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Scanning electron microscopy of Eimeria tenella infection and subsequent repair in chicken caeca
J. COMP. PATH. 1975. VOL. 85.
571
SCANNING
EIMERIA
ELECTRON MICROSCOPY OF INFECTION AND SUBSEQUENT REPAIR IN CHICKEN CAECA
TE.iVELLA
BY
D. R. WITLOCK, Department cf Poultry
H. D. DANFORTH, Science, University
of Georgia,
and M. D. RUFF Athens, Georgia
30602,
U.S.A.
INTRODUCTION
Several workers have reported the changes seen with a light microscope during an Eimeria tenella infection (Tyzzer, 1929; Davies, Joyner and Kendall, 1963; Long, 1973). The mucosal morphology of the normal chicken caeca and some of the damage seen with the aid of the scanning electron microscope (SEM) during an E. tenella infection have been reported (Witlock, Lushbaugh, Danforth and Ruff, 1975). However, little is known about surface changes which occur in the progress of an E. tenetla infection and subsequent tissue repair. The purpose of this study was to investigate these changes. MATERIALS
AND
METHODS
Fifty White Leghorn cockerels 2-weeks of age were each infected orally with 100 000 oocysts of E. tenella. Two birds were killed daily from day 1 to day 21 pastinfection (PI), the caecal tissuesremoved and designated area 1 (the mid caecal pouch region) and area 2 (3 to 5 mm. from the blind end of the pouch). On days 0 and 21 two uninfected control birds were killed and the caecal tissueswere fixed in 2 per cent. glutaraldehyde-0.1 M phosphate buffer for 1.5 h., washed 3 to 5 times at 5 min. per washin 0.1 M phosphate buffer pH 7.4 with 5 per cent. sucrose,dehydrated in an ascending ethanol series(70, 80, 95 and 100 per cent.) and critical point dried (Boyde and Wood, 1969). The dried specimenswere mounted on specimen stubs with conductive silver paint and gold coated in a Hummer vacuum coater for 4 min. at 10 mA. This tissue was gently removed from the stub after viewing by the SEM, washed several times in propylene oxide, and infiltrated and embedded in Epon plastic. One l,trn. sections were taken from this material, mounted on microscope slides, stained with Paragon D and examined with the light microscope. RESULTS
L7ninfected (2 Weeks of Age) The surface of the mid caecal pouch region (area 1) was characterized by protruding collar structures (Fig. 1) which were the opening to the crypts or glands of the caeca. Area 2 (Fig. 2) was composed of flattened collar structures which were also opening to the glands of the caeca. No gross morphological differences were observed between the 2 week and 5 week controls (Figs 23-24).
Early Phase of Infection (Days 0 to 4 PI) The collar structures in area 1,3 days PI (Fig. 3) appeared swollen and lacked distinct borders when compared with the uninfected collars (Fig. 1). By 4 days
572
Fig.
D.
R.
WITLOCK
et
al.
1. SEM micrograph of area 1, mid caecal pouch, P-week-old uninfected chicken. Note protruding collars. x 300. Fig. 2. SEM micrograph of area 2, the distal tip of the caeca, 2-week-old uninfected chicken. Note flattened surface of collar. x 300. Fig. 3. Area 1, 3 days PI with Eineria tenella.Note (arrows) swelling of collar. x 140. Fig. 4. Area 1, 4 days PI. Note (arrows) ridges of epithelial cells forming at top of collars. x650.
E. tenella INFECTION : ELECTRON
MICROSCOPY
Fig. 5. Area 1, 4 days PI, light micrograph of similar tissue shown in Fig. 4. Ridges are composed of dissociating epithelial cells (arrows). x 78. Fig. 6. Area 2, 4 days PI, ridges (arrow) are present in this area. x 230. Fig. 7. Area 1, 6 days PI, cell sloughing (arrow) resulted in denuded surface. x 140. Fig. 8. Area 1, 8 days PI, mucosal surface completely denuded. x 260.
574
D.
R.
WITLOCK
et
cd.
PI (Fig. 4) ridg es were seen on the collars from infected tissue. Tissue sections of a similar specimen showed that these ridges were composed of cells disassociated from the collar matrix (Fig. 5). The surface of area 2 by day 4 PI (Fig. 6) was only slightly altered from the flattened surface seen in the uninfected tissue (Fig. 2). The mucosa of the infected tissue had distinct ridges bordering the collar structures, but they were not as prominent as those observed in area 1, 4 days PI (Fig. 4).
Late Phase of Infection (Days 5 to 8 PI) In area 1, most damage to the host tissue occurred by day 6 PI (Fig. 7). The sloughing of cells was usually complete by day 8 PI (Fig. 8) resulting in a surface completely denuded of epithelia. The exposed surface was composed of the connective tissue of the tunica propria. The ridges surrounding the flat collars in area 2 were further enlarged by the 6th day PI (Fig. 9) when compared to day 4 PI (Fig. 6). The ridges now protruded above the surface of the collars and formed a cup shape with the collar lumen at the bottom. Ruptured cells, possibly oocyst extrusion sites, were visible on the ridges. In area 2 cell sloughing was usually complete by day 8 PI.
Early Phase of Tissue Repair (Days 8 to 12) In early repair of areas 1 and 2, epithelial cells emerged from deep within the crypts (Fig. 10) and proliferated across the surface of the tunica propria (Fig. 11). During this time cells from relatively undamaged collar structures were also growing across the stripped surface (Fig. 12). By day 12 PI the damaged surfaces were, in general, covered with a layer of epithelia (Fig. 13). Distinct collars were seen and large amounts of cellular debris were found on the surface of the newly formed epithelium. In area 2 which had a lighter infection, the repair processes were evident as early as day 8 PI (Fig. 14) and progressed at a more rapid rate than in area 1. Epithelial cells from damaged crypts often formed a mushroom-like structure of cells on the mucosal surface. Cells from crypts moved across the connective tissue to form a protective sheet of epithelium (Fig. 15). Cells at the periphery of the advancing sheet had a flattened appearance and rounded edges (Figs 15 and 16). A higher magnification (Fig. 16) showed these cells turning under as the sheet advanced across the damaged areas. The brush border (fuzzy coat) was visible on the epithelial cells (Fig. 16). The surface of area 2 was almost reorganized by day 12 PI (Fig. 17). The collar structures were prevalent although ridges were still present on the surface adjacent to the collars.
Late Phase of Tissue Repair (Days 12 to 21) Most of the surfaces in area 1 were covered by epithelia by day 14 PI. In regions which had been extensively damaged, the epithelial repair was incomplete (Fig. 18). However, in less damaged areas the surface was nearly reconstructed, although cellular debris was still present between the collars
E. tenella
INFECTION
: ELECTRON
MICROSCOPY
Fig. 9. Area 2, 6 days PI, ridges have formed a cup shape with the collar lumen at base of cup. x 260. Fig. 10. Area 1, 8 days PI, high magnification of crypt opening. Note (arrow) epithelial cells in process of emerging from crypt. X 2500. Fig. 11. Area I,8 days PI, epithelial cells growing from crypt and spreading across tunica propria. x 750. Fig. 12. Area 1, 8 days PI, epithelial cells from undamaged collars moving to cover tunica propria. x 750.
576
D.
R.
WITLOCK
et al.
Fig. Fig.
13. Area 1, 12 days PI, tunica propria has been covered with a layer of epithelia. x 260. 14. Area 2, 8 days PI, epithelial cells from undamaged portions have covered tunica propria. tolt;Jarrow) cells emerging from crypts have raised above surface forming a mushroom shape.
Fig.
15. Area 2, 8 days PI, note epithelial cells at periphery of moving epithelial sheet are flattened. x 840. 16. Area 2, 8 days PI, greater magnification of Fig. 15. Note rounded edges of flattened cells. Brush border (fuzzy coat) is also visible. x 3 100.
Fig.
E. tenella
Fig.
INFECTION:
ELECTRON
MICROSCOPY
17. Area 2 (distal tip of caeca), 12 days PI, surface regenerated. Note (arrow) ridges on surface adjacent to the collars. x 260. Fig. 18. Area 1 (mid caecal pouch), 14 days PI, cells from newly formed collars (arrow) moving across damaged surface. x 650. Fig. 19. Area 1, 14 days PI, lightly damaged area. Collar structure partially restored although large amounts of debris present. x 250. Fig. 20. Area 2, 14 days PI. The collar structures are present, ridges of debris and cells still present. x 150.
578
Fig. Fig. Fig. Fig.
D.
21. 22. 23. 24.
Area Area Area Area
1, 2, 1, 2,
21 21 21 21
days days days days
PI, PI. PI, PI,
R.
surface is partially Surface completely uninfected control. uninfected control.
WITLOCK
repaired. repaired x 260. x 290.
et
cd.
Ridges of debris still present. x 130. to resemble the uninfected. x 270.
E. tenella
INFECTION:
ELECTRON
MICROSCOPY
579
(Fig. 19). By day 21 PI (Fig. 21) th e surface of the previously infected caeca was approaching the normal configuration. Although debris was still present, the surface was reorganized to such an extent that infected tissue from area 1 (Fig. 21) and area 2 (Fig. 22) resembled that seen in the normal uninfected area 1 (Fig. 23) and area 2 (Fig. 24). I n area 2, the repair process was nearly complete by day 14 PI (Fig. 20) although a few ridges were still present. DISCUSSION
Studies with the SEM give a new insight into the damage and repair process of the epithelial surface following Eimeria tenella infections. Some of the phenomenon reported previously from tissue sections were also visible on the surface of the caecal mucosa. The acute swelling of the mucosa reported by Tyzzer (1929) was seen in both areas 1 (mid caecal pouch) and 2 (distal tip of caeca). In an infected chicken, extensive tissue damage occurs by day 5 PI, with haemorrhage and the sloughing of epithelial cells (Tyzzer, 1929). The second generation schizonts are found throughout the interglandular tissues of the caecal mucosa (Tyzzer, 1929; Davies et al., 1963), and are associated with the caecal haemorrhage. Sloughing was seen in area 1 at day 6 PI (Fig. 7) and in some cases the surface was completely denuded by day 8 PI (Fig. 8). Damage appeared most severe in the mid-portion of the caeca. Eimeria tenella is primarily parasitic in the epithelial lining of the caeca (Tyzzer, 1929) with the dilated or mid portion more extensively involved (Tyzzer, 1929; Ripsom, Johnson and Herrick, 1949). The reparative process in the caeca was similar to that described for the healing of the small intestine by Florey and Harding ( 1935) and surface wounds by Arey (1936). In our study, the cells of the crypts could be seen to emerge from the crypt mouths and begin their migration across the damaged tissue. At the same time, sheets of epithelia from undamaged portions of the caecum were migrating across the connective tissue to cover the damaged area. The cells at the edge of the migrating epithelial sheets were flattened as had been described by Florey and Harding (1935). Whether or not the cell movements were due to amoeboid processes (Arey, 1936) or from the pressure exerted from increased mitotic activity occurring at the base of the crypt is not clear, but evidence for both mechanisms was seen. Migrating sheets of cells appear not to have covered functional crypts, but rather to have been diverted around the crypt mouth, while cells emerging from the crypt have a mushroom-shaped appearance. A distinct delineation between the cells of the crypt and those of the flattened migrating sheet was observed. Similar structures composed of granulation tissue have been reported by McMinn (1969). The distal tip of the caeca appeared to repair damage faster than did the mid portion (area 1) based on the conditions of the two areas in inoculated birds compared with their respective uninoculated controls at 21 days PI. Whether the difference in repair time is due to the degree of infection each of these areas achieved, or whether the flat mucosa found in area 2 has a faster cell turnover time is not clear. The work of Tyzzer (1929) and Ripsom et al. (1949) indicated that the mid portion (area 1) is usually more heavily infected
580
D.
or damaged than is the distal tip have shown that the flat mucosa when compared to other forms 1969; Loehry, Croft, Singh and 1970; Symons, 1965). The flat caeca is similar to that seen in disease (Witlock et al., 1975).
R.
WITLOCK
et
d.
of the caeca (area 2). Likewise, several workers configuration has a faster cell turnover time of intestinal surfaces (Loehry and Creamer, Creamer, 1969; Asquith, Johnson and Cooke, mucosal surface of the uninoculated chicken some parasitic infections and human coeliac
SUMMARY
The progressive tissue damage during an Eimeria tenella infection and the subsequent repair processes of the caecal mucosa were observed with the scanning electron microscope (SEM). Distinct ridges began to form around the collars about the third day post-infection (PI) in the mid caecal pouch and the distal tip of the caeca. These ridges persisted until the 6th day PI in the tip of the caeca. Damaged tissue had sloughed by the 8th day PI and there was some indication of early repair. Initially, new epithelial cells emerged from the crypts to cover the damaged tissue and simultaneously epithelial cells migrated from the undamaged tissue across the injured area. Repair of the damaged tissue was usually completed by the 21st day for the distal tip and iater for the mid caecal pouch. The process of repair in the caeca was similar to healing in the mammalian small intestine and surface epithelial wounds. REFERENCES
Arey, L. B. (1936). W ound healing. Physiological Reviews, 16, 3271106. Asquith, P., Johnson, A., and Cooke, W. T. (1970). Scanning electron microscopy of normal and celiac mucosa. American Journal of Digestive Diseases, 15, 5 1l-52 1. Boyde, A., and Wood, C. (1969). Preparation of animal tissuesfor surface scanning electron microscopy. Journal of Microscopy, 90, 221-249. Davies, S. F. M., Joyner, L. P., and Kendall, S. B. (1963). Coccidiosis, Oliver and Boyd, Edinburgh and London. Florey, H. W., and Harding, H. E. (1935). The healing of artificial defects of the duodenal mucosa. Journal of Pathology and Bacteriology, 40, 21l-2 18. Loehry, C. A., and Creamer, B. (1969). Three dimensional structure of the rat small intestinal mucosa related to mucosal dynamics. Part III. Mucosal structure and dynamics in the rat infected with the nematode .N$postron&us brasiliensis. Gut, 10, 118-120. Loehry, C. A., Croft, D. N., Singh, A. K., and Creamer, B. (1969). Cell turnover in the rat small intestinal mucosa; an appraisal of cell loss. Part II. Cell loss in rats with an abnormal mucosa. Gut, 10, 16-18. Long, P. L. (1973). Pathology and pathogenicity of coccidial infections. In The Coccidia D. M. Hammond and P. L. Long, Eds. University Park Press,Baltimore and Butterworths, London. McMinn, R. M. H. (1969). Tissue Repair. p. 247-265, Academic Press,New York and London. Ripsom, C. A., Johnson, C. A., Herrick, C. A. (1949). Some host-parasite relationshipsbetween the chicken and its protozoan parasite Eimeria tenella. Annals of the flew York Academy of Science, 52, 496-50 1. Symons, L. E. A. (1965). Kinetics of the epithelial cells, and morphology of villi and crypts in the jejunum of the rat infected by the nematode .h@postronglus brasiliensis.
Gastroenterology, 49, 158-168.
E.
tenella
INFECTION:
ELECTRON
MICROSCOPY
581
Tyzzer, E. E. (1929). Coccidiosis in gallinaceous birds. American Journal of Hygiene, 10, 269-349. Witlock, D. R., Lushbaugh, W. B., Danforth, H. D., and Ruff, M. D. (1975). Scanning electron microscopy of the cecal mucosa in Eimeria tenella infected and uninfected chickens. Avian Diseases, 19, 293-304. [Received for publication,
March
25th, 19751
571
SCANNING
EIMERIA
ELECTRON MICROSCOPY OF INFECTION AND SUBSEQUENT REPAIR IN CHICKEN CAECA
TE.iVELLA
BY
D. R. WITLOCK, Department cf Poultry
H. D. DANFORTH, Science, University
of Georgia,
and M. D. RUFF Athens, Georgia
30602,
U.S.A.
INTRODUCTION
Several workers have reported the changes seen with a light microscope during an Eimeria tenella infection (Tyzzer, 1929; Davies, Joyner and Kendall, 1963; Long, 1973). The mucosal morphology of the normal chicken caeca and some of the damage seen with the aid of the scanning electron microscope (SEM) during an E. tenella infection have been reported (Witlock, Lushbaugh, Danforth and Ruff, 1975). However, little is known about surface changes which occur in the progress of an E. tenetla infection and subsequent tissue repair. The purpose of this study was to investigate these changes. MATERIALS
AND
METHODS
Fifty White Leghorn cockerels 2-weeks of age were each infected orally with 100 000 oocysts of E. tenella. Two birds were killed daily from day 1 to day 21 pastinfection (PI), the caecal tissuesremoved and designated area 1 (the mid caecal pouch region) and area 2 (3 to 5 mm. from the blind end of the pouch). On days 0 and 21 two uninfected control birds were killed and the caecal tissueswere fixed in 2 per cent. glutaraldehyde-0.1 M phosphate buffer for 1.5 h., washed 3 to 5 times at 5 min. per washin 0.1 M phosphate buffer pH 7.4 with 5 per cent. sucrose,dehydrated in an ascending ethanol series(70, 80, 95 and 100 per cent.) and critical point dried (Boyde and Wood, 1969). The dried specimenswere mounted on specimen stubs with conductive silver paint and gold coated in a Hummer vacuum coater for 4 min. at 10 mA. This tissue was gently removed from the stub after viewing by the SEM, washed several times in propylene oxide, and infiltrated and embedded in Epon plastic. One l,trn. sections were taken from this material, mounted on microscope slides, stained with Paragon D and examined with the light microscope. RESULTS
L7ninfected (2 Weeks of Age) The surface of the mid caecal pouch region (area 1) was characterized by protruding collar structures (Fig. 1) which were the opening to the crypts or glands of the caeca. Area 2 (Fig. 2) was composed of flattened collar structures which were also opening to the glands of the caeca. No gross morphological differences were observed between the 2 week and 5 week controls (Figs 23-24).
Early Phase of Infection (Days 0 to 4 PI) The collar structures in area 1,3 days PI (Fig. 3) appeared swollen and lacked distinct borders when compared with the uninfected collars (Fig. 1). By 4 days
572
Fig.
D.
R.
WITLOCK
et
al.
1. SEM micrograph of area 1, mid caecal pouch, P-week-old uninfected chicken. Note protruding collars. x 300. Fig. 2. SEM micrograph of area 2, the distal tip of the caeca, 2-week-old uninfected chicken. Note flattened surface of collar. x 300. Fig. 3. Area 1, 3 days PI with Eineria tenella.Note (arrows) swelling of collar. x 140. Fig. 4. Area 1, 4 days PI. Note (arrows) ridges of epithelial cells forming at top of collars. x650.
E. tenella INFECTION : ELECTRON
MICROSCOPY
Fig. 5. Area 1, 4 days PI, light micrograph of similar tissue shown in Fig. 4. Ridges are composed of dissociating epithelial cells (arrows). x 78. Fig. 6. Area 2, 4 days PI, ridges (arrow) are present in this area. x 230. Fig. 7. Area 1, 6 days PI, cell sloughing (arrow) resulted in denuded surface. x 140. Fig. 8. Area 1, 8 days PI, mucosal surface completely denuded. x 260.
574
D.
R.
WITLOCK
et
cd.
PI (Fig. 4) ridg es were seen on the collars from infected tissue. Tissue sections of a similar specimen showed that these ridges were composed of cells disassociated from the collar matrix (Fig. 5). The surface of area 2 by day 4 PI (Fig. 6) was only slightly altered from the flattened surface seen in the uninfected tissue (Fig. 2). The mucosa of the infected tissue had distinct ridges bordering the collar structures, but they were not as prominent as those observed in area 1, 4 days PI (Fig. 4).
Late Phase of Infection (Days 5 to 8 PI) In area 1, most damage to the host tissue occurred by day 6 PI (Fig. 7). The sloughing of cells was usually complete by day 8 PI (Fig. 8) resulting in a surface completely denuded of epithelia. The exposed surface was composed of the connective tissue of the tunica propria. The ridges surrounding the flat collars in area 2 were further enlarged by the 6th day PI (Fig. 9) when compared to day 4 PI (Fig. 6). The ridges now protruded above the surface of the collars and formed a cup shape with the collar lumen at the bottom. Ruptured cells, possibly oocyst extrusion sites, were visible on the ridges. In area 2 cell sloughing was usually complete by day 8 PI.
Early Phase of Tissue Repair (Days 8 to 12) In early repair of areas 1 and 2, epithelial cells emerged from deep within the crypts (Fig. 10) and proliferated across the surface of the tunica propria (Fig. 11). During this time cells from relatively undamaged collar structures were also growing across the stripped surface (Fig. 12). By day 12 PI the damaged surfaces were, in general, covered with a layer of epithelia (Fig. 13). Distinct collars were seen and large amounts of cellular debris were found on the surface of the newly formed epithelium. In area 2 which had a lighter infection, the repair processes were evident as early as day 8 PI (Fig. 14) and progressed at a more rapid rate than in area 1. Epithelial cells from damaged crypts often formed a mushroom-like structure of cells on the mucosal surface. Cells from crypts moved across the connective tissue to form a protective sheet of epithelium (Fig. 15). Cells at the periphery of the advancing sheet had a flattened appearance and rounded edges (Figs 15 and 16). A higher magnification (Fig. 16) showed these cells turning under as the sheet advanced across the damaged areas. The brush border (fuzzy coat) was visible on the epithelial cells (Fig. 16). The surface of area 2 was almost reorganized by day 12 PI (Fig. 17). The collar structures were prevalent although ridges were still present on the surface adjacent to the collars.
Late Phase of Tissue Repair (Days 12 to 21) Most of the surfaces in area 1 were covered by epithelia by day 14 PI. In regions which had been extensively damaged, the epithelial repair was incomplete (Fig. 18). However, in less damaged areas the surface was nearly reconstructed, although cellular debris was still present between the collars
E. tenella
INFECTION
: ELECTRON
MICROSCOPY
Fig. 9. Area 2, 6 days PI, ridges have formed a cup shape with the collar lumen at base of cup. x 260. Fig. 10. Area 1, 8 days PI, high magnification of crypt opening. Note (arrow) epithelial cells in process of emerging from crypt. X 2500. Fig. 11. Area I,8 days PI, epithelial cells growing from crypt and spreading across tunica propria. x 750. Fig. 12. Area 1, 8 days PI, epithelial cells from undamaged collars moving to cover tunica propria. x 750.
576
D.
R.
WITLOCK
et al.
Fig. Fig.
13. Area 1, 12 days PI, tunica propria has been covered with a layer of epithelia. x 260. 14. Area 2, 8 days PI, epithelial cells from undamaged portions have covered tunica propria. tolt;Jarrow) cells emerging from crypts have raised above surface forming a mushroom shape.
Fig.
15. Area 2, 8 days PI, note epithelial cells at periphery of moving epithelial sheet are flattened. x 840. 16. Area 2, 8 days PI, greater magnification of Fig. 15. Note rounded edges of flattened cells. Brush border (fuzzy coat) is also visible. x 3 100.
Fig.
E. tenella
Fig.
INFECTION:
ELECTRON
MICROSCOPY
17. Area 2 (distal tip of caeca), 12 days PI, surface regenerated. Note (arrow) ridges on surface adjacent to the collars. x 260. Fig. 18. Area 1 (mid caecal pouch), 14 days PI, cells from newly formed collars (arrow) moving across damaged surface. x 650. Fig. 19. Area 1, 14 days PI, lightly damaged area. Collar structure partially restored although large amounts of debris present. x 250. Fig. 20. Area 2, 14 days PI. The collar structures are present, ridges of debris and cells still present. x 150.
578
Fig. Fig. Fig. Fig.
D.
21. 22. 23. 24.
Area Area Area Area
1, 2, 1, 2,
21 21 21 21
days days days days
PI, PI. PI, PI,
R.
surface is partially Surface completely uninfected control. uninfected control.
WITLOCK
repaired. repaired x 260. x 290.
et
cd.
Ridges of debris still present. x 130. to resemble the uninfected. x 270.
E. tenella
INFECTION:
ELECTRON
MICROSCOPY
579
(Fig. 19). By day 21 PI (Fig. 21) th e surface of the previously infected caeca was approaching the normal configuration. Although debris was still present, the surface was reorganized to such an extent that infected tissue from area 1 (Fig. 21) and area 2 (Fig. 22) resembled that seen in the normal uninfected area 1 (Fig. 23) and area 2 (Fig. 24). I n area 2, the repair process was nearly complete by day 14 PI (Fig. 20) although a few ridges were still present. DISCUSSION
Studies with the SEM give a new insight into the damage and repair process of the epithelial surface following Eimeria tenella infections. Some of the phenomenon reported previously from tissue sections were also visible on the surface of the caecal mucosa. The acute swelling of the mucosa reported by Tyzzer (1929) was seen in both areas 1 (mid caecal pouch) and 2 (distal tip of caeca). In an infected chicken, extensive tissue damage occurs by day 5 PI, with haemorrhage and the sloughing of epithelial cells (Tyzzer, 1929). The second generation schizonts are found throughout the interglandular tissues of the caecal mucosa (Tyzzer, 1929; Davies et al., 1963), and are associated with the caecal haemorrhage. Sloughing was seen in area 1 at day 6 PI (Fig. 7) and in some cases the surface was completely denuded by day 8 PI (Fig. 8). Damage appeared most severe in the mid-portion of the caeca. Eimeria tenella is primarily parasitic in the epithelial lining of the caeca (Tyzzer, 1929) with the dilated or mid portion more extensively involved (Tyzzer, 1929; Ripsom, Johnson and Herrick, 1949). The reparative process in the caeca was similar to that described for the healing of the small intestine by Florey and Harding ( 1935) and surface wounds by Arey (1936). In our study, the cells of the crypts could be seen to emerge from the crypt mouths and begin their migration across the damaged tissue. At the same time, sheets of epithelia from undamaged portions of the caecum were migrating across the connective tissue to cover the damaged area. The cells at the edge of the migrating epithelial sheets were flattened as had been described by Florey and Harding (1935). Whether or not the cell movements were due to amoeboid processes (Arey, 1936) or from the pressure exerted from increased mitotic activity occurring at the base of the crypt is not clear, but evidence for both mechanisms was seen. Migrating sheets of cells appear not to have covered functional crypts, but rather to have been diverted around the crypt mouth, while cells emerging from the crypt have a mushroom-shaped appearance. A distinct delineation between the cells of the crypt and those of the flattened migrating sheet was observed. Similar structures composed of granulation tissue have been reported by McMinn (1969). The distal tip of the caeca appeared to repair damage faster than did the mid portion (area 1) based on the conditions of the two areas in inoculated birds compared with their respective uninoculated controls at 21 days PI. Whether the difference in repair time is due to the degree of infection each of these areas achieved, or whether the flat mucosa found in area 2 has a faster cell turnover time is not clear. The work of Tyzzer (1929) and Ripsom et al. (1949) indicated that the mid portion (area 1) is usually more heavily infected
580
D.
or damaged than is the distal tip have shown that the flat mucosa when compared to other forms 1969; Loehry, Croft, Singh and 1970; Symons, 1965). The flat caeca is similar to that seen in disease (Witlock et al., 1975).
R.
WITLOCK
et
d.
of the caeca (area 2). Likewise, several workers configuration has a faster cell turnover time of intestinal surfaces (Loehry and Creamer, Creamer, 1969; Asquith, Johnson and Cooke, mucosal surface of the uninoculated chicken some parasitic infections and human coeliac
SUMMARY
The progressive tissue damage during an Eimeria tenella infection and the subsequent repair processes of the caecal mucosa were observed with the scanning electron microscope (SEM). Distinct ridges began to form around the collars about the third day post-infection (PI) in the mid caecal pouch and the distal tip of the caeca. These ridges persisted until the 6th day PI in the tip of the caeca. Damaged tissue had sloughed by the 8th day PI and there was some indication of early repair. Initially, new epithelial cells emerged from the crypts to cover the damaged tissue and simultaneously epithelial cells migrated from the undamaged tissue across the injured area. Repair of the damaged tissue was usually completed by the 21st day for the distal tip and iater for the mid caecal pouch. The process of repair in the caeca was similar to healing in the mammalian small intestine and surface epithelial wounds. REFERENCES
Arey, L. B. (1936). W ound healing. Physiological Reviews, 16, 3271106. Asquith, P., Johnson, A., and Cooke, W. T. (1970). Scanning electron microscopy of normal and celiac mucosa. American Journal of Digestive Diseases, 15, 5 1l-52 1. Boyde, A., and Wood, C. (1969). Preparation of animal tissuesfor surface scanning electron microscopy. Journal of Microscopy, 90, 221-249. Davies, S. F. M., Joyner, L. P., and Kendall, S. B. (1963). Coccidiosis, Oliver and Boyd, Edinburgh and London. Florey, H. W., and Harding, H. E. (1935). The healing of artificial defects of the duodenal mucosa. Journal of Pathology and Bacteriology, 40, 21l-2 18. Loehry, C. A., and Creamer, B. (1969). Three dimensional structure of the rat small intestinal mucosa related to mucosal dynamics. Part III. Mucosal structure and dynamics in the rat infected with the nematode .N$postron&us brasiliensis. Gut, 10, 118-120. Loehry, C. A., Croft, D. N., Singh, A. K., and Creamer, B. (1969). Cell turnover in the rat small intestinal mucosa; an appraisal of cell loss. Part II. Cell loss in rats with an abnormal mucosa. Gut, 10, 16-18. Long, P. L. (1973). Pathology and pathogenicity of coccidial infections. In The Coccidia D. M. Hammond and P. L. Long, Eds. University Park Press,Baltimore and Butterworths, London. McMinn, R. M. H. (1969). Tissue Repair. p. 247-265, Academic Press,New York and London. Ripsom, C. A., Johnson, C. A., Herrick, C. A. (1949). Some host-parasite relationshipsbetween the chicken and its protozoan parasite Eimeria tenella. Annals of the flew York Academy of Science, 52, 496-50 1. Symons, L. E. A. (1965). Kinetics of the epithelial cells, and morphology of villi and crypts in the jejunum of the rat infected by the nematode .h@postronglus brasiliensis.
Gastroenterology, 49, 158-168.
E.
tenella
INFECTION:
ELECTRON
MICROSCOPY
581
Tyzzer, E. E. (1929). Coccidiosis in gallinaceous birds. American Journal of Hygiene, 10, 269-349. Witlock, D. R., Lushbaugh, W. B., Danforth, H. D., and Ruff, M. D. (1975). Scanning electron microscopy of the cecal mucosa in Eimeria tenella infected and uninfected chickens. Avian Diseases, 19, 293-304. [Received for publication,
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