Cell-mediated immune responses of suckling pigs inoculated with attenuated or virulent transmissible gastroenteritis virus

Two litters of suckling pigs seronegative for transmissible gastroenteritis (TGE) virus were orally inoculated with live attenuated (P115) or virulent (M5C) strains of TGE virus. A third seronegative litter (controls) was given cell culture fluids from uninfected cells. Lymphocytes were collected from blood, spleen, mesenteric lymph nodes, and Peyer patches of euthanatized pigs at 0 day and approximately weekly until 26 days after exposure and at approximately 45 days after exposure. Sera were tested for virus-neutralizing antibody titers by use of plaque reduction. Lymphocytes were tested in a lymphocyte proliferation assay for uptake of [3H]thymidine after incubation with the homologous or the heterologous strain of inactivated TGE virus or uninfected cell culture fluids. Only pigs inoculated with virulent TGE virus developed clinical signs of TGE and shed virus. However, all pigs inoculated with TGE virus seroconverted at 6 days after exposure. Responses of lymphocytes from all sources from TGE virus-inoculated pigs peaked between 6 and 14 days after exposure. Pigs inoculated with virulent TGE virus had higher lymphocyte proliferative responses and neutralizing antibody titers than did pigs inoculated with attenuated TGE virus. Cessation of virus shedding coincided with the peak of lymphocyte proliferative responses. The highest responses were with intestinal lymphocytes (mesenteric lymph nodes and Peyer patches) from pigs inoculated with virulent TGE virus. The responses of intestinal lymphocytes from pigs inoculated with attenuated virus were not significantly different from those of pigs inoculated with cell culture fluid. Lymphocytes collected from all sources, except blood from M5C-inoculated pigs, had significantly (P less than 0.05) higher responses to the homologous than to the heterologous TGE virus stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses

Cross-protection studies between the feline infectious peritonitis (FIP) and the porcine transmissible gastroenteritis (TGE) viruses were conducted in cats, pigs and pregnant gilts. Cats vaccinated with TGE virus developed neutralizing antibodies against TGE virus and low titer antibody against FIP virus detected by an indirect fluorescent antibody technique but were not protected against a virulent FIP virus challenge. Baby pigs and pregnant gilts vaccinated with FIP virus did not develop detectable antibodies to TGE virus. Nevertheless, it appeared that vaccination of swine with FIP virus conferred some immunity against TGE virus infection. Seventeen-day-old pigs vaccinated with two doses of FIP virus had a 67% survival rate following a virulent TGE virus challenge, and 75% of the 3-day-old pigs suckling either FIP or TGE-virus-vaccinated gilts survived virulent TGE virus infection in contrast to 0% survival of baby pigs suckling unvaccinated gilts.     

Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination

A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus-neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do.     

Oral transmission of transmissible gastroenteritis virus by muscle and lymph node from slaughtered pigs

A study was conducted in the USA to determine whether transmissible gastroenteritis (TGE) virus could be transmitted from carcases of slaughtered pigs. Transmissible gastroenteritis virus was transmitted to 6-day-old piglets by dosing with homogenates of muscle and lymph node collected from 500 clinically normal pigs at the time of slaughter. All piglets in 2 separately housed litters showed clinical signs of TGE with 5 piglets dying within 10 d of oral dosing with homogenates. Transmissible gastroenteritis virus was isolated from 2 of these piglets and all piglets developed TGE antibody. Transmissible gastroenteritis virus was not isolated in tissue culture from muscle and lymph node homogenates, but was isolated from 4 (0.8%) of 500 tonsil samples collected from the same pigs. A survey of 250 serum samples provided an estimate of the prevalence of slaughtered pigs with TGE antibody of 34.8% in the sample population. The results indicate that carcases of some pigs from TGE endemic areas contain viable TGE virus, and that there would be a substantial risk of introducing TGE virus into Australia by the importation of uncooked pig meat from these areas.     

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.     

Isolation of transmissible gastroenteritis virus from pharyngeal swabs obtained from sows at slaughter.

A virologic survey was conducted to determine the frequency of transmissible gastroenteritis (TGE) virus infection in farm-raised sows. Pharyngeal swab specimens collected in an abattoir were examined for TGE virus by inoculation onto swine-testes cell culures. The virus was detected in 61 (3%) of a sample of 2,058 Iowa sows after slaughter. All TGE viral isolates, given orally to 2- or 3-day-old pigs, caused acute gastroenteritis and in some cases death. All pigs that recovered from illness had serum antibody to TGE virus.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of transmissible gastroenteritis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) using a detergent-solubilized antigen of purified virus was developed for detection of antibody against porcine transmissible gastroenteritis (TGE) virus in swine serum. The ELISA demonstrated antibody responses in pigs immunized intramuscularly with the attenuated TO-163 strain of TGE virus and in pigs orally infected with the virulent Shizuoka strain of the virus. The results of the ELISA were well correlated with those of the neutralization test. These results indicate the usefulness of the ELISA as a serological tool for TGE virus antibody.     

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.     

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.     

Effects of ambient temperatures on clinical and immune responses of pigs infected with transmissible gastro-enteritis virus

Two- to three-months-old pigs infected with transmissible gastroenteritis (TGE) virus showed no clinical response when housed at 30°C, but comparable infected pigs exposed to temperature changes from 30°C to 4°C following infection showed typical signs of TGE. Development of TGE-specific immune responses, as measured by blastogenic response of tissue lymphocytes, occurred at 3 days post-inoculation (DPI) in pigs held at 30°C, but not until 7 DPI in infected pigs held under the adverse conditions.Immunosuppression with corticosteroids resulted in a fall in circulatory T cells, lowering of non-specific blastogenic response of circulatory lymphocytes, and clinical signs of disease when immunosuppressed pigs were infected with TGE virus and held at 30°C. It is suggested that clinical responses to TGE virus infection may be affected by the influence of ambient temperatures on the immune responses of pigs.     

Recovery of transmissible gastroenteritis virus from chronically infected experimental pigs.

Transmissible gastroenteritis (TGE) virus was reisolated from pulmonary and intestinal tissues from 6 of 9 chronically infected experimental pigs (principals) necropsied 30 to 104 days after inoculation. Tissue homogenates (lung and small intestine) from the principals were prepared and inoculated into 3- to 5-day-old gnotobiotic pigs. The virus reisolated from the tissue homogenates produced a milder disease on 1st passage and a more severe disease on 2nd passage. The chronically infected experimental pigs (principals) developed serum-neutralization titers to TGE of 1:30 to 1:525. There appeared to be no relationship between serum titers and reisolation of TGE virus from the 9 principals. The persistence of virus in lung or intestine to 104 days indicates the recovered (or carrier) pig may be considered the primary source of TGE virus infection.     

Studies on Transmissible Gastroenteritis of Swine I. The Isolation and Identification of a Cytopathogenic Virus of Transmissible Gastroenteritis in Primary Swine Kidney Cell Cultures

Two virus isolates from transmissible gastroenteritis (TGE) of swine were adapted to grow in primary swine kidney cells. Growth of the virus was indicated by the resistance of the infected cells to the cytopathic effect of a virus diarrhea virus of cattle, and by the development of large round cells on the cell sheet.

Evidence that these virus isolates were TGE was obtained by the development of signs of the disease followed by death of exposed SPF pigs, or the resistance of the recovered pigs to further signs of disease when they were exposed to virulent TGE contained in virus bearing intestinal tissue.

The in vitro and in vivo serum neutralization tests, along with staining of infected cells by fluorescein conjugated TGE antiserum, gave further indication of the specific nature of the virus growing in the cell cultures.

     

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.

     

Mild transmissible gastroenteritis in pigs suckling vaccinated sows

A strain of transmissible gastroenteritis (TGE) virus of low virulence was isolated from 14-day-old pigs suckling sows vaccinated with an attenuated TGE vaccine. Diarrhea developed in suckling pigs approximately 14 days after farrowing in 4 farrowings; however, none of these pigs died from diarrhea. Diarrhea ceased after the 4th farrowing, when vaccination of sows was discontinued. Experimentally, both the field isolate and the vaccine strain were infective and in some instances lethal for 2-day-old pigs exposed orally; however, neither strain was as virulent as the Purdue strain.     

Clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows

Two federally licensed attenuated live transmissible gastroenteritis (TGE) virus vaccines (an IM vaccine and an oral-IM vaccine) and 1 nonlicensed nonattenuated live TGE virus vaccine were evaluated and compared in sows free of TGE virus-neutralizing antibodies. Litters from the sows were challenge exposed at 3 and 5 days of age, and results were combined according to the vaccine administered to the sows. The survivability of pigs suckling sows vaccinated with the nonattenuated vaccine was significantly (P less than 0.01) greater than that of pigs suckling sows vaccinated with the IM attenuated vaccine, significantly (P less than 0.05) greater than that of pigs suckling sows vaccinated with the oral-IM attenuated vaccine, and significantly (P less than 0.05) greater than that of pigs suckling sows that had not been vaccinated. The differences, however, between survivability of litters from sows vaccinated with the IM attenuated vaccine or the oral-IM attenuated vaccine and that of litters from the sows not vaccinated were not significant (P greater than 0.10). The nonattenuated TGE vaccine, although giving a higher level of protection than the attenuated vaccine, was eventually overwhelmed. Dexamethasone did not increase the incidence of diarrhea, and levamisole did not potentiate the lactogenic immunity in sows after given their first dose of the nonattenuated vaccine. Survivability in litters suckling sows that developed diarrhea after given their first dose of the nonattenuated vaccine was not greater than that in litters suckling sows that did not develop diarrhea. The best results were obtained when 3-day-old suckling pigs were challenge exposed with virulent TGE virus.     

Experimental Immunization of Sows with Inactivated Transmissible Gastroenteritis (TGE) Virus

Four sows were immunized with inactivated TGE virus. The virus was either propagated in pig kidney cell cultures, or obtained from the intestines of experimentally infected gnotobiotic pigs, and inactivated by treatment with β-propiolactone. The inactivated virus preparations were administered to sows by intramammary inoculation. Two sows received two inoculations; the other two sows received three inoculations of inactivated virus. The antibody responses in the serum and milk were determined and piglets nursing the sows were experimentally challenged with virulent TGE virus. Three inoculations of the virus preparation stimulated much higher levels of serum and milk antibody than did two inoculations. A schedule of three inoculations of sows with inactivated TGE virus was effective in stimulating protection against TGE for piglets nursing these sows.     

Detection of antibody against transmissible gastroenteritis virus of pigs by indirect immunoperoxidase antibody test

Immunoperoxidase intibody (IPA) test was developed for detecting antibody against transmissible gastroenteritis (TGE) virus of pigs. The IPA antibody titers in sera collected in the field from 82 pigs were approximately seven times higher than those obtained in a serum-neutralization test. The correlation between the TGE antibody concentrations in the IPA and serum neutralization tests was positive (r = +0.74). The IPA tests appears to have the potential for routine laboratory use for serologic diagnosis of TGE.     

Concurrent porcine rotaviral and transmissible gastroenteritis viral infections in a three-day-old conventional pig.

A 3-day-old suckling pig with diarrhea was necropsied, and immunofluorescent microscopic examination of the small intestinal mucosa, together with immune electron microscopic examination of the large intestinal contents, provided a presumptive diagnosis of a concurrent infection with transmissible gastroenteritis (TGE) virus and porcine rotavirus. Immunofluorescent microscopic, immune electron microscopic, and serologic data obtained from gnotobiotic pigs experimentally inoculated with the large intestinal contents of the suckling pig confirmed this diagnosis. Two gnotobiotic pigs, convalescent from previous TGE viral infections, became infected with porcine rotavirus only. However, another gnotobiotic pig, convalescent from a previous porcine rotaviral infection, became infected with TGE virus only, following inoculation with the large intestinal contents of the suckling pig.     

Lesions of transmissible gastroenteritis virus infection in experimentally inoculated pigs suckling immunized sows

Intestinal lesions of transmissible gastroenteritis (TGE) virus infection in conventionally reared pigs suckling either nonvaccinated, vaccinated, or previously infected sows were studied by scanning electron microscopy, light microscopy, and immunofluorescent microscopy for TGE viral antigen. Pigs were inoculated with virulent TGE virus when they were 5 or 21 days old and were euthanatized shortly after the onset of diarrhea or 96 hours after inoculation if no diarrhea developed. Pigs inoculated when they were either 5 or 21 days old and suckling nonvaccinated sows developed severe lesions, including swelling and necrosis of enterocytes and severe villus atrophy. Pigs inoculated when they were 5 days old and suckling sows vaccinated with attenuated vaccines developed less-severe villus atrophy, and those suckling sows immunized by exposure to nonattenuated TGE virus developed moderate or no villus atrophy. Pigs inoculated when they were 21 days old and suckling sows vaccinated with attenuated vaccines had severe villus atrophy, whereas those suckling sows immunized by exposure to nonattenuated virus had more-moderate villus lesions. Villus atrophy was inhibited to various degrees in pigs suckling immunized sows, depending in part on the antibody titer in the colostrum and milk.     

Quantitative transmissible gastroenteritis virus shedding patterns in lactating sows.

To test the role of sows in spreading transmissible gastroenteritis (TGE), 11 sows were intravenously, intranasally, or intramammarily inoculated with virulent virus within 5 days of farrowing. Six of the sows were separated from their offspring, and 5 were allowed to nurse their litters. All sows became clinically ill with sign of anorexia, depression, and fever that persisted until postinoculation day 4 or 5. They shed virus through milk, nasal secretions, and feces, with individual variations occurring in degree and duration of shedding in the 1st week after inoculation. Of 40 pigs separately fed milk samples from the 6 inoculated sows, 19 pigs (47.5%) became sick in 24 to 40 hours, and virus was isolated from them at necropsy. Of 43 pigs in the 5 litters that nursed exposed dams, all became sick with typical signs of TGE, and 29 (67.4%) died in 2 to 9 days. Sows given the single intramammary inoculation of virus developed statistically significant higher levels of TGE virus-neutralizing antibodies than did sows inoculated intravenously or intranasally.