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Experimental Ostertagia spp. infection of sheep: Populations and lesions in previously worm-free and previously exposed animals
Veterinary Parasitology, 2 (1976) 167--175 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
167
E X P E R I M E N T A L O S T E R T A G I A SPP. I N F E C T I O N O F S H E E P : P O P U L A TIONS AND LESIONS IN PREVIOUSLY WORM-FREE AND PREVIOUSLY EXPOSED ANIMALS
D.C. ELLIOTT and P.J.K. DURHAM*
Wallaceville Animal Research Centre, Research Division, Ministry of Agriculture and Fisheries, Private Bag, Upper Hutt (New Zealand) *Animal Health Reference Laboratory, Animal Health Division, Ministry of Agriculture and Fisheries, Private Bag, Upper Hutt (New Zealand) (Received 31 May 1976)
ABSTRACT
Eliiott, D.C. and Durham, P.J.K., 1976. Experimental Ostertagia spp. infection of sheep: Populations and lesions in previously worm-free and previously exposed animals. Vet. Parasitol., 2: 167--175. Sheep raised and maintained under worm-free conditions were subjected to one heavy exposure and several low-level exposures of infective Ostertagia spp. larvae. The infections were terminated by treatment with thiabendazole. These sheep and not previously exposed controls were then challenged with 400 000 or 20 000 Ostertagia spp. larvae and killed after a further 8 days. In the previously exposed sheep, worm numbers, rate of worm development, and pathological effect, were greatly reduced. No significant differences were seen between sheep aged 6 and 18 months at the time of challenge. INTRODUCTION
T h e c o u r s e o f n o r m a l d e v e l o p m e n t o f Ostertagia spp. in s h e e p has b e e n described b y T h r e l k e l d {1934}, A r m o u r et al. {1966), D e n h a m ( 1 9 6 9 ) and D u r h a m a n d Elliott (1976}. Elliott ( 1 9 7 4 a , b) f o u n d t h a t n u m b e r s o f a d u l t Ostertagia spp. in experim e n t a l l y i n f e c t e d s h e e p increased with increasing d o s e t o a m a x i m u m level, b u t d e c l i n e d at still higher doses. D u r h a m a n d Elliott {1976) n o t e d t h a t in s h e e p d o s e d 512 0 0 0 i n f e c t i v e Ostertagia spp. larvae, n o r m a l d e v e l o p m e n t o f the m a j o r i t y o f t h e parasites c o n t i n u e d until d a y 10 p o s t i n f e c t i o n (PI), a f t e r w h i c h t i m e a s u b s t a n t i a l loss o f w o r m s c o m m e n c e d . Severe a b o m a s a l lesions d e v e l o p e d a f t e r d a y 4 PI a n d w e r e still p r e s e n t at d a y 24 PI, b u t h a d s u b s i d e d b y d a y 66 PI. T h e results suggested possible h o s t resistance or o t h e r f a c t o r s a f f e c t i n g p a r a s i t e levels. T h e d e v e l o p m e n t o f a c q u i r e d resistance in animals t o various h e l m i n t h parasites is n o w well recognised, and t h e p r e s e n t s t u d y was designed to investigate resistance to challenge i n f e c t i o n in s h e e p p r e v i o u s l y e x p o s e d to Ostertagia spp.
168 MATERIALS AND METHOD Twenty-nine sheep, raised under worm-free conditions from birth, were used for the experiment. At the time of challenge, ten of these sheep were 18 m o n t h s and the remaining 19 were 6 months of age. Fourteen sheep were each given " e x p o s u r e " doses of Ostertagia larvae (O. c i r c u m c i n c t a 94%, O. trifurcata 6%) according to the following plan: On 8 November 1972, 8 000 larvae; from 15 November to 14 December, five doses of 2 000 larvae each at approximately weekly intervals; on 21 December, 320 000 larvae; from 12 January to 20 February 1973, seven doses of 2 000 larvae each at approximately weekly intervals. All animals were administered thiabendazole at 100 mg/kg orally 8 days following the final exposure dose. Fourteen days after the final exposure, challenge doses of 400 000 or 20 000 Ostertagia spp. larvae were administered to 11 previously exposed and 12 not previously exposed sheep, the lower dose being given only to some 6 m o n t h old animals. The remaining six sheep were non-challenged controls for the histopathological studies. All sheep were killed 8 days after challenge. Necropsy was performed on each animal, and the abomasal and small intestinal contents were removed separately. The worms were recovered from the contents of the abomasum and small intestine by sedimentation and sieving (53 p aperture). Sections of fundus, pylorus and abomasal lymph nodes were stained with haematoxylin-eosin and by the periodic acid-Schiff method. Worms were recovered from the abomasal mucosa by peptic digestion (Herlich, 1956). Statistical analyses were performed on transformed figures, using log (1 + n/100) for actual counts, or angle = arc-sin x/(percentage/100) for percentages. RESULTS AND DISCUSSION Parasitology Worm length distributions. -- Length distributions of worms from the nonexposed sheep are shown in Fig.1. In general, all Ostertagia spp. exceeding
5 mm in length represent adults, while worms fitting the description "early fourth stage" do not exceed 2 mm. For purposes of this experiment it was convenient to divide populations of the parasites in individual sheep into worms 2 mm in length and over, and those less than 2 mm. It is apparent from Fig.1 that in the not previously exposed groups the worms in the sheep administered 20 000 larvae were proportionately longer than those in the animals given 400 000. W o r m counts. -- Worm-count results are shown in Table I. Numbers include worms from digests of the abomasal wall and lumen of the abomasum, and from the small intestine. In both age groups of not previously exposed sheep given 400 000 larvae,
169
18-MONTH SHEEPCHALLENGED400,000
60.000t
'000001 ....
NIn
6-MONTH SHEEPCHALLENGED400~000
4O000
I .l
0 Ib_MO,NTH SHEEP C'NALLENGED;0,000'
6.000 t
,ooo I1 "°°°t O,
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MILLIMETRES Fig.1. L e n g t h d i s t r i b u t i o n of Ostertagia f r o m t h e n o n - e x p o s e d sheep, b a s e d o n g r o u p means. T h i n bars, u n d i f f e r e n t i a t e d w o r m s ; solid bars, male w o r m s ; clear bars, f e m a l e worms. H o r i z o n t a l axis, w o r m lengths; vertical axis, n u m b e r s o f worms.
worm counts were similar, and ranged from 52 to 80% of the number of larvae administered. Total numbers of worms in these groups were more than in the not previously exposed group which received 20 000 larvae per animal (P < 0.01). Adult worm numbers expressed as a percentage of the larvae given were not significantly higher in the not previously exposed sheep given 20 000 than in those given 400 000 larvae. In the not previously exposed groups, percentages of worms less than 2 mm were lower in the sheep given 20 000 larvae than in those given 400 000 larvae (P < 0.05). Total numbers of worms showed a reduction in the previously exposed, as compared with the not previously exposed groups (P < 0.01). This reduction applied to both sizes of worms in sheep given 400 000 larvae, but only to worms 2 mm and over in length in the sheep given 20 000 larvae. In the abomasal digests of the not previously exposed animals given 400 000 larvae, the highest count was 7 000, and numbers in the remainder were much lower. The highest count from a digest of an exposed sheep was 400. In one not previously exposed sheep given 400 000 larvae, 70 000 Ostertagia were found in the small intestine, but in the remainder of this group, numbers from this location did not exceed 7 500. The length distribution of worms in the small intestine and in the abomasum were similar for the two locations. The highest count from a small intestine of an exposed sheep was 300.
170
TABLEI
Ostertagia spp. counts of previously exposed and not previously exposed sheep, killed at 8 days after receiving challenge infections of 400 000 or 20 000 Ostertagia spp. larvae
Sheep aged 18 months challenged 400 000 larvae
"Previously exposed" groups
" N o t previously exposed" groups
< 2mm*
> 2ram*
Total
< 2ram
> 2ram
Total
500 3 700 6 000 1 000
-3 600 200
500 3 700 9 600 1 200
18 63 63 71
190 260 185 200
208 323 249 272
1 000 0--0.9%
3 800 0.1--2.4%
54 400 4.6--18%
209 100 46--65%
263 500 52--81%
74 29 43 38
215 255 204 207
289 284 248 246
Mean** 2 800 Percentage o f d o s e t 0.]--1.5% Sheep aged 1 200 6 months 12 800 challenged 60 900 400 000 larvae 4 500 Mean 19 900 Percentage of dose 0.3--15%
200 -600 100
l ]2 61 4
400 800 500 600
200 20 100 0--0.15% 0.3--15%
600 400 800 600
400 600 900 800
46 700 7--19%
Sheep aged 6 months challenged 20 000 larvae
200 3 100 7 200
-100 800
200 3 200 8 000
400 500 1 400 500
Mean Percentage of dose
3 500 1--36%
300 0--4%
3 800 1--40%
700 2--7%
*Length of worms. **Arithmetic mean. j" Number of worms found .
.
.
.
.
.
.
.
.
Number of infective larvae dosed
000 100 500 700
100 000 700 600
220 600 51--64% 16 10 17 17
300 900 200 000
15 400 54--86%
000 500 300 300
500 600 600 400
267 300 61--72% ]6 11 18 17
700 400 600 500
16 100 57--93~
100 X
1
Pathology L i t t l e d i f f e r e n c e was seen b e t w e e n t h e t w o age groups, and t h e f o l l o w i n g descriptions apply to both.
Control sheep. -- T h e r e w a s l i t t l e d i f f e r e n c e , g r o s s l y o r h i s t o l o g i c a l l y , bet w e e n e x p o s e d and n o n - e x p o s e d sheep. Grossly, the a b o m a s a l m u c o s a was p a l e p i n k in c o l o u r , w i t h a s m o o t h - t e x t u r e d s u r f a c e . M i c r o s c o p i c a l l y t h e fundic m u c o s a was well d i f f e r e n t i a t e d . A mild l y m p h o c y t i c i n f i l t r a t i o n w a s p r e s e n t in b o t h t h e s u p e r f i c i a l a n d b a s a l m u c o s a . In t h e l a t t e r l o c a t i o n it t e n d e d t o f o r m a t e n u o u s l i n e a d j a c e n t t o t h e m u s c u l a r i s m u c o s a , a n d w a s s l i g h t l y a c c e n t u a t e d a r o u n d t h e b l o o d vessels. T h e s u b m u c o s a o f b o t h f u n d u s a n d p y l o r u s a p p e a r e d n o r m a l a n d n o a b n o r m a l i t i e s w e r e s e e n in t h e lymph nodes.
171 Previously exposed sheep challenged with 400 000 larvae. -- Scattered whitish umbilicated nodules 1 to 2 m m in diameter were present in the fundic mucosa. There was a slight increase in l y m p h o c y t e numbers in the basal and superficial lymphocytic layers of the fundus, as compared with the controls. This was most obvious near the margins of the folds, where the leucocyte infiltrate included a proportion of plasma cells and small numbers of eosinophils and neutrophils. Mild oedema was present in the superficial area of the lamina propria at the margins of the folds. The basal mucosa at the region of the pylorus also had a mild increase in l y m p h o c y t e numbers, together with moderate numbers of plasma cells and occasional eosinophils. Cell differentiation was normal throughout the mucosa (Fig.2a, b), except at the margins of the folds, where some lack of differentiation, similar to that described by Armour et al. (1966) was evident. The submucosa was mildly oedematous and contained small numbers of lymphocytes and eosinophils in the pyloric region only. The abomasal lymph nodes were slightly enlarged, and microscopically showed mild lymphoid and reticuloendothelial hyperplasia. N o t previously exposed sheep challenged with 400 000 larvae. -- The abomasa of the previously unexposed sheep given this number of larvae had severe mucosal congestion, moderate fold oedema and a roughened mucosal surface, with evidence of early sloughing (Fig.2c, d, e). These abomasa showed generally the same type of reaction as did the previously exposed group, but to a much more severe extent. There was a higher proportion of plasma cells in the inflammatory response, particularly in the pyloric region. The superficial fundic mucosa showed marked congestion and poor differentiation in m a n y areas, particularly near the margins of the folds, where sloughing was also seen. Considerable congestion was also seen in the mucosa of the pyloric area, where patchy sloughing of the epithelium was evident close to coiled parasites. The parasites were located in dilated glands with flattened epithelial lining, and there was no local inflammatory cell accumulation. The submucosa was moderately oedematous and infiltrated by lymphocytes, with small numbers of plasma cells and eosinophils, but this reaction was mainly confined to the margins of the folds. Occasional parasitic granulomata were found in the fundic mucosa, and there was local lack of differentiation of adjacent fundic glands. Abomasal lymph nodes appeared similar to those in the previously exposed group, except for a slight increase of eosinophils in the medullary regions. Previously exposed sheep challenged with 20 000 larvae. -- Grossly the abomasa and their lymph nodes appeared normal. Microscopically little abnormality was seen except for a slight increase in the basal lymphocytic layer at the margins of the folds.
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173
Fig.2. a: Previously exposed sheep challenged with 400 000 Ostertagia larvae. The abomasal fold appears virtually normal. H.& E. b: Previously exposed sheep challenged with 400 000 larvae. The mucosa shows normal differentiation, the dark-staining parietal cells being easily recognisable in the deeper mucosa. H. & E. c: Not previously exposed sheep challenged with 400 000 larvae. The fundic mucosa is poorly differentiated, is infiltrated by inflammatory cells, and overlaid with inflammatory exudate. H. & E. d: Not previously exposed sheep challenged with 400 000 larvae. The submucosa of the abomasal fold is heavily infiltrated by inflammatory exudate. Inflammation and poor differentiation is also evident in the mucosa. H. & E. e: Not previously exposed sheep challenged with 400 000 larvae. The submucosa is poorly differentiated, and is infiltrated by inflammatory cells. H. & E.
N o t previously exposed sheep challenged with 20 000 larvae. - - T h e o n l y gross changes seen were occasional whitish u m b i l i c a t e d n o d u l e s in the a b o m a s a . M i c r o s c o p i c a l l y t h e f u n d u s h a d a m o d e r a t e increase in t h e n u m b e r o f cells in t h e basal and superficial l y m p h o c y t e layers, w h i c h was m o s t evident near the margins o f t h e folds. T h e p y l o r u s s h o w e d a mild increase in l y m p h o i d cells. The l y m p h n o d e s s h o w e d n o o b v i o u s change, either grossly o r m i c r o s c o p i cally. DISCUSSION T h e reason f o r the design a d o p t e d in t h e p r e s e n t e x p e r i m e n t was t h a t the results o f a previous s t u d y ( D u r h a m and Elliott, 1 9 7 6 ) i n d i c a t e d (1) t h a t in
174 the abomasa of the previously exposed sheep, any lesions resulting from the dose of 320 000 larvae would have healed by the time chosen to administer the challenge; (2) that 2 000 larvae/week would produce little reaction but might maintain antigenic stimulation of the host; (3) that at 8 days following challenge of the sheep not previously exposed, gross lesions would be well in evidence but substantial elimination of worms would not have commenced. In the present study, certain well-marked differences were evident between the previously exposed and the previously non-exposed groups at eight days following challenge. The previously exposed sheep had greatly reduced total numbers of worms, comparatively few of which had developed beyond 2 mm in length. The fact that similar results were obtained with both age groups challenged with 400 000 larvae indicates that no substantial age effect was in evidence. In the not previously exposed sheep, the length distribution patterns of the worm populations {see Fig.l) indicate a continuum of developmental stages, from the early-fourth to the early-adult stage. This implies that the early-fourth-stage worms may not necessarily have been arrested in development. The more-advanced state of development of worms in the low dose-rate, as compared with the high dose-rate groups, probably represents no more than a slight retardation at the higher rate, due to crowding. The worms found in the small intestine were probably undergoing expulsion although it seems that in high infections some O~stertagia may overflow to the d u o d e n u m (Durham and Elliott, 1976). The type of pathological change seen in the abomasa was similar in all challenged groups, and similar to that reported previously in sheep examined at comparable time intervals following infection (Sommerville, 1954; Armour et al., 1966; Durham and Elliott, 1976). However, the severity of the effect was greatly reduced in the animals previously exposed to Ostertagia and coincided with the lowered rate of establishment or early removal of worms. The explanation of this failure of establishment or early loss could possibly be due to immune response through cell-mediated immunity (Kelly, 1973). However, this seems an unlikely explanation for the reduced rate of establishment in the present experiment, since the cellular response usually evident at 8 days after challenge was comparatively reduced in the previously exposed sheep. A more likely explanation is the role of humoral antibody. Although IgA antibody is c o m m o n l y found in many mucous secretions, available evidence suggests that helminths do not markedly stimulate increased IgA production {Jones et al., 1970). The more likely immunoglobulin would be of the reaginic type, equivalent to the human IgE. This antibody has been shown to be important in many helminth infections {Sinclair, 1970), including Ostertagia (Hogarth-Scott, 1969}. It has been suggested that the antibody fraction involved in sheep is an IgG1A subclass (Hogarth-Scott, 1969; Curtain and Anderson, 1971, 1972}, increased secretion of which had been demonstrated in plasma cells following Ostertagia infection. This may form the basis of an immediate hypersensitive response, mediated by the products of the induced
175
m a s t c e l l d e g r a n u l a t i o n ( M u r r a y , 1 9 7 2 } , as a c o n s e q u e n c e o f w h i c h t h e p a r a s i t e s d o n o t e n t e r o r e s t a b l i s h s u c c e s s f u l l y in t h e a b o m a s a l m u c o s a . ACKNOWLEDGEMENTS The authors thank Dr K.J.A. Revfeim of the Biometrics Section, New Zealand Ministry of Agriculture and Fisheries, for the statistical analyses and Mr A.W. Barkus and Mr J.A. Bellamy, Wallaceville Animal Research Centre, New Zealand Ministry of Agriculture and Fisheries, for the figures and photographs. REFERENCES Armour, J., Jarrett, W.F.H. and Jennings, F.W., 1966. Experimental Ostertagia circumcincta infections in sheep: development and pathogenesis of a single infection. Am. J. Vet. Res., 27: 1267--1278. Curtain, C.C. and Anderson, N., 1971. Immunocytochemical localization of the ovine immunoglobulins IgA, IgG1, igG1A and IgG2: effect of gastrointestinal parasitism in the sheep. Clin. Exp. Immunol., 8: 151--162. Curtain, C.C. and Anderson, N., 1972. Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep. Int. J. Parasitol., 2: 449--457. Denham, D.A., 1969. The development of Ostertagia circumcincta in lambs. J. Helminthol., 43: 299--310. Durham, P.J.K. and Elliott, D.C., 1976. Experimental Ostertagia spp. infection of sheep: Development of worm populations and lesions resulting from different dose-levels of larvae. Vet. Parasitol., 2: 157--166. Elliott, D.C., 1974a. Experimental Ostertagia infection of sheep: worm populations resulting from single larval doses. N . Z . J . Exp. Agric., 2: 103--107. Elliott, D.C., 1974b. Experimental Ostertagia infection of sheep: worm populations resulting from single and multiple larval doses. N.Z.J. Exp. Agric., 2 : 1 0 9 - - 1 1 3 . Herlich, H., 1956. A digestion method for post-mortem recovery of nematodes from ruminants. Proc. Helminthol. Soc. Wash., 23: 102--103. Hogarth-Scott, R.S., 1969. Homocytotropic antibody in sheep. Immunology, 16: 543--548. Jones, V.E., Edwards, A.J. and Ogilvie, B.M., 1970. The circulating immunoglobulins involved in protective immunity to the intestinal stage of Nippostrongylus brasiliensis in the r a t Immunology, 18: 621--633. Kelly, J.D., 1973. Mechanisms of immunity to intestinal helminths. Aust. Vet. J., 49: 91--97. Murray, M., 1972. Immediate hypersensitivity effector mechanisms. II. In vivo reactions. In: E.J.L. Soulsby (Editor), Immunity to Animal Parasites. Academic Press, New York, N.Y. and London, pp. 155--190. Sinclair, I.J., 1970. The relationship between circulating antibodies and immunity to helminthic infections. Adv. Parasitol., 8: 97--138. Sommerville, R.I., 1954. The histotropic phase of the nematode parasite, Ostertagia circumcincta. Aust. J. Agric. Res., 5: 130--140. Threlkeld, W.L., 1934. The life history of Ostertagia circumcincta. Va. Agric. Exp. Stn Tech. Bull., 52, 24 pp.
167
E X P E R I M E N T A L O S T E R T A G I A SPP. I N F E C T I O N O F S H E E P : P O P U L A TIONS AND LESIONS IN PREVIOUSLY WORM-FREE AND PREVIOUSLY EXPOSED ANIMALS
D.C. ELLIOTT and P.J.K. DURHAM*
Wallaceville Animal Research Centre, Research Division, Ministry of Agriculture and Fisheries, Private Bag, Upper Hutt (New Zealand) *Animal Health Reference Laboratory, Animal Health Division, Ministry of Agriculture and Fisheries, Private Bag, Upper Hutt (New Zealand) (Received 31 May 1976)
ABSTRACT
Eliiott, D.C. and Durham, P.J.K., 1976. Experimental Ostertagia spp. infection of sheep: Populations and lesions in previously worm-free and previously exposed animals. Vet. Parasitol., 2: 167--175. Sheep raised and maintained under worm-free conditions were subjected to one heavy exposure and several low-level exposures of infective Ostertagia spp. larvae. The infections were terminated by treatment with thiabendazole. These sheep and not previously exposed controls were then challenged with 400 000 or 20 000 Ostertagia spp. larvae and killed after a further 8 days. In the previously exposed sheep, worm numbers, rate of worm development, and pathological effect, were greatly reduced. No significant differences were seen between sheep aged 6 and 18 months at the time of challenge. INTRODUCTION
T h e c o u r s e o f n o r m a l d e v e l o p m e n t o f Ostertagia spp. in s h e e p has b e e n described b y T h r e l k e l d {1934}, A r m o u r et al. {1966), D e n h a m ( 1 9 6 9 ) and D u r h a m a n d Elliott (1976}. Elliott ( 1 9 7 4 a , b) f o u n d t h a t n u m b e r s o f a d u l t Ostertagia spp. in experim e n t a l l y i n f e c t e d s h e e p increased with increasing d o s e t o a m a x i m u m level, b u t d e c l i n e d at still higher doses. D u r h a m a n d Elliott {1976) n o t e d t h a t in s h e e p d o s e d 512 0 0 0 i n f e c t i v e Ostertagia spp. larvae, n o r m a l d e v e l o p m e n t o f the m a j o r i t y o f t h e parasites c o n t i n u e d until d a y 10 p o s t i n f e c t i o n (PI), a f t e r w h i c h t i m e a s u b s t a n t i a l loss o f w o r m s c o m m e n c e d . Severe a b o m a s a l lesions d e v e l o p e d a f t e r d a y 4 PI a n d w e r e still p r e s e n t at d a y 24 PI, b u t h a d s u b s i d e d b y d a y 66 PI. T h e results suggested possible h o s t resistance or o t h e r f a c t o r s a f f e c t i n g p a r a s i t e levels. T h e d e v e l o p m e n t o f a c q u i r e d resistance in animals t o various h e l m i n t h parasites is n o w well recognised, and t h e p r e s e n t s t u d y was designed to investigate resistance to challenge i n f e c t i o n in s h e e p p r e v i o u s l y e x p o s e d to Ostertagia spp.
168 MATERIALS AND METHOD Twenty-nine sheep, raised under worm-free conditions from birth, were used for the experiment. At the time of challenge, ten of these sheep were 18 m o n t h s and the remaining 19 were 6 months of age. Fourteen sheep were each given " e x p o s u r e " doses of Ostertagia larvae (O. c i r c u m c i n c t a 94%, O. trifurcata 6%) according to the following plan: On 8 November 1972, 8 000 larvae; from 15 November to 14 December, five doses of 2 000 larvae each at approximately weekly intervals; on 21 December, 320 000 larvae; from 12 January to 20 February 1973, seven doses of 2 000 larvae each at approximately weekly intervals. All animals were administered thiabendazole at 100 mg/kg orally 8 days following the final exposure dose. Fourteen days after the final exposure, challenge doses of 400 000 or 20 000 Ostertagia spp. larvae were administered to 11 previously exposed and 12 not previously exposed sheep, the lower dose being given only to some 6 m o n t h old animals. The remaining six sheep were non-challenged controls for the histopathological studies. All sheep were killed 8 days after challenge. Necropsy was performed on each animal, and the abomasal and small intestinal contents were removed separately. The worms were recovered from the contents of the abomasum and small intestine by sedimentation and sieving (53 p aperture). Sections of fundus, pylorus and abomasal lymph nodes were stained with haematoxylin-eosin and by the periodic acid-Schiff method. Worms were recovered from the abomasal mucosa by peptic digestion (Herlich, 1956). Statistical analyses were performed on transformed figures, using log (1 + n/100) for actual counts, or angle = arc-sin x/(percentage/100) for percentages. RESULTS AND DISCUSSION Parasitology Worm length distributions. -- Length distributions of worms from the nonexposed sheep are shown in Fig.1. In general, all Ostertagia spp. exceeding
5 mm in length represent adults, while worms fitting the description "early fourth stage" do not exceed 2 mm. For purposes of this experiment it was convenient to divide populations of the parasites in individual sheep into worms 2 mm in length and over, and those less than 2 mm. It is apparent from Fig.1 that in the not previously exposed groups the worms in the sheep administered 20 000 larvae were proportionately longer than those in the animals given 400 000. W o r m counts. -- Worm-count results are shown in Table I. Numbers include worms from digests of the abomasal wall and lumen of the abomasum, and from the small intestine. In both age groups of not previously exposed sheep given 400 000 larvae,
169
18-MONTH SHEEPCHALLENGED400,000
60.000t
'000001 ....
NIn
6-MONTH SHEEPCHALLENGED400~000
4O000
I .l
0 Ib_MO,NTH SHEEP C'NALLENGED;0,000'
6.000 t
,ooo I1 "°°°t O,
, i
I~,
n,~
,
H ,
MILLIMETRES Fig.1. L e n g t h d i s t r i b u t i o n of Ostertagia f r o m t h e n o n - e x p o s e d sheep, b a s e d o n g r o u p means. T h i n bars, u n d i f f e r e n t i a t e d w o r m s ; solid bars, male w o r m s ; clear bars, f e m a l e worms. H o r i z o n t a l axis, w o r m lengths; vertical axis, n u m b e r s o f worms.
worm counts were similar, and ranged from 52 to 80% of the number of larvae administered. Total numbers of worms in these groups were more than in the not previously exposed group which received 20 000 larvae per animal (P < 0.01). Adult worm numbers expressed as a percentage of the larvae given were not significantly higher in the not previously exposed sheep given 20 000 than in those given 400 000 larvae. In the not previously exposed groups, percentages of worms less than 2 mm were lower in the sheep given 20 000 larvae than in those given 400 000 larvae (P < 0.05). Total numbers of worms showed a reduction in the previously exposed, as compared with the not previously exposed groups (P < 0.01). This reduction applied to both sizes of worms in sheep given 400 000 larvae, but only to worms 2 mm and over in length in the sheep given 20 000 larvae. In the abomasal digests of the not previously exposed animals given 400 000 larvae, the highest count was 7 000, and numbers in the remainder were much lower. The highest count from a digest of an exposed sheep was 400. In one not previously exposed sheep given 400 000 larvae, 70 000 Ostertagia were found in the small intestine, but in the remainder of this group, numbers from this location did not exceed 7 500. The length distribution of worms in the small intestine and in the abomasum were similar for the two locations. The highest count from a small intestine of an exposed sheep was 300.
170
TABLEI
Ostertagia spp. counts of previously exposed and not previously exposed sheep, killed at 8 days after receiving challenge infections of 400 000 or 20 000 Ostertagia spp. larvae
Sheep aged 18 months challenged 400 000 larvae
"Previously exposed" groups
" N o t previously exposed" groups
< 2mm*
> 2ram*
Total
< 2ram
> 2ram
Total
500 3 700 6 000 1 000
-3 600 200
500 3 700 9 600 1 200
18 63 63 71
190 260 185 200
208 323 249 272
1 000 0--0.9%
3 800 0.1--2.4%
54 400 4.6--18%
209 100 46--65%
263 500 52--81%
74 29 43 38
215 255 204 207
289 284 248 246
Mean** 2 800 Percentage o f d o s e t 0.]--1.5% Sheep aged 1 200 6 months 12 800 challenged 60 900 400 000 larvae 4 500 Mean 19 900 Percentage of dose 0.3--15%
200 -600 100
l ]2 61 4
400 800 500 600
200 20 100 0--0.15% 0.3--15%
600 400 800 600
400 600 900 800
46 700 7--19%
Sheep aged 6 months challenged 20 000 larvae
200 3 100 7 200
-100 800
200 3 200 8 000
400 500 1 400 500
Mean Percentage of dose
3 500 1--36%
300 0--4%
3 800 1--40%
700 2--7%
*Length of worms. **Arithmetic mean. j" Number of worms found .
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Number of infective larvae dosed
000 100 500 700
100 000 700 600
220 600 51--64% 16 10 17 17
300 900 200 000
15 400 54--86%
000 500 300 300
500 600 600 400
267 300 61--72% ]6 11 18 17
700 400 600 500
16 100 57--93~
100 X
1
Pathology L i t t l e d i f f e r e n c e was seen b e t w e e n t h e t w o age groups, and t h e f o l l o w i n g descriptions apply to both.
Control sheep. -- T h e r e w a s l i t t l e d i f f e r e n c e , g r o s s l y o r h i s t o l o g i c a l l y , bet w e e n e x p o s e d and n o n - e x p o s e d sheep. Grossly, the a b o m a s a l m u c o s a was p a l e p i n k in c o l o u r , w i t h a s m o o t h - t e x t u r e d s u r f a c e . M i c r o s c o p i c a l l y t h e fundic m u c o s a was well d i f f e r e n t i a t e d . A mild l y m p h o c y t i c i n f i l t r a t i o n w a s p r e s e n t in b o t h t h e s u p e r f i c i a l a n d b a s a l m u c o s a . In t h e l a t t e r l o c a t i o n it t e n d e d t o f o r m a t e n u o u s l i n e a d j a c e n t t o t h e m u s c u l a r i s m u c o s a , a n d w a s s l i g h t l y a c c e n t u a t e d a r o u n d t h e b l o o d vessels. T h e s u b m u c o s a o f b o t h f u n d u s a n d p y l o r u s a p p e a r e d n o r m a l a n d n o a b n o r m a l i t i e s w e r e s e e n in t h e lymph nodes.
171 Previously exposed sheep challenged with 400 000 larvae. -- Scattered whitish umbilicated nodules 1 to 2 m m in diameter were present in the fundic mucosa. There was a slight increase in l y m p h o c y t e numbers in the basal and superficial lymphocytic layers of the fundus, as compared with the controls. This was most obvious near the margins of the folds, where the leucocyte infiltrate included a proportion of plasma cells and small numbers of eosinophils and neutrophils. Mild oedema was present in the superficial area of the lamina propria at the margins of the folds. The basal mucosa at the region of the pylorus also had a mild increase in l y m p h o c y t e numbers, together with moderate numbers of plasma cells and occasional eosinophils. Cell differentiation was normal throughout the mucosa (Fig.2a, b), except at the margins of the folds, where some lack of differentiation, similar to that described by Armour et al. (1966) was evident. The submucosa was mildly oedematous and contained small numbers of lymphocytes and eosinophils in the pyloric region only. The abomasal lymph nodes were slightly enlarged, and microscopically showed mild lymphoid and reticuloendothelial hyperplasia. N o t previously exposed sheep challenged with 400 000 larvae. -- The abomasa of the previously unexposed sheep given this number of larvae had severe mucosal congestion, moderate fold oedema and a roughened mucosal surface, with evidence of early sloughing (Fig.2c, d, e). These abomasa showed generally the same type of reaction as did the previously exposed group, but to a much more severe extent. There was a higher proportion of plasma cells in the inflammatory response, particularly in the pyloric region. The superficial fundic mucosa showed marked congestion and poor differentiation in m a n y areas, particularly near the margins of the folds, where sloughing was also seen. Considerable congestion was also seen in the mucosa of the pyloric area, where patchy sloughing of the epithelium was evident close to coiled parasites. The parasites were located in dilated glands with flattened epithelial lining, and there was no local inflammatory cell accumulation. The submucosa was moderately oedematous and infiltrated by lymphocytes, with small numbers of plasma cells and eosinophils, but this reaction was mainly confined to the margins of the folds. Occasional parasitic granulomata were found in the fundic mucosa, and there was local lack of differentiation of adjacent fundic glands. Abomasal lymph nodes appeared similar to those in the previously exposed group, except for a slight increase of eosinophils in the medullary regions. Previously exposed sheep challenged with 20 000 larvae. -- Grossly the abomasa and their lymph nodes appeared normal. Microscopically little abnormality was seen except for a slight increase in the basal lymphocytic layer at the margins of the folds.
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173
Fig.2. a: Previously exposed sheep challenged with 400 000 Ostertagia larvae. The abomasal fold appears virtually normal. H.& E. b: Previously exposed sheep challenged with 400 000 larvae. The mucosa shows normal differentiation, the dark-staining parietal cells being easily recognisable in the deeper mucosa. H. & E. c: Not previously exposed sheep challenged with 400 000 larvae. The fundic mucosa is poorly differentiated, is infiltrated by inflammatory cells, and overlaid with inflammatory exudate. H. & E. d: Not previously exposed sheep challenged with 400 000 larvae. The submucosa of the abomasal fold is heavily infiltrated by inflammatory exudate. Inflammation and poor differentiation is also evident in the mucosa. H. & E. e: Not previously exposed sheep challenged with 400 000 larvae. The submucosa is poorly differentiated, and is infiltrated by inflammatory cells. H. & E.
N o t previously exposed sheep challenged with 20 000 larvae. - - T h e o n l y gross changes seen were occasional whitish u m b i l i c a t e d n o d u l e s in the a b o m a s a . M i c r o s c o p i c a l l y t h e f u n d u s h a d a m o d e r a t e increase in t h e n u m b e r o f cells in t h e basal and superficial l y m p h o c y t e layers, w h i c h was m o s t evident near the margins o f t h e folds. T h e p y l o r u s s h o w e d a mild increase in l y m p h o i d cells. The l y m p h n o d e s s h o w e d n o o b v i o u s change, either grossly o r m i c r o s c o p i cally. DISCUSSION T h e reason f o r the design a d o p t e d in t h e p r e s e n t e x p e r i m e n t was t h a t the results o f a previous s t u d y ( D u r h a m and Elliott, 1 9 7 6 ) i n d i c a t e d (1) t h a t in
174 the abomasa of the previously exposed sheep, any lesions resulting from the dose of 320 000 larvae would have healed by the time chosen to administer the challenge; (2) that 2 000 larvae/week would produce little reaction but might maintain antigenic stimulation of the host; (3) that at 8 days following challenge of the sheep not previously exposed, gross lesions would be well in evidence but substantial elimination of worms would not have commenced. In the present study, certain well-marked differences were evident between the previously exposed and the previously non-exposed groups at eight days following challenge. The previously exposed sheep had greatly reduced total numbers of worms, comparatively few of which had developed beyond 2 mm in length. The fact that similar results were obtained with both age groups challenged with 400 000 larvae indicates that no substantial age effect was in evidence. In the not previously exposed sheep, the length distribution patterns of the worm populations {see Fig.l) indicate a continuum of developmental stages, from the early-fourth to the early-adult stage. This implies that the early-fourth-stage worms may not necessarily have been arrested in development. The more-advanced state of development of worms in the low dose-rate, as compared with the high dose-rate groups, probably represents no more than a slight retardation at the higher rate, due to crowding. The worms found in the small intestine were probably undergoing expulsion although it seems that in high infections some O~stertagia may overflow to the d u o d e n u m (Durham and Elliott, 1976). The type of pathological change seen in the abomasa was similar in all challenged groups, and similar to that reported previously in sheep examined at comparable time intervals following infection (Sommerville, 1954; Armour et al., 1966; Durham and Elliott, 1976). However, the severity of the effect was greatly reduced in the animals previously exposed to Ostertagia and coincided with the lowered rate of establishment or early removal of worms. The explanation of this failure of establishment or early loss could possibly be due to immune response through cell-mediated immunity (Kelly, 1973). However, this seems an unlikely explanation for the reduced rate of establishment in the present experiment, since the cellular response usually evident at 8 days after challenge was comparatively reduced in the previously exposed sheep. A more likely explanation is the role of humoral antibody. Although IgA antibody is c o m m o n l y found in many mucous secretions, available evidence suggests that helminths do not markedly stimulate increased IgA production {Jones et al., 1970). The more likely immunoglobulin would be of the reaginic type, equivalent to the human IgE. This antibody has been shown to be important in many helminth infections {Sinclair, 1970), including Ostertagia (Hogarth-Scott, 1969}. It has been suggested that the antibody fraction involved in sheep is an IgG1A subclass (Hogarth-Scott, 1969; Curtain and Anderson, 1971, 1972}, increased secretion of which had been demonstrated in plasma cells following Ostertagia infection. This may form the basis of an immediate hypersensitive response, mediated by the products of the induced
175
m a s t c e l l d e g r a n u l a t i o n ( M u r r a y , 1 9 7 2 } , as a c o n s e q u e n c e o f w h i c h t h e p a r a s i t e s d o n o t e n t e r o r e s t a b l i s h s u c c e s s f u l l y in t h e a b o m a s a l m u c o s a . ACKNOWLEDGEMENTS The authors thank Dr K.J.A. Revfeim of the Biometrics Section, New Zealand Ministry of Agriculture and Fisheries, for the statistical analyses and Mr A.W. Barkus and Mr J.A. Bellamy, Wallaceville Animal Research Centre, New Zealand Ministry of Agriculture and Fisheries, for the figures and photographs. REFERENCES Armour, J., Jarrett, W.F.H. and Jennings, F.W., 1966. Experimental Ostertagia circumcincta infections in sheep: development and pathogenesis of a single infection. Am. J. Vet. Res., 27: 1267--1278. Curtain, C.C. and Anderson, N., 1971. Immunocytochemical localization of the ovine immunoglobulins IgA, IgG1, igG1A and IgG2: effect of gastrointestinal parasitism in the sheep. Clin. Exp. Immunol., 8: 151--162. Curtain, C.C. and Anderson, N., 1972. Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep. Int. J. Parasitol., 2: 449--457. Denham, D.A., 1969. The development of Ostertagia circumcincta in lambs. J. Helminthol., 43: 299--310. Durham, P.J.K. and Elliott, D.C., 1976. Experimental Ostertagia spp. infection of sheep: Development of worm populations and lesions resulting from different dose-levels of larvae. Vet. Parasitol., 2: 157--166. Elliott, D.C., 1974a. Experimental Ostertagia infection of sheep: worm populations resulting from single larval doses. N . Z . J . Exp. Agric., 2: 103--107. Elliott, D.C., 1974b. Experimental Ostertagia infection of sheep: worm populations resulting from single and multiple larval doses. N.Z.J. Exp. Agric., 2 : 1 0 9 - - 1 1 3 . Herlich, H., 1956. A digestion method for post-mortem recovery of nematodes from ruminants. Proc. Helminthol. Soc. Wash., 23: 102--103. Hogarth-Scott, R.S., 1969. Homocytotropic antibody in sheep. Immunology, 16: 543--548. Jones, V.E., Edwards, A.J. and Ogilvie, B.M., 1970. The circulating immunoglobulins involved in protective immunity to the intestinal stage of Nippostrongylus brasiliensis in the r a t Immunology, 18: 621--633. Kelly, J.D., 1973. Mechanisms of immunity to intestinal helminths. Aust. Vet. J., 49: 91--97. Murray, M., 1972. Immediate hypersensitivity effector mechanisms. II. In vivo reactions. In: E.J.L. Soulsby (Editor), Immunity to Animal Parasites. Academic Press, New York, N.Y. and London, pp. 155--190. Sinclair, I.J., 1970. The relationship between circulating antibodies and immunity to helminthic infections. Adv. Parasitol., 8: 97--138. Sommerville, R.I., 1954. The histotropic phase of the nematode parasite, Ostertagia circumcincta. Aust. J. Agric. Res., 5: 130--140. Threlkeld, W.L., 1934. The life history of Ostertagia circumcincta. Va. Agric. Exp. Stn Tech. Bull., 52, 24 pp.