution, and a coverslip placed on top of the tubes. The tubes were then centrifuged for 2· 5 minutes at 95 "g, after which the covers lips were removed and the adhering droplet washed with distilled water into a conical test tube which was then respun at 95 g for two minutes. The supernatant was siphoned off and the resultant eggs, clean and free from debris, were suspended in distilled water. An estimate of the numbers of eggs recovered was made by counting the numbers of eggs present in five 10 pI samples. The final volume of solution was adjusted to give between 200 and 300 eggs ml: I.

Parasite strains A summary of the parasite strains used in this study together with the origin and susceptibility to anthelmintics is given in Table 1. The anthelmintic sensitivities of these strains have either been previously published, or determined at the Central Veterinary Laboratory, Weybridge by means of a controlled 198

Detection of anthelmintic resistance TABLE 1: Parasite strains used in the study Strain

Origin

H/CS H/CDS O/CS

Weybridge Utrecht Weybridge Field isolate UK Utrecht

H/CR H/CDR

O/CR DICC

Field isolate UK '\ Field isolate UK

H/CU

Louisiana USA

H/CO

White River KRTZ South Africa

Resistance

Thiabendazole Fenbendazole Thiabendazole Thiabendazole Oxfendazole Levamisolet Fenbendazole Levamisole Albendazole Mebendazole Closantel Ivermectin

E050*

0·027 0·08 0·053 0·246 0·44 0·79 0·29 , ·42

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nematode larvae was based on that described by Hubert and Kerboeuf (1984), the major constituents of which were Earle's balanced salt solution and yeast extract. One gram of yeast extract (Difco Laborator~es) wa~ added to 90 ml of 0- 85 per cent physiological saltne and autoclaved for 30 minutes at 125°C. The medium was made up of one part lOx Earle's balanced salt solution (Flow Laboratories) to nine parts yeast extract solution. The final pH was adjusted to neutrality by adding sodium bicarbonate solution.

, ·43

Prefix refers tothe following parasite species H/C Haemonchus contortus R Resistant O/C Ostertagia circumcincts S Susceptible * Egg hatch assay data in #o'g ml- 1 thiabendazole t Resistance to levamisole after laboratory selection

anthelmintic efficacy test as recommended by Prichard et al (1980). The two susceptible strains (H/CS and O/CS) were originally isolated from the field in the UK over 20 years ago and have been maintained by periodic passage through worm-free animals. The strain of H contortus (H/CDS), originated from the Central 'Veterinary Institute, Lelystad and was obtained from the University of Utrecht. The thiabendazole resistant strain of H contortus (H/CR) was a composite strain comprised of seven field populations isolated from the south east of England (Cawthorne and Cheong 1984). The thiabendazole resistant strain of Ostertagia circumcincta (O/CR) was isolated from a flock of sheep maintained at the Central Veterinary Laboratory, Weybridge (Cawthorne and Whitehead 1983). Strain O/CC was isolated from a farm in Norfolk (M. A. Taylor and K. R. Hunt, unpublished data). The benzimidazole resistant strain of H contortus (H/CDR) was a laboratory selected strain produced by Dr J. H. Boersema in Utrecht (Dr M. Roos, personal communication). The levamisole/fenbendazole resistant strain of H contortus (H/CU), obtained from the University of Massachusetts, was a field isolate from the Baton Rouge, area of Louisiana (Hembry et aI1986). The benzimidazole/ivermectinlclosantel resistant strain of H contortus (a/co) was a field isolate obtained from the Veterinary Research Institute, Onderstepoort, South Africa (Van Wyk and Malan . 1988). The strain is reported cross resistant to several benzimidazole anthelmintics. Culture system The nutritive medium used for the culture of

Larval development test methodology A range of dilutions of thiabendazole were prepared as described by Cawthorne and Whitehead (1983). Levamisole and ivermectin solutions were prepared by dilution of commercially available preparations in distilled water as detailed by Taylor (1989). The final concentrations used in assays were 0'1,0'3 and 0-5 p,g ml- 1 for thiabendazole; 0-5,1, 2· 5, 5, 10 and 20 p..g ml- 1 for levamisole and 0·1 O' 3, 0- 5, 0-8, 1, 3, 5, 8, 10, 50 and 100 ng ml" i ivermectin. Agar plates were prepared by pouring agar (Oxoid 20 g 1- I) up to a depth of O' 5 em in plastic Petri dishes of 5 em diameter. The nematode egg suspension was incubated at 27°C for 24 hours. After this time the majority of nema tode eggs had hatched to first stage larvae. One ml of the suspension was then poured on to the agar plates, followed by O' 99 ml of nutritive medium and 10/AI of anthelmintic solution to give a final volume of 2 ml allowing optimal development in a shallow layer of water. An untreated control plate was produced by substituting distilled water for the anthelmintic solution. The plates were then incubated at 27°C for six days in a large covered glass Petri dish seale~ .with Parafilm, to maintain a high relativ; humidity and prevent the plates from drying out. Larvae were recovered by washing the surface of the agar into a conical centrifuge tube and centrif~ging at 95 g for two minutes. The supernatant was SIphoned off, and the larvae resuspended in distilled water. Recovered third stage larvae were counted and identified after staining with Lugol's iodine. Data analysis All tests were run in duplicate and the numbers of live larvae corrected by estimating the percentage rec~very in control wells. Data were plotted on a semilog scale and analysed by linear regression.

Results Results for benzimidazole, levamisole and iver-

M. A. Taylor

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TABLE 2: Larval development test: minimum inhibitory concentrations (MIC) for benzimidazole anthelmintics MIC

Strain

Parasite species Benzimidazole susceptible strains

H/CS H/CDS O/CS H/CR H/CDR

O/CR DICC H/CU H/eO

H contortus H contortus 0 circumcincte

Benzimidazole resistant strains H contortus

H contortus o circumcincte o circumcincta H contortus

H conrortus

(",gml- 1 thiabendazole ~0

.1

~O·1 ~o· 1

0·5 0·5 >0·5 >0·3 >0·5 >0·5

mectin larval development tests, as determined against 'various susceptible and resistant parasite strains, are given in Tables 2, 3 and 4, respectively. Percentage recovery of third stage larvae compared to eggs in culture on the control plates was generally less than 50 per cent for both H contortus and 0 circumcineta. This recovery was lower than that reported by .Hubert and Kerboeuf (1984) for Trichostrongylus colubriformis and 0 cireumeincta. There appeared to be no linear dose-response relationship between numbers of third stage larvae recovered and anthelmintic concentration. Results are therefore expressed as the minimum concentration required to completely inhibit development of larvae to infective third stage larvae over the incubation period (minimum inhi bitory concentration). Hatched first stage larvae of benzimidazole susceptible strains failed to develop at the lowest concentration used, that is, 0·1 J.Lg ml ' ! thiabendazole. In many cases dead or dying first stage larvae were present on the plate after seven days incubation. In contrast larvae from benzimidazole resistant strains had developed to third stage larvae after the seven day incubation period (Table 2). Levamisole susceptible strains failed to develop at 2· 5 J.Lg ml- I levamisole or above, whereas resistant strains were able to develop at higher concentrations TABLE 3: Larval development test: minimum Inhibitory concentrations (MIC) for levamlsole MIC

Strain

Parasite species

(",g ml- 1 levamisole)

Levamisole susceptible strains H/CS

OICS CIC H/CO

H contortus o circumcincta C curticei H contortus

Levamisole resistant strains O/CC H/CU

o circumcincta H conrortus

2-5

0-5 0·5

2-5

>5 >20

(Table 3). Ivermectin susceptible H contortus (H/CS and H/CU) larvae failed to develop at relatively low concentrations of ivermectin (0-1 and 0·.8 ng ml- 1) , compared to the South African ivermectin resistant strain of H eontortus (H/CO) which required a concentration of 8 ng rnl" to inhibit development. Ivermectin susceptible 0 eircumcincta (Dies) larvae were able to develop at higher drug concentrations than H contortus (Table 4). No resistant strain was available for comparison. Discussion

The larval development test was assessed against a number of nematode parasite strains which were either susceptible or resistant to one or more anthelmintic groups. As well as appearing sensitive and easy to perform, the test also had the advantage that it could be used to detect resistance to members of the three main broad spectrum anthelmintic groups. Coles et al (1988) using their larval development test as a screen for benzimidazole and levamisole resistance, proposed that a normal dose response curve existed for benzimidazole anthelmintics, but not for levamisole. Further work with this methodology indicated that there was no linear dose-response relationship with ivermectin (Giordano et aI1988). In this study there appeared to be no relationship between the number of larvae recovered and the concentration of any of the three anthelmintics used. The requirement for undeveloped eggs (Coles and Simpkin 1977) for in vitro egg hatch assays has been a major limitation to their application in routine diagnosis of benzimidazole resistance. A number of techniques have been described to avoid the problems often associated with screening for benzimidazole resistance in the field. Whitlock et al (1980) described a method involving the recovery of worm eggs by sugar flotation, and their incubation with a range of concentrations of anthelmintic in McCartney flasks before transportation back to the laboratory. A different approach was to prevent the development of nematode eggs by storing faecal samples on ice (Smith-Buijs and Borgsteede 1986) or under anaerobic conditions (Hunt and Taylor 1989). TABLE 4: Larval development test: minimum inhibitory concentrations (MIC) for ivermectin MIC

Strain

Parasite species Ivermectin susceptible strains

(ng ml- 1 ivermectin)

H/CS O/CS H/CU

H contortus o circumcincra H contortus

0·1 8·0 0-8

H/CD

Ivermectin resistant strain H comonus

a·o

Detection of anthelmintic resistance The larval development test as well as being able to differentiate between benzimidazole susceptible and resistant strains of nematodes, offered a number of advantages over the in vitro egg hatch assay. First, because the test relies on the effects of the anthelmintic on growth of first stage larvae, the age of faecal sample is not as critical as with the egg hatch assay. Anthelmintic sensitivities can therefore be carried out on eggs derhed from faecal samples that may be up to seven days old. Secondly, identification and speciation of resistant individuals is more easily performed on the third stage larvae, than on eggs or first stage larvae. A number of other known benzimidazole susceptible strains of sheep nematodes have been used in the test and all failed to develop at O· 1 Ilg ml : I thiabendazole (M. A. Taylor, unpublished data). The larval development test may therefore offer the opportunity of screening for benzimidazole resistance in the field because susceptible individuals, even from a mixed population, appear not to develop in the presence of thiabendazole. Sensitivity may be improved by screening samples from animals treated 10 to 14 days previously so that selection of resistant individuals in the population has already taken place. Only a narrow range of thiabendazole concentra.. tions were used compared to the levamisole and iver.. mectin assays. This range was found to give comparable results (M. A. Taylor, unpublished data) to the range of anthelmintic concentrations normally used in this laboratory for the in vitro egg hatch assay (Cawthorne and Whitehead 1983). It is accepted that using a wider range of anthelmintic concentrations would have given more accurate minimum inhibitory concentration values. Several in vitro techniques have been described which measure the paralysing effect of certain anthelmintics. A larval paralysis test described by Martin and LeJambre (1979) was able to distinguish between strains of nematodes susceptible or resistant to levamisole and ivermectin. Donald (1983) and Waller and Prichard (1986) have suggested that the subjectivity of determining whether a larva was paralysed or not, and the parabolic dose-response of levamisole, makes interpretation of the results of this technique difficult. The slow insidious onset of paralysis observed with ivermectin, produced similar problems of interpretation. Dobson et ill (1986) developed a levamisole egg hatch assay based on the paralysis of the first stage larvae within the egg. The assay is complex to perform requiring close monitoring of egg development so that .addition of the anthelmintic can be made close to the point of hatch. Eggs also require to be at similar stages of development necessitating the collection and storage of fresh faecal samples, so that an accurate assessment of development time can be made. Folz et al (1987) described the use of a micro . .

201

motility meter and suggested that it could be used as a means of extending the principle of the larval paralysis test by assessing motility indices at different concentrations and plotting a dose-response curve from which an LD50 value could be calculated. All of these techniques for determining anthel.. mintic sensitivity to anthelmintics with a paralysing mode of action require third stage larvae in relatively large numbers. In contrast, the larval development tests for both levamisole and ivermectin require smaller numbers of first stage larvae. They also appear to be much simpler to perform and interpret with results available within one week of faecal collection. Where resistance is suspected, the species involved can be readily identified on larval morphology. It was noticeable that first stage larvae were very sensitive to ivermectin and much lower ranges of drug concentrations were required for this assay than motility tests on third stage larvae (M. A. Taylor, unpublished observations). With H contortus, the minimum inhibitory concentration of ivermectin was significantly higher for the ivermectin resistant strain, compared to the susceptible strains used in the assays. Interestingly, the susceptible strain of 0 circumcincta had a similar minimum inhibitory concentration of ivermectin to that of the ivermectin resistant H contortus strain. However, it is recognised that few if any conclusions can be drawn from these observations because of the lack of other ivermectin resistant strains of either parasite species. In conclusion, the larval development test appears to have the potential for use as an in vitro screen for detecting the presence of anthelmintic resistance in the field. The indications are that it can be run with any anthelmintic to which resistance is suspected. Further investigations, particularly with levamisole and ivermectin resistant field isolates, will be required before the test can be calibrated and standardised for all anthelmintic groups. Acknowledgements The assistance of Mr K. R. Hunt and Miss C. A. Wilson is gratefully acknowledged. Parasite isolates were kindly supplied by the University of Massachusetts (Dr G. C. Coles), the University of Utrecht (Dr M. Roos) and the Veterinary Research Institute, Onderstepoort (Dr Van Wyk). References CAWTHORNE, R. J. G. & CHEONG,- F. H. (1984) Veterinary Record 114, 562-564 CAWTHORNE, R. J. G. & WHITEHEAD, J. D. (1983) Veterinary Record 112, 274-277 COLES, G. C. & SIMPKIN, K. G. (1977) Research in Veterinary Science 22, 386-387

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mintic Drugs. CSIRO, Division of Animal Health, Canberra, Australia. pp 13-27 PRICHARD, R. K., HALL, C. A., KELLY, J. D. MARTIN, I. C. A. & DONALD, A. D. (1980jAuSiialian Veterinary Journal 56,239-252 SMITH·BUIJS, C. M. C. & BORGSTEEDE, F. H. M. (1986) Research in Veterinary Science 40,4-7 TAYLOR, M. A. (1989) PhD thesis, University of London TAYLOR, M. A. & HUNT, K. R. (1989) Veterinary Record 125, 143-147 VAN WYK, J. A. & MALAN, F. S. (1988) Veterinary Record 123, 226-228 VAN WYK, J. A., MALAN, F. 5., GERBER, H. M. & ALVES, R. M. R. (1987) Onderstepoort Journal of Veterinary Researcb 54, 143-146 WALLER, P. J. (1985) Resistance in Nematodes to Anthelmintic Drugs. CSIRO, Division of Animal Health, Canberra, Australia. pp 1-11 WALLER, P. J. & PRICHARD, R. K. (1986) Chemotherapy of Parasitic Diseases. Ed W. C. Campbell and R. S. Rew. New York, Plenum Press. pp 339-362 WHITLOCK, H. V., KELLY, J.'D., PORTER, C. J., GRIFFIN, D. L. & MARTIN, I. C. A. (1980) Veterinary Parasitology 7,215-232

Received October 16 1989 Accepted February 21, 1990 7