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In vitro field screening for anthelmintic resistance in strongyles of sheep and horses
Veterinary Parasitology, 7 (1980) 2 1 5 - 2 3 2 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
IN VITRO FIELD SCREENING FOR ANTHELMINTIC STRONGYLES OF SHEEP AND HORSES
RESISTANCE
215
IN
H.V. WHITLOCK, J.D. KELLY*, C.J. PORTER, D.L. G R I F F I N and I.C.A. MARTIN**
Departments of Veterinary Pathology and Veterinary Physiology **, University of Sydney, Sydney, N.S.W. 2006 (Australia) (Accepted 4 March 1980)
ABSTRACT Whitlock, H.V., Kelly, J.D., Porter, C.J., Griffin, D.L. and Martin, I.C.A., 1980. In vitro field screening for anthelmintic resistance in strongyles of sheep and horses. Vet. Parasitol., 7: 215--232. Simplified in vitro field methods are described for the detection and assay of benzimidazole-resistance in sheep trichostrongylids and horse strongyles. Worm eggs are recovered from fresh faeces, within 1 hour of collection, by flotation in sugar solution. The separated eggs are then incubated for 20--24 hours at 27--30°C in solutions o f thiabendazole in distilled water ranging from 0.1 to 1.1 p.p.m, for sheep trichostrongylids and from 0.05 to 0.5 p.p.m, for horse strongyles. Under controlled conditions, eggs from thiabendazole-susceptible individuals of both sheep and horse nematodes rarely hatch at thiabendazole concentrations of 0.1 p.p.m. Eggs from resistant individuals will hatch at 0.1 p.p.m, and above. Semi-quantitative estimates of the level of resistance can be determined by measuring the % egg-hatch at varying concentrations of thiabendazole. A field method for selecting test animals with low egg-counts, and an in vitro method for the culture of eggs or first-stage larvae to third stage for identification are described.
INTRODUCTION Detection of benzimidazole (BZ) resistant strains of gastro-intestinal nematodes {Kelly and Hall, 1979a) has been aided by the development and u s e o f in v i t r o t e c h n i q u e s w h i c h a r e b a s e d o n p r o c e d u r e s f o r a s s a y o f n e m a t o d e egg h a t c h a b i l i t y ( L e J a m b r e e t al., 1 9 7 0 ) . T h e s e m e t h o d s i n c l u d e t h o s e o f L e J a m b r e ( 1 9 7 6 ) , C o l e s a n d S i m p k i n { 1 9 7 7 ) a n d H a l l e t al. ( 1 9 7 8 a ) , a n d r e l y o n t h e r e c o v e r y o f w o r m eggs f r o m f a e c e s f o l l o w e d b y i n c u b a t i o n i n s e r i a l c o n c e n t r a t i o n s o f a q u e o u s s o l u t i o n s o f B Z a n t h e l m i n t i c s in o r d e r t o d e t e r m i n e t h e 5 0 % l e v e l o f egg e m b r y o n a t i o n a n d / o r h a t c h a b i l i t y . S u c h m e t h o d s a r e d e s i g n e d p r i m a r i l y f o r l a b o r a t o r y use. F a e c e s a r e c o o l e d to 5--10°C during warm weather to retard development because the egg-hatch *Present address: Officer in Charge, Victorian Department of Agriculture, Regional Veterinary Laboratory, Bendigo, Victoria 3550, Australia. 0304-4017/80/0000--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
216 assay depends on the susceptibility of fresh eggs to BZ anthelmintics through anaerobic metabolic pathways. As eggs develop, aerobic metabolism predominates and they become refractory to BZ anthelmintics. The maximum period for maintenance of egg susceptibility to BZ anthelmintics is suggested as 4 h (Coles and Simpkin, 1977). To minimise this risk period, the methods described in this paper require the recovery of eggs immediately after rectal collection, and are designed for field processing. Low post-treatment egg counts are detected by a modified egg-counting method. A simple mixing, sieving and flotation m e t h o d for the recovery of eggs free of faecal debris is also described, which requires 5.--10 min to perform and recovers between 60 and 70% of the eggs present. The entire test preparation may be completed within an hour. The methods are designed for initial field detection or semi-quantitative assay of suspected resistant strains as a preliminary to detailed investigation by more sensitive methods. MATERIALS AND METHODS
Apparatus (i) Faecal egg-count method (Fig. 1) Universal* egg-counting chamber (Fig. lf). Measuring and mixing equipment for preparation of faecal suspensions (Fig. la--e). Saturated salt solution. Wash bottle for dispensing saturated salt solution.
(ii) Recovery of eggs from faeces (Figs. 2--7) 250-ml plastic measuring cylinder. Mixing plunger (Fig. 2). 25 cm X 25 cm squares of surgical gauze {0.5--1.0 mm aperture weave). Sugar solution, specific gravity 1.24. 250-ml plastic beakers. 220-ml, 100-ml and 60-ml flat McCartney, screw-cap bottles; a small hole is drilled through the centre of each screw cap. Wash bottle containing distilled water. Heavy d u t y plastic bags (to contain cold water during hot weather). 10-ml disposable syringes.
(iii) Preparation of thiabendazole (Thibenzole**) test solutions Stock solution 1:1000 (0.57 ml of 17.6% w/v suspension Thibenzole in 100 ml distilled water; add several drops of N 10 HC1 to clear solution). Each *J.A. Whitlock and Co., 17 Northbrook St, Bexley, N.S.W. 2207, Australia. **Thibenzole --Registered Trademark, Merck Sharp and Dohme (Aust.) Pty. Ltd.
217 test batch is made up from a freshly prepared thiabendazole (TBZ) solution of 1:100 000 (1 ml of stock solution to 99 ml of distilled water). Note: Test solutions are best prepared from pure TBZ where available. Chemical analysis may be used for assay of concentrations. Biological tests of each batch by screening against known susceptible and/or resistant strains have proved satisfactory.
(iv) In vitro culture and examination o f tests Incubation facilities, 27 ° C. 1.0% aqueous iodine. Larval examination slide. (v) Culture of first-stage larvae to third stage (Fig. 11) Whatman filter papers No. 1, 50 mm diameter. Buchner filter apparatus. Beeswax: solid paraffin mixture, 1 : 3. Wide-mouthed glass jars, approx. 50 ml and 100 ml capacity. Cotton wool. Sodium hypochlorite. Method for counting low levels o f nematode eggs in faeces (Universal technique) A 3-ml volume of fresh faeces is measured by displacement of 3 ml of water contained in a 10-ml disposable syringe which has been cut level at the zero calibration (Fig. la). Seven ml of water is placed in the syringe and faeces are added to bring the meniscus to the zero calibration. The measured faeces is then ejected into the mixing cylinder (Fig. l b ) containing 50 ml of flotation solution. Saturated salt solution may be used. The faecal suspension is prepared by gentle mixing with a plunger (Fig. lc) constructed from a flat disc, 3 mm thick, attached to a thin rod. The disc is 3 mm less in diameter than the internal diameter of the cylinder and is used for mixing suspensions of sheep faeces. A similar disc, but 6 mm less in diameter than the cylinder, is attached to the opposite end of the rod and is used for mixing suspensions o f horse faeces. A tube (Fig. l d ) , having a sieve of 1.0 mm aperture mesh across one end and a flange at the other end, is used to sieve a portion of the mixed suspension by inserting the tube with a mixing action into the cylinder. A sample of the sieved suspension is then transferred with the aid of a pipette (Fig. l e ) to the egg-counting chamber (Fig. lf). The Universal slide has four counting chambers. Each chamber has a counting area of 0.5 ml which is sub-divided into 5 counting strips of 0.1 ml. The eggs are counted under low-power magnification (× 40 or 50) and the factors applicable to the 1:20 dilution are as follows: × 200 for eggs in 0.1 ml; × 100 in 0.2 ml; × 40 in 0.5 ml; × 10 for four chambers of the slide (2.0 ml).
218
f
a
b
c
d
e
f Fig. 1. Universal egg-counting technique. Apparatus and procedures for the preparation of faecal suspensions (a--e). The Universal egg-counting slide (f).
Method for recovery of eggs from faeces A technique for the recovery of clean eggs from faeces for the culture of first-stage n e m a t o d e larvae of sheep (Whitlock, 1959) has been adapted for the recovery of eggs for in vitro BZ assay. The technique for use with sheep is as follows: (1) Faeces are collected directly from the rectum and added immediately to 120 ml of water in a 250-ml measuring cylinder so that approximately 20 g of faeces is measured by displacement (when the meniscus reaches the 140-ml calibration). Two or three pellets from 10--15 sheep may be used for flocktesting (Fig. 2). (Use cold water during hot weather.) (2) A smooth faecal suspension is prepared with the aid of the mixing rod, using a gentle pumping action (Fig. 3). (3) A double layer of surgical gauze is placed over a 250-ml beaker and the faecal suspension is poured through the gauze (Fig. 4). (4) The gauze containing the coarse faecal debris may be wrapped into a ball and squeezed to ensure m a x i m u m recovery of faecal suspension. (5) An equal volume of sugar solution (SG = 1.24) is mixed with the sus-
219
pension. The sugar solution may also be pre-cooled during hot weather. The faecal debris may also be mixed with the 120 ml of sugar solution and the sieving process repeated to recover a greater number of eggs. (6) Two dry, flat-sided McCartney bottles (100 ml) are filled with the faecal suspension. Air is excluded and as many bubbles as possible are floated out. The bottles are capped and placed horizontally, flat side down, for 30 min (Fig. 5). During hot weather, the jars should be placed on plastic bags containing cold water to retard development of eggs. Larger quantities of faecal suspension are processed where egg counts are very low, especially for the quantitative assay. Six- or eight-ounce bottles may be used. (7) After 30 min flotation, the bottle is held in the horizontal plane. The screw cap is removed, and the contents are carefully poured o u t of the bottle, avoiding turbulence and air bubbling in the neck of the bottle (Fig. 6). (8) Debris is washed from the b o t t o m flat surface with the aid of a wash bottle containing distilled water. Care is taken n o t to disturb the eggs adhering to the upper surface of the bottles (Fig. 7). These procedures are followed for recovery of clean eggs from horse faeces, except that 30--40 g are used to prepare the faecal suspension. Additional quantities of faeces m a y be processed when egg counts are very low.
Method for culture o f first-stage larvae to third-stage infective larvae As part of the in vitro BZ assay, it is sometimes desirable to harvest firststage larvae (L1) from the TBZ test solutions and culture them through to the third stage (L3) in order to facilitate species identification. The following technique is used for this purpose: Part of the 0.1-p.p.m. culture, after 24 h incubation at 27--30°C (see in vitro screening procedures), may be retained {without iodine treatment) for culture to L3. The first-stage larvae are washed free of TBZ using a 50-mm Figs. 2--7. R e c o v e r y o f w o r m eggs from faeces. Fig. 2. Faeces are measured by displacement in a calibrated cylinder containing water. Fig. 3. The faecal suspension is prepared by mixing w i t h a disc attached to a thin rod. Fig. 4. Coarse debris is r e m o v e d by passing the faecal suspension through c o t t o n gauze. Fig. 5. The faecal suspension is m i x e d with sugar solution and placed in flat sided bottles for 30 min. Figs. 6--7. The bottles are emptied so that eggs are retained in the film adhering to the upper inside wall o f the bottle. Fig. 8. E m b r y o n a t e d and u n d e v e l o p e d H a e m o n c h u s c o n t o r t u s eggs. Fig. 9. Motile first-stage H. c o n t o r t u s larvae.
O
222
a
b
c
d
Fig. 10. First-stage larvae of sheep nematodes. (a) Ostertagia circumcincta, total length 360--433 um (mean 399 urn), tail short and pointed, larvae broad at oesophagus. (b) Trichostrongylus colubriformis, total length 350--412 um (mean 381 urn), tail short and pointed. (c) Haernonchus contortus, total length 340--381 um (mean 362 urn), tail tapered, "medium" length. (d) Strongyloides papiUosus, total length 237--268 um (mean 253 urn), short larvae, pointed tail faintly visible. d i a m e t e r No. 1 W h a t m a n filter p a p e r , distilled w a t e r , a B u c h n e r filter a n d negative pressure. T h e filter p a p e r is c a r e f u l l y r e m o v e d f r o m t h e B u c h n e r f u n n e l and a p a p e r o f t h e s a m e d i a m e t e r is p l a c e d o v e r t h e larvae t o f o r m a sealed e n v e l o p e b y ringing t h e t w o p a p e r s at t h e edges w i t h a w a r m p a r a f f i n - b e e s w a x m i x t u r e . T h e e n v e l o p e is rolled g e n t l y i n t o a t u b e b e f o r e t h e sealingm i x t u r e solidifies. A small l a y e r o f c o t t o n w o o l is p l a c e d in t h e b o t t o m o f a suitable widem o u t h jar so t h a t faeces f r o m a w o r m - f r e e sheep, e i t h e r f r e s h l y c o l l e c t e d o r held in cold storage ( 2 - - 5 ° C ) , m a y be c o a r s e l y b r o k e n d o w n a n d p a c k e d
IN VITRO FIELD SCREENING FOR ANTHELMINTIC STRONGYLES OF SHEEP AND HORSES
RESISTANCE
215
IN
H.V. WHITLOCK, J.D. KELLY*, C.J. PORTER, D.L. G R I F F I N and I.C.A. MARTIN**
Departments of Veterinary Pathology and Veterinary Physiology **, University of Sydney, Sydney, N.S.W. 2006 (Australia) (Accepted 4 March 1980)
ABSTRACT Whitlock, H.V., Kelly, J.D., Porter, C.J., Griffin, D.L. and Martin, I.C.A., 1980. In vitro field screening for anthelmintic resistance in strongyles of sheep and horses. Vet. Parasitol., 7: 215--232. Simplified in vitro field methods are described for the detection and assay of benzimidazole-resistance in sheep trichostrongylids and horse strongyles. Worm eggs are recovered from fresh faeces, within 1 hour of collection, by flotation in sugar solution. The separated eggs are then incubated for 20--24 hours at 27--30°C in solutions o f thiabendazole in distilled water ranging from 0.1 to 1.1 p.p.m, for sheep trichostrongylids and from 0.05 to 0.5 p.p.m, for horse strongyles. Under controlled conditions, eggs from thiabendazole-susceptible individuals of both sheep and horse nematodes rarely hatch at thiabendazole concentrations of 0.1 p.p.m. Eggs from resistant individuals will hatch at 0.1 p.p.m, and above. Semi-quantitative estimates of the level of resistance can be determined by measuring the % egg-hatch at varying concentrations of thiabendazole. A field method for selecting test animals with low egg-counts, and an in vitro method for the culture of eggs or first-stage larvae to third stage for identification are described.
INTRODUCTION Detection of benzimidazole (BZ) resistant strains of gastro-intestinal nematodes {Kelly and Hall, 1979a) has been aided by the development and u s e o f in v i t r o t e c h n i q u e s w h i c h a r e b a s e d o n p r o c e d u r e s f o r a s s a y o f n e m a t o d e egg h a t c h a b i l i t y ( L e J a m b r e e t al., 1 9 7 0 ) . T h e s e m e t h o d s i n c l u d e t h o s e o f L e J a m b r e ( 1 9 7 6 ) , C o l e s a n d S i m p k i n { 1 9 7 7 ) a n d H a l l e t al. ( 1 9 7 8 a ) , a n d r e l y o n t h e r e c o v e r y o f w o r m eggs f r o m f a e c e s f o l l o w e d b y i n c u b a t i o n i n s e r i a l c o n c e n t r a t i o n s o f a q u e o u s s o l u t i o n s o f B Z a n t h e l m i n t i c s in o r d e r t o d e t e r m i n e t h e 5 0 % l e v e l o f egg e m b r y o n a t i o n a n d / o r h a t c h a b i l i t y . S u c h m e t h o d s a r e d e s i g n e d p r i m a r i l y f o r l a b o r a t o r y use. F a e c e s a r e c o o l e d to 5--10°C during warm weather to retard development because the egg-hatch *Present address: Officer in Charge, Victorian Department of Agriculture, Regional Veterinary Laboratory, Bendigo, Victoria 3550, Australia. 0304-4017/80/0000--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
216 assay depends on the susceptibility of fresh eggs to BZ anthelmintics through anaerobic metabolic pathways. As eggs develop, aerobic metabolism predominates and they become refractory to BZ anthelmintics. The maximum period for maintenance of egg susceptibility to BZ anthelmintics is suggested as 4 h (Coles and Simpkin, 1977). To minimise this risk period, the methods described in this paper require the recovery of eggs immediately after rectal collection, and are designed for field processing. Low post-treatment egg counts are detected by a modified egg-counting method. A simple mixing, sieving and flotation m e t h o d for the recovery of eggs free of faecal debris is also described, which requires 5.--10 min to perform and recovers between 60 and 70% of the eggs present. The entire test preparation may be completed within an hour. The methods are designed for initial field detection or semi-quantitative assay of suspected resistant strains as a preliminary to detailed investigation by more sensitive methods. MATERIALS AND METHODS
Apparatus (i) Faecal egg-count method (Fig. 1) Universal* egg-counting chamber (Fig. lf). Measuring and mixing equipment for preparation of faecal suspensions (Fig. la--e). Saturated salt solution. Wash bottle for dispensing saturated salt solution.
(ii) Recovery of eggs from faeces (Figs. 2--7) 250-ml plastic measuring cylinder. Mixing plunger (Fig. 2). 25 cm X 25 cm squares of surgical gauze {0.5--1.0 mm aperture weave). Sugar solution, specific gravity 1.24. 250-ml plastic beakers. 220-ml, 100-ml and 60-ml flat McCartney, screw-cap bottles; a small hole is drilled through the centre of each screw cap. Wash bottle containing distilled water. Heavy d u t y plastic bags (to contain cold water during hot weather). 10-ml disposable syringes.
(iii) Preparation of thiabendazole (Thibenzole**) test solutions Stock solution 1:1000 (0.57 ml of 17.6% w/v suspension Thibenzole in 100 ml distilled water; add several drops of N 10 HC1 to clear solution). Each *J.A. Whitlock and Co., 17 Northbrook St, Bexley, N.S.W. 2207, Australia. **Thibenzole --Registered Trademark, Merck Sharp and Dohme (Aust.) Pty. Ltd.
217 test batch is made up from a freshly prepared thiabendazole (TBZ) solution of 1:100 000 (1 ml of stock solution to 99 ml of distilled water). Note: Test solutions are best prepared from pure TBZ where available. Chemical analysis may be used for assay of concentrations. Biological tests of each batch by screening against known susceptible and/or resistant strains have proved satisfactory.
(iv) In vitro culture and examination o f tests Incubation facilities, 27 ° C. 1.0% aqueous iodine. Larval examination slide. (v) Culture of first-stage larvae to third stage (Fig. 11) Whatman filter papers No. 1, 50 mm diameter. Buchner filter apparatus. Beeswax: solid paraffin mixture, 1 : 3. Wide-mouthed glass jars, approx. 50 ml and 100 ml capacity. Cotton wool. Sodium hypochlorite. Method for counting low levels o f nematode eggs in faeces (Universal technique) A 3-ml volume of fresh faeces is measured by displacement of 3 ml of water contained in a 10-ml disposable syringe which has been cut level at the zero calibration (Fig. la). Seven ml of water is placed in the syringe and faeces are added to bring the meniscus to the zero calibration. The measured faeces is then ejected into the mixing cylinder (Fig. l b ) containing 50 ml of flotation solution. Saturated salt solution may be used. The faecal suspension is prepared by gentle mixing with a plunger (Fig. lc) constructed from a flat disc, 3 mm thick, attached to a thin rod. The disc is 3 mm less in diameter than the internal diameter of the cylinder and is used for mixing suspensions of sheep faeces. A similar disc, but 6 mm less in diameter than the cylinder, is attached to the opposite end of the rod and is used for mixing suspensions o f horse faeces. A tube (Fig. l d ) , having a sieve of 1.0 mm aperture mesh across one end and a flange at the other end, is used to sieve a portion of the mixed suspension by inserting the tube with a mixing action into the cylinder. A sample of the sieved suspension is then transferred with the aid of a pipette (Fig. l e ) to the egg-counting chamber (Fig. lf). The Universal slide has four counting chambers. Each chamber has a counting area of 0.5 ml which is sub-divided into 5 counting strips of 0.1 ml. The eggs are counted under low-power magnification (× 40 or 50) and the factors applicable to the 1:20 dilution are as follows: × 200 for eggs in 0.1 ml; × 100 in 0.2 ml; × 40 in 0.5 ml; × 10 for four chambers of the slide (2.0 ml).
218
f
a
b
c
d
e
f Fig. 1. Universal egg-counting technique. Apparatus and procedures for the preparation of faecal suspensions (a--e). The Universal egg-counting slide (f).
Method for recovery of eggs from faeces A technique for the recovery of clean eggs from faeces for the culture of first-stage n e m a t o d e larvae of sheep (Whitlock, 1959) has been adapted for the recovery of eggs for in vitro BZ assay. The technique for use with sheep is as follows: (1) Faeces are collected directly from the rectum and added immediately to 120 ml of water in a 250-ml measuring cylinder so that approximately 20 g of faeces is measured by displacement (when the meniscus reaches the 140-ml calibration). Two or three pellets from 10--15 sheep may be used for flocktesting (Fig. 2). (Use cold water during hot weather.) (2) A smooth faecal suspension is prepared with the aid of the mixing rod, using a gentle pumping action (Fig. 3). (3) A double layer of surgical gauze is placed over a 250-ml beaker and the faecal suspension is poured through the gauze (Fig. 4). (4) The gauze containing the coarse faecal debris may be wrapped into a ball and squeezed to ensure m a x i m u m recovery of faecal suspension. (5) An equal volume of sugar solution (SG = 1.24) is mixed with the sus-
219
pension. The sugar solution may also be pre-cooled during hot weather. The faecal debris may also be mixed with the 120 ml of sugar solution and the sieving process repeated to recover a greater number of eggs. (6) Two dry, flat-sided McCartney bottles (100 ml) are filled with the faecal suspension. Air is excluded and as many bubbles as possible are floated out. The bottles are capped and placed horizontally, flat side down, for 30 min (Fig. 5). During hot weather, the jars should be placed on plastic bags containing cold water to retard development of eggs. Larger quantities of faecal suspension are processed where egg counts are very low, especially for the quantitative assay. Six- or eight-ounce bottles may be used. (7) After 30 min flotation, the bottle is held in the horizontal plane. The screw cap is removed, and the contents are carefully poured o u t of the bottle, avoiding turbulence and air bubbling in the neck of the bottle (Fig. 6). (8) Debris is washed from the b o t t o m flat surface with the aid of a wash bottle containing distilled water. Care is taken n o t to disturb the eggs adhering to the upper surface of the bottles (Fig. 7). These procedures are followed for recovery of clean eggs from horse faeces, except that 30--40 g are used to prepare the faecal suspension. Additional quantities of faeces m a y be processed when egg counts are very low.
Method for culture o f first-stage larvae to third-stage infective larvae As part of the in vitro BZ assay, it is sometimes desirable to harvest firststage larvae (L1) from the TBZ test solutions and culture them through to the third stage (L3) in order to facilitate species identification. The following technique is used for this purpose: Part of the 0.1-p.p.m. culture, after 24 h incubation at 27--30°C (see in vitro screening procedures), may be retained {without iodine treatment) for culture to L3. The first-stage larvae are washed free of TBZ using a 50-mm Figs. 2--7. R e c o v e r y o f w o r m eggs from faeces. Fig. 2. Faeces are measured by displacement in a calibrated cylinder containing water. Fig. 3. The faecal suspension is prepared by mixing w i t h a disc attached to a thin rod. Fig. 4. Coarse debris is r e m o v e d by passing the faecal suspension through c o t t o n gauze. Fig. 5. The faecal suspension is m i x e d with sugar solution and placed in flat sided bottles for 30 min. Figs. 6--7. The bottles are emptied so that eggs are retained in the film adhering to the upper inside wall o f the bottle. Fig. 8. E m b r y o n a t e d and u n d e v e l o p e d H a e m o n c h u s c o n t o r t u s eggs. Fig. 9. Motile first-stage H. c o n t o r t u s larvae.
O
222
a
b
c
d
Fig. 10. First-stage larvae of sheep nematodes. (a) Ostertagia circumcincta, total length 360--433 um (mean 399 urn), tail short and pointed, larvae broad at oesophagus. (b) Trichostrongylus colubriformis, total length 350--412 um (mean 381 urn), tail short and pointed. (c) Haernonchus contortus, total length 340--381 um (mean 362 urn), tail tapered, "medium" length. (d) Strongyloides papiUosus, total length 237--268 um (mean 253 urn), short larvae, pointed tail faintly visible. d i a m e t e r No. 1 W h a t m a n filter p a p e r , distilled w a t e r , a B u c h n e r filter a n d negative pressure. T h e filter p a p e r is c a r e f u l l y r e m o v e d f r o m t h e B u c h n e r f u n n e l and a p a p e r o f t h e s a m e d i a m e t e r is p l a c e d o v e r t h e larvae t o f o r m a sealed e n v e l o p e b y ringing t h e t w o p a p e r s at t h e edges w i t h a w a r m p a r a f f i n - b e e s w a x m i x t u r e . T h e e n v e l o p e is rolled g e n t l y i n t o a t u b e b e f o r e t h e sealingm i x t u r e solidifies. A small l a y e r o f c o t t o n w o o l is p l a c e d in t h e b o t t o m o f a suitable widem o u t h jar so t h a t faeces f r o m a w o r m - f r e e sheep, e i t h e r f r e s h l y c o l l e c t e d o r held in cold storage ( 2 - - 5 ° C ) , m a y be c o a r s e l y b r o k e n d o w n a n d p a c k e d