Direct immunoelectron microscopy of transmissible gastroenteritis virus with immunoglobulins G and A and guinea pig complement

Porcine colostral immunoglobulin (Ig)G and IgA, isolated from transmissible gastroenteritis virus-infected sows, were compared by direct immunoelectron microscopy. It was estimated, using antibodies with a less than a twofold difference in virus-neutralizing activity, that IgG was 500 times more efficient than was IgA for coating transmissible gastroenteritis virions. Guinea pig complement enhanced the antibody coating with IgG, but did not increase virus-neutralizing activity of IgG or IgA.     

Antigenicity of structural components from porcine transmissible gastroenteritis virus

Pregnant sows were inoculated with inactivated transmissible gastroenteritis virus and with preparations of virus surface projections and subviral particles derived by detergent treatment of the virus. Neutralising antibody was demonstrated in serum and colostrum from animals that received whole virus or preparations of surface projections whereas subviral particles failed to stimulate neutralising antibody formation. Similar results were obtained with serum from rabbits inoculated with whole virus and structural components. All three preparations stimulated the formation of agglutinating antibodies, as demonstrated by sedimentation analysis and filtration studies with radiolabelled virus.The immunoglobulin classes responsible for neutralising antibody activity in sows inoculated by the intramammary route were examined. In each case where the immunoglobulin class was determined, IgG was found. One sow that received surface projections also had IgA with neutralising activity in her colostrum. In contrast, infection of sows with live whole virus resulted in neutralising antibody of the IgG, IgM and IgA classes.     

The effect of interferon induction in parturient sows and newborn piglets on resistance to transmissible gastroenteritis.

High titers of interferon were found in the serum and milk of three sows treated two days after farrowing with polyinosinic:polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose, but circulating interferon was not found in the piglets suckled by these sows. When two treated sows and their suckling piglets were exposed to infection with transmissible gastroenteritis virus eight hours after treatment, the sows showed no signs of disease, although they developed circulating interferon in response to the virus infection. The piglets suckled by the treated sows developed signs of transmissible gastroenteritis which were identical to those seen in a control litter of piglets suckled by an untreated sow. Piglets treated at two days of age with the polyinosinic:polycytidylic acid complex showed a delay in onset of clinical signs when exposed to infection with transmissible gastroenteritis virus, compared with untreated control piglets. When two sows were treated with the polyinosinic:polycytidylic acid complex before farrowing, neither circulating interferon nor activated natural killer cells were found in the piglets after birth.     

Efficacy of vaccination of sows with serologically related coronaviruses for control of transmissible gastroenteritis in nursing pigs

Three groups of pregnant sows were vaccinated at 8 and 2 weeks before parturition with tissue culture-adapted feline infectious peritonitis (FIP) virus, porcine transmissible gastroenteritis (TGE) small-plaque (SP) virus from a persistently infected cell line, or noninfected cell culture fluids (controls). Pigs nursing vaccinated sows were orally challenge exposed with virulent TGE virus when they were 1 to 3 days old. The morbidity of the nursing pigs was 48% in the SP-TGE group, 82% in the FIP group, and 93% in the controls. The survival rate among the nursing pigs was 77% in the SP-TGE groups, 48% in the FIP group, and 14% in the controls. Virus-neutralizing antibodies of immunoglobulin A were detected in colostrum and milk of the SP-TGE group, but not in the FIP or control groups.     

A double-protease-resistant variant of transmissible gastroenteritis virus and its ability to induce lactogenic immunity

A virus resistant to 2 major intestinal proteases (trypsin and alpha-chymotrypsin) was derived from the attenuated Purdue strain of transmissible gastroenteritis virus. Its enzymatic stability was confirmed, in vitro, by exposure to proteolytic enzymes and to porcine intestinal fluids. Vaccination of 5 seronegative pregnant sows with the variant virus by a series of 2 oral and 1 IM inoculations resulted in high titers of neutralizing antibody in serum and colostrum. The mean antibody titer in milk whey decreased 44-fold within 1 week after parturition. At 3 days of age, the 40 pigs delivered by these sows were challenge exposed orally with virulent transmissible gastroenteritis virus. Pigs nursing the 5 vaccinated sows underwent a relatively mild clinical course of illness. The average mortality of these 40 pigs was 33%. Thirty-six pigs which had been raised by 4 nonvaccinated sows had a more severe illness, greater daily weight loss, and higher mortality (92%).     

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.     

The postulated role of feeder swine in the perpetuation of the transmissible gastroenteritis virus.

Clinical, immunofluorescence and histopathological observations were found to be an efficient approach for the confirmation of the diagnosis of transmissible gastroenteritis in feeder swine. Two cases are reported to exemplify how feeder swine exposed to points of concentration such as holding areas, sales barns and auctions can play an important role in the epizootiology of transmissible gastroenteritis. A third field case is reported as an example of an outbreak of transmissible gastroenteritis beginning in feeder swine and then spreading to baby pigs on the farm. All baby pigs died that were born during the acute phase of the outbreak in the feeder swine. Baby pigs born shortly after the clinical signs had abated in the herd, and from sows that had been exposed orally to virulent transmissible gastroenteritis virus and vaccinated with a commercial transmissible gastroenteritis vaccine ten days before farrowing, survived. This was explained by a combination of a decrease in the amount of virus shed in the environment and the immunity induced in the sows. These observations of field outbreaks of transmissible gastroenteritis combined with recently reported experimental studies lend strong support to the hypothesis of a reservoir for transmissible gastroenteritis virus in feeder pigs. This reservoir would be based principally on the transmission of the virus on a continuous basis from the feces of recently infected pigs to susceptible pigs. Clinical signs of transmissible gastroenteritis in such pigs are difficult to recognize or absent and this contributes to the importance of the reservoir in the field.     

Experimental Immunization of Sows with Cell-cultured TGE Virus

Five sows were inoculated with a cell-cultured, cytopathic strain of the virus of transmissible gastroenteritis (TGE). Two sows were inoculated intramuscularly, and three by the intramammary route. The response was measured by the neutralizing antibody titers in the serum and the milk, and by the protection against experimental challenge of piglets nursing the sows. There were no marked differences in the serum or milk antibody titers resulting after the two methods of inoculation, although milk titers at the time of challenge were higher after intramammary inoculation. Piglets nursing sows inoculated by the intramammary route were protected to a greater extent than those nursing sows inoculated intramuscularly.     

The detection of transmissible gastroenteritis viral antibodies by immunodiffusion.

Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of othet viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroeneritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus.     

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.     

Colostral IgA, IgG, and IgM-IgA fractions as fluorescent antibody for the detection of the coronavirus of transmissible gastroenteritis.

Colostrum from sows and gilts inoculated with virulent transmissible gastroenteritis virus was fractionated into the 3 major immunoglobulin classes, IgA, IgG, and IgM-IgA fractions, by chromatographic and gel-filtration procedures. Each fraction was assayed for purity with rabbit anti-porcine serum and rabbit monospecific anti-porcine IgG, anti-porcine IgA, and anti-porcine IgM. These analyses showed that the IgG and IgA fractions were pure. The IgM fraction contained some IgA in the polymeric form and was designated the IgM-IgA fraction. Each Ig was assayed for virus-neutralizing activity on swine testes cells by the plaque-reduction method before and after conjugation with fluorescein isothiocyanate. On the basis of activity per milligram of protein, the virus-neutralizing titers were 1:641, 1:44, and 1:6.8 for the IgA, IgG, and IgM-IgA fractions respectively; the fluorescent antibody titers were 1:31.3, 1:0.1, and 1:15.6, respectively, for the same Ig.     

Relationship among transmissible gastroenteritis virus antibody titers in serum, colostrum, and milk from vaccinated sows, and protection in their suckling pigs

We studied the antibody responses to transmissible gastroenteritis (TGE) in serum, colostrum, and milk from sows vaccinated with 2 attenuated (1 IM and 1 oral-IM) and 1 nonattenuated live vaccines and the relationship of these responses with the survivability of the sow's suckling pigs after challenge exposure with virulent TGE virus. Contrary to previous studies, the anti-TGE virus-neutralizing geometric mean titers (GMT) in the milk of sows vaccinated with attenuated vaccines at 3 and 5 days of lactation were similar to that found in the colostrum. Colostral and serum antibody titers were highest in sows given 2 injections of the IM attenuated vaccine. Half of the sows given the oral-IM attenuated vaccine did not seroconvert after 2 oral doses. Only sows vaccinated with the nonattenuated live vaccine had milk GMT that remained high for 21 days after farrowing. The linear relationship between colostral GMT and percentage of survivability of suckling pigs challenge exposed at 3 days of age was significant (P less than 0.05), although the relationship between serum GMT and percentage of survivability and the relationship between milk GMT and percentage of survivability were not significant (P greater than 0.10). The linear relationship between colostral (P less than 0.10) or pre-challenge exposure milk (P less than 0.05) GMT and percentage of survivability of suckling pigs challenge exposed at 5 days of age was significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Upper respiratory infection of lactating sows with transmissible gastroenteritis virus following contact exposure to infected piglets.

Ten breeding sows were left in direct contact with their newborn piglets that had been experimentally infected with transmissible gastroenteritis (TGE) virus. All sows became infected with the virus. The sows developed fever and showed mild clinical signs of the disease for a few days. The sows excreted virus in the nasal secretion, feces, and milk during the acute febrile phase of illness. Virus was isolated from the nasal secretion of one sow as early as 20 hours after contact exposure to the infected piglets. At necropsy, the virus was more frequently isolated from the tissues of the upper respiratory tract than from small intestines; this finding indicated that the TGE coronavirus replicated in the upper respiratory tract and induced an acute respiratory infection in susceptible adult swine. Neutralizing antibody was present in the sera 8 sows after 12 to 36 days during the convalescent period. From these results, we conclude that susceptible sows in direct contact with ill piglets can become infected and by excreting virus can serve as a source of TGE virus for other susceptible pigs on the premises.     

Immune response in sows given transmissible gastroenteritis virus or canine coronavirus

Twelve pregnant sows were inoculated oral-nasally 8 weeks before farrowing with attenuated transmissible gastroenteritis virus (TGEV), tissue culture-adapted canine coronavirus (CCV), or fluids from mock-infected culture (controls). A 2nd dose of the same inoculum, one-half oral-nasally and one-half intramammarily, was given 6 weeks later. Neutralizing antibodies for TGEV and CCV were demonstrated in sera, colostrum, and milk whey from the virus-vaccinated sows. Homologous geometric mean neutralizing titers were generally 4-fold higher than were heterologous titers. After challenge exposure of the nursing pigs with virulent TGEV, average morbidity and mortality for the pigs were 81% and 34% (mortality range = 11% to 63%), respectively, in the TGEV-vaccinated group; 83% and 39% (mortality range = 15% to 83%), respectively, in the CCV-vaccinated group; and 97% and 84% (mortality range = 78% to 100%), respectively, in the controls. Seemingly, sera from swine exposed to CCV could test serologically positive for TGEV-neutralizing antibody, and TGEV and CCV share at least 1 common neutralizing determinant that may be involved in protection.     

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.     

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.     

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.     

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.     

Experimental inoculation of cats with human coronavirus 229E and subsequent challenge with feline infectious peritonitis virus.

Minimal-disease cats exposed to live human coronavirus 229E developed homologous antibody responses that suggested little or no replication of the virus in inoculated animals. Oronasal and subcutaneous inoculation of coronavirus 229E did not elicit an antibody response by heterologous (transmissible gastroenteritis virus, canine coronavirus) neutralization or by heterologous (transmissible gastroenteritis virus) kinetics-based enzyme-linked immunosorbent assay. No clinical signs attributable to coronavirus 229E were seen in inoculated cats. Although the number of animals in each of the five experimental groups was small (n = 2), antibodies produced in response to the virus did not appear to sensitize cats to subsequent feline infectious peritonitis virus challenge, but neither did they cross-protect cats against the challenge dose.     

Antibody-dependent and spontaneous cell-mediated cytotoxicity against transmissible gastroenteritis virus infected cells by lymphocytes from sows, fetuses and neonatal piglets.

The purpose of this study was to investigate the ontogeny in the pig of effector lymphocytes mediating antibody-dependent and spontaneous cell-mediated cytotoxicity against target cells infected with transmissible gastroenteritis virus. The same activities were also studied in sows in late pregnancy and early lactation. Peripheral blood lymphocytes and intraepithelial lymphocytes collected from piglets during the first week of life failed to mediate cytolysis as determined by chromium release assays, but significant activities developed during the second week and usually increased further by the sixth week. Peripheral blood lymphocytes collected from fetuses on the 109th gestation day failed to produce spontaneous cell-mediated cytotoxicity and only reacted in antibody-dependent cell-mediated cytotoxicity when contaminated with macrophages. The cytotoxic activities of peripheral blood lymphocytes from sows were lowest at parturition. It was concluded that impaired lymphocyte cytotoxicity in newborn piglets and parturient sows may contribute to their relatively high susceptibility to transmissible gastroenteritis.