Ovine coccidiosis: Pathology of Eimeria ovinoidalis infection

OVINE COCCIDIOSIS: PATHOLOGY OF EIMERIA 0 V7NOZDALZS INFECTION M. W. GREGORY and JANET CATCHPOLE Central

Veterinary

Laboratory,

New Haw, Weybridge,

Surrey KT15 3NB, UK

(Received 28 May 1986) Abstract-GmzoRu M. W and CATCHPOLE J. 1987. Ovine coccidiosis: pathology of Eimeria ovinoidulis infection. International Journalfor Parasitology 17: 1099-l 111. Doses of sporulated oocysts of Eimeria ovinoidalis ranging from 10’ to 5 X 1 Oh were given to 25 housed lambs aged between 5 and 13 weeks, most of which had been reared coccidia-free. Some had received an “immunizing” dose 3-4 weeks earlier. Lambs were killed between 8 and 21 days after inoculation (DAI) and the tissues were examined histologically. Doses higher than 1 Oh caused extensive loss of epithelial cells in the lower jejunum both from the surface and from the crypts at 10 DA1 when first-generation meronts were mature. Doses of 10’ oocysts or more caused diarrhoea from about 13 DA1 in both first and second infections; this was associated with massive invasion of the caecal epithelium by second-generation meronts and gamonts. Destruction of crypt stem cells by these stages led to denudation of the caecal mucosa, resulting in haemorrhagic enteritis, dehydration and delayed healing or death. INDEX KEY WORDS: Coccidiosis; sheep; lambs; diarrhoea; Eimeria ovinoidalis; pathology; Peyer’s patch.

INTRODUCTION Eimeria ninakohlyukimovae the goat, but was assumed McDougald (1979) showed

was first to affect

described in sheep as well.

that the species affecting sheep was distinct from that affecting goats, so he renamed the sheep species E. ovinoidulis. This organism can be highly pathogenic in laboratory lambs (Catchpole, Norton & Joyner, 1976) and in feedlots (Fitzgerald & Mansfield, 1978). In Britain it has shown an association with field cases of coccidiosis (Gregory, Joyner, Catchpole & Norton, 1980). The endogenous stages have been described in detail by Wacha, Hammond & Miner (1971). Sporozoites emerging from the oocysts give rise to a first generation of meronts that reach about 300 iurn in diameter 10 days after inoculation (DAI). They are referred to here as giant meronts, and occur mainly in the lamina propria of the lower jejunum. Merozoites from this stage move on to the large intestine where they produce small second-generation meronts. The progeny of this stage give rise to micro and macrogamonts that in turn give rise to oocysts, which first appear in the faeces at about 12 DAI. Observations on pathogenicity and gross pathology have been published (Lotze, 1954; Svanbaev, 1967; Catchpole et al., 1976; Fitzgerald & Mansfield, 1978). Wacha et al. (197 1) also recorded minor observations on hostcell reactions within and around the giant meronts. Apparently no work has been published on the mechanism of pathogenesis. As a contribution to this 1099

dysentery;

enteritis;

intestine;

enteropathy;

subject, we present observations on the histopathological changes that followed the oral administration of various numbers of sporulated oocysts to coccidiafree lambs and conventionally reared lambs. MATERIALS

AND METHODS

Animals and infecting organisms. Coccidia-free lambs were obtained by removing Dorset-horn lambs from the dam at birth and rearing them artificially in wire-floored cages as described by Catchpole et al. (1976). Oocyst output was monitored three times weekly from 10 days of age until 10 days after inoculation; thereafter total collections of faeces were examined daily. The conventional lambs were suckled normally and reared under worm-free conditions indoors. Pure isolates of oocysts were originally obtained by picking out small numbers from field samples. These were cultured by inoculating orally into coccidia-free lambs, using a syringe and ball-ended cammla. Oocysts were harvested over the period of maximum output by sieving of faeces, followed by washing and floating in saturated salt (NaCl). After further washing they were suspended in 2% potassium dichromate, allowed to sporulate at 27’C and then stored at 4’C. A haemocytometer was used to assess the number of oocysts per ml of suspension. Percentage sporulation was assessed by counting 100 oocysts at 1000 X magnification. Long-term storage was achieved by freezing of sporocysts in liquid nitrogen (Norton & Joyner, 1968). Experimental designs. Histological material was used from several experiments, most of which were not designed with histopathology in mind. Details of inocula and days of autopsy are given in Table 1. The two animals H 13 & 14 were from experiments involving E. weybridgensis which

M. W. GREGORY and J. CATCHPOLE

1100

TAELE ~-PARTICULARS OF ANIMALS AND INOCULA USED IN THE VARIOUSEXPERIMENTS

Oocysts

of

E. ovinoidalis Lamb No.

Expt. No.

Age (weeks)

A

7

14

B

7 6

9 9

C

808 826

Status at start

inoculated 2nd

1st

1st inoc

10h

8

10” 104

10

4.4 x 100 5x 106

10 10

5x 5x

13 6

Autopsy: days after

5

7

a

10”

10

E

1 2

9 IO

a a

-2 x IO’ -2 x 102

17 20

F

1 2 3 5 6 7

10 11

1 6 11

9

G

H

Key:

11 10 15

12 1s I6 17 18 19

6 7 10 10 10 8 6

13 14

10 7

c (day 26)

14 21 13

10

40 14 10 12 12 11 14 11 -

12

10‘

31 1s

11

IO’ 101 10’

-10’ 101 1 OJ 104

105

10’ 10.’ 10’ 10

w (day 68)

39 46 38 21 42 18

IO’ 10’ 10’ 10’ 10’ -101

IO’ IO

a a

Adventious infection

12

D

10

2nd inoc

Previous inoculations (and day of autopsy)

f (day 42)

c (slight) c (slight) o+c

17

w(day31) w (day 15)

a = coccidia-free b = conventionally-reared o = E. bakuensis (syn. E. ovina)

c = E. crandallis f = E. faurei w = E. weybridgensis had to be abandoned because of adventitious infection in other animals. Eimeria weybridgensis tends not to affect the same parts of the gut as E. ovinoidalis (see Norton, Joyner & Catchpole, 1974). Processing the tissues. In the early experiments (A, B, E & F) lambs were killed by intravenous pentobarbitone; tissue specimens were removed within a few minutes of death and fixed in Serra’s fluid (formahn 6 parts, alcohol 3 parts, acetic acid 1 part) or formal sublimate (saturated mercuric chloride 9 parts, formalin 1 part) and embedded in paraffin wax. In these experiments tissues were taken at 1 m intervals

throughout the small intestine, and from the caecum, colon and ileo-caecal lymph node (i.e. the hook-shaped node at the end of the chain of mesenteric lymph nodes, near the ileocaecal junction). In later experiments (C, D, G & H) tissues were taken under deep anaesthesia using intravenous pentobarbitone, fixed in Millonig’s buffered formalin (MBF) as described by Carson, Martin & Lynn (1973). embedded in methacrylate and sectioned at 3 and Ipm. In these experiments, tissues were taken from the ileum, caecum, colon and ileo-caecal lymph node. The duodenum and jejunum were also sampled in Experiment H. In all experi-

FIG. 1. First generation (giant) meronts in the ileal lamina propria. The one to the right is at an early stage; the largest one is almost mature but is already invaded by macrophages; the elongated one between them contains merozoites ready for release. Lamb C808 (10 DAI) ileum, methacrylate 1 pm TB. Bar = 50 pm. FIG. 2. Second generation meronts in the epithelium of caecal crypts. The round mononucleate and oligonucleate bodies (arrows) are probably immature meronts. No host reaction is obvious at this stage. Lamb Cl5 (12 DAI) caecum, methacrylate, 3 pm H&E. Bar = 20ym. FIG. 3. Gamonts

in the epithelium of a caecal crypt. Both micro-(d) and macro-(Q) gamonts are seen. at various maturation. Lamb H 14 (15 DAI) caecum, methacrylate I pm TB. Bar = 10 pm.

stages of

Eimeria

ovinoiddis

in lambs

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M. W.

GREGORY

men&, scrapings of the mucosa were examined fresh under a coverslip at the time of autopsy, to look for coccidial forms. Sections for light microscopy were stained by routine methods includmg haematoxyhn and eosin (H&E) periodic acid Schiff (PAS). toluidine blue (TB), methylene blue/basic fuchsin (MB/BF), chromotrope, and Schmorl’s lipofuscin reaction.‘ For scanning electron microscopy (SEM) tissues fixed in MBF were impregnated with osmium tetroxide using thiocarbohydrazide (TCH), as described by Mahck, Wilson & Stetson (1975) but using only one TCH cycle. They were then critical-point dried from acetone using carbon dioxide. lightly coated with gold and examined under a Cambridge Stereoscan S4-10 and a JEOL JSM T200. RESULTS Clinical and pathological findings fell roughly into

two categories: those occurring before 11 DAI, associated with giant meronts (Fig. 1) in the small intestine, and those occurring on or after 11 DAI, associated with small meronts (Fig. 2) and gamonts (Fig. 3) in the large intestine. Changes associated with giant meronts Lambs that were given only 1CYoocysts or less and killed before day 14 showed no clinicai signs, and no giant meronts or macroscopic changes were detected in the tissues. In lambs that were given 5 X 104 or more oocysts and killed before 14 DAI, giant meronts were seen in the small intenstine as pin-point white spots visible to the naked eye. Where very large numbers were present (i.e. following inocula of 10h or more) at 10 DAI, they were accompanied by congestion, oedema of the intestinal wall, and petechiation of the mucosa. In lamb B7 the damage to the lower small intestine was so severe at 10 DA1 that liquid. bloody diarrhoea was passed; the lamb collapsed and was kilted on humane grounds. Giant meronts in the mucosa appeared to cause three kinds of host reaction: leukocyte infiltration, crypt hyperplasia and epithelial loss. Leokocyte infiltration affected giant meronts individually: a corona of eosinophils and neutrophils surrounded the meront (Fig. 4). In many cases the parasite was invaded by host cells including macrophages. Unaffected meronts were seen alongside others that were in the process of destruction by host cells (Fig. 5). The end result of such attack appeared to be a small mass of macrophage-hike cells representing an extinct giant meront. The proportion of giant meronts under attack by leukocytes varied between 0.4% and about 10% up to 11 DAI. By 14 DA1 small numbers only were present in some lambs, and most of these contained host cells. Crypt hyperplasia (i.e. increased length of crypts) and villus atrophy (shortening of villi) were seen to varying extents at all stages. They were often localised. Epithehal cell loss was seen in very heavy infections at 10 DA1 where there were large numbers of mature giant meronts (Figs. 6-l 0); it was particularly marked in lamb B7 in which free merozoites were also abundant. Epithelial cells in the crypts as well as on

and J.

CATCHPOLE

the surface appeared to be sIoughing. The walls of affected crypts were attenuated, and cell debris (apparently of epithelial rather than leukocyte origin) was seen in the lumen (Figs. 9-10). This process is referred to here as crypt atrophy. In the small intestine of lamb B7, surface epithelial loss was seen at 5 m from the pylorus, and from 6 m there were enormous and increasing numbers of giant meronts. These were associated with haemmorhagic inflammation, loss of surface epithelium and crypt atrophy. In the most heavily infected parts, the architecture was completely destroyed and replaced by masses of giant meronts in a st~ctureless haemo~hagic layer that merged with an overlying diphthe~tic membrane. In lambs with moderate numbers of giant meronts, significant epithelial cell loss was not seen. Some iambs showed varying degrees of lymphocyte depletion in the follicles of the ileal Peyer’s patch, and some showed crypts within the domes (see below). The distribution of giant meronts was investigated in detail in 3 lambs (A7, D5 and B7). They were absent from the first 45-70X of the small intestine. Thereafter their concentration increased rapidly to a maximum between 5 and 1 m from the ileo-caecal junction. The highest density recorded was 265 per mm of section (lamb B7, see above). Three lambs that had received 10” or more oocysts showed giant meronts in the caecal mucosa. In two cases (one of which was a pure infection) a small immature giant meront was seen in a mesenteric lymph node on day 10. Reactions to second-generation rneronts arrd gamotzts Second-generation meronts were present at 11 and 12 DAI; gamonts were first seen at 12 DAL Both stages were seen in epithelial cells of the large intestine; they appeared to favour crypt cells in the caecum. Their appearance coincided with the onset of diarrhoea in most lambs. This stage of the infection was characterised by a caecum that was almost empty, and was contracted to about half its normal diameter. The caecal wall was hyperaemic, oedematous and greatly thickened. In some cases the mucosa was haemorrhagic. These changes extended into the colon, and decreased gradually in severity towards the rectum. The ileum showed some hyperaemia and atrophy of the Peyer’s patch. The ileo-caecal lymph node tended to be enlarged and in some cases haemorrha~c. Histologically, the presence of second-generation meronts and gamonts was associated with epithelial cell loss in the large intestine, both on the surface and in the crypts (Fig. 11). Detachment and death appeared to affect cells en masse, whether infected with coccidia or not. There appeared to be movement of cells across what remained of the basement membrane (Fig. 12). The outcome was that crypts were filled with debris from dead and dying epithelial cells and leukocytes, together with coccidial stages and mucin. In some this debris was calcified. Crypt

Eimeria ovinoidalis in lambs

FIG. 4. Host reaction to giant meronts. An aura of leukocytes (mostly neutrophils) has formed around the parasite but and the apparent depletion of epithelial cells in the crypts in the appc :ars not to have invaded it. Note the crypt hypcrplasia lower left corner of the picture. Lamb F3 (13 DAI) j e j unum, paraffin, 4 pm, chromotrope. Bar = 50 pm. FIG. 5. Host reaction to giant meronts. The one on the left is in the process of destruction by leukocytes (mostly neutrophils in tt ris case). That on the right appears to have elicited no host reaction. Lamb F3 (13 DAI) jejunum, paraffin, 4 ,um, chromotrope. Bar = 50 pm. FIG. 6. Host reaction to giant meronts. The surface epithelium is disorganised, with clumps and strands of cells extending into the lumen and apparently detaching (short arrow). The lamina propria shows hyperaemia and diffuse leukocyte infill nation, and there is leukocyte debris within crypts and in the gut lumen (long arrows). Lamb C826 (10 DAI) ileum, methacrylate 3 ,um H&E. Bar = 50 pm. FIG. 7. Host reaction to giant meronts. Scanning electron micrograph of an area similar to that seen in Fig. 6. Note thevillus atro phy, the crypt mouths on the surface (arrows), and, in the foreground, the apparent disintegration of the surface epithelium. Lamb C808 (10 DAI) ileum, Bar = 1 SO pm.

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M. W. GREGORYand J. CATCHPOLE

Eimeriu ovinoiclalis in lambs walls were seen that had broken down completely and released their contents into the lamina propria. Necrotic cell debris was seen around many crypts whose walls appeared to be unbroken (Fig. 13). Macrophage-like cells were abundant around the bases of crypts, and appeared in many places to be penetrating the basement membrane and phagocytosing dead and dying epithelial cells (Fig. 11). Sections from the caecum of some lambs showed little or no epithelial tissue left; the mucosa was occupied by granulation tissue and covered by a thin layer of fibrin (Fig. 14). Tissue repair By 17 DAI, parasite multiplication had largely subsided. The only sign of coccidial infection was the presence of oocysts in the lamina propria. Where pockets of epithelial tissue remained it formed hyperplastic crypts, some of them cystic, and gave rise to sheets of spreading surface epithelium (Fig. 16). On days 20 and 21 crypts were still hyperplastic and occasionally cystic. Scattered oocysts were seen in the lamina propria and (in one lamb) in the ileo-caecal lymph node (Fig. 15). Miscellaneous observations Eosinophils. Varying degrees of diffuse eosinophil infiltration of the lamina propria were seen in all lambs including Cl9 which was coccidia-free. No consistent relationship to coccidial infection was detected. In all lambs the eosinophils near the tips of the villi tended to have simple nuclei, while those near the bases of the villi showed the classical bilobed nuclei. Globule leukocytes. These tended to be abundant in the ileal Peyer’s patch, but again a consistent relationship to coccidial infection was not obvious. They were particularly numerous in the caecum of lamb D5, one of only three lambs in which giant meronts were seen in the caecum. Crypts in the domes of Peyers patches. In 12 of the 24 lambs examined, crypt-like structures were seen in the domes of the ileal Peyer’s patch. Some contained cell debris, apparently dead eosinophils and neutrophils; others contained yellow-brown amorphous material that stained positively with PAS and with Schmorl’s lipofuscin stain; some contained both. Many were cystic, with no apparent outlet (Fig. 17); in others the crypt opened into the gut lumen near the

1105

base of the dome (Figs. 18 and 19). The crypt epithelium was in many cases attenuated on the side nearest the dome epithelium (Fig. 18); in some cases it was disrupted, with the crypt contents released into the lamina propria (Fig. 19). This appeared to occur frequently: the yellow-brown material was present in most Peyer’s patch domes in the ileum, both free and within macrophages. It was rarely seen in the lamina propria outside the domes. The dome epithelium tended to be cuboidal with few intraepithelial lymphocytes (Figs. 17-20). Another structure which in a single section could be confused with a crypt, was the cleft (Fig. 20). Clefts were relatively shallow, had no contents, and arose from the top of the dome. Serial sections showed them to be cleft-like rather than tubular in structure. DISCUSSION

At least two distinct pathological processes appeared to be causing the disease signs observed: one in the small intestine, in response to large numbers of giant meronts and merozoites, and one in the large intestine in response to large numbers of secondgeneration meronts and gamonts. Both involved loss of epithelium from the mucosal surface and from the crypts. The former required infecting doses of the order of 10h oocysts, and is probably of rare occurrence in the field. The latter followed doses as low as lO”oocysts, and is thought to be the main pathogenic process in naturally-occurring coccidiosis due to E. ovinoidalis (see final paragraph, below). The changes observed, both major and minor, will now be discussed in roughly chronological order. Leukocyte reactions. A mixed leukocyte reaction was directed at individual giant meronts, and this has been remarked on by other authors (Rat & Willson, 1959; Chineme & Njoku, 1978). Wacha et al. (1971) found that over 90% of giant meronts were invaded by eosinophils, neutrophils and macrophages by the time they reached maturity. They used conventionallyreared lambs that had probably encountered the organism previously, which suggested that invasion by host cells might be a reflection of acquired immunity. We found little evidence of such a relationship. One conventional lamb (B7) showed 2.5% of giant meronts invaded; two others (C808 & 826) showed none. Sections from one lamb (H13) that received two pure infections of E. ovinoidalis at an interval of 20 days showed four giant meronts, none of which

FIG. 8. Scanning electron micrograph of a giant meront protruding through a gap in the epithelium. The gap appears to have resulted from sloughing of epithelial cells. Note the smooth surface of the meront, and the shapes of merozoites that can be seen within it.The arrow indicates what is thought to be a free merozoite. Lamb C808 (10 DAI) ileum. Bar = 10 pm. FIG. 9. Host reaction to giant meronts. In this massive infection there is no surface epithelium. Crypt cells are detaching and migrating into the lumen. The worst affected areas were left with little or no epithelial tissue. A mass of merozoites and cell debris is seen in the lumen (arrow). Similar lesions have been reported in field outbreaks. Lamb B7 (10 DAI) ileum, paraffin, 4 pm H&E. Bar = SO pm. FIG. 10. Higher

magnification

of the same section as in Fig. 9, showing the migration into the lumen. Bar = 20 pm.

of epithelial

cells from the crypt walls

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M. W. GREGORYand J. CATCHPOLE

FIG. 11. Host reaction to gamonts. The surface epithelium has been shed, and the crypts are disintegrating. There appears to be widespread detachment of crypt epithelial cells, whether infected or not. Macrophage-like cells appear to be active around the bases of crypts. Lamb G17 (14 DAI) caecum, methacrylate, 1 ,nm MB/BF. Bar = 20 pm. FIG. 12. Breakdown of caecal crypt. The epithelium is attenuated and disintegrating; the lumen is occupied and cells are apparently moving through the basement membrane. Lamb Cl 7 (14DAl) caecum, methacrylate Bar= lOpurn.

by cell debris. 1 pm MB/BF.

FIG. 13. Crypt atrophy in the caecum. Cell debris is seen in crypt lumina and in the lamina propria around the crypt bases. resulting apparently from apoptosis of epithelial ceils. Lamb G6 (14 DAI) caecum, methacrylate, 3 pm H&E. Bar = 20 pm.

Eimeria ovinoidalis in lambs

FIG. 14, Damage caused by gamonts in the large intestine. There is no identifiable epitbelial tissue left in this part of the section. The surface is covered by a thin layer of fibrin, and the mucosa consists of gr~ulation tissue. In this case there is no evidence of necrosis or bacterial invasion. Lamb F6 (17 DAI) caecum. Paraffin 4 pm H&E. Bar = 50 Frn. FIG. 15. Oocysts

(arrows)

in a mesenteric lymph node. They appear to be enclosed in multinucleate (20 DAI) ileocaecal lymph node, paraffin 4 pm H&E. Bar = 20 pm.

giant cells. Lamb E2

FIG. 16. Tissue repair in large intestine. Another part of the same section as Fig. 14 showing the healing process. An isolated residuum of stem ceils has led to the formation of a group of crypts. Some have failed to reach the surface, and haved formed cysts; others have given rise to a spreading sheet of surface epithelium. The submucosa is oedematous. Lamb F6 (17 DAI) caecum. Paraffin 4 flrn H&E. Bar - 100 pm.

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M. W. GREGORYand J. CATCHPOLE

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Eimeria ovinoidalis in lambs

showed any leukocyte reaction. On the other hand, lamb D5 received a single pure infection, and 1.3% of its giant meronts were invaded by host cells. Even 8 days after a pure infection (lamb A7) 0.5% of giant meronts were invaded. Some aspects of the globule leukocyte reaction have been dealt with by Gregory & Nolan (1981) including references to some of the animals reported here (their lamb G07.7 = our F7). The large number of globule leukocytes in the caecum of lamb DS was unexpected, and may have been related to the presence of giant meronts in the caecum of this animal. We have seen monolobular nuclei in eosinophils at the tips of villi in other sheep, including those free of coccidia. The phenomenon was seen under various fixation and staining methods. It may be a feature of sheep in general, that has not been reported before. Villus atrophylclypt hyperplasia. These reactions were seen in the small intestine, apparently in response to giant meronts and merozoites. They are common reactions of the small intestine to various stimuli, including coccidial infection (Pout, 1967) and immunopathological reactions (Ferguson, 1979). Crypt atrophy in the small intestine. This reaction is not easy to understand. It occurred 10 days after massive inoculations ( lo6 oocysts or more). This was the day before the first appearance of second generation meronts in the caecum. It thus coincided roughly with the release of first generation merozoites. But these merozoites are not known to infect cells in the small intestine. It is possible that they migrated through epithelial cells and destroyed some of them in the process. On the other hand, the picture had features in common with that produced by certain cytotoxic drugs: increased apoptosis and decreased mitosis (Bennet, Harrison, Bishop, Searle & Kerr, 1984). This suggests that a toxin might be produced by the giant meronts. Such a toxin could serve the parasite by hastening the departure of surface epithelium, thereby easing the escape of merozoites. We have found no description of crypt atrophy as such in the literature. Marsh & Tunnicliff (1941) mentioned the phenomenon in their report of two outbreaks of acute coccidiosis in adult ewes. They found masses of giant meronts in the small intestine, accompanied by “destruction of intestinal glands”. [Gamonts were not seen. The organism was identified

as “Globidium gibuthi”, because the authors did not recognise the giant meronts as part of coccidian life cycles, despite the fact that Moussu & Marotel(1902) had accurately described them as such nearly 40 years earlier.] There have been other reports of severe disease attributed to giant meronts in the small intestine of sheep and goats (Rat & Willson, 1959; Mugera & Bitakaramire, 1968). Depletion of lymphocytes in the ileal Peyer’s patch.

This was seen in most of the severely ill animals. These cells are mainly B-cells, most of which are probably destined to migrate to the intestinal larnina propria for production of immune globulins (Bienenstock & Befus, 1980; Larsen & Landsverk, 1986). The depletion may therefore be a manifestation of hastened migration. T-cell areas were not affected. We have seen B-cell depletion in gut-associated lymphoid tissue of rabbits with severe coccidiosis (Gregory & Catchpole, 1986). Crypts in the domes of the ileal Peyer’s patch. We have found no reference in the literature to these structures. They were particularly noticeable in four lambs, three of which had received “immunizing” inoculations. The lipofuscin-like material that was apparently derived from such crypts, was generally present in ileal Peyer’s patch domes whether crypts were seen in them or not. Two further observations may be relevant: firstly, crypts over Peyer’s patches tended to contain more leukocyte debris than crypts in non-Peyer’s patch mucosa; secondly, very few of the domes seen contained many intraepithelial lymphocytes. In some animals the domes themselves were small or absent. Each dome is normally surrounded by a ring of crypts. It could be that antigenic stimulation from the gut contents results in sudden swelling of the domes, or indeed in the formation of domes. Dome epithelium is perhaps particularly averse to allowing breaks in its continuity, so crypts thus covered over would be left with no outlet. Their content would then be released into the dome, or into the lumen via a side outlet at the base of the dome. Epithelial destruction in the large intestine. The

damage to the large intestine is perhaps easier to explain than that in the small intestine, because the cells that are lost are target-cells of the parasite. The last meront generation infects the epithelial cells lining

FIG. 17. Cystic crypt within a dome of the ileal Peyer’s patch. The brownish content appeared to be mainly lipofuscin, which was also present in the lamina propria of the domes but was rarely seen elsewhere. Lamb A7 (8 DAI) ileum, paraffin 4 pm H&E. Bar = 50 ,~rn. FIG. 18. Crypt in dome of ileal Peyer’s patch. The crypt lumen communicates with the gut lumen, and the lipofuscin-like contents appear to be escaping. Note the attenuation of the crypt epithelium towards the dome surface (arrow). Lamb A7 (8 DAI) ileum, paraffin, 4 pm H&E. Bar = 30 pm. FIG. 19. Crypt in dome of ileal Peyer’s patch. Note the breakdown of the crypt epithelium at the upper extremity, and release of lipofuscin-like contents into the lamina propria of the dome. Note also the cuboidal dome epithelium and virtual absence of intra-epithelial lymphocytes. Lamb F6 (17 DAI) ileum, parafftn 4 pm H&E. Bar = 30 pm. FIG. 20. Cleft in dome of ileal Peyer’s patch. At first glance these structures appear are shallow clefts. Lamb F3 (13 DAI) ileum, paraffin, 4 pm H&E. Bar = 50 pm.

to be crypts, but serial sections

show they

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M. W. GREGORYand J. CATCHPOLE

the crypts in the large intestine. The progeny of these meronts-the gamonts-then attack what remains of the crypt epithelium. Infected stem cells evidently ceased to multiply effectively. All infected cells were shed, either prematurely or when the parasites within them matured. It appeared that non-parasitized cells were also shed, which suggests that the mechanism of cell loss may be similar to that seen in massive infections in the small intestine, discussed above. In that case it could properly be called crypt atrophy. Whatever the mechanism, the outcome was that all the cells in a crypt-including the stem cells-may be destroyed. If there are large numbers of parasites, all the crypts in considerable areas of mucosa may be destroyed. Meanwhile the surface epithelium has been shed, either because it has reached the end of its lifespan or because of the coccidial infection. Without the crypt stem cells, the epithelium cannot regenerate. So the lesion can only heal by spread of epithelium from areas where stem cells have been left intact. Healing is therefore very slow, and the animal is liable to succumb to dehydration and/or bacterial invasion before healing is complete. The pathogenic mechanism is apparently closely parallel to that of E. fluvestens in rabbits (Gregory & Catchpole, 1986). Similar pathological pictures are caused by other agents that destroy crypt stem cells, such as some parvoviruses and ionising radiation. Relevance to naturally-occurring disease. In the above experiments we saw that the clinical signs resulting from damage to the small intestine by giant meronts could be similar to those resulting from damage to the large intestine by gamonts. The former required massive intake of oocysts and were associated with negative faecal oocyst output. Such cases apparently do occur in the field but are probably rare. The latter occurred after low and moderate oocyst input and were associated with high oocyst output. However, affected animals recovered slowly, so diarrhoea persisted long after oocyst output dropped to low levels (Catchpole & Gregory, unpublished). Eimeria ovinoidalis is the only species that has consistently shown high pathogenicity in experimental infections (Catchpole et af., 1976; Catchpole & Gregory. 1985). We therefore think it probable that most field outbreaks of ovine coccidoisis are attributable principally to damage in the large intestine caused by gamonts of E. ovinoidulis. Acknowledgements-We thank Valerie Hyde and Audrey Newman for the care of the animals; M. A. Peirce and Ann Nolan for the histological sections.

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