-Postweaning multisystemic wasting syndrome (PMWS) is a recently emerged disease affecting pigs. Type 2 porcine circovirus (PCV2) has been associated with this syndrome although other factors are required in association with this virus for PMWS expression. The aim of this study was to investigate whether general immunostimulation (injections of keyhole limpet hemocyanin emulsified in incomplete Freund adjuvant and of thioglycollate medium) could strengthen the severity of PMWS in six-week-old specific-pathogen-free (SPF) piglets transfected with pure tandem-cloned PCV2 DNA by the intramuscular route. Non-immunostimulated piglets transfected with the viral clone did not present clinical signs but only mild pathological microlesions characteristic of PMWS. These piglets seroconverted and high viral genome loads and infectious titers were detected in the lymphoid organs at the end of the trial. Mild-to-moderate forms of PMWS were generally observed in the immunostimulated transfected piglets, as well as one severe form for a piglet (8003) which died. These piglets with mild-to-moderate forms had higher DNA loads than the transfected-only animals. Thus, viral replication was enhanced by immunostimulation. This is the first time that clinical PMWS has been reported in an SPF immunostimulated piglet infected with a pure inoculum consisting of tandem-cloned PCV2 DNA. This result confirms that PCV2 is the agent of PMWS and that immunostimulation could enhance PMWS in SPF piglets transfected with a PCV2 DNA clone. porcine circovirus type 2 / immunostimulation / tandem-cloned DNA / PMWS

Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed. The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator. Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype. Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L. sakei. A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L. sakei by two successive crossovers. A strain deleted of the lacLM operon encoding the ␤-galactosidase of L. sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation of atkYB. Results show that the atkYB operon is induced by small concentrations of CuSO 4 (30 to 40 M) but not when CuSO 4 is omitted or added at higher concentrations.The P-type ATPases are a large family of enzymes so named because of a phospho-aspartate intermediate in the ATPdriven cation transport cycle. A wide range of different cations has been demonstrated to serve as substrates for P-type ATPases of procaryotes (39). All P-type ATPases function as cation pumps, either for uptake, efflux, or exchange. Bacteria have developed specific genes for resistance to the toxic ions of heavy metal elements (see reference 40 for a review). In the chromosome of Lactobacillus sakei, downstream from the 3Ј end of ptsI encoding enzyme I of the phosphotransferase system, two open reading frames (ORFs) were observed (41). They showed sequence similarity to a copper efflux P-type ATPase and its negative regulator, which have been cloned from Enterococcus hirae (30,31). Expression of these proteins is regulated by copper in E. hirae (29,42). L. sakei is naturally found on meat and meat products, but little information is known about the requirement and sensitivity of this species for metal ions. The requirement for manganese by some bacteria, including lactic acid bacteria, is known (34). It was previously shown that the L-lactate dehydrogenase of L. sakei is activated by manganese and cadmium salts (15), and a dipeptidase activated by cobalt and manganese has been purified (25).Although several genes have now been cloned from L. sakei (14) and some genetic tools are emerging (2, 3, 18, 22), the molecular biology techniques specific for this species are still poorly developed. For example, no straightforward reporter gene system has been developed that would help in the analysis of gene regulation. The aim of the present work was to use the promoter of the atkYB locus to identify its possible regulation by heavy metals in order to develop a reporter gene system for L. sakei. Several genes have been used as reporter genes in lactic acid bacteria, such as the luciferase genes in Lactococcus lactis (5); the ...

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Post-weaning multisystemic wasting syndrome (PMWS) is a complex disease syndrome in swine, affecting nursery and fattening pigs. Although ongoing evidence suggests that porcine circovirus type-2 (PCV2) is the causal agent of PMWS, the host immune system appears to have a crucial role in the PMWS pathogenesis of PCV2-affected pigs. Owing to difficulties in producing a biologically pure form of PCV2 devoid of the other viral agents commonly present in swine tissues, we decided to use a tandem-cloned PCV2 DNA providing highly pure grade reagent in order to monitor the virulence of PCV2 alone or with an immunostimulating co-factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). A single intramuscular injection of tandem-cloned PCV2 DNA into 5-week-old piglets produced plasmid to viral genome progeny and infectious particles as early as 8 days post-injection in all the organs tested (the lung, the tonsil and the inguinal, mesenteric, bronchial and upper-right axial lymph nodes). The initial plasmid load was not detected with the help of primers designed to specifically detect the acceptor plasmid, thus confirming the replication of the viral genome. Despite the presence of a high level of PCV2 genome copies in the lymphoid organs--the tonsil and the lung--and the presence of infectious particles, no detectable clinical manifestations or pathological lesions were observed in the transfected pigs over the period of observation, regardless of whether they had been co-injected with plasmid containing GM-CSF DNA or had received plasmid containing PCV2 DNA alone. GM-CSF encoding DNA injection had no significant effect on viral replication or on the production of viral particles and appearance of the disease.