Experimental infection of young rabbits with a rabbit enteric coronavirus.

The clinical signs and lesions caused by the rabbit enteric coronavirus (RECV) were studied in young rabbits orally inoculated with a suspension containing RECV particles. The inoculated animals were observed daily for evidence of diarrhea. Fecal samples and specimens from the small intestine and from the gut associated lymphoid tissue (GALT) were collected from 2 h to 29 days postinoculation (PI) and processed for immune electron microscopy (IEM) and light microscopy. Coronavirus particles were detected in the cecal contents of most inoculated animals from 6 h to 29 days PI. Lesions were first observed 6 h PI and were characterized by a loss of the brush border of mature enterocytes located at the tips of intestinal villi and by necrosis of these cells. At 48 h PI, short intestinal villi and hypertrophic crypts were noted. In the GALT, complete necrosis of the M cells as well as necrosis of the enterocytes lining the villi above the lymphoid follicules with hypertrophy of the corresponding crypts were observed in all the animals. Five inoculated rabbits had diarrhea three days PI. The presence of RECV particles in the feces of the sick animals and the microscopic lesions observed in the small intestine suggested that the virus was responsible for the clinical signs. A few inoculated rabbits remained free of diarrhea. Fecal material collected at postmortem examination contained RECV particles. The results suggest that the virus could also produce a subclinical infection.     

Fatal cryptosporidiosis in quail

Young quails kept in wire-floored cages experienced high mortality, beginning at age 5 days, from diarrhea that was unresponsive to antibiotic therapy. At necropsy, the small intestine had clear fluid content and the cecum was distended by brown foamy fluid. Histopathologic findings in the small intestine were shortened villi with detached enterocytes at the tip. Cryptosporidium sp. (confirmed by electron microscopy) were numerous in the microvillous border. Neither bacterial nor viral pathogens were detected. No infection was established in day-old chickens gavaged with feces and intestinal contents from infected quails. Thorough cleansing of the cages followed by application of commercial bleach prevented recurrence.     

Pathology of natural rotavirus infection in clinically normal calves

During a longitudinal study of the epidemiology of rotavirus infection in a calf rearing unit, excretion of virus in faeces was detected by enzyme-linked immunosorbent assay in 40 of 48 (83 per cent) unweaned calves aged between three days and five weeks. Fifty per cent of the infected calves had no clinical signs of disease. Enterocytes containing rotavirus antigen and intestinal lesions were found in all of 12 clinically normal calves selected for necropsy between days 1 and 4 of virus excretion. Stunting and fusion of villi, exfoliation, disarrangement and vacuolation of enterocytes and the presence of cuboidal enterocytes were observed in infected calves but not in rotavirus-free control calves. Lesions predominated in the upper small intestine, where rotavirus was most abundant, especially on the first two days of virus excretion. The numbers of enterocytes infected with rotavirus diminished before the lesions resolved.     

Invasion of enterocytes in cultured porcine small intestinal mucosal explants by Salmonella choleraesuis.

Porcine small intestinal explants maintained in vitro were inoculated with Salmonella choleraesuis to study the characteristics of its invasion of enterocytes. The explants were fixed at selected intervals for up to 12 hours after inoculation and examined by conventional light microscopy, immunoperoxidase staining, and transmission electron microscopy. Although there was diffuse loss of villous enterocytes during the first hour of incubation, the villi were reepithelialized by the end of 2 hours of culture, and the mucosal epithelium remained intact and appeared to be viable through 12 hours of culture. Intraepithelial S choleraesuis were not detected before 6 hours after inoculation, but after 12 hours of incubation, bacteria were numerous within enterocytes. Ultrastructurally, penetration of the brush border by S choleraesuis resulted in focal loss of microvilli. Bacteria were endocytosed into membrane-bound vacuoles where most remained, but a few were free within the cytoplasm of enterocytes. Invasion of the explants closely resembled that described for live animal and cell culture models of Salmonella spp invasion.     

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.     

INTESTINAL CRYPTOSPORIDIOSIS IN A KID GOAT

SUMMARY: A 2-week-old Angora kid goat developed diarrhoea and was treated with parenterally administered antibiotics and saline. The kid rapidly became moribund and died within 6 hours of the first signs of illness. No bacterial or viral agents were detected in the intestinal content. On histological examination large numbers of small haematoxylin-staining bodies were detected associated with the brush border of small intestinal enterocytes. Associated with these bodies there was variable shortening of intestinal villi, some congestion and focal leucocyte infiltration in the lamina propira of the villi and focal epithelial erosions, sloughing or cell transition to more immature enterocyte forms. The location and features revealed by light microscopy and scanning and transmission electron microscopy indicate these bodies are cryptoporidia.     

Scanning electron microscopy of intestine of gnotobiotic piglets infected with porcine rotavirus.

The development of intestinal lesions caused by the porcine rotavirus were studied in six day old gnotobiotic piglets by scanning electron microscopy. The onset of diarrhea followed an incubation period of 17 to 31 hr. The first detectable lesion was observed in the ileum at 12 hr postinfection, a few hours before the onset of diarrhea. At this time enterocytes appeared swollen and began to separate from each other. Seventeen hours after the onset of diarrhea, lesions were quite severe jejunum and ileum. Enterocytes were detaching from the lamina propria leaving denuded areas. Microvilli were sparse on the cell surfaces and there was marked villous atrophy. Regeneration of ileal mucosa was evident at 4.8 days after the onset of diarrhea. Nine days after recovery from diarrhea the intestinal villi had returned to near its normal structure but there remained some evidence of mucosal damage.     

Effects of dietary change and rotavirus infection on small intestinal structure and function in gnotobiotic piglets

The combined effects of weaning and rotavirus infection on small intestinal structure and function and on growth rate were studied in 28 gnotobiotic piglets. There was little damage by rotavirus to the proximal small intestine, some damage to the mid small intestine and relatively severe damage to the distal small intestine; villi were stunted, crypts lengthened and activities of all brush border enzymes decreased. The damage was short-lived despite the synchronisation of rotavirus infection with simulated weaning. There was no evidence of persistent damage to the small intestine and growth rate was unaffected.     

Ultrastructure of Pre- and Postcolostral Enterocytes of the Newborn Calf

Ten calves were used to elucidate the ultrastructure of enterocytes before and 24 h after colostral intake. Tissue samples were obtained from duodenum, jejunum (5 locations) and ileum. Protein A-gold technique was applied to immuno-electron-microscopically demonstrate colostral IgA. The prominent feature of the precolostral enterocytes are intra-cytoplasmic vacuoles. The frequency of vacuoles increases from cranial jejunum to ileum and from the villi bases to the tips. The appearance of absorptive vacuoles after colostral administration correlates with the incidence of precolostral empty vacuoles. Bovine IgA was detected in absorptive vacuoles and within the intestinal lumen of postcolostral calves. In addition to a diffuse IgA labelling of most vacuoles, a few corresponding enterocytic vacuoles labelled inhomogenously or negatively. This study demonstrates morphologically that the main site of colostral absorption is the middle-to-caudal region of the small intestine. Immunoelectron microscopy of IgA labelling provides indications of a selective IgA absorption in addition to pinocytosis.     

Bacterial colonization and morphology of the intestine in porcine Escherichia coli enterotoxemia (edema disease)

Twenty-one pigs were divided into three groups. Pigs in one group were inoculated with the intestinal contents which included bacteria from a pig with edema disease. Pigs in another group were inoculated with a culture of Escherichia coli serogroup O 139:K12(B):H1 isolated from the aforementioned contents, and pigs in a third group served as uninoculated controls. The infection was similar following both inocula. Enterotoxemia developed in 11 of the 14 pigs allowed to survive for more than two days. The onset varied from two to seven days after inoculation. There were maximal viable counts of E. coli in the intestine from the second day post-inoculation and thereafter. In frozen and paraffin sections, as well as by scanning electron microscopy, the organisms were seen on the surface of the small intestinal epithelium where they formed either isolated colonies or continuous layers. They colonized the lower small intestine more intensely than the upper section. The intestinal epithelium and the villi of infected pigs were indistinguishable morphologically from the tissues of three uninoculated control pigs. The diarrhea which was observed in controls and inoculated pigs before inoculation and the villus atrophy in controls and inoculated pigs indicated a preexisting infection with at least one other agent.     

Enteropathogenicity of Dutch and German avian reoviruses in SPF white leghorn chickens and broilers

The enteropathogenicity of avian reoviruses (ARVs), isolated from chickens affected with malabsorption syndrome (MAS) from The Netherlands and Germany was studied. In the first trial seven different ARVs isolated from MAS cases were inoculated in 1-day-old specific pathogenic free (SPF) white leghorns. The pathogenicity was compared with 2 ARVs isolated from cases of tenosynovitis, namely reference strain S1133 and a Dutch strain. Although a difference in the severity of the clinical disease was observed, all reoviruses could induce vacuolar degeneration and sloughing of the epithelium of the small intestine at day 2 post inoculation (PI) till day 7 PI. Two Dutch and one German ARV derived from MAS causing the most severe intestinal lesions at day 2 PI, were further studied in the second trial using SPF broilers. These reoviruses did not cause weight gain depression in the broilers although lesions in the small intestine were present from day 1 up to day 4 PI and were more severe than in the white leghorn chickens. In one of the inoculated groups apical denuded villi were already present at day 1 PI. At day 7 PI the small intestine of the infected broilers appeared to be normal. Reovirus antigen was detected in the cytoplasm of the enterocytes at the tip and middle section of the affected villi both in layers and in broilers. To study the role of intestinal CD4+ and CD8+ T-cells and macrophages/monocytes in the pathogenesis of ARV, the numbers of these cells of the jejunal villi of one infected and the control broiler groups were compared. CD4+ T-cells were detected in low numbers and only in the infected broiler group at day 14 PI. The numbers of CD8+ T-cells and macrophages/monocytes were significantly higher in the infected broiler group than in the control broiler group at day 7 and 14 PI and at day 7 PI respectively. Our study indicates that the reovirus alone cannot induce intestinal lesions as found in MAS chickens. Moreover, CD8+ T-cells may play a major role in the pathogenesis and or reovirus clearance in the small intestine.     

Anatomy,Microbes, and Fiber: Small Versus Large Intestine

Nutrient absorption occurs from the intestinal surfaces. Strategy in nutrient recovery by the small intestine is opposite to that of the large intestine. The duodenum, jejunum, and ileum have a very expansive mucosa by virtue of villi from the wall and microvilli on enterocytes, whereas the cecal body dominates the large intestine with its mucosa providing minimal surface area exposure to the lumen. Refluxive motility and villi movement in the small intestine provide ready convective contact between lumen contents and the unstirred water layer at the villi surfaces while contractile elements move microvilli to enhance contact after transfer into this surface. Refluxive motility in the colon gently separates fine from coarse particulates of small intestinal indigesta using urine moved from the urodeum. Cecal entry is restricted to fluid and fines by narrow orifices and protruding villi. Microbes in the small intestine are suppressed and reflect a bird's environment, whereas rapid motility favors aerobes because of oxygen exchange with the wall. Microbes in the large intestine are highly concentrated with low oxygen levels that support anaerobes. Plant fiber that has high proportions of cellulose lends to coarse particulates after feed manufacturing, whereas fiber high in hemicellulose-pectin combinations disintegrate and may be partially soluble. Fiber generally acts as a dietary diluent with coarse particulates being more rapidly evacuated than soluble nonstarch polysaccharides or fines. Soluble nonstarch polysaccharides that increase viscosity of the small intestine's luminal contents adversely affect live performance because of impaired efficiency of convection, reduced rate of exchange with the mucosa, and an expanded microbial population. Both nonstarch soluble fiber and the fine particulates are readily fermented to volatile fatty acids in the ceca that contribute to metabolizable energy.     

Development of villus atrophy in the small intestine of sheep infected with Trichostrongylus rugatus

Pairs of sheep infected with 120 000 larvae of Trichostrongylus rugatus were killed at intervals from 2 to 56 days after infection (DAI). Worms were located in tunnels in the epithelium of villi or upper intestinal crypts at all stages of development. Villus atrophy developed progressively until 16 DAI, when surface microtopography, characterised by subtotal villus atrophy, stabilised. Most severe lesions were in the first 3 m of small intestine where the density of nematodes was highest. Discontinuities in the epithelium and effusion of inflammatory cells and tissue fluids into the lumen were rare. No effects of infection on body-weight gain, appetite or serum total protein and albumin were evident in the first 20 days of infection. However, it was concluded that T. rugatus fundamentally resembled T. colubriformis and T. vitrinus in the response it elicited in the intestine of sheep, and must be considered potentially pathogenic.     

Studies on the pathogenesis of Salmonella heidelberg infection in weanling pigs

Experiments were conducted to define the pathogenic potential of Salmonella heidelberg in weanling pigs. Oral inoculation with S heidelberg resulted in severe catarrhal enterocolitis with accumulation of large amounts of fluid in the small intestine and colon. Salmonella heidelberg was demonstrated, with fluorescence microscopy and bacteriologic cultural techniques, to colonize the ileum, to invade ileal mucosal enterocytes, and to reach mesenteric lymph nodes and extraintestinal tissues by 8 hours. In 5 pigs, intestinal loops were surgically prepared and inoculated with S heidelberg (to determine its invasiveness). Microscopically, there were atrophy of villi, erosion of enterocytes, and neutrophilic infiltration in the lamina propria. Ultrastructurally, intracellular bacteria were demonstrated in villous and cryptal enterocytes, as well as in macrophages of the lamina propria. Bacteria were morphologically intact, occurred free and membrane-bound and caused no detectable cytotoxic effect to the cell.     

Pathology of experimental CV777 coronavirus enteritis in piglets. I. Histological and histochemical study

Sixteen cesarean-derived colostrum-deprived piglets were infected oronasally with CV777 coronavirus on the second or third day of life. Two uninfected piglets were controls. They were killed at 96 and 120 hours after birth. After an incubation period of 22 to 36 hours, all principals showed severe diarrhea. The principals were killed between 12 and 120 hours after infection. Exfoliation of enterocytes were seen first in the piglet killed 24 hours after infection (two hours after the diarrhea began). From that time on, shortening and fusion of villi was present in all small intestinal parts. Affected cells showed vacuolation. The histochemical study showed that infected piglets had decreased activity of all four enzymes studied. The light microscope showed no lesions in the absorptive colonic epithelium. The significance of the lesions in relation to intestinal dysfunction is discussed, and lesions are compared to those of transmissible gastroenteritis and porcine rotavirus infection.     

Pathology of infectious diarrhea of infant rats (IDIR) induced by an antigenically distinct rotavirus

Suckling rats were inoculated with a group B rotavirus to determine the progression of the morphologic changes induced in the intestine by this virus. Several changes were observed by light microscopy 1 day after viral inoculation: shortening of small intestinal villi, villous epithelial necrosis, and villous epithelial syncytia. The lesions were most often present in the distal small intestine, although other small intestinal segments were affected to a lesser degree. By day 3 post-inoculation, epithelial necrosis, and syncytia were no longer present; however, the villous epithelium was disorganized and irregularly vacuolated, and intestinal crypt epithelium was hyperplastic. Alterations in villous height to crypt depth ratios were present in portions of the small intestine for the remainder of the 12-day study period. Epithelial syncytia appeared to form by the breakdown of the lateral interdigitating membranes of the absorptive villous epithelium. Viral particles, abundant in the syncytia, appeared to form from amorphous or reticular arrays of viral precursor material. Group B rotaviral antigens, as detected by indirect immunofluorescence, were present in large amounts in the small intestinal villous epithelium only on the first day after viral inoculation. These studies show that two important diagnostic features of group B rotaviral infections of rats, epithelial syncytia and viral antigen as determined by immunofluorescence, are present only on the first day of disease. These findings should be taken into consideration when attempting to diagnose disease induced by this agent.     

Lysosomal activity in enterocytes in the small intestine in piglets experimentally infected with Isospora suis

The lysosomal activity of enterocytes of the small intestine mucosa was investigated in gnotobiotic and conventional piglets experimentally infected on the first day after birth (DAB) by the oocysts of the coccidia Isospora suis. A method of the proof of beta-D-glucuronidase (EC.3.2.1.31.) activity was used to demonstrate lysosomes. The piglets were infected by different infection doses of oocysts (100,000 oocysts in gnotobiotic piglets and 200,000 oocysts in conventional piglets). In the gnotobiotic infected piglets the activity of beta-D-glucuronidase in enterocyte lysosomes was investigated in the period from day 3 to day 11 after infection (DAI) and in the infected conventional piglets in the period from day 2 to day 10 after infection. Comparing the control piglets, the group of gnotobiotic piglets at the age of 2-5 days and the group of conventional piglets at the age of 4-7 days, the higher activity of beta-D-glucuronidase was demonstrated in the lysosomes of intestinal mucosa enterocytes in the gnotobiotic control piglets (+5.30 of the average density value, Dx). In the infected gnotobiotic and conventional piglets the pattern of beta-D-glucuronidase activity was found to have three stages in the course of this infection. Two stages can be characterized by a great increase in the enzyme activity (DAI 3-9 in gnotobiotic piglets, DAI 2-3 and 7-9 in conventional piglets. The third stage, which is manifest mainly in the conventional infected piglets, is characterized by a marked decrease in the activity of beta-D-glucuronidase, reaching the level of control findings (DAI 10 and mainly 11 in gnotobiotic piglets. DAI 4-6 and 10 in conventional piglets). A topographical picture shows that the two stages of increase and the stage of beta-D-glucuronidase activity decrease occur in the whole small intestine without any predisposition defect of the enzyme in the different sections of the small intestine.     

Canine parvovirus enteritis 3: Scanning electron microscopical features of experimental infection

A group of 10-week-old puppies was orally inoculated with canine parvovirus of faecal origin. Scanning electron microscopy was used to study and compare the surface topography in both control and inoculated animals. In control dogs the villi were tall and finger-like in shape and numerous irregular transverse circumferential grooves were present on the surface. At higher magnification, the outlines of individual epithelial cells and depressions, interpreted as goblet cells, could be discerned. In the inoculated dogs, scanning electron microscopy changes were first seen at six days after inoculation. The small intestinal mucosa was covered by a thick layer of mucus. The underlying villi were stunted and had lost their surface features. In some instances there was loss of the luminal epithelium, exposing the lamina propria. In addition, there was dilation of the circumvillar basins and the crypt mouths. There appeared to be regenerative changes by day 7 after inoculation. The surface of the small intestinal mucosa was still covered by a thick layer of mucus. Where villi could be discerned, they were short and pointed and transverse grooves could be seen on their surface. There was some hypertrophy of the intervillus ridges. The changes in the surface topography of the small intestinal mucosa following canine parvovirus infection are compared to those seen in enteric infections in other species and the similarity of the lesion to that seen following sublethal irradiation is discussed.     

Colostral transmission of maedi visna virus: sites of viral entry in lambs born from experimentally infected ewes

Maedi visna virus (MVV) vertical transmission in sheep via infected colostrums is a very important route of infection in lambs. To verify colostral transmission and to study early viral entry in lambs, colostrum samples, and small intestine and mesenteric lymph nodes of lambs born from experimentally infected ewes were examined by histopathology, immunohistochemistry (IHC) and in situ hybridisation (ISH) studies. In particular, newborn lambs were naturally fed maternal colostrum and humanely killed at 10, 24, 48, 72, 96 h and 7 and 10 days after birth; two caesarian-derived lambs served as uninfected controls. No lesions suggestive of MVV infection were found, but marked immunoreactions for MVV capsid antigen (CA, p28) were detected in lambs fed maternal colostrum and in macrophages cultured from colostrum. IHC results in lambs suggest an initial viral absorption by intestinal epithelial cells at the tip of the villi, passage to mononuclear cells in the lamina propria and involvement of ileum Peyers' patches and mesenteric lymph nodes, with different staining patterns depending on infection times. ISH on intestinal sections of the 72 h lamb revealed the presence of proviral DNA in epithelial cells at the tip of the villi, suggesting a role for these cells in early MVV replication. The results contribute to knowledge about the pathogenesis of ovine lentivirus infection suggesting that the small intestine and mesenteric nodes are the sites of entry and propagation of MVV in lambs fed colostrums from infected ewes.     

Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy.

Enteric infection with an attaching and effacing Escherichia coli was diagnosed in a puppy with protracted diarrhea. Extensive colonization of the small intestinal mucosa was observed by light and scanning electron microscopy and characteristic lesions of bacterial attachment of the brush border of the enterocytes were demonstrated by transmission electron microscopy. The E. coli strain isolated from the small intestine belonged to serotype O49:H10, did not produce any known E. coli enterotoxin or cytotoxin, was not invasive, and was negative for the known fimbrial colonization factors produced by animal and human enterotoxigenic E. coli. A positive immunoperoxidase reaction was obtained on the bacteria attached to the enterocytes with an anti-E. coli O49 antiserum.