Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid.

Intestinal explants were maintained for weeks in a growth medium containing collagenase for progressive digestion to derive finite cell lines from the ileum (64 lines) or from the colon (8 lines) of a boar. Two ileal cell lines retaining either a fibroblastic or an epithelioid morphology have been used to derive heteroploid cell lines (IPI-1 and IPI-2) immortalized by transfection with an SV40 plasmid (pSV3-neo). The IPI-1 cells were found of fibroblastic lineage. The IPI-2 cell line gave rise to morphologically heterogeneous colonies ranging from typical epithelial cells to colonies of more-elongated cells. A crisis occurred during subcultivation of IPI-2 leading to the isolation of the IPI-2I cell line with a 24 h doubling time and a 21% plating efficiency. Epithelial nature of IPI-2I cells was supported by ultrastructural analysis of the cell monolayers. Differentiated cells were found to express microvilli at the apical cellular membrane and desmosomes connecting adjacent cells. Stable epithelioid phenotypes were obtained only from the IPI-2I cell line by multiple subcloning. These cells were found to express characteristics of both epithelial and mesenchymal cells by positive immunostaining with monoclonal antibodies reacting either with keratin 18 filament of simple epithelia or with vimentin filament typical in vivo of mesoderm. The lack of villin expression and the absence of transepithelial resistance have to be related to a poor differentiated state of this cell line. All these immortalized cell lines were permissive to the replication of microorganisms pathogenic for pig (Salmonella chloleraesuis, Salmonella typhimurium and tissue culture-adapted strains of transmissible gastroenteritis virus). The collection of finite and continuous cell lines will help to develop in vitro methods for long-term propagation of freshly isolated epithelium or three-dimensional organ culture in pig. In addition, the IPI-2I cell line provides a new model to study the conversion from a transformed to a nontransformed phenotype as incorporation of 2% dimethyl sulfoxide in the growth medium to repress large tumor antigen expression led to the progressive disappearance of cytokeratin 18 positive cells with, over a week, the death of the surviving vimentin-positive cells.

Host cell protein tyrosine kinases are activated during the entry of Listeria monocytogenes. Possible role of pp60c-src family protein kinases.

Listeria monocytogenes is able to invade a wide range of cell types by inducing its own internalization. Little is known, however, about the host cell proteins affecting the entry process which involves triggering the host cell signal transduction mechanism. We report here that entry of L. monocytogenes strains (serotypes 4b and 1/2a) into Caco-2 cells induced tyrosine phosphorylation of several host cell proteins including pp60c-src substrates. Using specific synthetic peptide substrates, we showed that L. monocytogenes activates, as early as 5 min after bacteria-cell contact, the pp60c-src family-related (srcFR) proteins by an inlAB-dependent pathway. The activation of srcFR proteins seems to be crucial in the entry of L. monocytogenes into Caco-2 cells. Indeed, specific inhibition of the srcFR signal by herbimycin A blocked the entry of L. monocytogenes strains. Taken together, our data show that L. monocytogenes enhances cell tyrosine phosphorylations and activates the pp60c-src family-related proteins by an inlAB-dependent pathway.

Cell proliferation enhances entry of Listeria monocytogenes into intestinal epithelial cells by two proliferation-dependent entry pathways.

Bacterial entry into intestinal host cells is the result of a fairly sophisticated manipulation of host cell machinery by the pathogens. To study further the potential cell target of Listeria spp., the in-vitro entry of L. monocytogenes strains into intestinal cells was examined in relation to the metabolism, proliferation and differentiation of the cells by the alamarBlue assay, [3H] thymidine incorporation, and brush border-associated enzyme activities, respectively. The study showed that cell metabolism was not involved in the entry of L. monocytogenes in three cell models (two human and one porcine). On the other hand, entry was closely related to the proliferation process and poorly related to the differentiation state of the cells. The use of L. monocytogenes mutants lacking invasion proteins showed that InlA and InlB acted in synergy to mediate the entry of L. monocytogenes into proliferative cells, whereas InlA alone seemed to be involved in the entry into non-proliferative cells. These two entry pathways could correspond to the two cellular processes used by L. monocytogenes to enter proliferative and non-proliferative cells, as suggested by the use of cytochalasin D, nocodazole, chloroquine and monodansylcadaverine. Taken together, we propose a hypothesis in which the entry of L. monocytogenes is mediated by interaction between randomly distributed E-cadherin on the surface of proliferative cells. In contrast, the entry into non-proliferative cells may involve pp60c-src, a proto-oncogenic tyrosine kinase signal that modifies E-cadherin localisation. In conclusion, these results suggest that L. monocytogenes may preferentially enter crypt cells in vivo by a microfilament-dependent process, whereas the few bacteria that infect villus cells enter by an E-cadherin-internalin interaction that mediates microtubule-dependent endocytosis.

Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice.

Some Listeria monocytogenes strains not related to clinical cases have been found to exhibit a low virulence level in mice as well as in an in vitro test using Caco-2 cells. The purpose of this study was to validate a new in vitro test of virulence based on a plaque-forming assay (PFA) using a HT-29 cell monolayer with 118 Listeria strains. The use of HT-29 cells in 96-well tissue culture plates allowed the testing of 30 strains per day and providing results in 24 h. In addition. statistical analyses demonstrated the reproducibility and repeatability of the PFA. No quantitative relationship was observed between the virulence of the strains and the hemolytic titer or the cytotoxic effects on HT-29 cells. In contrast, good agreement was observed between virulence assessed after subcutaneous (SC) infection and virulence obtained by PFA. Three groups of L. monocytogenes strains (avirulent, hypovirulent and fully virulent) were established by comparison of the clinical origin of the strains, the number of immunocompetent contaminated mice and the numbers of Listeria strains recovered in the spleen after SC infection. With one exception, i.e. a clinical case of L. seeligeri (sensitivity 0.98), the PFA successfully detected the virulent strains only (specificity 1). Decision-tree algorithms performed by SAS and S-Plus demonstrated that this tissue culture assay discriminated between the avirulent and hypovirulent strains and the virulent strains. This test could therefore be an alternative to in vivo tests, allowing grading of virulence.

Invasion of Salmonella enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids.

Fermentation reactions in the caeca of chickens, the predominant place for Salmonella colonization, result in high concentrations of short-chain fatty acids (SCFA). Thus Salmonella bacteria are in close contact with SCFA during their life cycle. A study was carried out to analyse the effects of SCFA on invasion of Salmonella enteritidis in an avian intestinal epithelial cell line. Preincubation of S. enteritidis for 4 h in growth media supplemented with various concentrations of propionate or butyrate resulted in decreased invasion compared to bacteria, preincubated in nonsupplemented media, and to bacteria, preincubated in media supplemented with formate or acetate. Incubation of the S. enteritidis bacteria in media supplemented with mixtures of SCFA mimicking the in vivo caecal concentrations resulted in increased invasion compared with butyrate-exposed bacteria, but equal invasion compared with nonexposed bacteria. Increasing the butyrate concentration in these mixtures did not modify invasion compared with the original mixtures.

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.

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.

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.

Low persistence of a large-plasmid-cured variant of Salmonella enteritidis in ceca of chicks.

In order to estimate the contribution of Salmonella in the persistence of this bacterium in chicks, we compared the persistence of a Salmonella enteritidis strain and its plasmid-cured variant in a chicken asymptomatic carrier state model. After oral inoculation, colonization with the plasmid-cured strain was significantly reduced (P < 0.001) in the ceca of chicks from the third week postinoculation and persisted for a shorter period than the wild-type strain. Moreover, numbers of S. enteriditis-infected livers were also significantly lower (P < 0.01) for the plasmid-cured strain compared with the wild-type strain from the third to the seventh week postinoculation. No difference in spleen colonization was observed. These results did not correlate with any in vitro difference in attachment, entry to, or intracellular multiplication of bacteria within intestinal or macrophage avian cell lines.

Interactions of Salmonella enterica subsp. enterica serovar Muenchen with intestinal explants of the turtle Trachemys scripta scripta.

Salmonella infections in reptiles, in contrast to those in birds and mammals, are limited to the intestinal tract. In this study, interactions of a strain of Salmonella enterica subsp. enterica serovar Muenchen (SEEM) with intestinal explants of the turtle Trachemys scripta scripta were examined by scanning electron microscopy (SEM). Adhesion and invasion in the chelonian intestinal explants at 30 degrees C and 37 degrees C were evaluated quantitatively. For purposes of comparison, the invasive capacity of SEEM in the continuous avian epithelial cell line DIV-1 at 30 degrees C and 37 degrees C was determined. Small numbers of M-like cells were found in the ileum of the turtles. The bacteria adhered mainly to the mucus of the intestinal explants. Only small numbers of salmonellae were associated with epithelial cells. Higher numbers of bacteria adhered at 30 degrees C than at 37 degrees C. Epithelial damage, embedding of bacteria in the epithelial surface and a ruffling-like process were noted only at 37 degrees C. Minimal numbers of salmonellae invaded the explants at 30 degrees C and 37 degrees C. Invasion of DIV-1 cells was greater at 37 degrees C than at 30 degrees C. The study suggested that the intestinal mucous layer provides an important site of colonization for salmonellae in the chelonian host and protects the underlying epithelial cells.

Histocompatible miniature pig (d/d haplotype): generation of hybridomas secreting A or M monoclonal antibody.

Two Hypoxanthine/Aminopterin/Thymidine-sensitive cell sublines (L142 and L231) have been derived from independent lymphoblastoid cell lines of B lineage. After propagation for more than 100 population doublings (1 year) in culture, these cells still retained a doubling time between 19 to 20 hours, near diploïdy and relatively low (L142) and high (L231) secretion rate of M immunoglobulins. Near diploid hybrid cells were easily generated with leukocytes from the spleen, the gut lamina propria or the mesenteric lymph nodes of pigs immunized against the transmissible gastroenteritis virus. Both the tumor sublines and the B cells were derived from histocompatible miniature pigs (d/d haplotype). Demonstration of fusion between the tumor sublines and B-cells was supported by the selection of hybridomas making the antigen-specific heavy (alpha isotype) and light chains from the B cell parent as well as the heavy and light chains of the lymphoblastoid parent. Moreover, some hybridomas were found to secrete only class A (dimeric) or class M immunoglobulins (0.2-10 micrograms/ml). Forty hybridomas secreted antibodies reactive in a virus-enzyme-linked cell immunoassay against cell-bound antigens and two were found to produce an antibody active only against the infected cell monolayer. Construction of intraspecies hybridoma can be used to perpetuate lymphocyte subsets useful for the study of the porcine immune system.

[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.

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.

[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.

Cell immortalization enhances Listeria monocytogenes invasion.

Recent outbreaks of human listeriosis have emphasized the importance of food in the etiology of epidemic listeriosis, suggesting that the gastrointestinal tract is the natural site of entry for Listeria monocytogenes into the organism. L. monocytogenes invasion of finite cell lines derived from the porcine ileum exhibited a 100-fold lower penetration level, without any intracellular multiplication, when compared to CaCo-2 cells, a widely used in vitro model for L. monocytogenes invasion. Same results were obtained with both pig kidney primary cells and mouse kidney finite cell lines. To demonstrate that cell immortalization enhances L. monocytogenes invasion, finite cell lines from porcine ileum and from murine kidney were immortalized by Simian virus 40 (SV40) large T oncogene. Unlike their untransformed counterparts, the immortal cells obtained were invaded by L. monocytogenes, as observed for CaCo-2 cells as well as for spontaneously immortal human (HeLa) and murine (3T3) cell lines. Extensive electron microscopy examinations of porcine epithelioid cells infected by L. monocytogenes showed numerous bacteria within the immortal cells, whereas neither intracellular bacteria nor any bacterial antigen were revealed inside finite cell lines. These data suggested that L. monocytogenes were not destroyed inside finite cell lines but only poorly entered the finite or primary cells. Speculating that L. monocytogenes invasion is under control of differentiation or proliferation of the cells, only an enterocyte subset at a defined state of differentiation or expressing particular receptors could be invaded in vivo.

Effects of hypothermia on the survival and cryopreservation of minipig ileal cells and Chinese hamster ovary cells.

Temperature of culture can be used to modulate cellular metabolism for improving small intestinal cell culture and cryopreservation. An hypothermia pretreatment (2 days at 25 degrees C and 3 hours recovery at 37 degrees C) improved hamster cell survival to freeze-thaw damage (p < 0.01) but decreased the survival of 2 immortal pig ileal cell lines even though epithelioid IPI-2I cells were more tolerant to hypothermia than IPI-1 fibroblasts. Epithelioid cells survived 3 days at 25 degrees C with unaltered expression of cytokeratin-18 whereas colonies of fibroblasts did not survive more than a day at 25 degrees C (p < 0.001). These results suggest that hypothermia-tolerance of pig ileal cell lines might differ according to cell lineage calling for further experiments on small intestinal primary cell culture.

The loss of contact inhibition and anchorage-dependent growth are key steps in the acquisition of Listeria monocytogenes susceptibility phenotype by non-phagocytic cells.

We have previously demonstrated that intestinal and kidney finite cell lines were resistant to L monocytogenes invasion (ie allowed low bacterial entry and no intracellular multiplication) in contrast to the continuous cell lines which were susceptible to Listeria invasion (ie allowed high bacterial entry and intracellular multiplication) (Velge et al (1994a) Med Microbial Immunol 183, 145). The aim of this study was to discover whether epigenetic or genetic cellular modifications could convert L monocytogenes resistant cells into a susceptible phenotype and to determine the cellular steps involved in Listeria susceptibility. Among the 5-azacytidine treated finite cell lines, the untransformed immortal cell lines established remained resistant to L monocytogenes invasion whereas the weakly transformed continuous cell lines established were converted into a susceptible phenotype. Transfection of resistant cells by SV40 large T antigen induced only highly transformed continuous cell lines displaying a susceptible phenotype. Taken together these data show that cell transformation enhanced Listeria invasion. This conclusion was supported by the observation that L monocytogenes was able to induce cell foci within murine finite cell monolayers. This morphological cell transformation was completely reversible and required live bacteria inside cells. In conclusion, we may speculate that the L monocytogenes intracellular multiplication observed within cell foci could be explained by the loss of contact inhibition of the finite cell monolayer. Indeed, the loss of both contact inhibition and anchorage-dependent growth are the key steps involved in the L monocytogenes susceptibility phenotype.

Transmissible gastroenteritis coronavirus: surface antigens induced by virulent and attenuated strains.

Three strains of the transmissible gastroenteritis virus (TGEV) possessing different degrees of pathogenicity for piglets were examined for their capacity to express M and S glycoproteins on the infected cell surface using a microwell immunoperoxidase test. These two viral glycoproteins were easily detected on the plasma membrane of 0.1% paraformaldehyde-fixed swine testis (ST) or pig kidney (RP.D) cells which were infected with high-passaged Purdue-115 and low-passaged D-52 strains and a high-passaged attenuated (188-SG) mutant of TGEV. No significant differences were found between attenuated and virulent strains with regard to the viral antigen expression on the membrane of infected cells over a 14-h period.

Porcine retrovirus: optimal conditions for its biochemical detection.

The assay of reverse transcriptase (RT) activity was used to detect the presence of retrovirus in porcine cells. A set of optimal assay conditions was determined to design a sensitive, quantitative and reproducible RT assay for porcine systems. The template-primer poly(rA).oligo(dT) was an absolute requirement. The presence of Mn++ was indispensable, with an optimal concentration of 0.25 mM. Monocations (K+, Na+) at 50 mM greatly enhanced, but their high doses inhibited the reaction. The pH of the medium influenced very much the reaction, especially with non-purified virus samples, with which the RT activity was inhibited at pHs above 8.2. Non-ionic detergents at 1% enhanced several-fold the RT activity. It was also shown that porcine retrovirus could be spontaneously reactivated in porcine cell lines by in vitro long-term propagation and transmitted to pigs by inoculation with virus-producing cells.