Experimental chemotherapy of infection with Capillaria obsignata

J. COMP.

137

1965. VOL. 75.

PATH.

EXPERIMENTAL

CHEMOTHERAPY

OF INFECTION

CAPILLARIA

WITH

OBSIGNATA BY

C. C. NORTON Central

V&&my

and L. P. JOYNER

Luboratq,

Mini&y

of Agrkdture,

Wqbridge

INTRODUCTION

Recent survey data (Norton, 1964 ; Wakelin, 1964) have indicated an increase in the incidence of Cafiillaria obsignata. Until recently, infections with this nematode have proved dif%.rlt to eradicate and as soon as the results of our survey began to indicate the predominance of this one species, experiments were initiated to establish the conditions for experimental infections and to explore the techniques for assessingthe efficacy of drugs. Activity against CapiElaria infection in fowls has been recorded for Haloxon and Methyridine (Clarke, 1962 ; Thienpont and

Mortehnans,

1962; Geeraerts, 1962, 1964a, b; Hendriks,

1962; Broome,

1963)

and experiments were designed to defme and compare the efficacy of these compounds under experimental conditions.

MATERIALS

AND

METHODS

Control of coccidiosis. Several workers with C. obsignata have recorded coccidiosis as a secondary factor in their experiments and some have taken positive steps to control it. As a preventive measure, all experimental chickens used in these experiments received throughout O-0125per cent. 2,4 dinitro-o-toluamide (zoalene) in the diet. This appeared to be completely effective; oocysts were never observed in faecal flotations and no adverse effect upon the nematode was detected. Capillaria ova were cultured successfully from the faeces of experimentally infected chickens, but in the initial experiments further precautions were taken to avoid inoculation of oocysts by preparing suspensionsof ova to be used for the infection of chickens from the faeces of experimentally infected turkeys. Separation and concentration of the oua of Capillaria. Initially, suspensionsof ova were prepared by extraction of female worms, but the largest yields of ova were obtained by flotation from the faecesof experimentally infected birds by the following technique. (1) faeces containing at least 1,000 ova per gramme were collected and allowed to soak and break up in water. (2) after filtering through two wire screenshaving 100 and 200 meshesper linear inch respectively, the ova were allowed to sediment out in 3 litre beakersfor 3 hours. The supernatant fluid was syphoned off and the sediment was concentrated by centrifugation at 2,000 r.p.m. for 2 minutes. (3) further purification was effected by two flotations in saturated salt solution (NaCl), followed by sedimentation in water. (4) finally the ova were set to embryonate at 27°C. in shallow layers of distilled water in petri-dishes. After 10 days, the embryonated eggs were suspendedin 1 per cent. formalin and after standardization the suspensionwas stored at 4°C. until used. Contrary to the experience of Frazier (1962) agitation was not necessaryduring embryonation. About 95 per cent. of the ova regularly developed to the infective stage under the conditions described. The infective ova were inoculated directly

138

CHEMOTHERAPY

into the crops of experimental spring-loaded syringe.

OF

CAPILLARIA

INFECTION

birds by means of a ball-ended

cannula attached to a

Recovery of worms from experimental birds. In recovering the worms from freshly killed birds two difficulties were encountered. The first arosefrom the active secretion of mucus by the gut and the secondbecausethe worms were deeply engaged in the mucosa. In six experiments involving the recovery of approximately 11,000 adult worms from experimentally infected birds, a single washing by rubbing the mucosa between finger and thumb under running tap water removed only 59 per cent. of the worms. A further 41 per cent. emerged on incubation of the intestine in saline at 37°C. In subsequent experiments birds were starved overnight before sacrifice. The intestines were opened and soaked in physiological saline at 37°C. for 3 hours. The surface of the mucosa was then rubbed gently between the fingers under running tap water into a 200 mesh screen. Sodium bicarbonate was added to the saline

washings to dissolve the mucus. The washings were then passedthrough the same screen; the worms retained were transferred to a bowl and finally bottled with formalin for subsequentcounting. The recovered worms were counted individually except where the numbers exceeded 1,000 when a dilution counting technique was employed (Taylor 1934). Egg counting mt thods. Capillaria ova were counted by the methods described by Parfitt (1958). The modified MacMaster technique was used for counts exceeding 100 eggs per gramme of faeces. The salt flotation technique was used when the eggs were fewer than 100 per gramme. RESULTS

Characteristics

of Experimental

Infections

Symptoms. In general the most severe symptoms were observed in birds aged 2 to 3 weeks when large or very large dosesof ova were administered (5,000 to 100,000 ova per bird). In such birds the first symptoms were noted on the 12th day following infection when strands of white and pink mucus appeared in the faeces. Microscopical examination of this material revealed the presence of irregularly coiled larvae and immature worms (Fig. 2). In severe infections the strings of mucus were numerous and the entire faecal output was mucoid and watery in appearance. Sections of intestine at this stage showed the larvae to be closely applied to the mucosa and in many casesthe villar tissue was penetrated (Fig. 3). Immediately surrounding the embedded larvae cellular breakdown was observed and a limited eosinophilic cellular reaction was noted (Fig. 4). Larvae were largely confined to the middle region of the small intestine. At this stage of the infection the birds began to lose condition and appeared listless with wings drooping and feathers ruffled. They recovered in about two weeks and the faeces returned to normal within the same period. Retardation of growth sufficient to form the basis of experimentation was only demonstrable where large numbers of ova were given to young birds as illustrated in Fig. 1. Under these circumstances large numbers of larvae were eliminated and in practice it proved difficult to establish heavy infections, with an acceptably small degree of variation between individual birds. reaches sexual maturity about 3 weeks after Production of ova. C. obsignata inoculation of embryonated ova. Levine (1938) first observed ova in the faces of infected birds on the 2 1st day after inoculation. In some of the present experiments ova were, seen as early as the 19th day; usually however they first appeared in the droppings on the 20th day. Within 2 or 3 days the numbers of ova usually

C.

C.

NORTON

AND Fii.

L.

P.

139

JOYNER

1.

Normal

Control

C.obsignata

5

IO Time

Retardation of growth in young 100,000 ova of C. obsignata.

chicks

15 in Days experimentally

20 infected

25 with

25,000,

50,ooO

and

reached a peak which was about 1,000 ova per gramme of faeces. The highest production of ova (6,350 0.p.g.) was observed in birds which had received 100,000 embryonated ova. In most experiments the production of ova fell to a low level (1 to 10 0.p.g.) after about 6 days. Recovery of adull worms. It was evident that large numbers of the worms were eliminated as a result of the host’s reaction to the larval stages. The proportion of worms which reached maturity was inversely related to the size of the inoculum as shown in Table 1. Variation in the worm burdens of individual bids appeared greatest with the heavier inoculations. To design a “critical” chemotherapeutic experiment based upon recovery of adult worms it was desirable to establish as heavy an infection as possible with an acceptable variation in individual worm burdens. An inoculation of 2,000 embryonated ova seemed a reasonable compromise, and the following procedure was finally adopted for establishing experimental infections (1) thirty to forty tweweek-old chicks were inoculated individually with 2,000 embryonated ova. Zoalene was incorporated in the food at a concentration of 0.0125 per cent.

140

CHEMOTHERAPY

OF

CAPILLARIA

INFECTION

(2) lo,16 or 22 days later, when larvae, immature or mature worms respectively were present, the birds were randomized into groups of at least 8 to 10, one group serving as an untreated control to the others which received anthelmintic treatment. (3) On the 24th day after infection food was withheld from the birds in the afternoon. On the following morning the birds were sacrificed and the worms recovered by soaking the intestines in saline at 37°C. as described.

TABLE Number of birds

Number of ova inoculabd 200 1,000 2,000 5,000 10.000 25;OOO 50,000 100,000 The table in individual

1

Average number of uJ0rm.C

4

177 412.5 622 709.5 969.4 1,835 556.7 3,100

3: 2: 5 15 5

gives the average number worm burdens, according

Exberiment

Age of infection at treatment (days)

ACTIVITY

Dosage (mg./k;r.)

200

OF

METHYRIDINE

Method of ad&itration s/c

87.5 41.25 31.1 14.19 9.69 7.34 1.11 3.1

‘take’,

and the variation

2

AGAINST

No. birds per SW

y,& ‘take’

134-193 188523 220-1,325 510--1,131 150-2.325 350-31750 175--1,425 275-8,375

of worms recovered, the percentage to the number of ova inoculated.

TABLE THE

Range

C. OBSIB.VATd

Number

INFECTIONS

of worms recovered

-‘Treated Range

a

0

A

22

B

2 22

C

21 16 10

150 150 150

SIC s/c WC

a

8

8

0

D

10

150

s/c

10

E

2

133 333

Oral Oral

F

II 10

100 208 100

G

;i 21

E 100

AC.

00

Untreated Range

Au.

283

736

482.5

> 443

712

610

> 227

1025

0 8”

a 8

3;;

41: 753

21;:: 601.6

0 i

0 0.4 1.6

0

7

2.0

10 10

0 0

54 5

5.5 1.3

Oral Oral s/c

t 8

52 0 0

Oral Oral s/c

8” 8

14 t

_______ 388 1185

751.5 889 (9 birdsr

> 267

988

6278

814 449.1 :, 0.125 2.75

> 426

996

684.5

374 0 1

> 301

893

634.7 (6 birds)

84.1 0 0.125

C.

C.

NORTON

AND

L.

OF HALOXON

GIVEN

ORALLY

AGAINST

Age of

Experiment

infection at treatment (day4

c.

Number Dosage (mg./kg.)

birds Per group

0 9:

12 4;;

I

:o" 22

:: 60

t 8

1: 22

2;': 211

lo:.!5 132.1'

D

10

60

10

0

582

294.0

THE INACTIVITY OF COMPOUNDS AGAINST

Age of

J

A

infection at treatment (days)

Piperazine citrate

22

Phenothiazine (Phenovis)

22

22

320

756

538.25

182

708

501.25

388

1185

889 (9 birds)

>

A SERIES

OF CONVENTIONAL

C. obsignata

Number

NO.

Dosage

22

22

2;::5 263.4

AU.

4

TABLE DEMONSTRATING ANTHELMINTIC

Untreated Range

AU.

8 8"

Ex@rhent

INFECTIONS

of worms recowred

Treated Range

50 25 10

EXPERIMENTS

obsignatu

.NO.

22 :;

H

141

JOYNER

3

TABLE ACTIVITY

P.

biro% Pff .kmP

of worms recovered

Treated Range

Untreated Range Au.

Au.

200 400

8 8

90 212

768 1340

440.25 700.6

1 1 180

Coopex

400

8

62

417

203.5

J

Thiabendazole Bephenium hydroxynaph-

500

8

463

837

645

7

250

8

360

1254

661.5

283

1170

634.6

736

482.5

thoate

Ten experiments were carried out using the following 7 different anthehnintics : Methyridine [Promintic I.C.I.;2-(Pmethoxyethyl) pyridine] ; Haloxon [O,O di(2-chlorethyl) 0-(3-chloro-4-methylcoumarin-7-yl) phosphate] ; Piperazine citrate; Phenothiazine (‘Phenovis’ dispersable powder B.Vet. C) ; Coopex [a mixture of Phenothiazine and Coroxon (O,O-diethyl 0-3-chloro-4-methy-umbelliferyl phosphate)] ; Thiabendazole [2-(4’-thiazolyl)-benzimidazole] ; Bephenium hydroxynaphthoate. C

142

CHEMOTHERAPY

OF

CAPILLARIA

INFECTION

The red.~ for Methyridine, Haloxon and the remaining compounds are recorded in Tables 2, 3 and 4 respectively. Examination of these tables shows a fairly even recovery of worms from untreated birds. 81 were examined during the course of the experiments and the range of variation in individual worm burdens was 180 to 1,185 with an average infection of 609.4 worms. Piperazine citrate, Phenothiazine, Thiabendazole and Bephenium hydroxynaphthoate were shown to have no activity against mature C. obsignata (Table 4). When the results of experiment J (Table 4) were subjected to a “t” test the figures obtained for Coopex were shown to be probably significant (p
Although several workers have described the activity of compounds against experimental Capillaria obsignata infections in chicks, only brief details of the methods used and the properties of the infections have been published. The present studies have shown that this nematode may be maintained indefinitely in the laboratory by standard helminthological procedures with only minor modifications. Experiments can be carried out in a natural host, the chick, which may be reared worm-free without difficulty. C. obsignatu therefore appears to offer distinct advantages as an experimental tool for general helminthological studies. Control of coccidiosis is most important in the study of helminth infections in poultry because most methods for the separation of ova from faeces will also concentrate oocysts. To overcome this problem cultures of ova of C. obsignata to be used for infection of chickens, may be prepared from turkeys or pigeons (Levine, 1938) or by crushing washed adult worms (Wehr, 1939). The simplest method, however, is to use a coccidiostatic drug and for this purpose Zoalene, (3,5-dinitro no oocysts were seen in faecal extracts and o-toluamide), proved satisfactory;

C.

C.

NORTON

AND

L.

P.

JOYNER

143

there was no evidence of activity by the drug against the worm. The cultures of ova remained viable in 1 per cent. formalin for at least 1 year, but some loss of infectivity was noted and in order to obtain reproducible experiments after any appreciable period of storage, it would be advisable to renew the culture by passage through chicks. Our observations on the life-history of the parasite were in general agreement with those of previous authors (Levine, 1938; Wehr, 1939). Very heavy inoculations appear to be necessary to produce demonstrable lesions. Young chicks which received 100,000 ova and harboured up to 8,375 worms showed only occasional haemorrhagic spots in the intestine at post mortem examination. These heavy infections, however, produced a marked retardation of weight gains. In a survey carried out in 1959, Norton (1964) found that 10 per cent. of the birds infected with C. &sign&a carried more than 1,000 worms. Heavy worm burdens therefore occur in the field, but they appear to be difficult to set up in the laboratory. It is possible, however, that other factors, such as repeated infections, additional infections with other parasites or dietary deficiencies may have an effect on the number of worms which develop under practical conditions. A greater percentage of worms mature when the inoculum is reduced to 2,000 ova per bird, presumably because the inflammatory reaction in the mucosa resulting from the sudden introduction of larvae is relatively mild and fewer parasites are eliminated. Although the average worm burdens are not as great as those from birds which receive a high inoculum, the variation between individual birds is much less. Uniformity was judged to be a greater requirement for chemotherapeutic experiments than weight of infection and 2,000 ova was adopted as a standard inoculum. Initially, experiments were carried out to assess the activity of a series of known anthelmintics against adult C. obsignata. Where activity was demonstrated, further experiments were undertaken to study the effect upon immature worms. Under field conditions, methyridine [2-(P-methoxyethyl) pyridine] has proved effective against established infections when administered either subcutaneously or in the drinking water ( Thienpont, Bruynooghe and Mortelmans, 1963 ; Geeraerts, 1964a & b; Enneking, 1963). The most detailed laboratory data have been published by Broome (1963) who used C. obsignata ova supplied by us, and reported that doses of 100 mg. methyridine per kg. body weight injected subcutaneously completely eradicated 10 to 21-day-old infections. Against 3-day-old infections, however, the activity of the drug was slightly reduced. In the present series of experiments complete eradication of mature infections was achieved with subcutaneous doses of 150 mg. per kg.; 100 mg. per kg, was not quite so effective. Against immature infections 10 days old, subcutaneous doses of 150 mg. per kg. still proved effective, but the worms were not completely eradicated. There was evidence therefore that young worms were less responsive to treatment. This was particularly apparent when the drug was given orally, when 100 mg./kg. removed 86.75 per cent. of adult worms, but only 34.4 per cent. of lo-day-old larvae. From the present data therefore methyridine appeared to be not quite so effective as in the experiments of Broome (1963). This may be because our infections were heavier and the variability was somewhat less. Haloxon is an organophosphorus compound 0,O di-(2-chlorethyl) 0-(3-chloro-

144

CHEMOTHERAPY

OF

CAPILLARIA

INFECTION

4-methylcoumarin-7-yl) phosphate original1 described by Brown, Hollingshead, Kingsbury and Malone (1962). Clarke (1962) demonstrated its high efficacy against mature C. obsignata infections. Doses of 25 mg. to 50 mg. per kg. given orally removed 98.8 and 99.8 per cent. of worms respectively. The activity of these doses is largely confirmed by the present data which also demonstrate that the immature worms are not removed so efficiently. Broome (1962) suggested that methyridine acts by a paralysing effect upon the nematode muscles and it is likely that organophosphorus compound acts in the same way. Retention of the adult worm probably depends upon the muscular activity of the worm itself, which therefore would be easily eliminated in the presence of paralytic drugs. The immature stages, however, are in much more intimate contact with the tissue of the host and, thus, less likely to be dislodged by simple paralysis of the worm. The remaining compounds were largely inactive against the adult worms although there was some effect with Coopex which was probably due to the organophosphorus component. The observed inactivity of Thiabendazole against adult C. obsignata is in agreement with the results of Long and Wakelin (1964). These authors, however, demonstrated a marked activity by this drug against immature stages which suggests a different type of activity from any observed in the present study. For a complete evaluation of activity it is therefore necessary to test against both adult and larval infections.

CONCLUSIONS

Experimental infections with Cupilluriu obsignutu may be established in chickens by the oral inoculation of embryonated ova. Some effects of such infections on the host, the development of the worms and a technique for their recovery from infected birds are described. For chemotherapeutic experiments the most suitable inoculum was found to be 2,000 ova. Approximately 30 per cent. of the worms inoculated were recovered at maturity 24 days after infection. With larger doses, the proportion of worms recovered was diminished. The techniques for the recovery of worms and ova described permit the cyclical maintenance of the parasites in experimental birds. The most active compound was found to be methyridine. Doses of 100 to 150 mg./kg. injected subcutaneously were effective against adult, immature and larval worms. Haloxon in doses of 50 to 60 mg./kg. was fully effective against adult worms, but larvae and immature stages were less sensitive. Piperazine citrate, phenothiazine, thiabendazole, and bephenium were inactive against adult C. obsignuta. The activity of Coopex, a mixture of phenothiazine and coroxon, was probably due to the organophosphorus component.

ACKNOWLEDGMENT

Part of this work is included in a thesis by one of the authors (C.C.N.) which was accepted in fulfilment of the requirements for admission to Fellowship of the Institute of Medical Laboratory Technology. Acknowledgements are due to the Council of the Institute for permission to publish this part of the data.

4

E

2 -r

2

Fig. 2.

L a r v a e of C. ob.\&atcr e n t a n g l e d in mucus, \nidcd in faeces of experimentally infcctcd chickens I.1 days after inoculation. 37.

Fig. 3.

I-:tr\,ar of C. ob,\&c~tcr lying bet\vccn \-illi a n d superficially e m b e d d e d in m u c o s a of small intestine of chicks 1 2 davs after inoculation. Haernatoxylin a n d eosin. ,; 90.

Fig. 4.

Cloiled larva of L ’. ohipntu in the l a m i n a propria s u r r o u n d e d by eosinophilic ~11s. S m a l l intestine of chick 1 4 days after inoculation. Haematoxylin a n d rosin. :~ 3 5 0 .

C.

C.

NORTON

AND

L.

P.

JOYNER

145

REFERENCES

Broome, A. W. J. (1962). Drugs, Parasites and Hosts, Ed., L. G. Goodwin and R. H. Nimmo-Smith, Churchill; Lond.; (1963). Vet. Rec., 75, 1326. Brown, N. C., Hollingshead, D. T., Kingsbury, P. A., and Malone, J. C. (1962). Nature,

Lond.,

194, 379.

Clarke! M. L. (1962). Vet. Rec., 74, 1431. Ennekmg. (1963). Prakt. Tierarzt., 44, 4. Fraszier, M. N. (1962). Poult. Sci., 41, 854. Geeraerts, J. (1962). Vlaams dier Tijdschr., 31, 105; (1964a). Ibid., 33, 33; (1964b). Ibid., 103. Hendriks, J. (1962). Tijdschr. Diergeneesk, 87, 314. Levine, P. P. (1938). J. Parasit., 24, 45. Long, P. L., and Wakelin, D. (1964). Brit. PouEt. Sci., 5, 187. Norton, C. C. (1964). J. Helminth, 38, 269. Parfitt, J. W. (1958). Lab. Pratt., 7, 353. Taylor, E. L. (1934). 1. Helminth., 12, 143. Thienpont, D., and Mortelmans, J. (1962). Vet. Rec., 74, 850. Thienpont, D., Bruynooghe, D., and Mortelmans, J. (1963). Ibid., 75, 1019. Wakelin, D. (1964). J. Helminth., 38, 191. Wehr, E. E. (1939). Tech. Bull. U.S. Dep. Agric., 679. [Received

for

publication,

September

1lth, 19641