[Risks of infection in xenotransplantation: what are they and how are they to be controlled?]. / Les risques infectieux en x¿enotransplantation, quels sont-ils et comment les ma¿itriser?

Porcine grafts are a potential source of pathogenic agents capable of contaminating the recipient. The spread of porcine retroviruses in the patient's tissues is a major risk that must be rigorously evaluated. The control of the microbiological state of the pig donor, concerning retroviruses and other pathogens, is the necessary condition for controlling this risk.

Specific cytotoxic lymphocyte response in swine against structural proteins of transmissible gastro-enteritis virus: a study using lymphoblastoid cell line and recombinant vaccinia virus.

To determine the specificity, if any, of cellular cytotoxicity against transmissible gastro-enteritis virus (TGEV) infected cells, we developed a test using B lymphoblasts from a MHC histocompatible (d/d haplotype) cell line (L14), as stimulating and target cells. These cells were previously infected with recombinant vaccinia virus including different TGEV structural genes, either the spike (vS), membrane (vM) or nucleoprotein gene (vN). Lymphocytes from a TGEV immunized (d/d) swine developed a cytotoxic activity after secondary in vitro stimulation in the presence of vS, vM or vN infected L14 cells. The cytotoxic activity was induced and directed against the homologous vS and vM infected cells but no cytotoxic activity occurred at all against vN infected cells. While vM infected cells induced a cytotoxic activity against vM infected cells only, vS infected cells stimulated a cross-reactive cytotoxic activity against vM and vN infected cells in addition to that against vS infected cells. This latter cytotoxicity may be due to an increase in a non-specific background of Natural Killer or lymphocyte activated killer activity, which is seen also after coculture with wild type vaccinia virus (vW) infected cells. Thus these results are of practical importance in two respects. First, lymphoïd B cell lines represent an excellent tool for determining which viral antigens are recognized by cytotoxic lymphocytes and second, they indicate the need to incorporate the M and S genes into a TGEV vaccine to induce cellular immunity against TGEV.

Transmissible gastroenteritis coronavirus: the development and applications of the fixed-cell immunoperoxidase technique.

Since the first demonstration in 1971 that solid-phase enzyme-linked immunosorbent assays (ELISAs) could be used for the quantitative determination of antigens and antibodies, this method has been widely applied in serodiagnosis of parasitic and infectious diseases. In addition to the classic ELISA variants using antigen or antibody to coat the plastic plates, there has recently been growing interest in the application of fixed-cell ELISA to research and diagnostic work on viral diseases. The authors discuss the development and applications of this technique to basic research and diagnosis of transmissible gastroenteritis, a highly contagious disease of swine. The success of this technique, as the name suggests, is largely due to the use of a suitable fixative, which preserves the antigenicity of the neo-synthesised viral proteins, and the presence of optimal conditions for viral antigen synthesis. In addition, various parameters are optimised, and this is discussed with reference to transmissible gastroenteritis virus. These parameters would help veterinarians and research workers to develop this technique in their own laboratories.

The use of PCR genome mapping for the characterisation of TGEV strains.

Previous studies on different transmissible gastroenteritis virus (TGEV) strains, including porcine respiratory coronavirus (PRCV), have identified regions within the genome that are polymorphic as regards insertions and deletions. For example the 672 base deletion within the S gene and multiple deletions 5', within and 3' of the ORF-3a gene were detected in strains of PRCV. The presence of deletions may be associated with a change in the virulence, attenuation or tissue tropism of the isolate. The Nouzilly (188-SG) TGEV vaccine strain was attenuated by passage of a cell culture adapted virulent isolate D-52 188 times through swine testis cells after treatment with gastric juice. PCR amplification with oligonucleotides, corresponding to known TGEV sequences, were used to analyse D-52 and 188-SG for genetic variation. Results with several pairs of oligonucleotides within the first 1565 nucleotides of the S gene did not identify a deletion within this region of the genome from either strain. However, oligonucleotides directed against the ORF-3a/3b region detected a deletion of about 250 nucleotides within the 188-SG genome but not in the D-52 genome. Since all the attenuated TGEV strains so far sequenced, PRCV, Miller SP and 188-SG, contained deletions within the ORF-3a/3b, it would suggest that this region of the TGEV genome is involved in regulating viral virulence.

Induction of lactogenic immunity to transmissible gastroenteritis virus of swine using an attenuated coronavirus mutant able to survive in the physicochemical environment of the digestive tract.

A transmissible gastroenteritis (TGE) coronavirus mutant (188-SG), selected as attenuated and resistant to acidity and proteases of the digestive tract of adult pigs, was used as vaccine ("Nouzilly strain") in sows to protect suckling piglets against a challenge exposure carried out with a highly virulent TGEV strain. The pregnant sows were immunized once (42-49 days before farrowing) or twice (42-49 and 7-15 days before farrowing) by the oral, intramuscular or conjunctival route with the 188-SG strain. Sows exposed to virulent TGEV in the field and experimentally infected sows (two oral inoculations during pregnancy) were used as positive controls leading to high protection. The neutralizing antibody response to vaccination and/or infection was studied in serum and milk. No protection against mortality was observed in the litters of (1) the nine seronegative, susceptible sows, with piglet mortality of 65/70, (2) the seven once orally vaccinated sows, with mortality of 44/54, (3) the seven sows vaccinated twice by the conjunctival route, with mortality of 55/76. Moderate protection was observed in (1) the eight sows vaccinated intramuscularly twice with piglet mortality of 36/90, (2) the seven orally and intramuscularly vaccinated sows with piglet mortality of 31/51. In of 3 contrast, improved protection was observed in (1) the 10 sows vaccinated twice orally, with piglet mortality of 23/95, (2) the four naturally infected sows with piglet mortality of 6/41, (3) the six sows experimentally infected with virulent TGEV with piglet mortality of 1/59. No correlation was found between neutralizing antibodies titers in serum and milk and protection rate of the piglets. The results indicate that relative protective lactogenic immunity against TGEV is induced only by repeated ingestion of the attenuated 188-SG strain of TGEV.

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.

Natural infection with the porcine respiratory coronavirus induces protective lactogenic immunity against transmissible gastroenteritis.

Our objective was to evaluate the level of passive protection against transmissible gastroenteritis (TGE) among 57 newborn piglets nursing from seven seropositive sows previously naturally infected with porcine respiratory coronavirus (PRCV). After challenge exposure we observed mortality rates of 44% for litters of seven PRCV-infected sows, 40% for litters of four sows orally immunized with the attenuated TGEV strain Nouzilly, and 91% for litters of seven seronegative susceptible sows. A blocking ELISA with two appropriate monoclonal antibodies distinguished serological responses of PRCV-infected sows from those of TGEV-immunized sows. The results suggest that natural infection of the sow with PRCV may induce a degree of protective lactogenic immunity against TGE.

Lactogenic immunity to transmissible gastroenteritis (TGE) of swine induced by the attenuated Nouzilly strain of TGE virus: neutralizing antibody classes and protection.

An attenuated TGE coronavirus mutant (Nouzilly strain) relatively resistant to acidity and digestive enzymes was used for immunization of 11 seronegative pregnant sows, mainly by the oral route. Nouzilly strain is able to induce a protective lactogenic immunity in suckling piglets against severe virulent TGE challenge. However the level of neutralizing activity in serum and milk of vaccinated sows at the time of challenge exposure is not correlated with passive protection rate. Separation of the immunoglobulin classes bearing neutralizing activity in serum and milk from vaccinated sows, by ion-exchange chromatography, failed to demonstrate any relationship between antibody level of these fractions and passive protection rate of piglets. Our results suggest that IgA neutralizing antibodies present in milk are not the only parameters to consider to explain the lactogenic immunity induced by the Nouzilly strain given by oral route.

Transmissible gastroenteritis (TGE) of swine: in vitro virus attachment and effects of polyanions and polycations.

Four transmissible gastroenteritis virus (TGEV) strains (Purdue-115, D-52, 188-SG and Gep-II) and two cell lines (swine testis-ST and pig kidney-RPD) were used to study virus attachment and cell susceptibility. Virus attachment was partially thermodependent and the rate varied, depending on the strain. Identical TGEV inocula produced a higher plaque number by plaque assay in the swine testis cell line (ST) than in the pig kidney cell line (RPD) but [3H]uridine-labelled virus was found associated equally well with both cell lines. A field TGEV strain (Gep-II), which was unable to multiply in cell cultures, appeared able to inhibit the attachment of radiolabelled cell-passaged virus. Therefore, the susceptibility to TGEV infection was apparently not determined at the virus-to-cell attachment stage. The attachment sites on the cell surface were specific, however, differences in TGEV attachment determinant between strains were not observed. Attachment of all the virus strains tested was enhanced by DEAE-dextran and inhibited by dextran sulfate, poly-L-lysine (PLL), poly-L-alpha-ornithine (PLO) and protamine sulfate.

[Influence of concanavalin A on the attachment of the coronavirus of transmissible gastroenteritis in cell cultures]. / Influence de la concanavaline A sur l'attachement du coronavirus de la gastro-entérite transmissible en culture cellulaire.

Effect of Concanavalin (ConA) on attachment of three strains of Transmissible Gastroenteritis coronavirus (TGE) of swine was investigated in cell culture. Whatever the virus strain, reduction of virus plaques number is observed when viral suspension is incubated with cells together or after addition of ConA. Intensity of inhibition is related with ConA concentration. ConA treatment of preinfected cell cultures has no effect on plaque formation. Addition of alpha-methyl-D-mannoside inhibits the ConA activity. Treatment of cells with metaperiodate has no effect on plaque formation and ConA activity. Our results suggest that ConA inhibits formation of plaques by TGE coronavirus.

Effect of deoxycholate, amphotericin B and fongizone on transmissible gastroenteritis coronavirus.

At a concentration of 2 µg/ml, neither amphotericin B nor deoxycholate had an inactivating effect upon transmissible gastroenteritis Coronavirus in-fectivity. However, amphothericin B stimulated plaque formatin in agarose and facilitated the entry of viral RNA into swine testis cells. The combination of amphotericin B + deoxycholate inactivated virus infectivity and induced a decrease in plaque diameter. Finally, in the presence of these agents, the production of infectious virus and interferon was unchanged.

A la concentration de 2 µg/ml, l'amphotéricine B et le désoxycholate n'ont pas d'effet inactivant sur le pouvoir infectant du coronavirus de la gastroentérite transmissible. En revanche, l'amphotéricine B stimule la formation des plages sous agarose et facilite l'entrée de l'ARN viral dans les cellules ST. L'association amphotéricine B + désoxycholate inactive le pouvoir infectant du virus et induit une diminution du diamètre des plages. Enfin, en présence de ces agents la production de virus infectieux et d'interféron n'est pas modifiée.

Étude comparée de trois souches du coronavirus de la gastroentérite transmissible: Conditions de la réplicationvirale et de la synthèse des antigènes structuraux.

Purdue-115 and D-52 strains of TGEV were compared with the 188-SG strain, which was obtained by means of a survivor selection process in gastric juice of adult pig. The 188-SG strain was characterized by (a) low infectivity, (b) delayed and restricted growth associated with low and delayed RNA synthesis, and (c) a high content of structural antigens. In contrast, Purdue-115 and D-52 strains were characterized by (a) high infectivity, and (b) a normal pattern of virus replication and RNA and structural antigen synthesis. Tunicamycin induced the inhibition of synthesis of El and E2 glycoproteins (detected by the ELISA test using monoclonal and polyclonal antibodies) as well as a significant reduction in the NP protein. The inhibitory effect of tunicamycin was influenced by the cell type and virus strain.

Nous avons réalisé l'étude comparative de la souche 188-SG et des souches Purdue-115 et D-52 de Coronavirus de la gastroentérite transmissible. Obtenue par sélection de survie dans du contenu gastrique de porc adulte, la souche 188-SG se distingue des deux autres par les propriétés suivantes: a) un faible pouvoir infectant; b) une multiplication tardive et modérée, liée à une synthèse tardive et faible d'ARN; et c) une richesse anormalement élevée en antigènes structuraux révélés par un test ELISA qui met en œuvre des anticorps monoclonaux et polyclonaux. La tunicamycine exerce un effet inhibiteur sur la synthèse des glycoprotéines E1 et E2. Parallèlement, on observe une réduction significative de la synthèse de la protéine NP non glycolysée. l'effet inhibiteur de la tunicamycine est modulé par des facteurs tenant d'une part au type de cellules utilisées et, d'autre part, à la souche de Coronavirus.

Detection of transmissible gastroenteritis coronavirus antigens by a sandwich enzyme-linked immunosorbent assay technique.

A new sandwich enzyme-linked immunosorbent assay, using monoclonal and polyclonal antibodies, was developed to detect transmissible gastroenteritis virus antigens from cell culture and from intestinal wash or feces obtained from experimentally infected pigs. This technique was shown to be suitable for the detection of virulent field strain unadapted to cell culture. Cross reactions had not been observed with other enteric pathogens, rotavirus, porcine epizootic diarrhea virus, and Escherichia coli.

Neutralizing secretory IgA and IgG do not inhibit attachment of transmissible gastroenteritis virus.

Secretory IgA (sIgA) and IgG from porcine milk and serum, respectively, [3H]uridine-labelled virus, swine testis and pig kidney cell lines were used to examine the neutralized virus-cell interaction. Transmissible gastroenteritis virus (TGEV), 99.99% neutralized by immunoglobulin, was able to attach to the cells. Moreover, sIgA enhanced virus attachment. However, the neutralized virus was unable to enter cells, as demonstrated by the action of proteinase K which removed it from the cell surface. It was also found that pre-attached virus was still neutralizable and that IgG and sIgA had similar TGEV-neutralizing capacities.

Transmissible gastroenteritis (TGE) of swine: survivor selection of TGE virus mutants in stomach juice of adult pigs.

Two transmissible gastroenteritis (TGE) virus mutants (188-SG and 152-SG) were obtained from a low-passage virus strain (D-52) by 188 and 152 cycles of stomach juice treatment and multiplication in cell culture. Compared to the high-passage Purdue-115 and the original D-52 strains, these mutants were more stable at pH 2.0, more resistant to pepsin and trypsin, and characterized by a small plaque phenotype. In vivo, the two mutants were not found to be virulent for 4-day-old piglets and sows after oral inoculation. To test induction of lactogenic immunity, the 188-SG mutant was administered orally to pregnant sows (6 or 7 weeks before parturition) followed by one intramuscular booster (1 week before parturition). After challenge with virulent TGE virus, piglet mortality 7 days after exposure was reduced (to 22%) as compared to the death rate in piglets from control sows (91%).

Conditioned taste aversion suppresses induction of delayed-type hypersensitivity immune reactions.

Conditioned taste aversion was induced in mice by pairing saccharin drinking with an intraperitoneal injection of lithium chloride, a toxic but nonimmunosuppressive drug. Conditioned mice showed not only suppressed saccharin drinking but also a 75% reduction in the induction of delayed-type hypersensitivity immune responses to low doses of sheep erythrocytes. This effect was observed with doses of lithium chloride which had no effect of their own on immune functions. In addition, a reduction in water consumption was not responsible for the reduced immune response of conditioned mice since the immune responses of water deprived mice did not differ from those of nondeprived mice. Conditioned mice exposed to saccharin had higher plasma levels of glucocorticoids than nonconditioned mice, suggesting that the experience of being reexposed to a taste paired with lithium chloride was perceived as aversive. These data demonstrate that alterations in immune functions can be induced by a conditioned taste aversion procedure independently of any immunosuppressive drug.

[Transmissible gastroenteritis: vaccination of sows in contaminated environment using inactivated virus in oil emulsion]. / Gastroentérite transmissible: essais de vaccination de la truie en milieu contaminé à l'aide du virus inactivé en émulsion huileuse.

An oil emulsion vaccine against TGE was prepared using a virus suspension produced in tissue culture and inactivated by formaline. Intramuscular vaccination trials were performed on sows in the field from two herds (167 sows) which had been infected by TGE virus more than one year previously. Considering qualities of immunity after natural infection, the objective of the work was to try to give a new impetus to immunity in such herds using vaccination as a booster. After two vaccine injections the serological pattern of the herd was modified: negative serological reaction disappeared, the proportion of highly positive serological reactions increased and higher homogeneity was observed in the distribution of levels of neutralizing antibody reactions. In contrast, using protection tests in piglets against virulent challenge, there was no evidence of significant differences between litters from vaccinated sows (86% of piglets were protected) and those of unvaccinated sows (89% of piglets were protected). Our results suggest that passive protection given to piglets by lactogenic immunity following natural infection could last all the economic life of the sow. The interest of the vaccination as a booster of mammary immune response and its epidemiological consequences are discussed.

[Transmissible gastroenteritis of swine: stability of coronavirus in gastric and intestinal contents]. / Gastroentérite transmissible du porc: stabilité du coronavirus dans le contenu gastrique et intestinal.

Low and high passaged cell culture strains of TGE coronavirus were examined for stability in gastric and small intestine juices collected in 3-6 month-old pigs killed at different times after last feeding. Results revealed high fragility of the TGE virus in these digestive liquids. Differences in stability were observed between strains of TGE virus. But no correlation could be made between the level of stability and cell passage status of the virus strain. We conclude that the stability of TGE coronavirus in gastric and small intestine juices could not be considered as a genetic marker of virulence. In future, it will be necessary to take into account these data concerning fragility of TGE coronavirus for improvement of oral vaccination methods in pregnant sows using a live virus vaccine.

[Induction of immunosuppression by dietary aversion acquired in the absence of immunosuppressive treatment]. / Induction d'une immunosuppression par une aversion alimentaire acquise en l'absence de traitement immunosuppreseur.

Conditioned taste aversion induced by either an immunosuppressive (cyclophosphamide) or non immunosuppressive (lithium chloride) drug reduced expression of a T cell mediated immune response that is highly susceptible to stress hormones. These results demonstrate that the proposed concept of conditioned immunosuppression really represents another example of how stress can alter normal regulation of cell mediated immune events.