The aetiology and diagnosis of calf diarrhoea.
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Biomedical subjects
Publications and source records attributed to S Tzipori.
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In an outbreak of diarrhea among 82 artificially reared red deer calves, 56 developed the disease and 20 subsequently died. During the outbreak 80% of diarrheal and 50% of apparently healthy calves excreted cryptosporidial oocysts in feces. The coincidence of infection with Crytosporidium and clinical diarrhea suggested a causal relationship. Histologic examination of intestinal sections from a necropsied deer calf showed lesions consistent with field and experimental cryptosporidiosis in other species. The deer Cryptosporidium subclinically infected newborn mice; in indirect immunofluorescence tests, it could not be distinguished from a calf Cryptosporidium.
Thirteen gnotobiotic lambs, aged from a few hours to 8 days, were inoculated orally with single infections of enterotoxigenic Escherichia coli (ETEC) (four animals), lamb rotavirus (five animals), and Cryptosporidium (four animals). Six gnotobiotic and two specific-pathogen-free lambs were co-inoculated with either rotavirus and ETEC (four animals), rotavirus and Cryptosporidium (two animals), or ETEC and Cryptosporidium (two animals). Lambs 4 days of age and older became only subclinically infected with either rotavirus, ETEC (08:K87:K99 ST+), or both enteropathogens given simultaneously. Six-day-old lambs inoculated with Cryptosporidium became extremely depressed, anorectic, and had intermittent diarrhea. There was no difference in the clinical manifestations, level of disaccharidase activity in the small intestine, or extent of histological damage between lambs inoculated with Cryptosporidium alone or together with either of the other two agents. The results indicate that under the conditions of these experiments, lambs become clinically resistant to infection with ETEC, rotavirus, or both agents together, by 4 days after birth, whereas lambs 2 days old or younger were clinically susceptible to infection by these agents. In contrast, they remained clinically susceptible to infection with Cryptosporidium up to at least 6 days of age. Cryptosporidium infections were not aggravated by coinfection with either ETEC or rotavirus.
Severe diarrhea which lasted 7 to 12 days occurred in 40 of 48 artificially reared lambs within 5 to 12 days of birth, and 16 of them died. Of 16 diarrheic fecal samples examined, 10 contained Cryptosporidium oocysts and 1 contained rotavirus, but no other known enteropathogen was detected. Upon histological examination, cryptosporidia were found in the ilea of three affected lambs, and in one of them, villous atrophy and fusion, with epithelial cross-bridging between villi, were present in distal small intestine. Diarrhea was induced in two specific pathogen-free lambs by oral inoculation with fecal homogenate containing Cryptosporidium oocysts. Both the small and large intestines became infected with the organism, and associated lesions included stunting, fusion, and deformities of villi in the distal small intestine, with replacement of columnar enterocytes by immature cuboidal cells. Subclinical infections were induced in newborn specific pathogen-free mice and rats. Judged by these data, the lamb-derived Cryptosporidium sp. is similar to those recovered from calves, deer, and humans.
Antibodies to cryptosporidium were detected by indirect immunofluorescence in over 80% of sera from 10 animal species, including humans.
Two to 5 days after oral inoculation with Cryptosporidium obtained from infected calves, 7 newborn specific-pathogen-free lambs became depressed and anorectic and developed diarrhea. Four moribund lambs were killed within 3 days after onset of illness, and the 3 other lambs died after protracted intermittent diarrhea and reduced milk intake, which lasted up to 14 days. The small and large intestines were heavily infected with Cryptosporidium, with the terminal portion of the ileum being the most severely affected. Histologic examination revealed villous atrophy, with considerable fusion and epithelial cross-bridging between villi. Lambs infected at 5 to 20 days of age had less severe clinical signs of disease, with intermittent diarrhea, reduced milk intake, and growth retardation. Lambs infected at 30 days of age became infected with the organism, but did not develop clinical signs of disease or growth retardation.
Piglets from five litters were doses orally with cryptosporidium originally derived from diarrhoeic calves. The piglets were either nursed by the sow, artificially reared after sucking colostrum, or weaned on to creep feed. Colostrum-fed, artificially reared piglets obtained from two litters and exposed in the first week of life developed clinical signs of inappetence, vomiting and diarrhoea and shed oocysts in the faeces. Histologically the parasite was observed throughout the small and large intestine attached to epithelial cell surfaces and its presence was associated with extensive mucosal damage, particularly in the posterior small intestine, stunting and fusion of villi, immaturity of villous epithelial cells and oedema with increased cellularity of the lamina propria. Piglets from two other litters, both sucking and colostrum-fed artificially reared, exhibited similar but milder clinical signs. Histological lesions were less severe and cryptosporidium infection less extensive. When weaned piglets were exposed they remained clinically healthy although histologically there was evidence of cryptosporidium attachment in the small intestine and minor mucosal damage. There appears to be a good correlation between the extent of intestinal infection, the degree of mucosal damage and the severity of clinical disease induced by cryptosporidium in piglets.
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Some aspects of postweaning diarrhoea (PWD) in a piggery during the first week after early weaning were investigated. A haemolytic enterotoxigenic strain of E. coli (O149:K88:H10) was regularly recovered from piglets with PWD while rotavirus was demonstrated on a number of occasions. Prior to weaning piglets were either free of, or shed very few, haemolytic E. coli in their faeces. However, all piglets were excreting haemolytic E. coli between 5 and 7 days after weaning. The role of rotavirus in PWD was unclear. There appeared to be a direct relationship between serum antibodies to rotavirus in the slow at farrowing and those of the piglets soon after birth. The decline of maternal neutralising antibody to rotavirus coincided with the immediate postweaning period (3 to 5 weeks after birth). This was followed by an increase in antibody levels, 5 to 8 weeks after birth. There was no significant difference in the growth rate between affected and unaffected piglets over a period of 120 days. Medication of water during the first week after weaning had no significant effect on the incidence of PWD in the herd. A change in both the weaner diet and the weaning procedure reduced piglet mortality associated with PWD by more than half.
A haemolytic enteropathogenic E. coli (WG) and pig rotavirus were isolated from a field case of postweaning diarrhoea in pigs. Four-week-old gnotobiotic piglets fed on milk diet were found to be extremely susceptible to infection with WG E. coli. Piglets were less susceptible to the infection immediately after the diet was changed from milk to dry food, and were almost completely resistant 4 days after the change to dry food. There was no difference in the clinical response to infection with WG E. coli when the piglets were fed either a high energy diet or low energy diet. Four-week-old piglets fed milk showed mild symptoms of diarrhoea when inoculated with pig rotavirus. Symptoms were more severe when piglets were inoculated immediately after the change from milk to dry food. Piglets inoculated 4 days after the change of diet showed no symptoms of diarrhoea at all. Under the conditions of these experiments the enteropathogenic E. coli produced a more serious disease than did pig rotavirus. Infection of 4-week-old gnotobiotic piglets with both agents given sequentially produced a diarrhoeal disease that was more severe than that produced by each agent separately.
An inexpensive system was developed for the routine derivation and maintenance of gnotobiotic piglets, calves and lambs. Isolator chambers to house these animals were welded in the laboratory from flexible, clear polyvinylchloride film. The isolators consisted of either a simple, closed cylindrical chamber, or for the larger animals, a fibreglass-reinforced plastic (FRP) tub surmounted by a flexible film canopy. The isolators were adaptable for a variety of purposes and most components were interchangeable between isolators. The assembled isolators were sterilised with peracetic acid. Over 300 gnotobiotic piglets, 29 gnotobiotic calves and 18 gnotobiotic or specific pathogen free lambs have been reared successfully by these techniques. Six colostrum deprived foals and a litter of conventional puppies have been successfully maintained in microbial isolation in these isolators. The FRP tub base isolators also proved suitable for housing gnotobiotic weaner piglets. No viral or bacterial cross contamination was detected between isolators housing experimentally infected animals.
Cryptosporidium, isolated from calves with diarrhea, infected, with or without causing enteritis, seven different species of animals.
Rotavirus RNA prepared from calf, pig, mouse, deer, foal and dog-adapted human isolates was compared using polyacrylamide gel electrophoresis. Reproducible differences in the RNA migration patterns were found between all isolates. There were 11 clearly resolved segments in the pig, mouse and foal samples. The calf rotavirus RNA and deer rotavirus RNA separated into 9 bands and 10 bands, respectively. The dog-adapted human virus migrated in 12 bands, and this probably results from the complex passage history of the original human rotavirus isolate.
A rotavirus, morphologically similar to other known rotaviruses, was demonstrated in the faeces of 5 foals with diarrhoea on two properties. Four of these 5 samples produced specific intracytoplasmic fluorescence in cell culture when reacted with calf rotavirus antiserum conjugate. Sixteen affected foals from both properties were depressed, did not suckle and became recumbent. Most had a watery diarrhoea which lasted for 3 days and resulted in some dehydration and loss of body condition. Sick foals were separated from their mothers following the onset of diarrhoea and given fluid therapy and antibiotics, but despite these measures 4 of 12 affected foals on one property died. Acute and convalescent sera were collected from 6 foals, and in each case a rise in the titres of complement fixing and neutralising antibodies was demonstrated. Faecal filtrates containing foal rotaviruses were fed to gnotobiotic piglets and the effects compared with those of other rotaviruses. Viral isolates from both properties produced an asymptomatic infection in piglets. This contrasts with the other rotaviruses isolated in Australia that have been shown to cause diarrhoea in gnotobiotic piglets.
A rotavirus isolated from a field outbreak of diarrhoea in artificially reared piglets was purified, filtered and administered orally to gnotobiotic and conventional piglets. Four successive passages of the virus in gnotobiotic piglets produced severe diarrhoea within 20 to 24 hours of administration. The diarrhoea lasted several days causing dehydration, emaciation, loss of body weight and some deaths. Virus was demonstrated in the faeces of the infected piglets by electron microscopy. Conventionally reared piglets developed little or no diarrhoea when given virus, whereas artificially reared piglets developed moderate to severe diarrhoea which lasted from 3 to 8 days with some deaths. No clinical disease was obvious in surviving piglets following challenge with the virus 10 or 17 days after initial infections.