Veterinary Parasitology, 7 (1980) 123--131 123 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE EFFECT OF PHENOTHIAZINE TREATMENT ON ARRESTED HAEMONCHUS CONTORTUS LARVAE IN SHEEP

LESLEY GRIFFIN*

School o f Clinical Veterinary Medicine, University of Cambridge, Cambridge (Great Britain) *Present address: Box 30020, Nalrobi, Kenya

(Accepted 11 October 1979)

ABSTRACT

Griffin, L., 1980. The effect of phenothiazine treatment on arrested Haemonchus contor- tus larvae in sheep. Vet. Parasitol., 7: 123--131.

An experimental infection of freshly cultured Haemonchus contortus larvae was given to housed, non-pregnant sheep in October. Phenothiazine was administered to all animals shortly after patency, but the egg output remained negative until mid-April indicating that removal of adult worms from the abomasum did not stimulate the resumption of development of arrested larvae. Worm burdens at post mortem suggest that adults were not being expelled to any great degree during the winter from the host, but this informa- tion is clouded by the difference in numbers of both adult worms and arrested larvae in sheep of different haemoglobin type. The results suggest that haemoglobin type may also be a factor in arrest of larvae as it is in the resistance of sheep to adult H. contortus.

INTRODUCTION

The temporary cessation of development has been recognised as a feature of many intestinal nematodes of man and animals, and the subject has been extensively reviewed (Michel, 1969, 1974; Schad, 1976). The phenomenon has been reported with many economically important species infecting domestic ruminants and more recently with species infecting man, indicating that the epidemiology of such infections is more complicated than earlier studies suggest.

Several factors have been implicated in inducing arrest. Environmentally induced arrest by which the parasite can survive when external conditions are unfavourable for development has been demonstrated with Ostertagia ostertagi and Cooperia oncophora in cattle (Smith, 1974; Michel et al., 1975), Obeliscoides caniculi in rabbits (Hutchinson et al., 1972), and Ancyl- ostoma caninum in puppies (Schad, 1974), all of which exhibited arrest under the stimulus of low temperature.

Host factors such as age, immunity or natural resistance have also been thought to induce arrest, as hosts exposed to repeated infection, and older

0304--4017/80/0000---0000/$02.25 © 1980 Elsevier Scientific Publishing Company

124

animals, show a high proport ion of arrested nematodes (Dineen et al., 1965; Michel, 1968; Kelly, 1973).

Genetic or density dependent factors related to the parasite have also been regarded as factors inducing arrest. A number of studies suggest that removal of adult worms from the host serves to stimulate development of arrested larvae (Roberts , 1957; Michel, 1969). This may be a means of preventing in- tense competi t ion and overcrowding among adult worms. Gibson (1953) treated housed horses with phenothiazine effective only against lumen dwelling adult nematodes and found an increase in egg ou tpu t after treat- ment following an initial period of negative counts. He treated the horses several times and each time there followed a period of increased egg produc- tion. He suggested that arrested forms resumed development to replace the adults as they were removed. This was supported by studies on Ostertagia species in sheep by Dunsmore (1963) who concluded that removal of adult worms triggered development among those arrested. Michel (1963) demon- strated that while adult worms were regularly removed from calves which were being infected daily with Ostertagia ostertagi no arrested forms devel- oped. However, when anthelminthic t reatment was s topped the number of arrested forms present increased.

To determine if the adult populat ion has a similar effect on arrested Hae- monchus contortus larvae in housed lambs a similar experiment to that des- cribed by Gibson (1953) was set up. Lambs were infected with H. contortus and shortly after pa tency phenothiazine was administered and egg ou tpu t moni tored for 8 months. Concurrently haematological parameters were mea- sured, the haemoglobin type of each animal determined, and the abomasal worm populat ion examined at three stages during the experiment.

MATERIALS AND METHODS

Experimental animals

Twenty nine-month-old clun lambs were used, ten of which were virgin fe- males and ten castrates. All were reared from birth on uncontaminated pas- ture and treated with thibendazole 3 weeks prior to the start of the study. They were housed in a partially covered concrete pen, cleaned daily and fed hay ad libitum.

Parasite strain

The parasite was obtained from Newcastle and was known to have a pro- pensity to arrest (Waller and Thomas, 1975). It was inoculated into a ram which was used as a donor. The larvae collected from the donor were not subjected to any artificial form of preconditioning.

125

Experimental design (Table I)

At the end of October 1977 each lamb was infected orally with approxi- mately 15,000 third stage H. contortus larvae. Twenty four days later when eggs were detected in the faeces eight lambs selected randomly were slaugh- tered and the number of adult and arrested worms present in the abomasum was counted (Group I).

TABLE I

Groups

I II III

Day 0

Day 24 post-infection

Day 26 post-infection

Week 24 post-infection

Week 30 post-infection

H°e.

killed

H.c. H.c.

phenothiazine phenothiazine

killed

killed

On day 26 post-infection the remaining lambs were treated with pheno- thiazine at 600 mg/kg to remove adult worms from the abomasa, the drug being ineffective against arrested forms. Two weeks after the first phenothia- zine t reatment any lambs still passing eggs in the faeces were given a second dose of phenothiazine to remove any surviving adults.

The egg ou tpu t was moni tored thereafter and when an increase occurred again a further six randomly selected animals were slaughtered and the abomasal worm populat ion determined (Group II). The experiment contin- ued for a further 6 weeks until the following June at which time the experi- ment was terminated, the remaining six lambs were slaughtered and the abomasal worm populat ion examined (Group III).

Parasitological techniques

Faeces from the donor ram were collected and cultured in large jars. The cultures were maintained at room temperature for 3 weeks, and the larvae collected using the Baerman technique, counted and used for infection immediately.

126

Faecal egg counts were made initially twice each week, later once per week by the modified McMaster technique.

At necropsy the abomasum of each lamb was removed, washed carefully into a bucket and made up to 2 1 with water at approximately 37°C. The opened abomasum was incubated in the water for about 1 h to allow larvae and adults to migrate from the mucosa into the water. The water was mixed well, 10% aliquots were removed, and adults and arrested stages of H. con- tortus counted.

Haematological observations

Weekly blood samples were collected from each lamb, the packed cell volume (PCV) was determined using a microhaematocri t centrifuge, plasma protein concentrat ion was measured using Atago refractometer, red blood count (RBC), mean corpuscular volume (MCV) and haemoglobin concentra- tion (Hb) were determined using a coulter counter, Model FN.

At the start of the s tudy the haemoglobin type of each lamb was deter- mined by electrophoresis on cellulose acetate paper.

RESULTS

Mean haematological parameters and faecal egg output throughout the study are shown in Fig. 1.

Faecal egg ou tpu t

Three weeks after initial infection the mean faecal egg ou tpu t increased from 0 to nearly 4000 eggs per gram (epg). Egg counts showed great varia- tion from 0 in three lambs to over 20,000 in one lamb. After phenothiazine t reatment the egg ou tpu t decreased to zero in most lambs, but three still showed a very low ou tpu t at 50 epg. However, this ou tpu t increased gradual- ly over the following 2 weeks. The mean count reached 1035 epg at week 6.5. Following a second dose of phenothiazine the egg ou tpu t of all lambs except two remained negative until week 23.5, the middle of April, when six out of the remaining 12 lambs had low counts. Thereafter all remaining lambs were passing steadily increasing numbers of eggs in their faeces until the end of the s tudy at week 33 in June. Two of the lambs showed inter- mi t tent low egg counts for 6 weeks in spite of the second dose of phenothia- zine, before the count decreased to barely detectable levels.

Haema tological observations

The mean PCV, RBC and haemoglobin concentrat ion decreased steadily during the prepatent period and more dramatically at the t ime when egg out- pu t was highest. Concurrently a compensatory increase in MCV occurred.

127

4o PCV%

3o ~ 4 / ~ ' - ' - ' " ~ * - - * - - ~ ' ~ ' o I - " ~ ' ~ ' ~ " x . / ° ~ o

, i i \RBC zOO ~ . / ° - - . . . , . . . i ~ ° ~ ° ~ i,,"

ae(-MCV ~,t ' i ,

( f \ ++l+ - m , ---.,'

++.~+ol~Hb g/loo ml

~+.ok " , , .I" ..,...~.,.___._.

.... I \ / s o~Plasmaprotein g/loo ml

. . . . . . . . .

e p g

Time in weeks

Fig. 1. The mean haemato log ica l indices o f 20 Clun lambs w i t h an expe r imen ta l i n fec t i on o f a p p r o x i m a t e l y 15,000 in fect ive H. contortus larvae, in re la t ion to mean faecal egg counts, t phenothiazine treatment.

4000

3 0 0 0

~CO0

I.)00

Following phenothiazine t reatment red cell indices were rapidly restored to normal, and remained as such for the next 12 weeks. Thereafter a slight de- crease in PCV, RBC and haemoglobin concentration was noticeable particu- larly towards the end of the study when egg ou tput increased. Concurrently an increase in MCV and a slight increase in plasma protein occurred.

Worm burden

The mean and individual worm burdens of the lambs in each group to- gether with haemoglobin type are shown in Table II. Animals in Group I showed a wide range in numbers of both adults and arrested stages recovered from the abomasa, ranging from 0 to 12,400 of the former with arrested lar- vae in numbers from zero to 3200. The mean level of arrest expressed as a percentage of the to ta l number of worms observed was about 22%.

Animals o f Group II showed lower worm burdens of both arrested and mature individuals, but the percentage arrest was similar to that of Group I

128

TABLE II

The numbers of arrested larvae and adult H. contortus recovered from lambs of known haemoglobin type at intervals after experimental infection with 15,000 L3 in October

Lamb Haemoglobin Adult Arrested Arrest

No. type " total seen %

Group I 1 B 12400 200 1.58 slaughtered 3 AB 0 800 100 24 days post 6 B 7800 0 0 infection 12 B 9300 1700 15.4

14 B 0 3200 100 17 B 5100 2300 31.0 19 B 200 1300 86.6 20 A 0 200 100

Mean 4350 1212 % arrest 21.79

Group II 2 AB 60 100 62.5 slaughtered 4 A 200 40 16.6 24 weeks p.i. 7 AB 320 40 11.1

8 A 40 40 50.0 13 AB 40 20 25.0 16 AB 40 0 0

Mean 116.6 40 % arrest 25.5

Group III 5 AB 600 0 0 slaughtered 9 AB 100 0 0 30 weeks p.i. 10 B 2900 200 6.4

11 AB 500 0 0 18 B 4500 0 0

Mean 1720 40 % arrest 2.2

(25%). The numbers of adult worms of Group III were substantially higher than of Group II, but the numbers arrested were zero in all but one indivi- dual. Consequently the percentage of arrested forms observed was only 2%.

Those lambs with haemoglobin type B showed higher numbers of adults or arrested forms in Group I and III than those with the A allele, either as the homozygote or the heterozygote.

DISCUSSION

The faecal egg ou tpu t was closely correlated with the haematological in- dices. As the egg count increased so emerged the typical blood picture of haemorrhagic anaemia caused by H. contortus . After t reatment when the egg count decreased to zero for several months the red cell indices recovered their normal levels. This suggests that remaining larvae were arrested in devel-

129

opment as even low worm burdens causing chronic disease cause a decrease in RBC, PCV and haemoglobin levels (Allonby and Urquhart, 1975).

Earlier work and current research by Connan (1971) using the same strain of H. c o n t o r t u s in untreated lambs maintained under the same conditions as described have shown that the larvae infecting lambs in September become arrested over the winter months and produce a patent infection in April. The increase in mean faecal egg output after removal of adult worms as described by Dunsmore (1963) and Gibson (1953) was not observed in H. contor tus . Indeed removal of adult worms was not followed immediately by increased faecal egg counts. The low numbers of eggs recorded from a very few animals may be due to the incomplete removal of adults in these individuals. There appeared to be a synchronous resumption of development as a steady in- crease in faecal egg output occurred in all remaining animals within the same week during April. The egg output increased over the following month until all animals showed a high count. This feature is supported by the very low numbers of arrested larvae in these animals at necropsy during June.

Blitz and Gibbs (1975 b) also suggested that the removal of adult stages of H. c o n t o r t u s has no effect on the resumption of development of arrested forms, but in contrast to the work described here, the authors reported that development occurred over a 2-month period and not en masse. As none of the lambs was pregnant during this experiment this synchronous resumption of development was clearly not dependent on periparturient physiological changes in the host, but supports earlier suggestions (Blitz and Gibbs, 1972 a) that arrest in H. c o n t o r t u s may be a diapause type of phenomenon charac- teristic of certain strains, an adaptation in the life cycle of the parasite to winter conditions with resumption regulated by the parasite itself. Indeed current work in which the same strain of parasite used to infect lambs in January did not show patent infections in April as did those used in Septem- ber (R.M. Connan, personal communication, 1979) may throw more light on this phenomenon.

The mean worm burden of Group II is substantially lower than the other two groups. The fact that it is lower than Group I may suggest that arrested larvae resume development throughout the winter in numbers too low to produce a demonstrable egg count, and the adults are expelled from the host. However, this cannot be reconciled with the fact that the mean worm burden of the animals in Group III was higher than that of Group II. The haemoglobin type of the experimental animals may have a bearing on this phenomenon. Seven of the eight animals in Group I were of type B, whereas all those of Group II were type A or AB, with two type B in Group III. It has been shown that sheep with type A haemoglobin have higher haematolo- gical parameters during an H. con tor tu s infection, and "self-cure" more readily than those with type B haemoglobin (Allonby and Urquhart, 1976), and that the former have a lower worm establishment than the latter (Altaif and Dargie, 1978). The findings in this experiment support the earlier obser- vations that resistance to H. con tor tu s may be correlated with haemoglobin

130

type, as those with the A allele have lower numbers of both adult and ar- rested forms of the nematode. This is particularly striking in Group III in which the two animals with type B have substantially higher worm burdens than the others which are of type AB. The distribution of haemoglobin types among the groups makes it difficult to match the numbers of arrested forms recovered at the start of winter (Group I) to the number of adults recovered during spring and summer (Groups II and III) to determine the possible loss of worms during the winter. However, considering those animals of haemo- globin type B, a similar number of adult forms of H. contortus were re- covered in June from Group III as of arrested forms recovered from the pre- vious October from Group I. This suggests that the arrested forms resumed development en masse in spring and not continuously throughout the winter to be expelled from the host. It is possible that arrest in H. contortus may normally be regulated by the parasite itself but that this may be modified by condit ions encountered within the host.

ACKNOWLEDGEMENTS

I would like to thank Dr. R. Connan for advice and assistance during the study. The research was supported by a grant from the Wellcome Trust.

REFERENCES

Allonby, E.W. and Urquhart, G.M., 1975. The epidemiology and pathogenic significance of haemonchosis in a Merino flock in East Africa. Vet. Parasitol., i: 129--143.

Allonby, E.W. and Urquhart, G.M., 1976. A possible relationship between haemonchosis and haemoglobin polymorphism in Merino sheep in Kenya. Res. Vet. Sci., 20: 212-- 214.

Altaif, K.I. and Dargie, J.D., 1978. Genetic resistance to helminths. The infl~-ence of breed and haemoglobin type on the response of sheep to primary infections with Hae- monchus contortus. Parasitology, 77: 161--175.

Blitz, N.M. and Gibbs, H.C., 1972 a. Studies on the arrested development of Haemonchus contortus in sheep. I. The induction of arrested development. Int. J. Parasitol., 2" 5--12.

Blitz, N.M. and Gibbs, H.C., 1972 b. Studies on the arrested development of Haemonchus contortus in sheep. If. The termination of arrested development and the spring rise phenomenon. Int. J. Parasitol., 2: 13--22.

Connan, R.M., 1971. The seasonal incidence of inhibition of development in Haemonchus eontortus. Res. Vet. Sci., 12: 272--274.

Dineen, J.K., Donald, A.D., Wagland, B.M. and Turner, J.H., 1965. The dynamics of the host parasite relationship. II. The response of sheep to primary and secondary infec- tion with Nematodirus spathiger. Parasitology, 55 : 163--171.

Dunsmore, I.D., 1963. Effect of the removal of an adult population of Ostertagia from sheep on concurrently existing larvae. Aust. Vet. J., 39: 359--463.

Gibson, T.E., 1953. The effect of repeated anthelminthic treatment with phenothiazine on the faecal egg counts of housed horses, with some observations on the life cycle of Tr ichonema spp. in t he horse . J. H e l m i n t ho l . , 27: 29- -40 .

131

Hutchinson, G.W., Lee, E.W. and Femando , M.A., 1972. Effects of variations in tempera- ture on infective larvae and their relationship to inhibited development of Obeliscoides cuniculi in rabbit . Parasitology, 65: 333--342.

Kelly, J.D., 1973. Immuni ty and epidemiology of helminthiasis in grazing animals. N.Z.Vet. J., 21 : 183--194.

Michel, J .F. , 1963. The phenomenon of host resistance and the course of infection of Os- tertagia ostertagi in calves. Parasitology, 53: 63--84.

Michel, J .F. , 1968. Immuni ty to helminths associated with the tissues. In: A.E.R. Taylor (Editor), Immuni ty to Parasites. Vol. 6. Syrup. British Society of Parasitology. Black- well, Oxford, pp. 67--89.

Michel, J .F. , 1969. The epidemiology and control of some nematode infections of grazing animals. Adv. Parasitol., 7 : 211--282.

Michel, J .F. , 1974. Arrested development of nematodes and some related phenomena. Adv. Parasitol., 12: 279--366.

Michel, J.F., Lancaster, M.B. and Hong, C., 1975. Arrested development of Ostertagia os- tertagi and Cooperia oncophora. J. Comp. Pathol., 84: 539--554.

Roberts, F.H.S., 1957. Reactions of calves in infestation with the stomach worm Hae- monchus placei. Aust. Vet. J., 35: 409--414.

Schad, G.A., 1974. Experimental ly induced arrested development of the parasitic larvae of hookworms. Proc. Third Int. Congr. Parasitol., 2: 772--773.

Schad, G.A., 1976. The role of arrested development in the regulation of nematode popu- lations. In- G.W. Esch (Editor), Regulation of Parasite Populations. Academic Press, New York, pp. 111--167.

Smith, H.J., 1974. Inhibi ted development of Ostertagia ostertagi, Cooperia oncophora and Nematodirus helvetianus in parasite free calves grazing fall pastures. Am. J. Vet. Res., 35: 935--938.

Waller, P.J. and Thomas, R.J., 1975. Field studies on inhibit ion of Haemonchus contortus in sheep. Parasitology, 71 : 285--291.