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Enterocolitis in piglets caused by Cryptosporidium sp. purified from calf faeces
Veterinary Parasitology, 11 (1982) 121--126 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
121
E N T E R O C O L I T I S I N P I G L E T S C A U S E D BY CR Y P T O S P O R I D I U M SP. PURIFIED FROM CALF FAECES
S. TZIPORI, M. SMITH, T. MAKIN and C. HALPIN Department of Agriculture, A ttwood Veterinary Research Laboratory, Mickleham Road, Westmeadows, Victoria 304 7 (Australia) (Accepted for publication 15 March 1982)
ABSTRACT Tzipori, S., Smith, M., Makin, T. and Halpin, C., 1982. Enterocolitis in piglets caused by Cryptosporidium sp. purified from calf faeces. Vet. Parasitol., 11: 121--126. Twelve gnotobiotic piglets were dosed with a bacteria-free calf faecal homogenate which contained Cryptosporidium oocysts. The infection induced severe enterocolitis in piglets when inoculated at 1 day of age, moderate diarrhoea at 7 days of age and a subclinical infection at 15 days of age. In piglets aged 3 days or less, the entire intestine was extensively infected with Cryptosporidium and the mucosa was severely damaged. In piglets 7 days of age or older, the upper small intestine was sparsely infected with the organisms, but the ileum and the large bowel were heavily infected with associated mucosal damage. INTRODUCTION I n v e s t i g a t i o n s i n t o t h e a e t i o l o g y o f i n f e c t i o u s enteritis in y o u n g animals and h u m a n s o v e r t h e last 10 y e a r s h a v e identified m a n y p o t e n t i a l p a t h o g e n s . E n t e r o p a t h o g e n i c b a c t e r i a and viruses a f f e c t i n g o n e species o f a n i m a l w e r e o f t e n s u b s e q u e n t l y r e p o r t e d t o o c c u r in others, and k n o w l e d g e o b t a i n e d f r o m studies carried o u t in o n e species was o f t e n utilized to i n t e r p r e t the m a n i f e s t a t i o n o f disease in others. T h e p r o t o z o o n , Cryptosporidium, has r e c e n t l y b e e n s h o w n t o be associated w i t h d i a r r h o e a in a n u m b e r o f a n i m a l species (Tzipori et al., 1981a, c) p a r t i c u l a r l y calves (l~orin et al., 1 9 7 8 ; M o o n et al., 1 9 7 8 ; P o h l e n z et al., 1 9 7 8 ; N a g y et al., 1 9 8 0 ; Snodgrass et al., 1 9 8 0 ; T z i p o r i et al., 1 9 8 0 b ) . Since Cryptosporidium has b e e n r e p o r t e d in piglets ( K e n n e d y et al., 1 9 7 7 ; Bergeland, 1 9 7 7 ) a n d i n f e c t i o n a p p e a r s to be p r e v a l e n t in t h e swine p o p u l a t i o n ( T z i p o r i a n d C a m p b e l l , 1 9 8 1 ) t h e role o f this a g e n t in piglet enteritis s h o u l d b e e x a m i n e d . Cryptosporidium a p p e a r s to lack h o s t specificity ( T z i p o r i et al., 1 9 8 0 a ) and t r a n s m i s s i o n o f t h e o r g a n i s m b e t w e e n calves a n d pigs is readily d e m o n s t r a t e d {Moon and B e m r i c k , 1 9 8 1 ; T z i p o r i et al., 1 9 8 1 d ) . H o w e v e r , t h e i n o c u l a c o n t a i n i n g t h e Cryptosporidium used in these e x p e r i m e n t s c o n t a i n e d o t h e r enteric m i c r o o r g a n i s m s . In this s t u d y we e x a m i n e the age s u s c e p t i b i l i t y
0304-4017/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
122 of unstressed piglets to infection with an inoculum containing calf Cryptosporidium oocysts which, we believe, was free of other microorganisms. MATERIALS AND METHODS
Inoculum The Cryptosporidium used in this experiment was derived from a calf with diarrhoea and after one passage in specific-pathogen-free (SPF) mice, it was fed to a day-old gnotobiotic (GB) calf (Tzipori et al., 1982). Four ml of faecal homogenate (20% v/v in phosphate-buffered saline) from this calf, which contained Cryptosporidium oocysts, were mixed with 6 ml of absolute alcohol. Bacterial sterility was checked 2 h later after which the following antibiotics were added: 200 mg benzyl penicillin, 200 mg streptomycin sulphate B.P. and 40 mg gentamycin sulphate. The mixture was fed to 2-, 3-day-old GB piglets. The gut contents of Piglet 3 (Table I) were homogenised (20% v/v in phosphate-buffered saline), dispensed into 4-ml aliquots and stored at 4°C. The aliquots were used to inoculate 10 GB piglets. TABLE I C l i n i c a l r e s p o n s e o f t w e l v e g n o t o b i o t i e p i g l e t s 3 i n o c u l a t e d w i t h Cryptosporidium o r i g i n a l l y d e r i v e d from a calf
Pig n o .
1 2 3 4 5 6 7 8 9 10 11 12
A g e at inoculation (days)
Incubation (days)
Duration (days)
Anorexia
Diarrhoea
Anorexia
1 1 3 3 7 7 7 7 7 7 15 15
3 2 1 4 0 4 4 3 0 0 0 0
3 3 2 4 3 4 4 3 3 4 0 0
1 3 3 3 0 0 1 3 1 1 0 0
Diarrhoea
Killed after inoculation (days)
Excretion of oocysts in the faeces (days)
1 2 2 4 3 3 3 4 2 3 0 0
4** 43** 4*** 10 8 7 8 9 10 7 5 26
2 2 2 7 7 4 4 3 3 2 2 6
*Two uninoculated GB piglets from the same litter remained clinically normal and no oocysts were d e t e c t e d in t h e i r f a e c e s . , *Died. ***Killed when moribund.
Oocyst count A known volume of gut content homogenate (5 t~l) from Piglet 3 was smeared onto a microscope glass slide, stained with Giemsa and the number of oocysts present in the smear was counted. The number of oocysts per 4 ml inoculum was estimated to be between 20 X 103 and 35 X 103.
Experimental procedure Fourteen piglets derived from 2 litters were reared under gnotobiotic conditions as described elsewhere (Makin and Tzipori, 1980). The piglets were housed in pairs and inoculated at the age of: 1 day old (Piglets 1 and 2),
123 3 days old (3 and 4), 7 days old (5--10) and 15 days old (11 and 12). The piglets were observed 3 to 4 times daily for signs of illness, faecal swabs were taken once or twice daily until the piglets were killed, and the milk intake was recorded. Two piglets were kept as uninoculated controls.
Necropsy The piglets were anaesthetised at certain intervals after inoculation (Table I) and portions of gut were taken before the animals were killed. Sections were taken for histology, transmission and scanning electron microscopy (EM), and lactase analysis from 5 equally spaced sites along the small intestine and 2 sites from the large intestine (Tzipori et al., 1981b).
• "icrobiology Inocula, daily faecal samples and gut contents of killed piglets were examined routinely for the presence of enteric viruses and bacteria as described previously (Tzipori et al., 1980b). Cryptosporidium oocysts were detected by Giemsa staining of faecal smears. RESULTS Treatment of the calf faecal homogenate with absolute alcohol (60% final concentration), resulted in an inoculum which contained Cryptosporidium oocysts, b u t following bacterial sterility checks was believed to be free of other microorganisms. Some of the oocysts lost their smooth outline during the treatment, but the majority remained morphologically intact. Piglets 3 and 4, which were fed the alcohol-treated inoculum, became unsteady on their feet and remained depressed for 4--5 h, after which time they recovered completely. Faecal swabs obtained twice daily from these 2 piglets showed that they were bacteria-free, and 3 days after inoculation Cryptosporidium oocysts were the only organisms detected in the faeces.
Clinical observation An incubation period of 1--4 days was observed in piglets that developed clinical signs. The onset of diarrhoea and anorexia, the duration of illness and the excretion of oocysts in the faeces are summarised in Table I. Piglets 1 and 2, inoculated 6 h after birth, vomited 2 and 3 days after inoculation, respectively. They suffered severe diarrhoea that resulted in dehydration and the death of Piglet 1, and the near death of Piglet 2. The severity of illness of Piglets 3 and 4 was similar to that seen in Piglets 1 and 2. Piglets 5--10, inoculated at 7 days of age developed moderate diarrhoea for 2--4 days and none of these piglets died. Piglets 11 and 12, aged 15 days when inoculated, remained clinically healthy although Cryptosporidium oocysts were detected in their faeces 3 days after inoculation. The 2 uninoculated piglets remained clinically healthy and no oocysts were detected in their faeces throughout the observation period.
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124
Histopathological findings The extent of Cryptosporidium infection observed in the intestine by light microscopy varied from slight to heavy depending on time of necropsy after inoculation and the intestinal site examined. In Piglets 1, 2 and 3, heavy infection of the brush border of the enterocytes extended from the d u o d e n u m to the rectum. In Piglets 4--11 the infection was either patchy or absent in the first 3 sites, but heavy in the ileum and large intestine. Piglet 12, which was killed 26 days after inoculation, was free of infection. In the ileum and large bowel there was no difference in the degree of infection between piglets inoculated at the age of 1 or 15 days. The organisms coated the entire villi, but were in higher concentrations at the tips and in some sections of the ileum and in the large intestine cryptosporidia were observed embedded in the crypts. Mucosal lesions of stunted and fused villi, oedema and cellular infiltrations of the lamina propria, were most consistently observed in the middle and lower small intestine These changes increased in severity towards the terminal ileum where the loss of healthy vacuolated villous cells and their replacement by immature low enterocytes was more evident. No bacterial adherence was observed in any of the gut sections. Transmission and scanning EM studies have revealed organisms with internal structures consistent with Cryptosporidium which have previously been documented in experimentally-inoculated piglets (Tzipori et al., 1981d). The 2 uninoculated piglets which were killed aged 10 or 15 days, had long slender villi throughout the small intestine with no evidence of inflammation or adherence of microorganisms to the brush-border of enterocytes.
Measurement of lactase activity Table II gives details of measurement of lactase activity at 5 different sites along the small intestine. It is apparent from the table that piglets inoculated at 3 days of age had the lowest level of activity, followed by piglets inoculated at 7 days. All 3 groups of piglets, however, were well below that recorded for the control piglets. TABLE n
Lactase activity ( m e a n s -+SE) a t 5 different sites equally spaced f r o m the p y l o r o u s region to t e r m i n a l i l e u m c o l l e c t e d f r o m 10 i n f e c t e d and 2 u n i n o c u l a t e d piglets Small intestinal sites ( m e a n lactase a c t i v i t y / ~ m o l e s / m i n / g w e t w t . )
Killed
No. of pigs/ group
1
2
3
4
5
3 7
7,13 14--17
2 6
15 C**
20,41 * 10,15
2 2
2.02 6.90 (+1.93) 8.19 14.12
1.74 7.42 (-+2.47) 10.93 19.33
2.34 4.82 (-+2.43) 8.69 17.74
0.43 5.13 (-+1.89) 3.48 13.33
1.94 2.98 (-+2.34) 1.72 4.56
Age ( d a y s ) w h e n
Inoculated
* A l t h o u g h Pig 12 w a s 41 d a y s old w h e n it was killed t h e values w e r e similar to its litter m a t e , i n d i c a t i n g possible residual d a m a g e . * * C o n t r o l piglets.
125 DISCUSSION During the last two years, transmission of Cryptosporidium to a variety of animal species has been reported (Tzipori et al., 1980a; ~,,oon and Bemrick, 1981). In these transmission experiments the inocula used, although shown to be free of other known enteropathogens, contained other microorganisms which rendered the results less conclusive. As far as we are aware this is the first experiment where an inoculum has been used which, we believe, contained only Cryptosporidium oocysts. The results confirmed previously reported observations that Cryptosporidium is capable of inducing diarrhoea in some mammalian species. The bacteria-free Cryptosporidium also induced enterocolitis in calves (Tzipori et al., 1982). Alcohol was selected to purify Cryptosporidium because, of all the disinfectants that were found to be ineffective against the oocysts, it was the least toxic to piglets (Campbell et al., 1982). There has been an a t t e m p t in this experiment to quantitate the inoculum by estimating the number of oocysts present in small volumes (5 ~) of diluted samples. The technique can be further improved by using a graduated microscopic slide where a number of squares can then be nominated for counting. This technique is probably only useful in assessing the infectious dose of faecal homogenate; gut contents or ileal scrapings, no doubt, contain intermediate stages of the life cycle which may also be infectious and not be expressed in terms of infectious dose. It appears from this experiment that unstressed piglets, devoid of maternal protection, become clinically resistant to infection with Cryptosporidium within 15 days. It remains to be seen whether clinical cryptosporidiosis in older piglets, like infection with other enteropathogens, can be precipitated by stress factors such as early weaning. Clinical illness attributed to cryptosporidiosis in pigs, however, does not appear to be c o m m o n , probably because of the prevalence of infection in pigs (Tzipori and Campbell, 1981; Tzipori et al., 1981d) which could mean that enough maternal antibody is received by the newborn piglet to protect it through a short period of susceptibility. The mucosal changes observed in GB piglets after inoculation with Cryptosporidium, were consistent with previous, more extensive descriptions of experimental infection of conventional piglets (Tzipori et al., 1981d). The extent of infection and damage in the ileum and large bowel did not vary between piglets inoculated at the age of 1 or 15 days. The difference was the involvement of the upper small intestine in the younger piglets, which could be associated with the manifestation of clinical disease in this age group due to probable impairment of digestion as well as absorption. In older piglets where the ileum was most severely affected the membrane bound enzymes were, to a great extent, spared and therefore these piglets suffered malabsorption only. This is also evident from the levels of lactase activity measured in the older piglets; although they were lower than the controls, they were considerably higher than in the 3-day-old piglets.
126 ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k Ms. J u l l B i l l i n g t o n a n d Ms. K a r e n W i l s o n f o r t e c h n i c a l a s s i s t a n c e a n d Ms. M a r g a r e t P a r k i n s o n f o r t y p i n g t h e m a n u s c r i p t . T h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m t h e A u s t r a l i a n Pig I n d u s t r y R e s e a r c h Committee.
REFERENCES Bergeland, M.G., 1977. Necrotic entertis in nursing piglets. Proc. Am. Assoc. Vet. Lab. Diagn., 20: 151--158. Campbell, I., Tzipori, S., Hutchison, G. and Angus, K.W., 1982. The effect of disinfectant on survival of Cryptosporidium oocysts. Vet. Rec., in press. Kennedy, G.A., Kreitner, G.L. and Strafuss, A.C., 1977. Cryptosporidiosis in three pigs. J. Am. Vet. Med. Assoc., 170: 348--350. Makin, T. and Tzipori, S., 1980. Inexpensive techniques for the production and maintenance of gnotobiotic piglets, calves and lambs. Aust. Vet. J., 56: 353--359. Moon, H.W. and Bemrick, W.J., 1981. Faecal transmission of calf cryptosporidia between calves and pigs. Vet. Pathol., 18 : 248--255. Moon, H.W., McClurkin, A.W., Isaacson, R.E., Pohlenz, J., Skartvedt, S.M., Gilette, K.G. and Baetz, A.L., 1978. Pathological relationships of rotavirus Escherichia coli and other agents in mixed infections in calves. J. Am. Vet. Med. Assoc., 193: 577--583. Morin, M., Larivi~re, S., Lalier, R., Begin, M., Roy, R. and Ethier, R., 1978. Neonatal calf diarrhoea; pathology and microbiology of spontaneous cases in dairy herds and incidence of the enteropathogen implicated as aetiological agents. Proc. 2nd Symp. Neonatal Diarrhoea. (Veterinary Infectious Discases Organisation), Saskatoon, pp. 347--365. Nagy, B., Anthal, A. and Lakner, J., 1980. Significance of intestinal cryptosporidiosis in calf diarrhoea. Proc. 2nd Int. Symp. Vet. Lab. Diagn., Switzerland, pp. 431--434. Pohlenz, J., Moon, H.W., Chenille, N.F. and Bemrick, W.J., 1978. Cryptosporidiosis as a probable factor in neonatal diarrhoea in calves. J. Am. Vet. Med. Assoc., 171: 452--457. Snodgrass, D.R., Angus, K.W., Gray, E.W., Keir, W.A. and Clerihew, L.W., 1980. Cryptosporidia associated with rotavirus and Escherichia coli in an outbreak of calf scour. Vet. Rec., 106: 458--459. Tzipori, S. and Campbell, I., 1981. Prevalence of Cryptosporidium antibodies in ten species of animal. J. Clin. Microbiol., 14: 455--456. Tzipori, S., Angus, K.W., Campbell, I. and Gray, E.W., 1980a. Cryptosporidium: Evidence for a single species genus. Infect. Immunol., 30: 884--886. Tzipori, S., Campbell, I., Sherwood, D., Snodgrass, D.R. and Whitelaw, A., 1980b. An outbreak of calf diarrhoea attributed to cryptosporidial infection. Vet. Rec., 107: 579--580. Tzipori, S., Angus, K.W., Campbell, I. and Clerihew, L., 1981a. Diarrhoea in lambs due to Cryptosporidium infection. J. Clin. Microbiol., 14: 100--105. Tzipori, S., Angus, K.W., Gray, E.W. and Campbell, I., 1981b. Diarrhoea in lambs experimentally-infected with Cryptosporidium isolated from calves. Am. J. Vet. Res., 42: 1400--1404. Tzipori, S., Angus, K.W., Campbell, I. and Sherwood, D., 1981c. Diarrhoea in young deer associated with Cryptosporidium. J. Infect. Dis., 144: 170--175. Tzipori, S., McCartney, E., Lawson, G.H.K., Rowland, A.C. and Campbell, I., 1981d. Experimental inoculation of piglets with Cryptosporidium. Res. Vet. Sci., 31: 358--368. Tzipori, S., Angus, K.W., Sherwood, D. and Campbell, I., 1982. Experimental Cryptosporidium in calves: clinical manifestation and pathological findings. Vet. Rec., in press.
121
E N T E R O C O L I T I S I N P I G L E T S C A U S E D BY CR Y P T O S P O R I D I U M SP. PURIFIED FROM CALF FAECES
S. TZIPORI, M. SMITH, T. MAKIN and C. HALPIN Department of Agriculture, A ttwood Veterinary Research Laboratory, Mickleham Road, Westmeadows, Victoria 304 7 (Australia) (Accepted for publication 15 March 1982)
ABSTRACT Tzipori, S., Smith, M., Makin, T. and Halpin, C., 1982. Enterocolitis in piglets caused by Cryptosporidium sp. purified from calf faeces. Vet. Parasitol., 11: 121--126. Twelve gnotobiotic piglets were dosed with a bacteria-free calf faecal homogenate which contained Cryptosporidium oocysts. The infection induced severe enterocolitis in piglets when inoculated at 1 day of age, moderate diarrhoea at 7 days of age and a subclinical infection at 15 days of age. In piglets aged 3 days or less, the entire intestine was extensively infected with Cryptosporidium and the mucosa was severely damaged. In piglets 7 days of age or older, the upper small intestine was sparsely infected with the organisms, but the ileum and the large bowel were heavily infected with associated mucosal damage. INTRODUCTION I n v e s t i g a t i o n s i n t o t h e a e t i o l o g y o f i n f e c t i o u s enteritis in y o u n g animals and h u m a n s o v e r t h e last 10 y e a r s h a v e identified m a n y p o t e n t i a l p a t h o g e n s . E n t e r o p a t h o g e n i c b a c t e r i a and viruses a f f e c t i n g o n e species o f a n i m a l w e r e o f t e n s u b s e q u e n t l y r e p o r t e d t o o c c u r in others, and k n o w l e d g e o b t a i n e d f r o m studies carried o u t in o n e species was o f t e n utilized to i n t e r p r e t the m a n i f e s t a t i o n o f disease in others. T h e p r o t o z o o n , Cryptosporidium, has r e c e n t l y b e e n s h o w n t o be associated w i t h d i a r r h o e a in a n u m b e r o f a n i m a l species (Tzipori et al., 1981a, c) p a r t i c u l a r l y calves (l~orin et al., 1 9 7 8 ; M o o n et al., 1 9 7 8 ; P o h l e n z et al., 1 9 7 8 ; N a g y et al., 1 9 8 0 ; Snodgrass et al., 1 9 8 0 ; T z i p o r i et al., 1 9 8 0 b ) . Since Cryptosporidium has b e e n r e p o r t e d in piglets ( K e n n e d y et al., 1 9 7 7 ; Bergeland, 1 9 7 7 ) a n d i n f e c t i o n a p p e a r s to be p r e v a l e n t in t h e swine p o p u l a t i o n ( T z i p o r i a n d C a m p b e l l , 1 9 8 1 ) t h e role o f this a g e n t in piglet enteritis s h o u l d b e e x a m i n e d . Cryptosporidium a p p e a r s to lack h o s t specificity ( T z i p o r i et al., 1 9 8 0 a ) and t r a n s m i s s i o n o f t h e o r g a n i s m b e t w e e n calves a n d pigs is readily d e m o n s t r a t e d {Moon and B e m r i c k , 1 9 8 1 ; T z i p o r i et al., 1 9 8 1 d ) . H o w e v e r , t h e i n o c u l a c o n t a i n i n g t h e Cryptosporidium used in these e x p e r i m e n t s c o n t a i n e d o t h e r enteric m i c r o o r g a n i s m s . In this s t u d y we e x a m i n e the age s u s c e p t i b i l i t y
0304-4017/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
122 of unstressed piglets to infection with an inoculum containing calf Cryptosporidium oocysts which, we believe, was free of other microorganisms. MATERIALS AND METHODS
Inoculum The Cryptosporidium used in this experiment was derived from a calf with diarrhoea and after one passage in specific-pathogen-free (SPF) mice, it was fed to a day-old gnotobiotic (GB) calf (Tzipori et al., 1982). Four ml of faecal homogenate (20% v/v in phosphate-buffered saline) from this calf, which contained Cryptosporidium oocysts, were mixed with 6 ml of absolute alcohol. Bacterial sterility was checked 2 h later after which the following antibiotics were added: 200 mg benzyl penicillin, 200 mg streptomycin sulphate B.P. and 40 mg gentamycin sulphate. The mixture was fed to 2-, 3-day-old GB piglets. The gut contents of Piglet 3 (Table I) were homogenised (20% v/v in phosphate-buffered saline), dispensed into 4-ml aliquots and stored at 4°C. The aliquots were used to inoculate 10 GB piglets. TABLE I C l i n i c a l r e s p o n s e o f t w e l v e g n o t o b i o t i e p i g l e t s 3 i n o c u l a t e d w i t h Cryptosporidium o r i g i n a l l y d e r i v e d from a calf
Pig n o .
1 2 3 4 5 6 7 8 9 10 11 12
A g e at inoculation (days)
Incubation (days)
Duration (days)
Anorexia
Diarrhoea
Anorexia
1 1 3 3 7 7 7 7 7 7 15 15
3 2 1 4 0 4 4 3 0 0 0 0
3 3 2 4 3 4 4 3 3 4 0 0
1 3 3 3 0 0 1 3 1 1 0 0
Diarrhoea
Killed after inoculation (days)
Excretion of oocysts in the faeces (days)
1 2 2 4 3 3 3 4 2 3 0 0
4** 43** 4*** 10 8 7 8 9 10 7 5 26
2 2 2 7 7 4 4 3 3 2 2 6
*Two uninoculated GB piglets from the same litter remained clinically normal and no oocysts were d e t e c t e d in t h e i r f a e c e s . , *Died. ***Killed when moribund.
Oocyst count A known volume of gut content homogenate (5 t~l) from Piglet 3 was smeared onto a microscope glass slide, stained with Giemsa and the number of oocysts present in the smear was counted. The number of oocysts per 4 ml inoculum was estimated to be between 20 X 103 and 35 X 103.
Experimental procedure Fourteen piglets derived from 2 litters were reared under gnotobiotic conditions as described elsewhere (Makin and Tzipori, 1980). The piglets were housed in pairs and inoculated at the age of: 1 day old (Piglets 1 and 2),
123 3 days old (3 and 4), 7 days old (5--10) and 15 days old (11 and 12). The piglets were observed 3 to 4 times daily for signs of illness, faecal swabs were taken once or twice daily until the piglets were killed, and the milk intake was recorded. Two piglets were kept as uninoculated controls.
Necropsy The piglets were anaesthetised at certain intervals after inoculation (Table I) and portions of gut were taken before the animals were killed. Sections were taken for histology, transmission and scanning electron microscopy (EM), and lactase analysis from 5 equally spaced sites along the small intestine and 2 sites from the large intestine (Tzipori et al., 1981b).
• "icrobiology Inocula, daily faecal samples and gut contents of killed piglets were examined routinely for the presence of enteric viruses and bacteria as described previously (Tzipori et al., 1980b). Cryptosporidium oocysts were detected by Giemsa staining of faecal smears. RESULTS Treatment of the calf faecal homogenate with absolute alcohol (60% final concentration), resulted in an inoculum which contained Cryptosporidium oocysts, b u t following bacterial sterility checks was believed to be free of other microorganisms. Some of the oocysts lost their smooth outline during the treatment, but the majority remained morphologically intact. Piglets 3 and 4, which were fed the alcohol-treated inoculum, became unsteady on their feet and remained depressed for 4--5 h, after which time they recovered completely. Faecal swabs obtained twice daily from these 2 piglets showed that they were bacteria-free, and 3 days after inoculation Cryptosporidium oocysts were the only organisms detected in the faeces.
Clinical observation An incubation period of 1--4 days was observed in piglets that developed clinical signs. The onset of diarrhoea and anorexia, the duration of illness and the excretion of oocysts in the faeces are summarised in Table I. Piglets 1 and 2, inoculated 6 h after birth, vomited 2 and 3 days after inoculation, respectively. They suffered severe diarrhoea that resulted in dehydration and the death of Piglet 1, and the near death of Piglet 2. The severity of illness of Piglets 3 and 4 was similar to that seen in Piglets 1 and 2. Piglets 5--10, inoculated at 7 days of age developed moderate diarrhoea for 2--4 days and none of these piglets died. Piglets 11 and 12, aged 15 days when inoculated, remained clinically healthy although Cryptosporidium oocysts were detected in their faeces 3 days after inoculation. The 2 uninoculated piglets remained clinically healthy and no oocysts were detected in their faeces throughout the observation period.
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124
Histopathological findings The extent of Cryptosporidium infection observed in the intestine by light microscopy varied from slight to heavy depending on time of necropsy after inoculation and the intestinal site examined. In Piglets 1, 2 and 3, heavy infection of the brush border of the enterocytes extended from the d u o d e n u m to the rectum. In Piglets 4--11 the infection was either patchy or absent in the first 3 sites, but heavy in the ileum and large intestine. Piglet 12, which was killed 26 days after inoculation, was free of infection. In the ileum and large bowel there was no difference in the degree of infection between piglets inoculated at the age of 1 or 15 days. The organisms coated the entire villi, but were in higher concentrations at the tips and in some sections of the ileum and in the large intestine cryptosporidia were observed embedded in the crypts. Mucosal lesions of stunted and fused villi, oedema and cellular infiltrations of the lamina propria, were most consistently observed in the middle and lower small intestine These changes increased in severity towards the terminal ileum where the loss of healthy vacuolated villous cells and their replacement by immature low enterocytes was more evident. No bacterial adherence was observed in any of the gut sections. Transmission and scanning EM studies have revealed organisms with internal structures consistent with Cryptosporidium which have previously been documented in experimentally-inoculated piglets (Tzipori et al., 1981d). The 2 uninoculated piglets which were killed aged 10 or 15 days, had long slender villi throughout the small intestine with no evidence of inflammation or adherence of microorganisms to the brush-border of enterocytes.
Measurement of lactase activity Table II gives details of measurement of lactase activity at 5 different sites along the small intestine. It is apparent from the table that piglets inoculated at 3 days of age had the lowest level of activity, followed by piglets inoculated at 7 days. All 3 groups of piglets, however, were well below that recorded for the control piglets. TABLE n
Lactase activity ( m e a n s -+SE) a t 5 different sites equally spaced f r o m the p y l o r o u s region to t e r m i n a l i l e u m c o l l e c t e d f r o m 10 i n f e c t e d and 2 u n i n o c u l a t e d piglets Small intestinal sites ( m e a n lactase a c t i v i t y / ~ m o l e s / m i n / g w e t w t . )
Killed
No. of pigs/ group
1
2
3
4
5
3 7
7,13 14--17
2 6
15 C**
20,41 * 10,15
2 2
2.02 6.90 (+1.93) 8.19 14.12
1.74 7.42 (-+2.47) 10.93 19.33
2.34 4.82 (-+2.43) 8.69 17.74
0.43 5.13 (-+1.89) 3.48 13.33
1.94 2.98 (-+2.34) 1.72 4.56
Age ( d a y s ) w h e n
Inoculated
* A l t h o u g h Pig 12 w a s 41 d a y s old w h e n it was killed t h e values w e r e similar to its litter m a t e , i n d i c a t i n g possible residual d a m a g e . * * C o n t r o l piglets.
125 DISCUSSION During the last two years, transmission of Cryptosporidium to a variety of animal species has been reported (Tzipori et al., 1980a; ~,,oon and Bemrick, 1981). In these transmission experiments the inocula used, although shown to be free of other known enteropathogens, contained other microorganisms which rendered the results less conclusive. As far as we are aware this is the first experiment where an inoculum has been used which, we believe, contained only Cryptosporidium oocysts. The results confirmed previously reported observations that Cryptosporidium is capable of inducing diarrhoea in some mammalian species. The bacteria-free Cryptosporidium also induced enterocolitis in calves (Tzipori et al., 1982). Alcohol was selected to purify Cryptosporidium because, of all the disinfectants that were found to be ineffective against the oocysts, it was the least toxic to piglets (Campbell et al., 1982). There has been an a t t e m p t in this experiment to quantitate the inoculum by estimating the number of oocysts present in small volumes (5 ~) of diluted samples. The technique can be further improved by using a graduated microscopic slide where a number of squares can then be nominated for counting. This technique is probably only useful in assessing the infectious dose of faecal homogenate; gut contents or ileal scrapings, no doubt, contain intermediate stages of the life cycle which may also be infectious and not be expressed in terms of infectious dose. It appears from this experiment that unstressed piglets, devoid of maternal protection, become clinically resistant to infection with Cryptosporidium within 15 days. It remains to be seen whether clinical cryptosporidiosis in older piglets, like infection with other enteropathogens, can be precipitated by stress factors such as early weaning. Clinical illness attributed to cryptosporidiosis in pigs, however, does not appear to be c o m m o n , probably because of the prevalence of infection in pigs (Tzipori and Campbell, 1981; Tzipori et al., 1981d) which could mean that enough maternal antibody is received by the newborn piglet to protect it through a short period of susceptibility. The mucosal changes observed in GB piglets after inoculation with Cryptosporidium, were consistent with previous, more extensive descriptions of experimental infection of conventional piglets (Tzipori et al., 1981d). The extent of infection and damage in the ileum and large bowel did not vary between piglets inoculated at the age of 1 or 15 days. The difference was the involvement of the upper small intestine in the younger piglets, which could be associated with the manifestation of clinical disease in this age group due to probable impairment of digestion as well as absorption. In older piglets where the ileum was most severely affected the membrane bound enzymes were, to a great extent, spared and therefore these piglets suffered malabsorption only. This is also evident from the levels of lactase activity measured in the older piglets; although they were lower than the controls, they were considerably higher than in the 3-day-old piglets.
126 ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k Ms. J u l l B i l l i n g t o n a n d Ms. K a r e n W i l s o n f o r t e c h n i c a l a s s i s t a n c e a n d Ms. M a r g a r e t P a r k i n s o n f o r t y p i n g t h e m a n u s c r i p t . T h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m t h e A u s t r a l i a n Pig I n d u s t r y R e s e a r c h Committee.
REFERENCES Bergeland, M.G., 1977. Necrotic entertis in nursing piglets. Proc. Am. Assoc. Vet. Lab. Diagn., 20: 151--158. Campbell, I., Tzipori, S., Hutchison, G. and Angus, K.W., 1982. The effect of disinfectant on survival of Cryptosporidium oocysts. Vet. Rec., in press. Kennedy, G.A., Kreitner, G.L. and Strafuss, A.C., 1977. Cryptosporidiosis in three pigs. J. Am. Vet. Med. Assoc., 170: 348--350. Makin, T. and Tzipori, S., 1980. Inexpensive techniques for the production and maintenance of gnotobiotic piglets, calves and lambs. Aust. Vet. J., 56: 353--359. Moon, H.W. and Bemrick, W.J., 1981. Faecal transmission of calf cryptosporidia between calves and pigs. Vet. Pathol., 18 : 248--255. Moon, H.W., McClurkin, A.W., Isaacson, R.E., Pohlenz, J., Skartvedt, S.M., Gilette, K.G. and Baetz, A.L., 1978. Pathological relationships of rotavirus Escherichia coli and other agents in mixed infections in calves. J. Am. Vet. Med. Assoc., 193: 577--583. Morin, M., Larivi~re, S., Lalier, R., Begin, M., Roy, R. and Ethier, R., 1978. Neonatal calf diarrhoea; pathology and microbiology of spontaneous cases in dairy herds and incidence of the enteropathogen implicated as aetiological agents. Proc. 2nd Symp. Neonatal Diarrhoea. (Veterinary Infectious Discases Organisation), Saskatoon, pp. 347--365. Nagy, B., Anthal, A. and Lakner, J., 1980. Significance of intestinal cryptosporidiosis in calf diarrhoea. Proc. 2nd Int. Symp. Vet. Lab. Diagn., Switzerland, pp. 431--434. Pohlenz, J., Moon, H.W., Chenille, N.F. and Bemrick, W.J., 1978. Cryptosporidiosis as a probable factor in neonatal diarrhoea in calves. J. Am. Vet. Med. Assoc., 171: 452--457. Snodgrass, D.R., Angus, K.W., Gray, E.W., Keir, W.A. and Clerihew, L.W., 1980. Cryptosporidia associated with rotavirus and Escherichia coli in an outbreak of calf scour. Vet. Rec., 106: 458--459. Tzipori, S. and Campbell, I., 1981. Prevalence of Cryptosporidium antibodies in ten species of animal. J. Clin. Microbiol., 14: 455--456. Tzipori, S., Angus, K.W., Campbell, I. and Gray, E.W., 1980a. Cryptosporidium: Evidence for a single species genus. Infect. Immunol., 30: 884--886. Tzipori, S., Campbell, I., Sherwood, D., Snodgrass, D.R. and Whitelaw, A., 1980b. An outbreak of calf diarrhoea attributed to cryptosporidial infection. Vet. Rec., 107: 579--580. Tzipori, S., Angus, K.W., Campbell, I. and Clerihew, L., 1981a. Diarrhoea in lambs due to Cryptosporidium infection. J. Clin. Microbiol., 14: 100--105. Tzipori, S., Angus, K.W., Gray, E.W. and Campbell, I., 1981b. Diarrhoea in lambs experimentally-infected with Cryptosporidium isolated from calves. Am. J. Vet. Res., 42: 1400--1404. Tzipori, S., Angus, K.W., Campbell, I. and Sherwood, D., 1981c. Diarrhoea in young deer associated with Cryptosporidium. J. Infect. Dis., 144: 170--175. Tzipori, S., McCartney, E., Lawson, G.H.K., Rowland, A.C. and Campbell, I., 1981d. Experimental inoculation of piglets with Cryptosporidium. Res. Vet. Sci., 31: 358--368. Tzipori, S., Angus, K.W., Sherwood, D. and Campbell, I., 1982. Experimental Cryptosporidium in calves: clinical manifestation and pathological findings. Vet. Rec., in press.