Gastroenteritis In Pigs Research Articles

Raw potato starch diet supplement in weaned pigs could reduce Salmonella Typhimurium infection by altering microbiome composition and improving immune status.

Salmonella enterica serovar Typhimurium (ST) is one of the causative agents of gastroenteritis in pigs. Pigs fed a diet supplemented with raw potato starch (RPS) have improved gut health by the alteration of the microbiota composition and production of short-chain fatty acids (SCFAs). This study aimed to evaluate the effects of RPS supplementation in reducing infection severity and fecal shedding in ST-infected pigs. The weaned experimental pigs were divided into two groups: CON (n = 6) fed a corn/soybean-based diet and TRT (n = 6) supplemented with 5% RPS. After 21 d, the pigs were inoculated with ST, and their body weight, clinical signs, and fecal shedding of ST were monitored for 14 d. At 14 d post-inoculation (dpi), the jejunum, cecum, ileum, and colon tissues were collected from euthanized pigs, and histopathological lesions and cytokine gene expression were compared. Additionally, blood samples at 2 dpi were analyzed for gene ontology enrichment. Moreover, the gutmicrobiome was analyzed using 16S rRNA metagenomic sequencing, and the SCFA concentration was measured using gas chromatography. The average daily weight gain was significantly higher in TRT than in CON during the ST infection period; however, histopathological lesion scores were significantly lower in TRT than in CON. The relative abundance of nine genera of butyrate- and acetate-producing bacteria significantly increased in TRT compared with that of only two acetate-producing bacteria in CON. Among the genes involved in the immune response, IL-18 expression level was significantly lower in the jejunum and colon in TRT than in CON. Furthermore, Reg3γ expression was significantly different in the cecum and colon of both groups. The diet supplemented with RPS in weaned pigs could result in predominance of butyrate- and acetate-producing bacteria, reducing the severity of ST infection by improving the immune status.

Coronaviruses: modern taxonomy and research chronology

The classification of coronaviruses began in 1968 when five viruses (IBV, MHV, B814, 229E, and OC43) were united into an independent group, “coronaviruses”, based on the characteristic morphology of the virions. In 1971, the genus Coronavirus was formed, and in 1975, the family Coronaviridae, which in 1996 was included in the order Nidovirales. In 2009, the Coronaviridae family was divided into subfamilies. In 2018, new taxa of viruses were created – region, suborder, and subgenus, and in 2019 – kingdom, type, and class. According to the International Committee on Taxonomy of Viruses, issue No. 37 of 07.2021 (ratification of 03.2022), the family Coronaviridae belongs to the region Riboviria, kingdom Orthornavirae, phylum Pisuviricota, class Pisoniviricetes, order Nidovirales, suborder Cornidovirineae. The Coronaviridae family includes 54 viruses, which are grouped into three subfamilies (Letovirinae, Orthocoronavirinae, Pitovirinae), six genera (Alphaletovirus, Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus, Alphapironavirus) and 28 subgenera. Representatives of the most numerous subfamily Orthocoronavirinae infect various species of mammals and birds, causing various pathologies: respiratory and intestinal infections, polyserositis, myocarditis, hepatitis, nephritis, neuroinfections, immunopathology. In particular, viruses that cause infectious bronchitis in chickens, transmissible gastroenteritis in pigs, epidemic diarrhea in pigs, encephalomyelitis in pigs, coronavirus infection in cattle, and epizootic catarrhal gastroenteritis in minks are relevant for veterinary practice. The natural reservoirs of coronaviruses from the genera Alphacoronavirus and Betacoronavirus are bats and birds from the genera Gammacoronavirus and Deltacoronavirus. Particularly dangerous human coronaviruses are SARS-CoV, MERS-CoV and SARS-CoV-2, which cause emergent infections. The circulation of SARS-CoV, MERS-CoV, and SARS-CoV-2 among animals is shown. The natural reservoir of these viruses is bats, and the intermediate hosts for SARS-CoV are Himalayan civets, one-humped camels for MERS-CoV, and pangolins for SARS-CoV-2. The circulation of SARS-CoV-2 among different species of domestic and wild animals leads to the accumulation of mutations, which causes the adaptation of the virus to new hosts and ecological niches and its subsequent introduction into the human population.

Characterisation of ORF3, M, N and E Gene Sequences of Porcine Epidemic Diarrhoea Virus from Domestic Pigs in Poland.

Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen causing porcine epidemic diarrhoea and acute gastroenteritis in pigs of all ages. Previous analysis of the viral genome of PEDV in Poland was only based on the spike protein (S) gene sequences and no analysis of other genes has been performed. The aim of this study was to analyse the envelope (E), membrane (M) and nucleocapsid (N) protein and open reading frame 3 (ORF3) gene sequences. Viral RNA from 18 Polish pig faecal samples that were quantitative reverse transcription PCR-positive for PEDV was analysed in four genomic regions (E, M, N and ORF3). Phylogenetic analysis based on these regions' sequences revealed that Polish PEDV isolates were highly related and were clustered into group G2a across the four genes compared. Moreover, the Polish strains were located in distinct subclusters on the phylogenetic trees, which suggests the presence of at least three independently evolving PEDV genetic lines circulating in Poland. The occurrence of unique mutations in the sequences of Polish PEDV strains suggests that PEDV continues to undergo evolutionary processes, accumulating the mutations necessary for viral fitness in its natural hosts. The Polish PEDV strains differed genetically from the CV777 vaccine strain, suggesting the risk of relatively low vaccine efficacy if this strain is used. Our results promote a better understanding of the genetic diversity of PEDV field isolates in Poland and highlight the importance of molecular characterisation of PEDV field strains for the development of an effective vaccine against PEDV.

Sensor System Based on a Piezoelectric Resonator with a Lateral Electric Field for Virus Diagnostics.

A sensor system based on a piezoelectric resonator with a lateral electric field in the frequency range 6-7 MHz of the electric field for virus detection is described. Through use of the transmissible virus causing gastroenteritis in pigs and specific antibodies, the possibility of detecting the virus in suspension in real time was determined. It was found that the frequency dependence of the real and imaginary parts of the electrical impedance of such a resonator loaded with a virus suspension changes significantly after the addition of specific antibodies to the suspension. No changes are observed if the antibodies are not specific. Thus, the results obtained illustrate the possibility of detecting viruses in situ, directly in the liquid phase, if the change in the real or imaginary parts of the electrical impedance after the addition of antibodies is used as an analytical signal. The possibility of virus detection in the presence of foreign viral particles has been illustrated.

Experimental investigations of application of the water-steam оzone-containing gaseous mixture for prophylaxis and treatment of respiratory infectious diseases

Objective. Determination of the virus-cidal and bacterio-cidal action of the ozone-containing steam-water mixture, which was obtained, using apparatus POS-1, simulated on viral models of transmissive gastroenteritis of pigs of the coronaviruses family and polyresistant clinical strains of bacterial cultures, as well as adjustment of optimal parameters of ozone in the gaseous mixture content, which do not cause pathological changes in the organism’s organs and systems.
 Materials and methods. Apparatus POS-1, created by collective of Scientific-Methodical Centre «Medical Innovation Technologies»», was applied for production of the ozone-containing gaseous mixture. Virusological investigations were conducted on the base of the Institute of Epidemiology and Infectious Diseases named after L. V, Gromashevskiy. As a working material the models of virus of the pigs transmissive gastroenteritis of the coronaviruses family were applied. The series of bacteriological and experimental investigations were conducted on the base of Shalimov National Institute of Surgery and Transplantology NAMS of Ukraine. Polyresistant cultures in concentration of 108 colony-creating units per 1 ml were applied for bacteriological investigations. The experiment was conducted on white rats to study the gaseous ozone-containing mixture affection on living biological tissues.
 Results. Virusological investigations have shown, that in the 20 minutes exposition and a contact with extracellular virus the infection titer have lowered in 100 000 times, and in a 30 minutes exposition - a complete deactivation of virus have had occurred. Bacteriological investigations have revealed the complete absence of the cultures development after their processing with the ozone-containing mixture during 20 min. The results of swimming tests and behavioral reactions in white rats of control and experimental groups did not differ. Histological investigations of the respiratory organs tissues as well as of spleen, thyroid gland, kidneys and suprarenal glands did not reveal pathological effects of the ozone-containing mixture.
 Conclusion. Parameters of the ozone generation and delivery in the gaseous mixture content were studied and optimally selected. Investigations of the ozone-containing mixture affection on a viral strain of the coronaviruses family, as well as bacteriological investigations on polyresistant bacterial cultures have had confirmed its pronounced virus-cidal and bactericidal properties. Application of the ozone-containing mixture do not cause any pathological changes in the living organism organs and systems. This gaseous mixture may be used for prevention and treatment of respiratory infectious diseases of viral and bacterial genesis.

An immunoinformatics study: designing multivalent T-cell epitope vaccine against canine circovirus

BackgroundCanine circovirus is a deadly pathogen of dogs and causes vasculitis and hemorrhagic enteritis. It causes lethal gastroenteritis in pigs, fox, and dogs. Canine circovirus genome contains two main (and opposite) transcription units which encode two open reading frames (ORFs), a replicase-associated protein (Rep) and the capsid (Cap) protein. The replicase protein and capsid protein consist of 303 amino acids and 270 amino acids respectively. Several immuno-informatics methods such as epitope screening, molecular docking, and molecular-dynamics simulations were used to craft peptide-based vaccine construct against canine circovirus. ResultsThe vaccine construct was designed by joining the selected epitopes with adjuvants by suitable linker. The cloning and expression of the vaccine construct was also performed using in silico methods. Screening of epitopes was conducted by NetMHC server that uses ANN (Artificial neural networking) algorithm. These methods are fast and cost-effective for screening epitopes that can interact with dog leukocyte antigens (DLA) and initiate an immune response. Overall, 5 epitopes, YQHLPPFRF, YIRAKWINW, ALYRRLTLI, HLQGFVNLK, and GTMNFVARR, were selected and used to design a vaccine construct. The molecular docking and molecular dynamics simulation studies show that these epitopes can bind with DLA molecules with stability. The codon adaptation and in silico cloning studies show that the vaccine can be expressed by Escherichia coli K12 strain. ConclusionThe results suggest that the vaccine construct can be useful in preventing the dogs from canine circovirus infections. However, the results need further validation by performing other in vitro and in vivo experiments.

Bile acids LCA and CDCA inhibited porcine deltacoronavirus replication in vitro

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes gastroenteritis in pigs and no vaccines or antiviral drugs are available. Bile acids are active factors in intestines and influence the replication of enteric viruses. Currently, the role of bile acids on PDCoV replication is unknown. In this study, we tested the effects of different types of bile acids on the replication of PDCoV in cell culture. We found that physiological concentrations of bile acids chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) had antiviral activity against PDCoV in porcine kidney cell line (LLC-PK1) and porcine small intestinal epithelial cell line (IPEC-J2). In IPEC-J2 cells, CDCA and LCA inhibited PDCoV replication at post-entry stages by inducing the production of interferon (IFN)-λ3 and IFN-stimulated gene 15 (ISG15) via G protein-coupled receptor (GPCR). In summary, bile acids CDCA and LCA restricted PDCoV infection and LCA functioned through a GPCR-IFN-λ3-ISG15 signaling axis in IPEC-J2 cells. Our results may open new avenues for the development of antiviral drugs to treat PDCoV infection in pigs.

The N-Terminal Domain of Spike Protein Is Not the Enteric Tropism Determinant for Transmissible Gastroenteritis Virus in Piglets.

Transmissible gastroenteritis virus (TGEV) is the etiologic agent of transmissible gastroenteritis in pigs, and the N-terminal domain of TGEV spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. Here, we assembled a full-length infectious cDNA clone of TGEV in a bacterial artificial chromosome. Using a novel approach, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems efficiently and rapidly rescued another recombinant virus with a 224-amino-acid deletion in the N-terminal domain of the TGEV Spike gene (S_NTD224), which is analogous to the N-terminal domain of porcine respiratory coronavirus. S_NTD224 notably affected the TGEV growth kinetics in PK-15 cells but was not essential for recombinant virus survival. In animal experiments with 13 two-day-old piglets, the TGEV recombinant viruses with/without S_NTD224 deletion induced obvious clinical signs and mortality. Together, our results directly demonstrated that S_NTD224 of TGEV mildly influenced TGEV virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against TGEV. Importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research.

Патоморфологічні зміни за трансмісивного гастроентериту поросят

Transmisive gastroenteritis of pigs is currently registered in all countries of the world with intensive management of pig production, there are practically no large pig farms in which there is no transmissible gastroenteritis of pigs. Also the disease is widespread in pig farms of Ukraine. Transmissible gastroenteritis sick piglets in the first week after birth. During the initial appearance of the disease in a short time can be spread among pigs of all age groups with almost 100% death in 1 – to 10-day piglets and 4% among weaned piglets. In the literature adequately describes the etiology, pathogenesis, clinical picture and treatment of the disease, but pathological changes described not fully. The purpose of our research was to study the pathological morphology changes in transmissible gastroenteritis of pigs. To achieve this goal were the following objectives: to conduct a postmortem of sick pigs to transmissible gastroenteritis, to study macroscopic and microscopic changes in internal organs of pigs in this disease and to describe changes in the internal organs that were not previously documented. Work completed during 2016–2017 years on the basis of one of the private pig farms of industrial type Kyiv region and of the Department of pathological anatomy, National University of life and environmental Sciences of Ukraine, which was delivered to autopsy the dead animals. Postmortem 12 corpses of piglets that died from transmissible gastroenteritis conducted in the dorsal position by the method of partial evisceration, microscopic studies were performed according to standard techniques. When conducting pathological studies of pigs died from transmissible gastroenteritis we had installed the following changes: the mucous membrane of the stomach in a state of catarrhal inflammation, with hemorrhages and erosions; the mucous membrane of the small intestine has the symptoms of a catarrhal-hemorrhagic inflammation. Blind the colon and the intestines are found the superficial necrosis in the form of a scaly plaque. Microscopic studies found atrophy of the villi of the jejunum and the iliac colon. The pyknosis and lysis of nuclei, necrosis of individual epithelial cells. The destruction of the villi up to a section of the crypts. Smooth muscle cells of the muscle membrane was in a state of granular dystrophy.

Genomic Motifs as a Novel Indicator of the Relationship between Strains Isolated from the Epidemic of Porcine Epidemic Diarrhea in 2013-2014

Porcine epidemic diarrhea virus (PEDV) is a positive-sense RNA virus that causes infectious gastroenteritis in pigs. Following a PED outbreak that occurred in China in 2010, the disease was identified for the first time in the United States in April 2013, and was reported in many other countries worldwide from 2013 to 2014. As a novel approach to elucidate the epidemiological relationship between PEDV strains, we explored their genome sequences to identify the motifs that were shared within related strains. Of PED outbreaks reported in many countries during 2013–2014, 119 PEDV strains in Japan, USA, Canada, Mexico, Germany, and Korea were selected and used in this study. We developed a motif mining program, which aimed to identify a specific region of the genome that was exclusively shared by a group of PEDV strains. Eight motifs were identified (M1–M8) and they were observed in 41, 9, 18, 6, 10, 14, 2, and 2 strains, respectively. Motifs M1–M6 were shared by strains from more than two countries, and seemed to originate from one PEDV strain, Indiana12.83/USA/2013, among the 119 strains studied. BLAST search for motifs M1–M6 revealed that M3–M5 were almost identical to the strain ZMDZY identified in 2011 in China, while M1 and M2 were similar to other Chinese strains isolated in 2011–2012. Consequently, the PED outbreaks in these six countries may be closely related, and multiple transmissions of PEDV strains between these countries may have occurred during 2013–2014. Although tools such as phylogenetic tree analysis with whole genome sequences are increasingly applied to reveal the connection between isolates, its interpretation is sometimes inconclusive. Application of motifs as a tool to examine the whole genome sequences of causative agents will be more objective and will be an explicit indicator of their relationship.

ВЫДЕЛЕНИЕ И ИДЕНТИФИКАЦИЯ ВИРУСА ЭПИЗООТИЧЕСКОЙ ДИАРЕИ СВИНЕЙ ПРИ ВСПЫШКЕ В КРУПНОМ СВИНОВОДЧЕСКОМ ХОЗЯЙСТВЕ

Epizootic diarrhea fever (EDF), characterized by acute enteritis and hard intestinal disorders, is caused by the virus of the same group of С oronavirus ( Coronaviridae ) as the virus of transmissible gastroenteritis of pigs, coronavirus of cats and dogs, and human 229E strain. From 2005, at the territory of the Russian Federation the outbreaks of the disease were recorded, clinically similar to pig transmissible gastroenteritis. The infections caused a loss of piglets, in some cases up to 80 %. However, the sows in such farms have been vaccinated against the transmissible gastroenteritis of pigs. The aim of our study was to isolate in cell culture and identify an infectious agent that causes the outbreaks with diarrheal syndrome in pigs. From the pathological material of 3-5-day-old piglets with the pathology a RNA virus was isolated and identified as the virus of epizootic diseases of pigs. Virus infectivity in the fifth passage in Vero cell culture was 10 3,50 ТCID 50/sm 3. The isolate was named as BS-08. To date, there are 25 successive passages of the virus in cell culture. A citopatic effect is registered in 24-48 hours, and the infectious titer (titer of citopatic effect — TCE) makes 10 4,75-10 5,50 TCID 50/sm 3.

Desarrollo de una prueba de reacción en cadena de la polimerasa anidada para el diagnóstico de la gastroenteritis transmisible del cerdo

The aim of this study was to develop a nested polymerase chain reaction (nested PCR) for the rapid detection of transmissible gastroenteritis virus (TGEV) of pigs. The primers were designed on the basis of highly conserved regions of several TGEV sequences included in the analysis. External primers were used to amplify a fragment of the expected size (441 bp) in all the samples evaluated using reverse transcriptase polymerase chain reaction (RT-PCR), but with very low intensity. In the second amplification (nested PCR), internal primers were used to amplify a fragment of the expected size (168 bp), with good concentration. The performance of the test based on virus isolates in tissue culture and in clinical samples was judged good for the virological diagnosis of transmissible gastroenteritis of pigs.

Molecular characterization of a porcine sapovirus strain isolated from a piglet with diarrhoea: a case report

Porcine sapoviruses, members of the family Caliciviridae, have been considered as an aetiological agent of gastroenteritis in pigs. In this study, we analysed 251 faecal samples obtained from 3 to 90 day-old diarrhoeic pigs in the Czech Republic between January 2005 and June 2010 and tested them by negative staining electron microscopy for the presence of sapoviruses. Only one sample showed the presence of viral particles with characteristic sapovirus morphology. The presence of sapovirus (SaV) was confirmed by an RT-PCR assay with primers specific for the sapoviral RNA polymerase and capsid genes. Phylogenetic analysis based on a partial sequence of the RNA polymerase gene placed the new Czech isolate into the GVII genogroup of porcine sapoviruses; however, analysis of a portion of the capsid gene sequence classified the isolate as GIII of the genus Sapovirus. These contradictory findings indicate that recombinant porcine sapovirus was identified. According to our knowledge this is the first description of porcine sapovirus in domestic pigs in the Czech Republic

Molecular detection of porcine kobuviruses in pigs in Korea and their association with diarrhea

Kobuviruses are small, non-enveloped viruses with a single-stranded, positive-sense genomic RNA, belonging to the family Picornaviridae, a highly diverse family of important pathogens of human and other animals. Porcine kobuvirus has been found recently, and consequently, information about the virus is lacking. In this study, we identified porcine kobuviruses from pigs in Korea by RT-PCR, cloning and sequencing, and we showed the existence of genetic diversity among geographically separated porcine kobuviruses through genetic and phylogenetic analysis. Epidemiological studies of porcine kobuvirus linked to diarrhea indicated that porcine kobuvirus infections are endemic in diarrheic pigs in Korea. Statistical analysis of the porcine kobuvirus positive rate between diarrheic and healthy pigs as well as a survey for other enteric pathogens in diarrheic pigs suggests that porcine kobuvirus may play a role as a causative agent of gastroenteritis in pigs.

A new infectious disease challenge: Urbani severe acute respiratory syndrome (SARS) associated coronavirus.

Most community acquired pneumonias are bacterial and the hospitalised patient, often elderly, is rapidly rendered pathogen free by broad spectrum antibiotics. There is no particular threat of pathogen transmission to other patients or to hospital nurses or doctors. It was therefore an unpleasant and unexpected surprise when a cluster of hospital staff and trainee medical students in a Hong Kong hospital became ill with cough, breathlessness and a high temperature. These were all contacts with a 64-year-old doctor who had been admitted into the hospital with the initial diagnosis of community acquired pneumonia. This was the first indication in Hong Kong that a viral not bacterial infection, having apparently arisen in the nearby and adjacent province of Guangdong several months earlier, had spread to the colony.1 A WHO epidemiologist, C. Urbani, categorised the new clinical syndrome severe acute respiratory syndrome (SARS) in Vietnam in February and later died of the virus which is now named after him. As politicians are apt to tell us, the rest is history. Whilst acknowledging that the scientific and medical communities will be in the lower range of a long ladder of step by step learning, it is already clear that modern molecular virology can identify a new virus and devise molecular testing with speed. However, on the negative side there is international and national panic. It is important that the scientific community appreciates how a modern society in the 21st century reacts to an infectious disease threat. This information will be of value to the WHO, which has issued a template plan for preparation in the event of a global outbreak of a much more contagious and life threatening disease, namely emergent influenza A virus.

Elimination of transmissible gastroenteritis virus from a pig farm by culling and serological surveillance.

The first and only outbreak of transmissible gastroenteritis in pigs in Ireland occurred in 1984 in a 650-sow unit which was one of three such units owned by one enterprise which supplied pigs to a 12,000 head fattening unit in the same ownership. The depopulation of the clinically infected unit and extensive serological monitoring of pigs in the other units and in pig farms throughout the country has proved successful in the elimination of transmissible gastroenteritis infections from pigs in Ireland.

Transmissible gastroenteritis in pigs in south east Spain: prevalence and factors associated with infection.

In the Murcia region of south east Spain, epidemics of transmissible gastroenteritis-like disease have occurred in pigs every three years since 1980. In 1987 a sero-epidemiological survey was conducted to estimate the prevalence and spread of transmissible gastroenteritis virus (TGEV) infection among breeding pigs and farms, and to determine any association between the infection and herd size and geographical zone. The Murcia region was divided into four geographical zones and the farms were classified by size into four categories. The random sample was statistically representative of both the breeding stock and the farms in each geographical zone, and the analyses involved 6000 breeding pigs from 480 farms. The immunological techniques employed were the indirect ELISA and the blocking ELISA. The prevalence of seropositive breeding pigs was 1.27 +/- 0.28 per cent and the prevalence of infected farms was 5 +/- 1.94 per cent. On the infected farms, the prevalence of seropositive pigs ranged from 5 to 60 per cent. The infection occurred throughout the Murcia region, but a significant association (P < 0.01) was observed between the geographical zone and the prevalence of TGEV infection. Herds with > or = 50 breeding pigs had a greater prevalence (P < 0.01) of TGEV infection.

Solid phase immune electron microscopy for diagnosis of transmissible gastroenteritis in pigs

A serological trapping technique is described for detecting transmissible gastroenteritis (tge) virus in faeces. The technique involves the coating of electron microscope grids with protein A and specific tge virus antiserum. Optimal conditions for performing this solid phase immune electron microscopy technique were a concentration 250 µg ml−1 of protein A; 1:100 diluted rabbit anti-tge virus hyperimmune serum for coating the grids and overnight incubation with virus samples. The possibility of detecting coronavirus in crude faeces was highly improved with solid phase immune electron microscopy, compared with conventional negative staining electron microscopy, by specific trapping of virus and prevention of adsorption of contaminants. The sensitivity of the method was evaluated by coded investigation of a dilution series of stock virus mixed with different pools of faeces. The improvement of virus detection in faeces by solid phase immune electron microscopy, compared with standard electron microscopy was at least 100-fold. Faecal shedding of coronavirus by pigs infected with virulent and attenuated strains of tge virus was studied. Virus detection in faeces by a standard electron microscopy technique was not practical, since the virus was obscured by a large quantity of debris in the faeces. By using solid phase immune electron microscopy, however, the aspect of the specimens on the grids improved so much that, in addition to more common immunofluorescence, the technique might be useful as a diagnostic test for tge. Virus was detected in daily faecal samples from one or two days after experimental infection with virulent tge virus, until death in five out of eight animals. Unlike immunofluorescence, solid phase immune electron microscopy may be used for diagnosis in living animals.

Expression of porcine transmissible gastroenteritis virus genes in E. coli as beta-galactosidase chimaeric proteins.

Transmissible gastroenteritis virus (TGEV) causes gastroenteritis in pigs of all ages but has a high mortality in neonatal piglets resulting in severe economical losses in affected pig farms. In piglets, under two weeks of age, the first clinical sign is usually vomiting 18–24h after infection rapidly followed by a diarrhoea, resulting in loss of weight and dehydration; death usually occurs after 2–5 days (Garwes, 1982). The virus is enveloped, contains a single stranded RNA genome of positive polarity and three structural proteins; a 200 000 dalton surface glycoprotein (the spike or peplomer protein), a 30 000 dalton glycoprotein associated with the viral envelope (the integral membrane protein) and a basic 47 000 dalton phosphorylated protein associated with the viral RNA (the nucleoprotein). During replication of the virus in infected cells four subgenomic species of RNA are produced of which three have been shown to produce the three structural proteins (Millson et al. unpublished results). The fourth RNA species does not appear to produce a polypeptide detectable either in infected cells or by in vitro translation of the RNA though it has the capacity to produce a polypeptide of about 33 000 daltons. The nucleoprotein gene has been copied from the smallest subgenomic RNA species, shown by in vitro translation to produce the nucleoprotein (Millson et al. impublished results), into cDNA. The complete DNA sequence of the gene has been determined (Britton et al. unpublished results) and a 1.38kb fragment composed of 245 amino acids, corresponding to 68% of the complete gene product, has been fused to the 3′ end of the E. coli lacz gene. The fused genes produced a β-galactosidase-TGEV nucleoprotein fragment chimaeric protein in E. coli. The chimaeric protein has been purified and used to raise antibodies in mice which immune precipitated only the viral nucleoprotein confirming that the DNA sequence assumed to contain the nucleoprotein gene sequence does so. This method provides a useful source of TGEV antigen for virus diagnostic tests and viral protein for use in a research programme aimed at understanding the mechanisms of antigen processing and immune stimulation. The method is useful for confirming the presence of open reading frames (ORFs) and identifying the true product of genes on cDNA either suspected to contain a particular gene or for those previously identified by DNA sequencing.

Non specific defenses against transmissible gastroenteritis in pigs. Study of virus-lymphocyte interactions

Non specific defenses against transmissible gastroenteritis in pigs. Study of virus-lymphocyte interactions