Scanning electron, light, and immunofluorescent microscopy of intestine of gnotobiotic calf infected with calf diarrheal coronavirus.

Intestinal lesions in 2 gnotobiotic calves given (oral inoculation) calf diarrheal coronavirus were studied by scanning electron, light, and immunofluorescent microscopy. The calves were euthanatized at 34 and 73 hours after the onset of diarrhea. Lesions in the small intestine were similar to those reported in animals affected with transmissible gastroenteritis of swine. Small intestinal villi were shortened, some adjacent villi were fused, and villous epithelium was composed of low cuboidal to squamous cells. In the ansa spiralis coli, there were atrophy of the colonic ridges and marked differences in length and spacing of the microvilli on individual epithelial cells.     

Follicle associated epithelium of the gut associated lymphoid tissue of cattle

The morphology of the gut-associated lymphoid tissue of the small and large intestine in three gnotobiotic calves was examined by scanning and transmission electron microscopy, and the distribution of specialized membranous cells present in the follicle associated epithelium was defined. Isolated follicles remaining in the ileum of a cow after involution of the continuous Peyer's patch were examined by scanning electron microscopy. The presence of membrane-bound particles, reported to be exclusively associated with the continuous Peyer's patch, was investigated in other gut-associated tissue of the small and large intestine of the calf. The presence of two types of follicle associated epithelium in the small intestine of the calf was confirmed, and the follicle associated epithelium of the large intestine proved to be a homogeneous population of specialized membranous cells, similar to that of the continuous Peyer's patch of the small intestine. In the discrete Peyer's patches, some specialized membranous cells were completely hidden by adjacent enterocytes and could only be identified by cytoplasmic extensions into the intestinal lumen. In the proximal part of the continuous Peyer's patch, a transitional zone was detected where the follicle associated epithelium of some doomed villi was composed of a homogeneous population of specialized membranous cells, while the epithelium covering other doomed villi consisted of a mixture of absorptive and specialized membranous cells, usually only found in the discrete Peyer's patches. Membrane-bound particles were observed associated with gut-associated lymphoid tissue in the small and large intestine.     

The pathogenesis of enteric colibacillosis in neonatal unsuckled calves.

The development of pathological lesions in the small intestine of neonatal calves is described. Seven newborn calves were challenged orally with a known enteropathogenic strain of E coli 0101k?(A) and killed at varying times after inoculation. Adhesion of bacteria to the mucosa of the small intestine was observed in all calves. A few organisms were seen in the distal small intestine at three hours after inoculation and thereafter adhesion progressed anteriorly along the intestine in calves killed from six to 36 hours. In these calves pathological changes occurred between six and 12 hours after inoculation. Villi were stunted and thickened and the epithelial surface was irregular. A further calf, anaesthetised from five-and-a-half to 10 hours after inoculation and repeatedly sampled from the distal small intestine, developed similar lesions abruptly at nine hours after inoculation. Villus and crypt lengths in the challenged calves were compared with those in three normal uninoculated control calves.     

Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs.

Porcine rotavirus was shown to infect gnotobiotic pigs and induce an acute enteric disease clinically characterized by diarrhea, anorexia, depression, and occasional vomition. Onset of clinical signs correlated closely with the appearance of lesions within the small intestinal mucosa, and recovery from infection was associated with the regeneration of normal, functional villous epithelium. Villous atrophy, especially in the caudal two-thirds of the small intestine, was the consistent lesion observed in pigs with clinical signs of rotaviral infection. Villi were often short, blunt, and covered with cuboidal epithelial cells. Immunofluorescent microscopy methods demonstrated that the principal site of rotaviral replication was the villous columnar epithelial cells in the small intestine.     

Scanning electron, light, and transmission electron microscopy of intestine of gnotobiotic calf.

Adjacent areas of upper, middle, and lower parts of the small intestine and spiral colon from a 48-hour-old gnotobiotic calf were compared by scanning electron microscopy (SEM), light microscopy (LM), and transmission electron microscopy (TEM). As visualized by all 3 methods, small intestinal histologic features, except for apical location of villous epithelial cell nuclei in sections of upper and middle parts of small intestine, were similar to those described for other species. The colonic surface visualized by SEM was composed of flattened ridges separated by furrows into which opened the crypts of Lieberkühn. The epithelial surfaces of the ridges and the furrows had an extensive microvillous covering and scattered goblet cell openings.     

Scanning electron microscopy of the small intestine of a normal unsuckled calf and a calf with enteric colibacillosis.

Sections of the small intestine were taken under general anaesthesia from a normal calf and from a calf with enteric colibacillosis and examined by scanning electron and light microscopy. In the normal calf villi were long and oval throughout the intestine and in the challenged calf there was villous stunting and fusion in the distal small intestine.     

Isolation of coronaviruses from neonatal calf diarrhoea in Great Britain and Denmark

Coronaviruses were isolated from neonatal calves with diarrhoea in Great Britain and Denmark. They were serially passed in gnotobiotic calves which developed acute diarrhoea. Pathological lesions were found in the small and large intestines. Coronaviruses were demonstrated by electron microscopic examination of the faeces and intestinal contents, immunofluorescent staining of sections of small and large intestine and by isolation in tracheal organ cultures. In early passages of the British coronavirus, particles of about 30 nm in diameter were observed in the faeces by electron microscopy. These particles were removed from the coronavirus preparations by cross-protection experiments in calves. The coronaviruses were morphologically and antigenically similar to the bovine coronavirus isolated in the United States and the British virus was adapted to replicate in calf kidney cell cultures.     

Gut-associated lymphoid tissue in the large intestine of calves. II. Electron microscopy

Scanning electron microscopy of lymphoid tissue in the large intestine of three germfree calves (age 3, 6, and 7 days) revealed two different units: propria nodules and lymphoglandular complexes (LGC). Propria nodules had lymphoid tissue predominantly in lamina propria and were covered by distinct follicle-associated epithelium which lacked goblet cells; nodules were surrounded by wide crypts, which were also lined by follicle-associated epithelium towards the luminal side. Lymphoglandular complexes had lymphoid follicles in the tunica submucosa; epithelial diverticulae extended through the muscularis mucosae branching into the lymphoid nodule. In centers of lymphoglandular complexes, protrusions of lymphoid tissue were covered with distinct follicle-associated epithelium. By transmission electron microscopy cells compatible with M cells in the small intestine of calves and cells with characteristics of both enteroabsorptive and M cells were found. Follicle-associated epithelium of propria nodules and lymphoglandular complexes differed only in the relative frequency of cell types.     

Pathogenic Shiga toxin-producing Escherichia coli in the intestine of calves

The purpose of this study was to compare the pathological effects of Shiga toxin-producing Escherichia coli (STEC) that vary in their association with bovine and human disease. Shiga toxin-producing E. coli of serotypes associated with both dysentery in calves and hemolytic uremic syndrome (HUS) in humans (O5:H-, O26:H11, O111:H-, O113:H21) were compared with O157:H7 STEC, which are associated with HUS in humans but not with disease in calves. The STEC were administered orally to 80 day-old chicks and into ligated loops in the ileum and colon of four 2- to 6-day-old calves. Examination of the ceca of the chickens 10 d postchallenge showed no adherence or tissue abnormality for any isolate. The calves were euthanized 8 to 10 h postinoculation, and sections of the intestinal loops were examined by light microscopy, transmission and scanning electron microscopy, and immunohistochemistry. All strains showed consistent focal adherence associated with mild lesions in the colon. Attaching and effacing lesions were observed with the eae-positive strains. Ileal lesions were similar to the colonic ones but were sometimes severe, with marked polymorphonuclear leukocyte proliferation in the lamina propria. It is concluded that chickens were unsuitable for studying interaction of STEC with the intestine and that there was no difference in the interaction of the ligated calf intestine with STEC of serotypes associated with disease in calves compared with O157:H7 STEC.     

Cryptosporidiosis in calves during the period of milk nutrition

It has been found out by examining faeces of 83 calves from calf house in Uhercice (Breclav district) that 52 calves excreted coccidium oocysts, genus Cryptosporidium Tyzzer, 1907, during the period of milk nutrition. The calves excreted oocysts from the eighth day of age, and at the end of the milk nutrition period (43rd-47th ridium Tyzzer, 1907, during the period of milk nutrition. The calves excreted oocysts of cryptosporidia were found in two thirds of calves suffering from diarrheas. By histological examination of a 27-day-old calf, developmental stages of this coccidium were found on the surface of villus epithelium in the distal part of ileum. Villi were shortened and expanded, submucous connective tissue was penetrated by inflammatory infiltrate with large quantity of eosinophil granulocytes. For demonstration of oocysts in faeces, Breza flotation method combined with sedimentation and staining by modified Giemsa panoptic method were used.     

Intestinal lesions in specific-pathogen-free lambs associated with a cryptosporidium from calves with diarrhea

Small and large intestines of seven specific pathogen-free lambs infected with cryptosporidia from calves with diarrhea were examined by scanning and transmission electron microscopy and by light microscopy. The small intestine was infected in all the lambs, and the cecum and colon in three. Small intestinal alterations were severe villous atrophy and dilatation of the crypts of Lieberkühn. Epithelial cross-bridging between contiguous villi caused much villous fusion. Epithelial cells constituting the bridges were connected by desmosomal junctions, and were continuous with the epithelial coverings of the associated villi. The lamina propria was heavily infiltrated with neutrophil leukocytes. Infected crypts in cecum and colon were dilated and devoid of mucus-secreting cells, while the ridges between crypts were hypertrophied, and the lamina propria was infiltrated by neutrophils. Cell vegetations with adherent bacteria were present in the surface intestinal epithelium of two lambs infected for 11 and 14 days, respectively. No adherent bacteria were seen in any site in lambs killed up to six days post-inoculation.     

Natural infection with an attaching and effacing Escherichia coli in the small and large intestines of a calf with diarrhoea

Attaching and effacing Escherichia coli were identified in the small and large intestine of a calf with naturally occurring diarrhoea. The organisms were associated with intestinal lesions and were identified by immunoperoxidase staining and transmission and scanning electron microscopy, but they did not produce Shiga-like toxin (verotoxin).     

Lesions of gnotobiotic calves experimentally infected with a calicivirus-like (Newbury) agent

Fourteen gnotobiotic calves were killed 0.5 to ten days after infection with Newbury agent SRV-1 and the changes in small intestinal structure and function were assessed, qualitatively and quantitatively, by light microscopy, scanning and transmission electron microscopy, enzymology and xylose absorption. The first enterocytes detected as infected by immunoperoxidase were those on the sides of villi at the base. Subsequently exfoliation of degenerate enterocytes resulted in stunted villi; mucosal beta-galactosidase activity fell and there was xylose malabsorption. Small intestinal damage, first detected at 12 hours after infection but almost repaired by ten days, was restricted to the anterior half of the small intestine. In the distal small intestine, where no virus-induced damage occurred, villi lengthened--possibly due to increased mitosis of crypt cells stimulated by enteroglucagon release.     

Studies on the immunity of the calf to colibacillosis--VII: the experimental reproduction of enteric colibacillosis in colostrum-fed calves.

Eight newborn calves were challenged orally with a known enteropathogenic strain of Escherichia coli 0101 K?(A) and two to six hours later each calf was fed a minimum of three pints colostrum. All calves suffered from acute diarrhoea of varying severity within 24 to 48 hours of infection. Immunofluorescent and histological examination of the small intestine demonstrated adherence of the challenge organism to the epithelium and the presence of pathological lesions similar to those seen in colostrum-deprived calves with enteric colibacillosis. It was concluded that in order to be effective prophylactically, colostrum must be fed prior to infection.     

Observations on the Ultrastructural Appearance of Endocrine Cells of the Small Intestine of the Calf

The endocrine cells of the small intestine of neonatal calves are described, with the aid of transmission and scanning electron microscopy. It is demonstrated that endocrine cells have long microvilli, and are organized into two populations, similar to those of other species.     

Pathogenesis of corneal lesions caused by Moraxella bovis in gnotobiotic calves

Moraxella bovis was instilled into the conjunctival sac of gnotobiotic calves and corneas were sampled serially after infection. Lesions developed in seven of eight infected calves, but were absent in a noninfected control calf. Histologically, M. bovis was first seen in foci of swollen epithelium and within basal epithelial cells adjacent to ulcers. Corneal ulcers were severe in later stages of infection; fibrin deposits, neutrophils, and bacteria were present in the stromas. Examination of early lesions by scanning electron microscopy showed M. bovis in pits on the surfaces of dark epithelial cells, enmeshed in degenerate epithelial cells and within erosions and an ulcer; in later samples, bacteria were rare. Ultrastructurally, M. bovis was seen in surface pits in superficial epithelial cell processes and within swollen epithelial cells. In stroma, M. bovis was frequently seen among collagen fibrils, within neutrophil phagosomes, and associated with cellular debris. This study demonstrates that a virulent strain of M. bovis can invade bovine corneal epithelial cells and can cause keratitis in the absence of injurious ultraviolet irradiation or other known predisposing environmental factors.     

Quantitative observations on experimental reo-like virus (rotavirus) infection in colostrum-deprived calves.

Ten calves were challenged with one of two strains of reo-like virus (rotavirus). Changes in the daily faecal and urinary outputs were monitored and packed cell volume, plasma sodium, potassium and urea levels were measured. Faeces were examined for the presence of rotavirus by direct electron microscopy and immunofluorescence in cultures of PK(15) cells. All calves excreted rotavirus in the faeces for several days. Two calves remained clinically normal throughout the experiment, but in the remaining calves, faeces became mucoid in consistency and yellow-white in colour. In only two calves did the daily faecal output exceed 500 g with a fall in the dry matter content to less than 10 per cent. Slightly elevated blood urea levels and hyperkalaemia were the only changes observed in blood chemistry and these quickly returned to normal. Virus antigen was observed in the epithelial cells by immunofluorescence in the proximal and middle small intestine of calves. Pathological lesions occurred predominantly in the proximal small intestine of nine calves examined.     

Occurrence of 'attaching and effacing' lesions in the small intestine of calves experimentally infected with bovine isolates of verocytotoxic E coli

Nine calves (five colostrum-fed, four colostrum-deprived) were challenged with two field strains of Escherichia coli which produced either verocytotoxin 1 (VT1) or verocytotoxin 2 (VT2). Although three colostrum-fed calves had blood and mucus in their faeces, no diarrhoea was observed. Three of the four colostrum-deprived calves had diarrhoea and in two of them severe lesions were detected in the small intestine. Focal changes were detected in the colon of three calves. E coli were associated with the lesions in the small and large intestine and were shown by transmission electron microscopy to be intimately attached to the enterocyte surface with effacement of microvilli (attaching and effacing lesions). This is the first report of E coli which produce VT2 being associated with disease in calves.     

Effect of rotavirus and/or Escherichia coli infection on the aggregated lymphoid follicles in the small intestine of neonatal gnotobiotic calves

The effect of rotavirus and/or Escherichia coli infections on the follicle-associated epithelium (FAE or M cells) of the domes of the aggregated lymphoid follicles (ALF, or Peyer's patches) of gnotobiotic calves was evaluated by light, scanning electron, transmission electron, and immunofluorescence microscopies. Calf rotavirus (CRV) infection produced loss of FAE cell microvilli, and virions were observed in cytoplasmic vacuoles of FAE cells, as well as in intercellular spaces between FAE cells and lymphoid cells migrating through the dome epithelium. The CRV particles appeared to have entered the FAE cells by phagocytosis, with no subsequent cytoplasmic replication. Enterotoxigenic E coli (ETEC) induced more severe alterations including marked microvilli loss and ballooning in the FAE cells. There was no adhesion to, or colonization of FAE cells by ETEC, but bacteria were observed free or phagocytized within the dome and the germinal centers of the ALF. There were no ETEC observed in the cytoplasm of FAE cells. The presence of nonenterotoxigenic E coli (NETEC) in the intestine of calves had no effect on the intestinal FAE cells. The addition of NETEC to CRV infections did not enhance or modify in any way the response of FAE cells to the viral infection; however, the combination of CRV + ETEC produced severe necrosis of the FAE cells, and loss of dome epithelium of ALF.     

Prevalence and pathogenicity of Cryptosporidium andersoni in one herd of beef cattle

Over a 35-week period from January to July 2002, a breed of Hereford beef cattle (H) and their hybrids were monitored. Five hundred and ninety-nine individual fecal samples from calves and 96 samples from their mothers were examined. First excretion of Cryptosporidium andersoni oocysts in calves was found in the 9th week after the start of calving (a calf 63-day old). The prevalence of C. andersoni in calves ranged from 11.1 to 92.9% depending on age. The mean prevalence in their mothers was found to be 43.8%. The size of oocysts was 8.48 +/- 0.78 x 6.41 +/- 0.59 microm. Infection intensity in calves ranged from 32 000 to 4 375 000 oocysts per gram (OPG) and in mothers from 78 000 to 2 552 000 OPG. Three cases of abomasal cryptosporidiosis slaughtered at the age of 81, 157 and 236 days were examined histologically and ultrastructurally [transmission electron microscopy (TEM) and scanning electron microscopy (SEM)]. Cryptosporidium infection of the abomasum was located in the upper half of the mucosal glands in the plicae spirales of the fundus, corpus and near the ostium omasoabomasicum. Cryptosporidia were not located in the glandular epithelium of the pars pylorica in the abomasum minimally 10 cm from pylorus. Histopathological changes in the site of cryptosporidial infection in the abomasum had a non-inflammatory character and included distinctive dilatation of infected parts of the glands with atrophy and metaplasia of the glandular epithelial cells, goblet cell activation and mucus hyperproduction. The TEM revealed a relatively small number of Cryptosporidium life cycle stages attached to glandular epithelial cells. In SEM the inner mucosal abomasal surface appeared swollen but was never infected by cryptosporidia.