Evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.     

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.     

Multiplication of low and high cell culture passaged strains of transmissible gastroenteritis virus in organs of newborn piglets

Distribution and persistence of four different strains of transmissible gastroenteritis (TGE) virus in newborn piglets were compared.The piglets inoculated with high-passaged TO-163 strain did not show any clinical signs of TGE on any days postinoculation (DPI), but the piglets inoculated with one of the other three strains, SH-14, SH-164 or TO-16, had soft feces or diarrhea. In the latter cases, the virus was isolated mainly from respiratory organs, lymph nodes, and digestive tract on any DPI, but was rarely detected in the digestive tract of piglets inoculated with the TO-163 strain. The frequency of virus recovery from the tissues was the highest till 4 DPI in all of the piglets inoculated with one of the four virus strains, and it was markedly reduced thereafter in the piglets inoculated with high-passaged strains.The TO-163 strain was subjected to serial passage in newborn piglets for seven passages. There was no evidence of regained pathogenicity with advance in passage, and detection of virus was restricted to lymph nodes and lung of these piglets.In gnotobiotic piglets inoculated with the TO-163 strain, frequent virus recovery and high titers of virus from the tissues were obtained on up to the 4th DPI. The viruses in high titer were found in the digestive tract of some of the piglets; however, none of them showed any clinical signs of TGE.     

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.     

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.     

Lactogenic immunity to transmissible gastroenteritis (TGE) of swine induced by the attenuated Nouzilly strain of TGE virus: passive protection of piglets and detection of serum and milk antibody classes by ELISA

Piglets of eight sows vaccinated by different routes with the attenuated TGE mutant coronavirus, Nouzilly (N) strain, and piglets from two field seropositive sows were challenged with a virulent TGE strain. On the day of challenge and 10 days after challenge, milk and serum samples from sows were analysed for their level of neutralizing antibodies, total immunoglobulin classes and TGE antibody classes by an ELISA. No direct relationship was seen between the level of protection of the litters and the titres of the different antibody classes on the day of challenge. However, an inverse correlation was seen 10 days after challenge between protection and the level of TGE antibodies.     

Transmissible gastroenteritis (TGE) of swine: effect of age of swine testes cells culture monolayers on plaque assays of TGE virus.

A continuous line of swine testes cell culture monolayers was infected at various ages with both cell culture-adapted transmissible gastroenteritis (TGE) virus and tissue infected with TGE virus. Both produced increasing numbers of plaques as the cell monolayers aged from two to five days. Therefore, allowing the swine testes cell monolayer to age five to six days before inoculation should increase the likelihood of detecting TGE virus by plaque assay.     

Effect of experimentally-induced villus atrophy on adhesion of K88ac-positive Escherichia coli in just-weaned piglets

Three- to four-week-old, just-weaned piglets were infected with transmissible gastroenteritis (TGE) virus and the next day with K88ac+ enterotoxigenic Escherichia coli (ETEC). Histological examination of caudal jejunum and ileum of piglets killed 2-3 days after virus challenge (1-2 days after ETEC infection) revealed severe villus atrophy especially in the jejunum compared with controls (P less than 0.05). Four-5 days after TGE virus infection villus length increased and after 7 days it was near normal. Villi scraped from jejunal and ileal mucosa of the piglets were incubated in vitro with K88ac+ E. coli and the number of bacteria adhering to 250 micron villus brush border was counted. Attachment of bacteria to villi of piglets killed 2-3 days after TGE virus infection was significantly decreased in comparison with adhesion to villi of non-infected piglets or of piglets killed 7 days after the virus infection. Correlation between in vitro adhesion and villus height was 0.6649 (P less than 0.001). The results suggest that the experimentally-induced villus atrophy was attended with a temporarily diminished susceptibility of villus enterocytes to adhesion of K88ac+ E. coli.     

Electron Microscopy of Intestinal Epithelial Cells of Piglets Infected with a Transmissible Gastroenteritis Virus

An electron microscopic study of intestinal epithelial cells of neonatal piglets infected with transmissible gastroenteritis (TGE) virus revealed a unique parasite-host cell interaction. Entry of the TGE virus into intestinal epithelial cells of newborn piglets is mediated through a network of cytoplasmic tubules of plasmalemma origin. the tubules, called microcanaliculi, are morphologically distinct from endoplasmic reticulum and Golgi. In uninfected animals similar tubules appear to be responsible for the indiscriminate uptake of large quantities of macromolecules from colostrum during the first few days of life.

Thin-section profiles of plasmalemma invaginations resembled tubules or canals and frequently contained viral particles. TGE viral particles developed and accumulated within cytoplasmic vacuoles. Initially the vacuoles were continuous with the microcanaliculi formed by deep plasmalemma invagination. Mature viral particles were 60 to 85 mu in diameter with an electron dense doughnut-shaped nucleoid surrounded by a trilaminar membrane which resembled the vacuolar wall. Abundant evidence of viral effects was observed in absorbtive epithelial cells of the jejunum and ileum but not of the duodenum.

The ability of absorbtive intestinal epithelial cells to form deep cytoplasmic tubular invaginations is temporally related to the pathogenesis of TGE and may explain in part why pigs usually are fatally affected by TGE only during the neonatal period.

     

猪传染性胃肠炎病毒吉林株的分离鉴定及S基因的克隆与序列分析

利用PK-15细胞从临床上表现为腹泻症状的病死仔猪肠系膜淋巴结材料中分离获得1株病毒,对该病毒进行病毒形态学、PCR检测与动物回归试验等系统鉴定后,证实该分离株为猪传染性胃肠炎病毒(TGEV),命名为TGEVJL。利用PCR方法克隆出其S基因部分片段,并将该基因序列和推导的氨基酸序列与7个不同来源的TGEV毒株进行同源性和亲缘关系的比较分析,结果表明,各毒株间核苷酸和氨基酸的同源性分别为96.3%～99.3%和94.8%～98.8%;系统进化树结果表明,TGEVJL株与日本分离的TQ14毒株和西班牙分离的TOY56-165毒株亲缘较近,表明不同地区分离毒株的S基因差异不大。     

大型繁育猪场应用TGE弱毒疫苗和PED灭活疫苗的效果观察

某大型猪场临床仔猪见不明原因腹泻,发病率高,而对妊娠母猪普遍接种ＴＧＥ和ＰＥＤ疫苗后收到了明显的防病效果。     

The effect of interferon induction in newborn piglets on the humoral immune response to oral vaccination with transmissible gastroenteritis virus.

Groups of newborn piglets were vaccinated orally with a modified live transmissible gastroenteritis (TGE) virus vaccine at 3 days and 13 days of age, and treated with the synthetic interferon (IFN) inducer polyinosinic:polycytidylic acid (poly ICLC) at 2, 3 or 4 days of age. Control groups consisted of piglets which were vaccinated but not treated with poly ICLC, as well as piglets which were treated with poly ICLC but not vaccinated. Significantly higher mean IFN titres were produced in response to induction at 3 or 4 days of age than at 2 days, and the mean IFN titre of the vaccinated piglets treated with poly ICLC at 3 days of age was significantly higher than in the unvaccinated piglets which were treated at the same time. The mean TGE virus neutralizing antibody titres in the vaccinated piglets which were treated with poly ICLC on the day before vaccination were significantly lower than the mean titres in the untreated vaccinated piglets 10 and 14 days after the first dose of vaccine. The mean titres in the vaccinated piglets which were treated with poly ICLC at 3 or 4 days of age did not differ significantly from those in the untreated vaccinated piglets. The piglets which were treated with poly ICLC on the day after vaccination developed severe diarrhoea which persisted for 5-7 days.     

Activation of natural killer cells in newborn piglets by interferon induction

Natural killer (NK) cell activity in the peripheral blood lymphocytes (PBL) of newborn piglets, normally negligible, was stimulated by in vitro treatment with porcine type I interferon (IFN), and the NK activity of PBL from weaned piglets was augmented by the same treatment. Binding of the PBL to the PK-15 targets used in the single cell cytotoxicity assay for NK activity was not affected by age or by IFN treatment. When newborn piglets were treated with a single intravenous dose at 2 days of age of 0.5 mg/kg of polyinosinic:polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC), a synthetic IFN inducer, their IFN levels peaked at 6 h post-induction, and NK activity in their PBL peaked at 24 h post-induction at the level normally found in weaned piglets. The NK activity then declined until 7 days post-induction, when it increased again in a similar manner to that in untreated control piglets. Target-binding of the PBL was not affected by poly ICLC treatment of the piglets. Newborn piglets treated with poly ICLC and subsequently exposed to infection with transmissible gastroenteritis (TGE) virus showed a delay in onset of clinical signs of TGE compared with untreated control piglets. It was concluded that NK cells in newborn piglets can be activated by treatment of the piglets with poly ICLC, and that the presence of active NK cells is associated with some increase in resistance to challenge with TGE virus.     

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.     

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.

     

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.     

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.     

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)     

Immunogenicity in Aujeszky's disease virus structural glycoprotein gVI (gp50) in swine.

Aujeszky's disease virus (ADV) envelope glycoprotein gVI (gp50) was purified from virus-infected Vero cells by ion-exchange and immunoaffinity chromatography and its usefulness as a subunit vaccine was evaluated in active and passive immunization studies. Four-week-old piglets were immunized intramuscularly (IM) with purified gVI twice two weeks apart and challenged intranasally (IN) 10 days after the second immunization with 30 LD50 (10(8)PFU) of a virulent strain of ADV. Pigs, vaccinated with 100 micrograms of purified gVI, produced virus neutralizing antibodies and did not develop clinical signs after challenge exposure. The challenge virus was not isolated from nasal swabs and tonsils of gVI-vaccinated pigs, whereas non-vaccinated control pigs developed illness after challenge exposure with the same virulent ADV strain which was later recovered from their nasal swabs and tonsils. Pregnant sows vaccinated twice with purified gVI (IM) at a three week interval produced virus neutralizing antibodies in colostrum. Four-day-old sucking piglets born of vaccinated sows were passively protected by colostral antibodies against intranasal challenge with a lethal dose of virulent ADV. Sera from gVI-vaccinated pigs were distinguished from experimentally infected swine sera by their differential reactivity in enzyme-linked immunosorbent assay (ELISA) using four major viral glycoproteins (excluding gVI) as antigen purified by the use of lentil-lectin.     

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.