Lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses

The infectivity and pathogenicity to newborn pigs of antigenically related coronaviruses from pigs (transmissible gastroenteritis virus; TGEV), cats (feline infectious peritonitis virus; FIPV), and dogs (canine gastroenteritis virus; CGEV) were studied by light, scanning electron, and immunofluorescence microscopy. Hysterectomy-derived, 12-hour-old pigs were orally given tissue culture or frozen preparations of 6 coronavirus strains (3 porcine, 2 feline, and 1 canine). The pigs were killed at regular intervals between 24 and 144 hours after exposure. Virulent TGEV and virulent FIPV produced necrosis of villous epithelium, resulting in villous atrophy in the jejunum and the ileum. Similar, but less extensive and severe lesions, were produced by the 4 other viruses. Coronaviral antigens were identified by immunofluorescence in villous epithelial cells of pigs that had been inoculated with virulent TGEV, attenuated TGEV, virulent FIPV, and tissue culture-adapted FIPV. In contrast, coronaviral antigens were not induced by the small plaque variant TGEV and virulent CGEV in the villous epithelium, but rather in cells of the lamina propria and crypt epithelium.     

Pathogenicity of an attenuated strain of transmissible gastroenteritis virus for newborn pigs.

The pathogenicity of a cell culture-attenuated strain of transmissible gastroenteritis virus for newborn pigs was investigated. Newborn (1- to 2-day-old) pigs were orally given 2 x 10(6) plaque-forming units of attenuated virus. All pigs developed mild diarrhea, but deaths did not occur. As determined by immunofluorescence and villous atropy, infection of the small intestine was limited to the caudal 50 to 66%. Fluorescing cells and atrophic villi were seen from 2 to 3 days until 6 to 7 days after exposure. Attenuated virus-exposed pigs produced circulating virus-neutralizing antibodies detectable as early as 5 days after exposure. By contrast, all pigs orally given 1 x 10(2) pig infective doses of virulent transmissible gastroenteritis virus developed severe diarrhea, and almost all of those not killed died within 2 to 5 days after exposure. In the latter pigs, the entire length of the small intestine, except for the first 4 to 5 cm, was infected with virus by 24 to 36 hours after exposure.     

Effect of virus-induced destruction of villous epithelium on intestinal secretion induced by heat-stable Escherichia coli enterotoxins and prostaglandin E1 in swine

Villous atrophy and crypt hyperplasia were induced in the jejunal epithelium of thirteen 3-week-old pigs by inoculation with transmissible gastroenteritis virus. The responses (changes in net fluid movement) induced in ligated intestinal loops of these pigs by intraloop injections of prostaglandin E1 (PGE1) or Escherichia coli broth culture filtrates containing either or both E coli heat-stable enterotoxins (STa and STb) were compared with the responses induced by these preparations in littermates not inoculated with virus. Villous atrophy was associated with a marked decrease in response to preparations containing STa, STb, or STa + STb, but the response to PGE1 was undiminished. These results were consistent with the reports of others that the response to cyclic adenosine monophosphate-mediated secretogogues (PGE1) is a function of crypt epithelium; however, the present results also suggest that the secretory response to STa and to STb is dependent on the integrity of the villous epithelium. In the present study, loss of villous epithelium was associated with loss of response to STa and STb, but not to PGE1.     

Transmissible Gastroenteritis in Feeder Swine: Clinical, Immunofluorescence and Histopathological Observations

Eight feeder swine (four to six months of age) were inoculated orally with 200,000 to 500,000 pig infectious doses (PID) of the Purdue strain of transmissible gastroenteritis (TGE) virus. Biopsies obtained from their small intestines were examined histopathologically and by fluorescent antibody tissue section technique at intervals that included 24, 48, 72 and 96 hours postexposure, and similar examinations were carried out at necropsy 168 hours postexposure. Evidence of virus infection was demonstrated in all segments of the small intestine except the upper duodenum and the viral antigen was found only in the cytoplasm of the absorptive cells covering the villi. Although six of the eight pigs failed to show clinical signs of TGE, typical microscopic lesions of villous atrophy with replacement of columnar absorptive cells by cuboidal cells were observed in seven pigs, and TGE virus antigen was demonstrated in the intestinal cells of four of eight pigs during the first week postexposure. The infection was usually mild to moderate and focal in the pigs without clinical signs of the disease and more severe and extensive in the pigs with clinical signs of the disease variable in severity. It was concluded that TGE virus probably replicated in all feeder swine exposed, and that the presence or absence of clinical signs of TGE in these pigs was related to the severity and extent of the villous atrophy and columnar cell replacement induced in their small intestines.     

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.     

Age Dependent Resistance to Transmissible Gastroenteritis of Swine (TGE) I. Clinical Signs and Some Mucosal Dimensions in Small Intestine

Pigs were exposed to transmissible gastroenteritis (TGE) virus when three days old or when 21 days old. Diarrhea was earliest in onset, most frequent, most profuse and most prolonged in the youngest group. Pigs exposed when three days old also had a higher case fatality rate than those exposed when 21 days old. The histological response of both groups to exposure was atrophy of villi and hyperplasia of crypts in jejunum and ileum. However, from days three to seven post-exposure, when most fatalities occurred in the younger group, atrophy of villi was both more intensive and extensive in the younger group. Hyperplasia of crypts was also greater and more prolonged in the younger group. Regeneration of atrophic villi was more rapid in jejunum than ileum in both groups. Results were interpreted to indicate two populations, with different rates of regeneration, in the 21-day old group. Based on this interpretation, regeneration of villi was more rapid in one population from the 21-day old group than in the three-day old group.

The length of villi and depth of crypts in control pigs varied longitudinally (i.e. from site to site) in the intestine, within each age group. Length of villi and depth of crypts in control pigs also varied with age.

     

Neutralization of a transmissible gastroenteritis virus of swine by colostral antibodies elicited by intestine and cell culture-propagated virus.

Cross-protection studies of gilts exposed to 4 transmissible gastroenteritis viruses--Ilinois (field strain), Miller-3, Miller low passage (M-LP), and Miller high passage (M-HP) tissue culture-adapted--indicated that only the gilt vaccinated with Illinois strain was protected, along with its newborn pigs, against challenge exposure with field virus. Similar results were obtained when the 4 viruses were incubated in vitro with colostrum from each of the 4 vaccinated gilts and subsequently used to orally inoculate newborn pigs. However, when the colostrums were used to neutralize M-HP virus in cell cultures, the neutralization titers were similar, indicating that a close antigenic relationship existed among the viruses. Neutralization studies in cell cultures, using immunoglobulin (Ig) fractions derived from colostrums of sows exposed to Illinois and M-HP virus, indicated that Illinois virus elicited more neutralizing activity in IgA than in the IgG fraction and that M-HP virus elicited more IgG than IgA antibody activity. In another study, Illinois virus was treated with these Ig-enriched fractions and then inoculated into the lumen of the jejunum of 3-day-old pigs. Anti-Illinois IgA was the only class of antibody which prevented replication of the Illinois virus in the intestine. Similar intraintestinal inoculations were used to test invasiveness of untreated Illinois and M-HP viruses. It was demonstrated that Illinois virus caused marked effect on the intestine: shortening of the villi, intestinal distension, edema, and presence of accumulated intestinal fluid within 60 hours after inoculation. The M-HP virus grew in the intestinal cells without affecting the length of the villi. The degree of invasiveness of Illinois or M-HP virus may account for the difference in the antibody class elicited in the colostrums.     

Rotavirus as a cause of diarrhea in pigs.

A rotavirus (reovirus-like agent) was associated with diarrheal diseases occurring in 1- to 4-week-old suckling pigs in 8 herds and in weaned pigs in 2 herds. Transmissible gastroenteritis virus was also detected in 2 of these herds, as was enteropathogenic Escherichia coli in 5 herds. Morbidity was generally greater than 80% in pigs of the affected age group within these herds, and mortality from diarrhea ranged from 7 to 20%. The disease due to rotavirus in suckling pigs appeared similar to the syndrome commonly referred to as milk scours, white scours, or 3-week scours. Diarrhea and villous atrophy, resembling that seen in transmissible gastroenteritis, occurred in naturally infected pigs and in gnotobiotic pigs experimentally infected with rotavirus. Diagnosis was accomplished by immune electron microscopy of intestinal contents and by immunofluorescent staining of enterocytes. A massive infection of enterocytes with rotavirus was demonstrated by immunofluorescence, which helps explain the pathogenesis of this disease. The apparent rarity of clinical rotaviral infections in suckling pigs greater than 7 days old is probably due to the acquisition of passive immunity from immune sows.     

Effect of recombinant DNA-derived bovine alpha-1 interferon on transmissible gastroenteritis virus infection in swine

Recombinant DNA-derived bovine interferon alpha 1-1 (BoIFN) inhibited replication of both vesicular stomatitis virus and transmissible gastroenteritis virus in cultures of swine testicular cells. Newborn pigs were orally inoculated with BoIFN and subsequently had interferon in their gastric contents and serum; however, interferon was found only occasionally in intestinal washings. Incubation of BoIFN with gastric contents from a newborn suckling pig did not affect antiviral activity, whereas intestinal (small intestine) contents from the same animal inactivated BoIFN within 1 minute. Beginning at 6 hours of age, newborn, colostrum-deprived pigs were given 1 mg of BoIFN orally every 12 hours. These pigs were not protected against challenge exposure to virulent transmissible gastroenteritis virus at 48 hours of age; disease and mortality were similar for these pigs and for control pigs not given BoIFN prior to challenge exposure. The BoIFN did not impair growth rate of pigs and did not cause obvious disease or lesions.     

Diagnosis of Transmissible Gastroenteritis in Pigs by Means of Immunofluorescence

The fluorescent antibody (FA) test for the diagnosis of field outbreaks of transmissible gastroenteritis (TGE) in baby pigs was compared to other available means including: virus isolation by inoculation of test pigs, intestinal lesions especially villous atrophy, and clinical observations.

Immunofluorescent tests were done on frozen sections of the small intestine and it was possible to make a specific diagnosis within two hours after collecting samples. The results obtained with the FA test compared favorably with virus isolation from infected tissues. It was considered a more advantageous procedure as long as infected pigs were in a relatively early phase of the disease. Because of the variability of the lesions as related to the stage of infection, pathologic diagnoses were less satisfactory. Field diagnoses made on the basis of clinical signs were least reliable.

     

Antibody response in pigs inoculated with transmissible gastroenteritis virus and cross reactions among ten isolates.

Groups of two or three day old pigs were inoculated intravenously with cell culture grown transmissible gastroenteritis virus. A single or a multiple dosage schedule was used. The magnitude of immune response was measured in terms of serum neutralization indices. A single dose of relatively attenuated virus caused mild clinical signs of transmissible gastroenteritis infection in the pigs and induced a low level of antibody in the serum by the seventh day after inoculation. Repeated injections of virus at seven day intervals stimulated little increase in antibody titers. However, high serum antibody titers were obtained for all pigs if the time interval between injections was extended to 15 days. Sera obtained early after exposure to live transmissible gastroenteritis virus contained mainly IgM antibody whereas sera obtained later after exposure contained mainly IgG antibody. Ten plaque purified isolates of transmissible gastroenteritis virus, comprising eight American isolates, one Japanese isolate and one British isolate were indistinguishable by means of reciprocal plaque reduction neutralization tests.     

The pathogenesis of an enteric infection in pigs,experimentally induced by the coronavirus-like agent,CV 777

Sixteen 2–3-days-old caesarean-derived, colostrum-deprived piglets were each dosed oro-nasally with 2 ml of a bacteria-free filtrate containing 10⁴ pig-infectious-doses of CV 777. The piglets were killed at intervals of 12 to 120 h after infection. The coronvirus-like agent caused a local infection of the intestinal tract which resulted in villous atrophy, malabsorption and diarrhea. The pathogenesis of this infection was similar to that of transmissible gastroenteritis (TGE), a known coronaviral infection of pigs. However, where were some differences. By immunofluorescent staining, CV 777 antigens were not only detected in the epithelial cells covering the small intestinal villi, but also in the cells of the colonic surface epithelium. Occasional fluorescence was also seen in the small intestinal crypt epithelium, but the regenerative capacity of the crypts was not affected. The progress of intestinal epithelial cell infection by CV 777 was much slower than that in TGE, resulting in a longer incubation period and in less drastic epithelial cell destruction. The infection of regenerating cells occurred to a much higher degree during the late stage of a CV 777 infection than has been observed in TGE.     

Intrafetal inoculation of swine with transmissible gastroenteritis virus.

Fetuses in 3 sows were inoculated (intramuscularly) with transmissible gastroenteritis (TGE) virus on 95th, 77th, and 74th days of the gestation. At 15, 14, and 37 days later (or days when pigs were obtained by hysterectomy), there was evidence of intestinal localization of virus, with villous atrophy and subsequent repair. All intrafetal-inoculated pigs became serologic-positive for TGE. A noninoculated pig shown to be seropositive for TGE at 15 days of age (after hysterectomy) was resistant to challenge exposure with virulent TGE virus given on the 32nd day, in contrast to 3 seronegative littermates that developed typical disease when challenge exposed.     

The effect of adoptive transfer of mononuclear leukocytes from an adult donor on spontaneous cell-mediated cytotoxicity and resistance to transmissible gastroenteritis in neonatal piglets.

The purpose of this study was to attempt to establish spontaneous cell-mediated cytotoxicity effector activity in the intraepithelial lymphocytes of neonatal piglets by adoptive transfer of mononuclear leukocytes from an adult donor and to determine the effect of transfer on the resistance of piglets to transmissible gastroenteritis. Cytotoxicity was determined by a chromium release assay using PK-15 cells persistently infected with transmissible gastroenteritis virus as targets. The experimental animals were inbred miniature pigs, in which a high degree of uniformity in lymphocyte defined histocompatibility complex antigens was demonstrated by the mixed lymphocyte reaction. Adoptive transfer of 8 X 10(7)-4 X 10(8) adult pig leukocytes established effector activity in eight recipient piglets, and leukocytes labelled with fluorescein isothiocyanate homed to the epithelium of the small intestine. When four recipients of 5 X 10(8) adult leukocytes were challenged with transmissible gastroenteritis virus, the onset of diarrhea was delayed for 24 h and the diarrhea was usually milder than in four untreated control piglets. It was concluded that the adoptive transfer of leukocytes with spontaneous cell-mediated cytotoxicity effector activity, which homed to the small intestinal epithelium, may have contributed to an increased resistance to transmissible gastroenteritis.     

Transmissible Gastroenteritis in Feeder Pigs: Observations on the Jejunal Epithelium of Normal Feeder Pigs and Feeder Pigs Infected with TGE Virus

Light and electron microscopy findings in the jejunal mucosa of the normal feeder pig and feeder pigs infected with transmissible gastroenteritis (TGE) virus are reported. Villi in the mid jejunum of the normal feeder pig were elongated, finger shaped and covered with a layer of columnar absorptive cells with a well developed and regular brush border. Severe lesions of villous atrophy were present in all jejunal segments of feeder swine killed 96 hours post infection with TGE virus. Atrophic villi were covered by flat to cuboidal cells with a poorly developed brush border in some areas. In other segments, cells varied in appearance from sub-columnar to columnar type of near normal appearance.

The ultrastructure of the jejunal absorptive cells in the normal feeder pig was found to be similar to that described for the jejunal cells of other adult mammals. There were no significant indications of high pinocytotic activity. The epithelial cells covering the atrophic villi of TGE infected pigs had a fine structure similar to that described for the crypt cells, ranging in appearance from very immature to moderately differentiated cells. Microvilli were very short, decreased markedly in number and irregular in arrangement. The terminal web was poorly developed. Strands of rough endoplasmic reticulum were markedly diminished and an increase in free ribosomes was noted. The significance of these observations in explaining pathogenesis of TGE in feeder pigs is discussed.

     

Lesions of the Gastrointestinal Tract of Pigs Infected with Transmissible Gastroenteritis

Gross, subgross and histological lesions were studied in 103 pigs infected with transmissible gastroenteritis virus and killed at daily intervals for 14 days. Twenty-three pigs served as controls. Thirty-six pigs were given colchicine four hours prior to being killed in order to determine the mitotic activity in the gastrointestinal tract. The gross lesions consisted of dehydration, excessive milk curd in the stomach, focal hemorrhage in the submucosa of the diverticulum ventriculi of the stomach, fundic and pyloric congestion in severly dehydrated animals and thinning of the small intestinal wall. The major subgross lesion was a marked shortening of the villi in the lower duodenum, jejunum and ileum within 24 hours after exposure to the virus. Regrowth of the villi became evident on about the sixth day after infection. Histological examination of the small intestine revealed that the villus-height/crypt-depth ratio in the jejunum was reduced from 7:1 in normal pigs to less than 1:1 in infected pigs. Villous atrophy was less severe in the proximal duodenum and ileum. Cells covering the atrophic villi were flatened or cuboidal and did not have well defined brush borders. Inflammatory changes in the gastrointestinal tract were minimal at all stages of infection. Goblet cell numbers increased slightly in the recovery stage of the disease and small numbers of mononuclear cells accumulated in the lamina propria during regrowth of the villi. The number of metaphase nuclei in the small intestinal crypts of infected pigs was greater than in normal pigs.     

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.     

An outbreak of diarrhea in piglets caused by a coronavirus antigenically distinct from transmissible gastroenteritis virus

Coronavirus-like particles were visualized by electron microscopy in the intestinal contents of piglets during a diarrheal outbreak on a Quebec pig farm. The precipitating antigens of transmissible gastroenteritis virus were not detected in the intestinal contents of diarrheic animals by counter-immunoelectrophoresis. Insignificant antibody titers against transmissible gastroenteritis virus were demonstrated in the sera of convalescent pigs by indirect immunofluorescence and these sera did not react with transmissible gastroenteritis virus when tested by immunoelectron microscopy. The causative agent could not be isolated in cell cultures. It was concluded that a coronavirus antigenically distinct from transmissible gastroenteritis virus was responsible for the enteric problems observed on this farm. The outbreak was controlled after oral inoculation of adult pigs with infected intestinal contents.     

Infection of pigs with transmissible gastroenteritis virus from contaminated carcases

Sixteen 6-month-old pigs were exposed to transmissible gastroenteritis (TGE) virus by placing them in close contact with piglets infected at 1 week of age. Fourteen of the older pigs were slaughtered between 1 and 5 d after exposure to infection and their carcases dressed in simulated abattoir conditions. Samples of muscle, bone marrow and carcase lymph nodes were stored at -25 degrees C for at least 30 d and then homogenised and fed to groups of 1-week-old and 3-week-old pigs. Four of 12 one-week-old pigs died and TGE virus was isolated from intestinal contents of one of these. All pigs of both age groups developed neutralising antibody to TGE virus over the ensuing 4 w. The results indicate that carcases from pigs infected with TGE virus can represent a source of infection for susceptible pigs given access to them.     

Pathogenicity of experimental infection with 'pneumotropic' porcine coronavirus

Virus localisation and lesions were studied in 14-one-week-old piglets following combined intranasal-oral inoculation with a British isolate of 'pneumotropic' porcine coronavirus (PCV) and were compared with the effects of transmissible gastroenteritis virus (TGEV) infection in five piglets. Unlike TGEV-infected piglets, all PCV-inoculated piglets remained clinically healthy. Seroconversion was detected at seven days after inoculation. Mild bronchointerstitial pneumonia involving terminal airways was consistently present at two days after infection and thereafter. Both PCV and TGEV infected bronchiolar epithelium and alveolar macrophages but, unlike TGEV, replication by PCV in villous enterocytes was limited and did not cause villous atrophy.