Archives of Virology 55, 263--274 (1977)
Archives of Virology © by Springer-Verlag 1977
Rotavirus Infection in Lambs: Pathogenesis and Patholoqy By D. R. SNODGRASS,K. W. A~Gus, and E. W. G~A¥ Animal Diseases Research Association, Moredun Institute, Edinburgh, Scotland W'ith 11 Figures Accepted June 21, 1977
Summary Experimental lamb rotavirus infections were studied by immunofluoreseenee, histopathology and electron microscopy of tissues from infected gnotobiotic lambs killed at intervals from the incubation period to recovery. The rotavirus was demonstrated by imnmnofluoreseence only in epithelial ceils of villi in the small and large intestine, and virus antigen was most abundant during the incubation period. An increased enterocyte turnover rate was suggested by the rapid movement of virus-infected cells to the villus tip, and this increase m a y be one of the basic pathogenic mechanisms of rotavirus infection. Principal histopathological changes were shortening of villi and sloughing of epithelial cells. These were greatest in the middle and posterior small intestine at the onset of diarrhoea, but regeneration occurred within a few hours. Virus morphology in tissues -was similar to that reported in other species, and virus presence correlated well with histopathologieaI change.
Introduction The pathology of rotavirus infections has been studied in man (8), calves (10, 15), piglets (5) and mice (1). The techniques used have been immunofluorescenee, histopathology, and electron microscopy, but only the observations in calves (10, I5) have utilised all 3 techniques. In all species examinations have been made only during a limited period in the course of the clinical disease. Immunofinoreseenee studies demonstrated that rotaviruses infected the villous epithelial eells of the small intestine, and it was suggested that a wave of infection in these cells passed along the small intestine from the anterior end (10). Principal histopathologieal changes recorded were shortening of villi, loss of columnar cells from villi and their replacement with euboidal cells (5, 10). Ultrastrnctural studies revealed damage to the mierovilli of the villous epithelial cells and the presence of virus particles within eisternae of the rough endoplasmie retieulum (1, 5, 8, 15). This paper describes the pathology of lamb rotavirus infections, studied by immunofluoreseenee, histopathology and electron microscopy from the incubation period to recovery.
264
D . R . SNODGRASS, K. W. ANGUS, and E. W. GRAY:
Materials and Methods Animals E i g h t gnotobiotic lambs were infected orally w h e n 2 - - 4 days old w i t h 2 - - 3 ml of a bacteria-free 20 per cent faecal filtrate containing l a m b r o t a v i r u s from t h e second, third, or f o u r t h gnotobiotie l a m b passage (13). One l a m b was killed at each of t h e following hours after infection (p.i.) : 12, 18, 27, 42, 48, 72, 96 a n d t44. T w o gnotobiotic lambs were k e p t as u n i n f e c t e d controls ; one was killed at 4 and t h e other at 6 days of age.
Necrop~'y Procedures L a m b s were deeply anaesthetised -with sodium pentobarbitone. S e g m e n t s were o b t a i n e d from spiral colon, caecum and from 3 sites from small intestine: anterior, a p p r o x i m a t e l y 20 cm posterior to t h e pylorus; middle, a p p r o x i m a t e l y e q u i d i s t a n t from t h e pylorus a n d the ileo-caecal j u n c t i o n ; and posterior, a p p r o x i m a t e l y 20 cm anterior to t h e ileo-caecal junction. The lambs were t h e n killed by exsanguina~ion and gut contents collected. I n addition, tissues were t a k e n f r o m the a b o m a s a l fold, kidney, liver, lung, mesenteric l y m p h node, m y o c a r d i u m and spleen.
Histological and Ultrastructural Methods Segments of small intestine and some colon segments were fixed i m m e d i a t e l y by i m m e r s i o n in t per c e n t g l u t a r a l d e h y d e in p h o s p h a t e buffer (pI-I 7.4). Slices of mueosa 1 m m t h i c k were t a k e n for electron microscopy (EM) and t h e r e m a i n d e r of t h e tissue transferred to 10 per cent buffered formal-saline for histology. All o t h e r tissues were collected directly into formal-sMine. A d d i t i o n a l portions of all tissues were frozen in a CO~dsopentane freezing m i x t u r e prior to storage at - - 7 0 ° C for subsequent i m m u n e fluorescent (IF) examination. Following post-fixation in a modified Bouin's f i x a t i v e (190 parts sat. aq. picrie acid, 10 parts 40 per cent formaldehyde, 5 parts gla~iM acetic acid) tissues were processed to paraffin-wax, a n d 5 ixm sections cut and stained b y Mayer's h a e m a l u m and eosin (HE). Other stains used were Pollak's t r i c h r o m e and t h e periodic acid-Schiff (PAS) technique. Selected areas of mucosa were post-fixed in o s m i u m t e t r o x i d e and processed t h r o u g h graded alcohols for e m b e d d i n g in Araldite. Suitable areas for u l t r a t h i n sectioning were selected f r o m Giemsa-stained 1 p.m Araldite sections.
Immuno]hl.orescence F r o z e n tissues were m o u n t e d on m i c r o t o m e chucks a n d 6 ~xm sections cut on a cryostat. Tissues were stained w i t h gnotobiotic l a m b a n t i s e r u m to l a m b rotavirus, followed by fluorescein-conjugated rabbit anti-sheep globulin. Control sections were stained directly w i t h the conjugated globulin.
Virus Isolation F a e c a l samples collected daily a n d intestinal c o n t e n t s t a k e n a t necropsy were e x a m i n e d for rotavirus b y immunofluorescence on cell cultures (14).
Bacteriology F a e c a l swabs were t a k e n at intervals during infection and at necropsy. No bacteria were isolated from t h e lambs killed 12, 18, 27, 42 a n d 48 hours p.i. Staphylococcus epidermidis was isolated f r o m lambs killed at. 72 and 96 hem's p.i., a Microeoccus from t h e l a m b killed at 144 hours p.i., and a Bacillus sp. f r o m t h e two control lambs. No pathogenic significance is a t t a c h e d to any of these bacteria.
Results Clinical and Virological Studies T h e l a m b k i l l e d ]2 h o u r s p . i . w a s c l i n i c a l l y n o r m a l . T h e o t h e r s e v e n i n f e c t e d l a m b s d e v e ] o p e d d i a r r h o e a a t 1 5 - - 2 0 h o u r s p.i., a n d t w o of t h e s e w e r e also dull a n d s h o w e d a b d o m i n a l d i s c o m f o r t . All i n f e c t e d l a m b s h a d d i a r r h o e a u n t i l t h e
Pathogenesis of Lamb Rotavirus Infections
265
time of d e a t h except for the lamb killed 144 hours p.i. The control lambs were normal t h r o u g h o u t the experimental period. No rotavirus was detected in the faeces of the lambs before infection, t o r n virus was isolated from faeces of all diarrhoeic lambs and at necropsy from intestinal contents of all infected lambs except t h a t killed 144 hours p.i. No virus was isolated from the uninfected control lambs.
Immuno[luorescence Specific I F staining was deteete=l in the epithelial cells of the villi of the middle and posterior small intestine, and to a lesser exten~ in the anterior small intestine, caecum and colon (Table 1). Scattered cells in the lamina propria, presumably eosinophils, also fluoresced. The lamb killed 12 hours p.i. showed fluorescence in the epithelial cells covering the apical half or more of most villi in the middle and posterior small intestine (Fig. 1). B y 18 hours p.i., fluorescence was observed in cells only over the tips of the villi (Figs. 2 and 3). Thereafter single fluorescent cells appeared sporadically on villi until 96 hours p.i. (Fig. 4). No specific fluorescence was detected in a n y tissue other t h a n the intestine, nor in a n y tissue from the control lambs. I F staining was not evaluated in the spleen and mesenterie l y m p h node due to non-specific staining. Table 1. tmmuno]Iuorescent staining o/ lamb intestine/or rotavirus antigen Time killed (hours p.i.)
Anterior
Middle
Large intestine Posterior
Colon
Caecum
12
_
+ ÷ + + a
+ + + +
_
_
lS
÷++b ++c
+++ ++
++ +
++ ++
27
++ _
42
+
+a
_
_
+ +
48
--
+
+
..+
--
--
+
72
96 144 Control Control
a b ¢ a
Small intestine
--+
.
+
+
.
.
.
.
+
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+ + + + Continuous fluorescent epithelial cells present over at teasf distal half of villi + + + Continuous fluorescent epithelial cells present over tip or distal third of villi @+ Sporadic fluorescent epitheliM cells present in most villi + Sporadic fluorescent epithelial cells present in a few villi
Pathology Focal congestion of the middle small intestine was found in the lamb killed at 12 hours p.i., while a more diffuse congestion of the small intestine and colon was present in the lamb which was scouring when killed at 18 hours p.i. At 12 hours p.i., the anterior lobes of both lungs were deep pink and p a r t l y consolidated. Similar areas were seen in the anterior lobes of both lungs of the lamb killed 72 hours p.i. Histopathotogical lesions in small intestine at 12 hours p.i. were confined to middle and posterior sites, in which the villi were swollen and spatulate and the
266
D . R . SNODC
Archives of Virology © by Springer-Verlag 1977
Rotavirus Infection in Lambs: Pathogenesis and Patholoqy By D. R. SNODGRASS,K. W. A~Gus, and E. W. G~A¥ Animal Diseases Research Association, Moredun Institute, Edinburgh, Scotland W'ith 11 Figures Accepted June 21, 1977
Summary Experimental lamb rotavirus infections were studied by immunofluoreseenee, histopathology and electron microscopy of tissues from infected gnotobiotic lambs killed at intervals from the incubation period to recovery. The rotavirus was demonstrated by imnmnofluoreseence only in epithelial ceils of villi in the small and large intestine, and virus antigen was most abundant during the incubation period. An increased enterocyte turnover rate was suggested by the rapid movement of virus-infected cells to the villus tip, and this increase m a y be one of the basic pathogenic mechanisms of rotavirus infection. Principal histopathological changes were shortening of villi and sloughing of epithelial cells. These were greatest in the middle and posterior small intestine at the onset of diarrhoea, but regeneration occurred within a few hours. Virus morphology in tissues -was similar to that reported in other species, and virus presence correlated well with histopathologieaI change.
Introduction The pathology of rotavirus infections has been studied in man (8), calves (10, 15), piglets (5) and mice (1). The techniques used have been immunofluorescenee, histopathology, and electron microscopy, but only the observations in calves (10, I5) have utilised all 3 techniques. In all species examinations have been made only during a limited period in the course of the clinical disease. Immunofinoreseenee studies demonstrated that rotaviruses infected the villous epithelial eells of the small intestine, and it was suggested that a wave of infection in these cells passed along the small intestine from the anterior end (10). Principal histopathologieal changes recorded were shortening of villi, loss of columnar cells from villi and their replacement with euboidal cells (5, 10). Ultrastrnctural studies revealed damage to the mierovilli of the villous epithelial cells and the presence of virus particles within eisternae of the rough endoplasmie retieulum (1, 5, 8, 15). This paper describes the pathology of lamb rotavirus infections, studied by immunofluoreseenee, histopathology and electron microscopy from the incubation period to recovery.
264
D . R . SNODGRASS, K. W. ANGUS, and E. W. GRAY:
Materials and Methods Animals E i g h t gnotobiotic lambs were infected orally w h e n 2 - - 4 days old w i t h 2 - - 3 ml of a bacteria-free 20 per cent faecal filtrate containing l a m b r o t a v i r u s from t h e second, third, or f o u r t h gnotobiotie l a m b passage (13). One l a m b was killed at each of t h e following hours after infection (p.i.) : 12, 18, 27, 42, 48, 72, 96 a n d t44. T w o gnotobiotic lambs were k e p t as u n i n f e c t e d controls ; one was killed at 4 and t h e other at 6 days of age.
Necrop~'y Procedures L a m b s were deeply anaesthetised -with sodium pentobarbitone. S e g m e n t s were o b t a i n e d from spiral colon, caecum and from 3 sites from small intestine: anterior, a p p r o x i m a t e l y 20 cm posterior to t h e pylorus; middle, a p p r o x i m a t e l y e q u i d i s t a n t from t h e pylorus a n d the ileo-caecal j u n c t i o n ; and posterior, a p p r o x i m a t e l y 20 cm anterior to t h e ileo-caecal junction. The lambs were t h e n killed by exsanguina~ion and gut contents collected. I n addition, tissues were t a k e n f r o m the a b o m a s a l fold, kidney, liver, lung, mesenteric l y m p h node, m y o c a r d i u m and spleen.
Histological and Ultrastructural Methods Segments of small intestine and some colon segments were fixed i m m e d i a t e l y by i m m e r s i o n in t per c e n t g l u t a r a l d e h y d e in p h o s p h a t e buffer (pI-I 7.4). Slices of mueosa 1 m m t h i c k were t a k e n for electron microscopy (EM) and t h e r e m a i n d e r of t h e tissue transferred to 10 per cent buffered formal-saline for histology. All o t h e r tissues were collected directly into formal-sMine. A d d i t i o n a l portions of all tissues were frozen in a CO~dsopentane freezing m i x t u r e prior to storage at - - 7 0 ° C for subsequent i m m u n e fluorescent (IF) examination. Following post-fixation in a modified Bouin's f i x a t i v e (190 parts sat. aq. picrie acid, 10 parts 40 per cent formaldehyde, 5 parts gla~iM acetic acid) tissues were processed to paraffin-wax, a n d 5 ixm sections cut and stained b y Mayer's h a e m a l u m and eosin (HE). Other stains used were Pollak's t r i c h r o m e and t h e periodic acid-Schiff (PAS) technique. Selected areas of mucosa were post-fixed in o s m i u m t e t r o x i d e and processed t h r o u g h graded alcohols for e m b e d d i n g in Araldite. Suitable areas for u l t r a t h i n sectioning were selected f r o m Giemsa-stained 1 p.m Araldite sections.
Immuno]hl.orescence F r o z e n tissues were m o u n t e d on m i c r o t o m e chucks a n d 6 ~xm sections cut on a cryostat. Tissues were stained w i t h gnotobiotic l a m b a n t i s e r u m to l a m b rotavirus, followed by fluorescein-conjugated rabbit anti-sheep globulin. Control sections were stained directly w i t h the conjugated globulin.
Virus Isolation F a e c a l samples collected daily a n d intestinal c o n t e n t s t a k e n a t necropsy were e x a m i n e d for rotavirus b y immunofluorescence on cell cultures (14).
Bacteriology F a e c a l swabs were t a k e n at intervals during infection and at necropsy. No bacteria were isolated from t h e lambs killed 12, 18, 27, 42 a n d 48 hours p.i. Staphylococcus epidermidis was isolated f r o m lambs killed at. 72 and 96 hem's p.i., a Microeoccus from t h e l a m b killed at 144 hours p.i., and a Bacillus sp. f r o m t h e two control lambs. No pathogenic significance is a t t a c h e d to any of these bacteria.
Results Clinical and Virological Studies T h e l a m b k i l l e d ]2 h o u r s p . i . w a s c l i n i c a l l y n o r m a l . T h e o t h e r s e v e n i n f e c t e d l a m b s d e v e ] o p e d d i a r r h o e a a t 1 5 - - 2 0 h o u r s p.i., a n d t w o of t h e s e w e r e also dull a n d s h o w e d a b d o m i n a l d i s c o m f o r t . All i n f e c t e d l a m b s h a d d i a r r h o e a u n t i l t h e
Pathogenesis of Lamb Rotavirus Infections
265
time of d e a t h except for the lamb killed 144 hours p.i. The control lambs were normal t h r o u g h o u t the experimental period. No rotavirus was detected in the faeces of the lambs before infection, t o r n virus was isolated from faeces of all diarrhoeic lambs and at necropsy from intestinal contents of all infected lambs except t h a t killed 144 hours p.i. No virus was isolated from the uninfected control lambs.
Immuno[luorescence Specific I F staining was deteete=l in the epithelial cells of the villi of the middle and posterior small intestine, and to a lesser exten~ in the anterior small intestine, caecum and colon (Table 1). Scattered cells in the lamina propria, presumably eosinophils, also fluoresced. The lamb killed 12 hours p.i. showed fluorescence in the epithelial cells covering the apical half or more of most villi in the middle and posterior small intestine (Fig. 1). B y 18 hours p.i., fluorescence was observed in cells only over the tips of the villi (Figs. 2 and 3). Thereafter single fluorescent cells appeared sporadically on villi until 96 hours p.i. (Fig. 4). No specific fluorescence was detected in a n y tissue other t h a n the intestine, nor in a n y tissue from the control lambs. I F staining was not evaluated in the spleen and mesenterie l y m p h node due to non-specific staining. Table 1. tmmuno]Iuorescent staining o/ lamb intestine/or rotavirus antigen Time killed (hours p.i.)
Anterior
Middle
Large intestine Posterior
Colon
Caecum
12
_
+ ÷ + + a
+ + + +
_
_
lS
÷++b ++c
+++ ++
++ +
++ ++
27
++ _
42
+
+a
_
_
+ +
48
--
+
+
..+
--
--
+
72
96 144 Control Control
a b ¢ a
Small intestine
--+
.
+
+
.
.
.
.
+
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+ + + + Continuous fluorescent epithelial cells present over at teasf distal half of villi + + + Continuous fluorescent epithelial cells present over tip or distal third of villi @+ Sporadic fluorescent epitheliM cells present in most villi + Sporadic fluorescent epithelial cells present in a few villi
Pathology Focal congestion of the middle small intestine was found in the lamb killed at 12 hours p.i., while a more diffuse congestion of the small intestine and colon was present in the lamb which was scouring when killed at 18 hours p.i. At 12 hours p.i., the anterior lobes of both lungs were deep pink and p a r t l y consolidated. Similar areas were seen in the anterior lobes of both lungs of the lamb killed 72 hours p.i. Histopathotogical lesions in small intestine at 12 hours p.i. were confined to middle and posterior sites, in which the villi were swollen and spatulate and the
266
D . R . SNODC
