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Transmissible gastroenteritis in endemically infected breeding herds of pigs in East Anglia, 1981-85.

Endemic infection was a common sequel to primary outbreaks of transmissible gastroenteritis in large breeding herds of pigs in East Anglia. The main clinical features of the disease were diarrhoea affecting sucking piglets aged about six days or more, diarrhoea among recently weaned pigs and brief episodes of overt clinical recrudescence in part of the herd. Post mortem and bacteriological findings were often more suggestive of colibacillosis than transmissible gastroenteritis. In some herds, endemic infection remained clinically mild or inapparent for long periods. Evidence of endemic transmissible gastroenteritis infection was found in 43 (50.6 per cent) of 85 herds of pigs studied prospectively between 1981 and 1983. There was a significant correlation with herd size; the disease recurred during the 12 months after primary outbreaks in 36 (65.5 per cent) of 55 herds with over 100 sows compared with seven (23.3 per cent) of 30 herds with less than 100 sows (P less than 0.001). In the larger herds it occurred more commonly where finishers were kept (P less than 0.05). Sow morbidity and management factors during the primary outbreak had no statistically significant effect on the incidence of recrudescence. Epidemiological aspects of the findings are discussed with emphasis on the difficulties associated with the diagnosis and control of endemic transmissible gastroenteritis infection.

Animals↗

[Isolation and use of a farm-specific hyperimmune serum against transmissible gastroenteritis].

A specific stationary hyperimmune serum was obtained in a pigbreeding complex with a transmissive gastroenteritis infection via trifold (at a fourteen-day interval) i/m injection of sows with 5 cm3 each of undiluted virus (10(6) TCCPE50 per cm3). Tested were hyperimmunization programmes with pigs in the final fattening period. It was found that the use of undiluted virus led to the equation of sows in terms of their immunologic state and to the essential rise of the titer of humoral antibodies. In order to obtain high titer immune serum against transmissive gastroenteritis it is sufficient to proceed with the i/m injection of pigs or adult swine in the final stage of fattening at rising amounts (from 3 to 10 cm3) of attenuated virus of sufficiently high titer (10(6) TCCPE50 per cu. cm). The hyperimmune serum produced a very good prophylactic effect with newborn pigs on the same farm - the twofold oral administration of 5 to 10 cm3 on the day of birth and a couple of days later led to a drop of both morbidity and mortality rate as well as to the improvement of body development.

Animals↗

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.

Animals↗

Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains. Correlation with antigenicity and pathogenicity.

Transmissible gastroenteritis virus (TGEV) causes an economically important enteric disease of swine. Differences in the pathogenicity, antigenicity and tissue tropism have been observed among porcine coronaviruses. Although porcine respiratory coronavirus (PRCV) is antigenically similar but not identical to TGEV isolates, these respiratory coronaviruses differ markedly in pathogenicity and tissue tropism compared to TGEV isolates. Using a reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay, TGEV and PRCV isolates were assigned to several distinct groups. By RFLP analysis of the 5' region of the S gene, TGEV strains were differentiated into 4 groups using the restriction enzyme Sau3AI. A fifth Sau3AI group contained the PRCV isolates. These 5 groups correlated with antigenic groups previously defined using monoclonal antibodies in our laboratory. Several restriction enzymes could be used to differentiate the TGEV strains into Miller and Purdue types. Analysis of a PCR amplified product in the 3 and 3-1 genes indicated the RT/PCR-RFLP assay results for TGEV Miller strains could be correlated with lower virulence created by passage in cell culture.

Animals↗

Molecular cloning and phylogenetic analysis of ORF7 region of chinese isolate TH-98 from transmissible gastroenteritis virus.

Genomic RNA was extracted from a Chinese isolate of porcine transmissible gastroenteritis virus (TGEV) designated TH-98. Employing RT-PCR technique to amplify ORF7 sequence of TGEV, which located at the 3' end of TGEV genome and is poorly understood functionally so far. A recombinant named pPROEX HTc-hp was constructed via inserting ORF7 gene into prokaryotic expression vector pPROEX HTc. The recombinant was sequenced and compared the DNA and its deduced amino acid (aa) sequences with that of some reference strains after restriction endonuclease and PCR analysis. The ORF7 gene named hp gene (Genbank accession number: AY337931) consists of 237 bp in length encoding a hydrophobic protein (HP) of 78 aa with a molecular weight of 9.1 kDa. The sequences of hp gene and Hp protein share 89%-97% and 87%-96% homologous identities compared with 11 TGEV reference strains derived from other regions or countries respectively, which revealed that there are significant variation within-strains, even though the ORF7 region is relatively conservative. In addition, a phylogenetic tree based on these ORF7 DNA sequences was generated, and the tree topology suggests that possible recombination events happened in the evolutionary history of TGEV.

Animals↗

Differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex RT-PCR.

Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are highly contagious enteric diseases of piglets. The clinical signs of these diseases are very similar and include watery, yellowish diarrhea. Thus, the effective differential detection of TGE virus and PED virus is required. In the present study, a duplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the differential detection of TGE and PED viruses. The primers were designed for the S gene of each virus. RNA was extracted from the intestines and stool samples that were collected from the swine with diarrhea. The RT-PCR test could detect both TGE and PED viruses with 2 TCID50/200 microl. Among 90 clinical samples, 7 TGE viruses and 2 PED viruses were detected by the duplex RT-PCR. This duplex RT-PCR may be a useful diagnostic method for the rapid, specific, and sensitive differential detection of TGE and PED viruses using clinical samples.

Animals↗

Efficacy of a transmissible gastroenteritis coronavirus with an altered ORF-3 gene.

Serial passage of virulent transmissible gastroenteritis virus through cell culture reduced its virulence in 3-day-old piglets. Intramuscular inoculation of pregnant gilts with 2 doses of this modified-live virus elicited a level of lactogenic immunity that protected their nursing piglets against a lethal dose of challenge virus. Sequence analysis of a 637-bp fragment of the spike gene containing most of the aminopeptidase receptor and the 4 major antigenic sites from the original and the serially passed viruses were nearly identical. Gel analysis revealed that the fragment from the ORF-3 gene of virulent virus was smaller than the corresponding fragment from the serially passed virus. Sequence analysis of the fragment from the passed virus revealed that the sequence between nt 5310 and nt 5434 was replaced by a 636-bp fragment from the polymerase 1A gene. This replacement resulted in the loss of the CTAAACTT leader RNA-binding site and ATG start codon for the ORF-3A gene but it did not affect the ORF-3B gene.

Animals↗

Comparison of intestinal (Illinois strain) and cell culture-adapted (M-HP strain) viral populations of transmissible gastroenteritis of swine.

Intestinal and cell culture-adapted viral populations of transmissible gastroenteritis (TGE) of swine were compared by means of sucrose gradient centrifugation, immunnofluorescence, electron microscopy, immune electron microscopy, statistical analysis of the number of plaque-forming units, and ultraviolet sensitivity. Results indicated that the size range and general coronavirus morphologic characteristics were shared by both viral populations. Marked morphologic variations existed among particles from both populations. Unlike the cell culture-adapted virus, the Illinois virus of intestinal origin was infractions representing 2 bands of infectivity which were isolated by the sucrose gradient centrifugation method. The intestinal and cell culture-adapted TGE viruses were similar in antigenicity and in sensitivity to ultraviolet irradiation. There was no indication of a 2nd virus in addition to the coronavirus described as the cause of TGE.

Animals↗

Passive protection of piglets by recombinant baculovirus induced transmissible gastroenteritis virus specific antibodies.

Sera of pigs immunized with parts of the transmissible gastroenteritis virus (TGEV) spike (S) protein expressed by recombinant baculoviruses were tested, together with a TGEV hyperimmune antiserum, for their abilities to protect three-day-old piglets against TGEV infection. The piglets were infected with virulent TGEV and the sera were given orally 3 h before infection, together with the virus, and every 6 h postinfection during the 30 h of the experiment. Virus shedding was monitored by TGEV isolation from rectal swab samples. The sera containing antibodies induced by the complete S protein or the amino terminal half of the S protein showed protective properties, indicated by delayed onset of clinical signs and virus shedding, similar to the TGEV hyperimmune serum. Those immune sera containing antibodies induced by shorter recombinant proteins were not protective.

Animals↗

An indirect fluorescent antibody test for antibodies to transmissible gastroenteritis of swine.

The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transmissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20 degrees C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advantages over the virus neutralization test.

Animals↗

[Course of transmissible gastroenteritis on a swine-breeding farm].

Epizootiologic studies were carried out from the introduction of the transmissive gastroenteritis on the Enterprise up to the end of 1984. Clinical observations and morphologic survey were conducted on the infected animal farms. The diagnosis was based on both virologic and serologic investigations with routinely employed methods. The basic reason for the occurrence and spread of transmissive gastroenteritis on the Enterprise were shown to be omissions in the system of prevention measures with regard to infectious diseases, i. e., the functioning of farms as open units and the intense connections between them. The basic way of spreading the infection was shown to be the removal of animals from healthy to affected farms. The part played by other ways of infection transmission proved negligible, and the strict adherence to veterinary and sanitary requirements of protection guaranteed to a high extent the prevention of transmissive gastroenteritis on swine-breeding complexes and farms. It was also found that the disease assumed a stationary character on the infected farms, with periodic enzootic outbreaks in some of them associated with the nonobservance of feeding and raising technologies.

Animal Husbandry↗

Leukocyte migration-inhibition procedure for transmissible gastroenteritis viral antigens.

Swine exposed to transmissible gastroenteritis viral antigens developed humoral and cell-mediated immunity. Migration of leukocytes from exposed swine was inhibited in the presence of the sensitizing antigens, whereas migration of leukocytes from nonexposed swine was not inhibited in the presense of these same antigens. In virus-neutralization-positive animals, it was not possible to correlate degree of inhibition with virus-neutralization titer. Inhibition was observed 7 days after exposure and was found to persist for at least 35 days.

Animals↗

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.

Alkaline Phosphatase↗

Humoral and cellular responses in swine exposed to transmissible gastroenteritis virus.

Swine exposed to attenuated transmissible gastroenteritis virus had higher virus-neutralizing antibody titers than did swine exposed to virulent virus. The cellular response, measured by the direct leukocyte migration-inhibition (LMI) procedure, was greater in swine exposed to virulent virus than in swine exposed to the attenuated virus. Leukocytes from exposed swine were inhibited more in the LMI procedure in the presence of the homologeous sensitizing antigen than in the presence of the heterologous viral antigen. The humoral response measured by virus neutralizing reached a peak 21 days after exposure, and the cellular response measured by LMI reached a peak 28 days after exposure.

Animals↗

Comparison of the sialic acid binding activity of transmissible gastroenteritis coronavirus and E. coli K99.

Transmissible gastroenteritis coronavirus (TGEV) and Escherichia coli K99 are both enteropathogenic for pigs with infections being most severe in neonate animals. For both microorganisms, a sialic acid binding activity has been shown to be an essential pathogenicity factor. Here we demonstrate with haemagglutination and haemagglutination-inhibition assays that TGEV and E. coli K99 differ in their sialic acid binding activities with respect to the type and amount of sialic acid residues required on the erythrocytes surface as well as with respect to the type of sialoglycoconjugate preferentially recognized. Intestinal mucins from piglets (12-14 days old) and adult animals were shown to inhibit TGEV to the same extent. From our results we conclude that E. coli K99 and TGEV interact with different sialoglycoconjugates to establish an intestinal infection. The implications for the enteropathogenicity of TGEV are discussed.

Age Factors↗

Genome of porcine transmissible gastroenteritis virus.

The Purdue strain of transmissible gastroenteritis virus, a porcine coronavirus, was grown to titers of greater than 10(8) PFU/ml in a swine testicle cell line, and the RNA was isotopically labeled with [3H]uridine. The RNA was extracted from purified virus and was found to have the following properties. (i) It consisted primarily of a homogeneous large-molecular-weight species which electrophoretically migrated with an apparent molecular weight of 6.8 X 10(6) under denaturing conditions. (ii) It migrated electrophoretically at the same rate on nondenaturing gels before and after heat denaturation, suggesting that it does not consist of subunits. (iii) It was susceptible to pancreatic RNase A digestion in high (0.3 M) NaCl. (iv) It was polyadenylated to the extent that greater than 60% of the native RNA bound to oligodeoxythymidilic acid-cellulose under conditions of high (0.5 M) NaCl. RNA extracted from virions was infectious. This coronavirus can therefore be characterized as a positive-strand RNA virus.

Animals↗

Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus.

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.

Animals↗

Complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein 3b of US strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus.

Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60 and virulent TGEV Purdue and attenuated TGEV Purdue P115, together with the ISU-1 strain of porcine respiratory coronavirus (PRCV-ISU-1), a naturally occurring TGEV deletion mutant with an altered respiratory tropism and reduced virulence. Pairwise comparison at both the nucleotide (nt) and amino acid (aa) levels between virulent and attenuated TGEV strains identified a common change in nt 1753 of the spike gene, resulting in a serine to alanine mutation at aa position 585 of the spike proteins of the attenuated TGEV strains. Alanine was also present in this protein in PRCV-ISU-1. Particularly noteworthy, the serine to alanine mutation resides in the region of the major antigenic site A/B (aa 506-706) that elicits neutralizing antibodies and within the domain mediating the cell surface receptor aminopeptidase N binding (aa 522-744). Comparison of the predicted polypeptide products of ORF3b showed significant deletions in the naturally attenuated PRCV-ISU-1 and TGEV Miller M60; these deletions occurred at a common break point, suggesting a related mechanism of recombination that may affect viral virulence or tropism. Sequence comparisons at both genomic and protein levels indicated that PRCV-ISU-1 had a closer relationship with TGEV Miller strains than Purdue strains. Phylogenetic analyses showed that virulence is an evolutionarily labile trait in TGEV and that TGEV strains as a group share a common ancestor with PRCV.

Amino Acid Sequence↗