Serge Bernard

Host adapted serotypes of Salmonella enterica.

Salmonella constitutes a genus of zoonotic bacteria of worldwide economic and health importance. The current view of salmonella taxonomy assigns the members of this genus to two species: S. enterica and S. bongori. S. enterica itself is divided into six subspecies, enterica, salamae, arizonae, diarizonae, indica, and houtenae, also known as subspecies I, II, IIIa, IIIb, IV, and VI, respectively [1]. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or water contaminated by infected faeces. The pathogenicity of most of the distinct serotypes remains undefined, and even within the most common serotypes, many questions remain to be answered regarding the interactions between the organism and the infected host.Salmonellosis manifests itself in three major forms: enteritis, septicaemia, and abortion, each of which may be present singly or in combination, depending on both the serotype and the host involved. Although currently over 2300 serovars of Salmonella are recognized, only about 50 serotypes are isolated in any significant numbers as human or animal pathogens [2, 3] and they all belong to subspecies enterica. Of these, most cause acute gastroenteritis characterized by a short incubation period and a severe systemic disease in man or animals, characterized by septicaemia, fever and/or abortion, and such serotypes are often associated with one or few host species [4–6].It is the intention of this review to present a summary of current knowledge of these host-adapted serotypes of S. enterica. The taxonomic relationships between the serotypes will be discussed together with a comparison of the pathology and pathogenesis of the disease that they cause in their natural host(s). Since much of our knowledge on salmonellosis is based on the results of work on Typhimurium, this serotype will often be used as the baseline in discussion. It is hoped that an appreciation of the differences that exist in the way these serotypes interact with the host will lead to a greater understanding of the complex host–parasite relationship that characterizes salmonella infections.

Site-specific alteration of transmissible gastroenteritis virus spike protein results in markedly reduced pathogenicity

The pathogenicity of neutralization-resistant mutants of the enteric coronavirus transmissible gastroenteritis virus (TGEV) was examined in the newborn piglet. The parental virus (Purdue-115 strain), as well as several mutants selected using monoclonal antibodies (MAbs) directed to antigenic sites A and B, caused an acute enteritis with 100% mortality. By contrast, most of the site D (MAb 40.1) mutants exhibited a strongly reduced enteropathogenicity, leading to the survival of animals inoculated with up to 1000-fold the 100% lethal dose of parental virus. Such a phenotypical change was correlated with point mutations or a small deletion, all located within the S gene sequence coding for the Pro-145 to Cys-155 segment of the mature polypeptide. These observations suggest that an N-terminal subregion of the S molecule is an essential determinant for pathogenesis in TGEV infection.

Radioisotopic Imaging Allows Optimization of Adenovirus Lung Deposition for Cystic Fibrosis Gene Therapy

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

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

Abstract 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.

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.

Kinetics of the in vitro antibody response to transmissible gastroenteritis (TGE) virus from pig mesenteric lymph node cells, using the ELISASPOT and ELISA tests

Abstract A method is described for in vitro studies of viral humoral immune responses in the pig. After oral immunization with transmissible gastroenteritis (TGE) coronavirus, antibody production from primed mesenteric lymph node cells was revealed by an in vitro boost with viral antigen. For the latter the leukocytes were co-cultured with UV-inactivated virus using a variety of different methods of antigenic stimulation. Enumeration of specific antibody-secreting cells (ASC) and titration of secreted anti-virus antibodies were performed with ELISASPOT (using 3-amino 9-ethyl carbazole as the peroxidase chromogen) and ELISA tests respectively, according to the Ig isotype. The results showed a close relationship between ASC numbers and secreted antibody titres. The best in vitro antibody synthesis was observed when the sensitized cells were maintained in contact with virus during the whole culture period. Antibody responses were defined by a kinetic profile characterized by a narrow peak, with a maximum occuring after 4 and 6 days of culture and with the IgA response appearing earlier than the IgG. This methodology, which analyses specific antibody responses at the cellular level, may permit studies on the mechanisms of Ig isotype regulation. Extended to leukocytes from other organs of the immune system, it may also constitute an in vitro model to study antibody responses expressed in different lymphoid tissues of the pig.

Fixed-cell immunoperoxidase technique for the study of surface antigens induced by the coronavirus of transmissible gastroenteritis (TGEV)

Abstract An immunoperoxidase technique performed on the TGEV-infected cells was developed for detection of virus-induced antigens. The presence of M antigen of TGEV on the surface of infected cells was demonstrated by this technique. This finding is in contrast to the M protein of murine hepatitis coronavirus which migrates to the Golgi apparatus but is not transported to the plasma membrane. The time course of appearance M and S antigens on the surface of TGEV-infected cell can be studied by this technique.

Contribution of molecular biology to the study of the porcine interferon system

Abstract We have performed molecular studies on the pig interferon (IFN) system (i) to analyse the role played by endogenous IFN in neonatal viral enteritis such as transmissible gastroenteritis and possibly to obtain, via recombinant DNA technology, a new anti-infectious and immunomodulatory agent in this species, (ii) to characterize the structure and biological functions of the IFN-like antiviral activity produced by the porcine embryo at the time implantation in the uterus. By probing porcine genomic libraries with human and porcine IFN-α probes to isolate related genes, we have shown that the porcine IFN-α multigene family included, like several other mammalian species, two subfamilies of related but distinct genes. Class I subfamily contains at least 11 loci, located on chromosome no. 1, among which nine have been cloned and two (potentially functional) sequenced. Class II subfamily, which is specifically expressed by the embryo of ruminants before implantation, contains at least seven loci among which six have been cloned. One of the sequenced class I loci: PoIFN-α1 encodes a 189 amino acids (AA) preprotein. After removal of the sequence encoding the putative signal peptide (23 N-terminal AA) this gene was inserted into an Escherichia coli bicistronic expression vector allowing intracellular synthesis of mature porcine IFN-α1 (methionyl IFN-α1). Expression of the recombinant protein was optimized by insertion of a seven base pairs long random synthetic sequence in the intercistronic region, followed by cloning in E. coli and immunodetection of clones expressing high amounts of recombinant protein. The E. coli strain obtained produced high levels of a 18 000 Da protein exhibiting the same in vitro overall biological properties as leucocyte derived porcine IFN (LeuIFN). However, it had a stronger antiviral effect on porcine cells than LeuIFN. After immunoaffinity purification to a specific activity of 5−10 × 107 International Units (IU)/mg of protein, pharmacokinetic and pharmacological studies were realized to determine the in vivo half life of this rIFN-α in the pig. These experiments revealed no major toxic effects in newborn (given 5 × 106IU/kg) or adult (1 × 106IU/kg) pigs. A significant pyrogenic effect (+ 1.5°C) was noted only in the adults.

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

Summary 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.

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.

Abstract 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.