Putative probiotic Lactobacillus spp. from porcine gastrointestinal tract inhibit transmissible gastroenteritis coronavirus and enteric bacterial pathogens.

A total of 310 bacterial strains isolated from the porcine gastrointestinal tract were tested for their activity against transmissible gastroenteritis (TGE) coronavirus and other enteric pathogens. Based on activity, the strains Probio-38 and Probio-37 were selected as potential probiotics and identified as Lactobacillus plantarum Probio-38 and Lactobacillus salivarius Probio-37 respectively by 16S rRNA gene sequencing. Supernatants of these strains inhibited TGE coronavirus in vitro in ST cells, without any cytopathic effect even after 72 h of incubation. Both the strains exhibited high survival in synthetic gastric juice. The strains were resistant to 5% porcine bile and exhibited antimicrobial activity against all the 13 enteric bacterial pathogens tested. These strains also exhibited resistance to most of the antibiotics analyzed. The inhibition of transmissible gastroenteritis coronavirus and enteric bacterial pathogens as well as the bile tolerance, high survival in gastric juice, and the antibiotic resistance indicate that the two isolated bacterial strains are ideal probiotic candidates for animal application after proper in vivo experiments.

Antigenic and biological diversity among transmissible gastroenteritis virus isolates of swine.

Twenty-four field isolates of transmissible gastroenteritis virus (TGEV) were isolated and examined for antigenic and biological characteristics. Most TGEV isolates produced a typical cytopathic effect (CPE) in swine testis (ST) cell culture, which included a ballooning or lifting away of the infected cells from the cell monolayer with heavy granulation evident. Minor variations in CPE were observed with one isolate, IA-145. Protein profiles of the TGEV isolates as determined by SDS-PAGE were essentially identical, with the exception of the isolate IA-101. The TGEV isolate IA-101 presented a higher molecular mass M protein and lacked an N protein doublet that was present in all other TGEV isolates. The TGEV isolates were shown to be closely related antigenically by using hyperimmune sera in a virus neutralization (VN) test. Some antigenic diversity was detected by utilizing monoclonal antibodies (mAbs) in a VN test. Titers of the mAbs were highest with the homologous Miller TGEV, and one virus isolate, IA-156, was very poorly neutralized with the mAbs used in this study. Indirect immunofluorescence assay (IFA) results were similar to those obtained by the VN test. These studies show that some biologic and antigenic diversity exists among TGEV isolates.

A sero-epizootiological study of porcine respiratory coronavirus in Belgian swine.

A porcine respiratory coronavirus (PRCV), antigenically closely related to transmissible gastroenteritis virus (TGEV), appeared in the European swine population in 1984. The present serological study was performed to obtain insight into the epizootiology of PRCV and of TGEV. PRCV-induced neutralizing antibodies were found in 90.6 per cent of the 160 sera collected from sows at slaughter, demonstrating the enzootic appearance of PRCV in the Belgian swine population. A serological study of fattening swine on 33 farms revealed that 11 farms situated in an area with a high farm density (all farms within 4 km2) and 11 on 22 closed breeding-fattening farms situated in areas with a low farm density (only one to four farms per 12 km2) were infected with PRCV throughout the year, whereas the other 11 closed breeding-fattening farms were temporarily free of PRCV. PRCV disappeared from the farms mainly in spring and summer. All the 11 farms became reinfected in autumn or winter, indicating that PRCV is regularly reintroduced in farms in the colder seasons. There was no correlation between the herd size and the temporary disappearance of PRCV from farms. It was observed on some farms that PRCV could infect pigs shortly after weaning in the presence of declining maternal antibodies, indicating that PRCV can persist on a farm by regularly infecting newly weaned pigs. TGEV-specific antibodies were found in 7.6 per cent of the 160 sera from the slaughterhouse sows. TGEV-specific antibodies were also detected in sera from fattening swine of 5 of the above mentioned 33 farms. TGEV-outbreaks were not observed on these farms.(ABSTRACT TRUNCATED AT 250 WORDS)

The application of immunogold silver staining (IGSS) for the detection of transmissible gastroenteritis virus in fixed tissues.

Protein A-gold (PAG) and a primary porcine antiserum were used in immunogold silver staining (IGSS) for the detection of transmissible gastroenteritis virus (TGEV) in formalin-fixed paraffin-embedded tissue sections of small intestine originating from infected pigs. Immunogold electron microscopy was used to evaluate the reactivity of the prepared PAG marker with the specific porcine TGEV antiserum. Gold particles were closely associated with single virions and immune aggregates of TGEV. When IGSS, using PAG as the marker, was applied to tissue sections, dark staining of TGEV-infected villous enterocytes was observed. Background was low, allowing good visualization by light microscopy of the distribution of viral antigen. Two other gold conjugates, protein A/G-gold (PA/GG) and protein G-gold (PGG), were tested in IGSS. The labeling with PA/GG was comparable to that obtained with PAG. However, no staining was observed when PGG was used. The use of IGSS and PAG offers advantages and may represent a useful technique for the detection of other viral pathogens.

Intestinal protection against challenge with transmissible gastroenteritis virus of pigs immune after infection with the porcine respiratory coronavirus.

An infection of pigs with the porcine respiratory coronavirus (PRCV) induces antibodies which neutralize the enteropathogenic transmissible gastroenteritis virus (TGEV) and PRCV to the same titre. In the present study, 10-week-old seronegative pigs (n = 8), pigs immune following TGEV inoculation (n = 4) or pigs immune following aerosol (n = 8) or intragastric inoculation (n = 4) with PRCV were challenged with TGEV. Whereas TGEV-immune pigs were completely protected against challenge, all PRCV-immune pigs showed serological evidence of TGEV replication. Nevertheless, the aerosol or intragastric inoculation with PRCV primed the humoral immune system against TGEV and the TGEV challenge induced a secondary antibody response in most PRCV-immune pigs. Furthermore, all PRCV-immune pigs showed a decrease in the duration of the excretion of infectious TGEV (0-4 days) in comparison with the duration of the virus excretion by seronegative pigs (5-6 days).

A capture-enzyme immunoassay for rapid diagnosis of transmissible gastroenteritis virus.

Two enzyme immunoassays (EIA) were developed for the detection of swine transmissible gastroenteritis virus (TGEV) antigens. The 2 EIAs used the same detecting system, a monoclonal antibody conjugated to horseradish peroxidase, but used different capture systems including a monoclonal antibody (m-EIA) or a polyclonal antibody (p-EIA). The EIAs were compared with the fluorescent antibody test (FAT) and electron microscopy (EM) for the detection of TGEV in intestinal samples of experimentally inoculated gnotobiotic piglets and of conventional diarrheic pigs submitted for diagnosis. In the gnotobiotic piglets experimentally inoculated with TGEV, 81.8% (9/11) were positive for TGEV by p-EIA, and 72.7% (8/11) were positive by m-EIA. In comparison, 81.8% (9/11) were positive by FAT and 27.2% (3/11) were positive by EM. Three noninfected controls were negative by all tests. In the diagnostic samples, 86.0% (43/50) were positive by p-EIA, 68.2% (30/44) were positive by m-EIA, 28.6% (14/49) were positive by IFA, and 38.0% (19/50) were positive by EM. The m-EIA had a higher agreement with FAT and EM than did p-EIA.

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.

Sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus.

A porcine respiratory coronavirus (PRCV) was inoculated by aerosol into nine hysterectomy-derived and colostrum-deprived pigs at the age of one week. They were killed at different times after inoculation and tissues were sampled for virus isolation and immunofluorescence. Results indicate that virus replicated to high titres in the respiratory tract. Replication mainly occurred in alveolar cells but also in epithelial cells of nasal mucosa, trachea, bronchi, bronchioli, in alveolar macrophages and in tonsils. After primary replication in the respiratory tract, viraemia occurred. Virus also reached the gastrointestinal tract after swallowing. Subsequently, PRCV was observed to replicate in the ileum. The infection spread within a few days from the ileum to the duodenum. Replication in the small intestine remained limited to a few cells located in or underneath the epithelial layer of villi and, or, crypts. The cell type could not be identified. Virus was isolated from mesenteric lymph nodes in all pigs, but immunofluorescence was not observed. Results show that small changes in molecular structure between transmissible gastroenteritis virus and PRCV resulted in important changes in host cell tropism.

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus.

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.

[Thymus-dependent nature of the antigens of enteropathogenic viruses]. / Timus-zavisimaia priroda antigenov énteropatogennykh virusov.

The nature of antigens in enteropathogenic animal viruses--coronavirus--an agent of transmissive gastroenteritis of pigs (TGE) and pig enterovirus of serotype VI was studied. Basing on the results of the histomorphometric study of the white spleen pulp under the individual introduction of viruses and in combination with the lymphoid chalone it was established that the developing immune response has a thymus-dependent induction mechanism. The pharmacological stimulation of the T-system of animals suffering from virus gastroenteritis provides a positive therapeutic effect. The selective stimulation of the animal T-system may be considered as a promising trend in therapy of intestinal infections and as one of the possible ways to increase the immune response to the antigens of viral vaccines.

Transmissible gastroenteritis.

Transmissible gastroenteritis (TGE) virus, a coronavirus, causes an acute infection of the small intestine of the pig. The disease is rarely fatal in adult animals but can cause extensive mortality in the neonate. Since its first description in Britain in the 1950s, the disease has been epizootic but recently it has become established on some farms and an antigenically related respiratory virus has become endemic in the national herd over the past two years. Piglet immunity to TGE, which relies on passive protection from milk, has been impossible to achieve with vaccines and research has aimed at understanding the nature of the interaction between virus and the pig. Following infection of the pregnant sow, antibody-producing cells migrate to the mammary gland where they secrete virus neutralising antibodies into the milk; prospective vaccines will need to stimulate a similar response. The location and number of antigenic sites on the virus particle associated with neutralisation have been established with monoclonal antibodies and the role of the other viral genes in pathogenesis and immunity is being studied with genetic engineering techniques.

Identification of a 17000 molecular weight antigenic polypeptide in transmissible gastroenteritis virus-infected cells.

Pulse labelling of cells with [35S]methionine at different times after infection followed by SDS-PAGE was used to resolve and to identify polypeptides designated as specific to transmissible gastroenteritis virus (TGEV)-infected swine testicular (ST) cell cultures. The major TGEV structural proteins, with apparent molecular weights of 200,000 (200K), 47K and 30K were detected in radiolabelled cell extracts by 6 h postinfection. Additionally, a 17K major polypeptide was present in infected cells but not in mock-infected control cultures. Labelling with [3H]glucosamine revealed only the 200K and 30K proteins to be glycosylated. TGEV-primed porcine lymphocytes, secondarily stimulated in vitro with sucrose gradient-purified virus, produced antibody only to the two glycoproteins (gp) indicating that the 17K polypeptide is not a surface feature of the virion. Two pigs were infected oronasally with the virulent Miller strain of TGEV and their sera were analysed by immunoprecipitation. At 25 days postinfection convalescent sera responded strongly to gp30 and gp200 and there was a weak initial response to the 17K polypeptide. Serum immunoglobulins at 60 days postinfection reacted strongly to the 17K protein while the antibody response to gp30 was significantly reduced and that to gp200 was slightly reduced.

Isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis.

A porcine respiratory, non-enteric virus which is related to the coronavirus transmissible gastroenteritis virus (TGEV) has been isolated in pigs and in cell culture. The isolate was designated TLM 83. It has become very widespread and enzootic among the swine population in Belgium and in other swine raising countries. It causes an infection of the lungs and appears to spread by aerogenic route. It does not replicate in the enteric tract. The experimental infection in conventional and gnotobiotic pigs in isolation remains subclinical. The infection, either experimental or in the field, results in the formation of antibodies which neutralise the classical enteric TGEV. Based on this relationship, this virus is assumed to be a new TGEV-related porcine respiratory coronavirus or TGEV itself which has totally lost its tropism for the enteric tract.

[Demonstration of a transmissible gastroenteritis virus by contrast immunofluorescence]. / Dokazvane na virusa na transmisivniia gastroenterit chrez kontrastniia imunofluorestsenten metod.

Tested were a number of procedures to employ the immunofluorescence method for diagnosing transmissive gastroenteritis in pigs--impression preparations, frozen cross-sectioned material, and infected cell cultures of the established cell line SPEV. It was found that immunofluorescence microscopy was a dependable method for the express diagnosis of transmissive gastroenteritis. It could be employed in its three variants. The use of impression preparations, however, did not prove as dependable as the remaining ways of applying the method, and this drawback had to be compensated for with the study of a greater number of impression preparations taken from more pigs that had contracted the disease. It was also established that most promising and effective was to apply the method with the use of cell cultures infected with suspensions of organs. Cell cultures of the established SPEV cell line infected with material that contained the virus could produce dependable positive results in immunofluorescence investigations at the 24th hour following inoculation. This method could be employed for the express diagnosis of transmissive gastroenteritis.

[Isolation of the transmissible gastroenteritis virus in a continuous pig embryo kidney cell line]. / Izolirane na virusa na transmisivniia gastroenterit v postoiannata kletuchna liniia SPEV.

Attempts were made to isolate the virus of transmissive gastroenteritis in a permanent cell line SPEV in view of the diagnostics of the disease. Used were small intestines of pigs during the first 24 hours after the setting in of clinical symptoms. The successful isolation of the virus in a SPEV line depended on the methods employed to handle the material as well as on the ways of inoculating the cell cultures. In the conditions of the investigation most proper proved centrifugation of the organ suspensions and the suspension method of infection. The SPEV line, however, was shown to be insufficiently sensitive to isolate the virus of transmissive gastroenteritis. About 30 per cent only of the positive material could be used to demonstrate the virus following direct infection, while the study of the remaining (up to 62 per cent) material required additional passing. The SPEV line could replicate the virus after its infection with organ suspensions containing the virus, and this could be demonstrated through immunofluorescence investigations. The same method could likewise be employed in the express diagnosing of the disease. The passing of the virus strains in the SPEV line led to their attenuation. In order to retain the virulence of the strains passing should take place in nonimmune pigs or should alternate with the use of both pigs and cell cultures.

Replication of transmissible gastroenteritis coronavirus (TGEV) in swine alveolar macrophages.

Several strains of the enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) have been shown to replicate in alveolar macrophages maintained in vitro. A distinct cytopathic effect was observed at a multiplicity of infection greater than or equal to 0.1. Infected cells released infectious virus. The extent of both virus production and cell destruction was highly dependent upon the virus input. At low input, cell viability was affected only slightly, and a delayed and persistent virus production could be observed. TGEV infection of macrophages also led to a marked synthesis of type I interferon. Thus, the possibility that alveolar macrophages act as an extra-intestinal target for TGEV must be considered.

Identification of porcine transmissible gastroenteritis virus in house flies (Musca domestica Linneaus).

Transmissible gastroenteritis (TGE) virus was detected in house flies (Musca domestica Linneaus) by staining with specific fluorescent antibody. The flies were collected within a swine confinement facility in which TGE was enzootic. Laboratory-reared flies were infected experimentally with TGE virus and the virus was recovered from the insects for 72 hours after infection. The TGE virus was identified both by the fluorescent antibody technique and by isolation in cell culture. The nature of plaque formation in cell monolayers inoculated with the virus passaged through flies changed from a large plaque (4 mm or greater in diameter) to a small plaque (1 mm in diameter) over the period. Large plaques were observed early after infection and were attributed to TGE virus mechanically carried by the flies. Small plaques occurred 8 to 12 hours after infection and were considered to be produced by virus replicated in the dipterous cell.