J.Å. Robertsson

Salmonella typhimurium SL1479, an auxotrophic mutant strain having a complete block in the aromatic biosynthetic pathway and therefore requiring p-aminobenzoic acid and 2,3-dihydroxybenzoate not available in mammalian tissues, was given orally in a dose of 10(10) live bacteria to 4- to 5-week-old calves. Only a mild transient fever response was seen. Strain SL1479 was unable to colonize and persist in the calves for more than 2 weeks. In a vaccination experiment, groups of six calves were (i) orally vaccinated with the live S. typhimurium SL1479 strain, (ii) subcutaneously vaccinated with a heat-inactivated S. typhimurium SVA1232 strain with aluminum hydroxide as adjuvant, or (iii) not vaccinated, to serve as controls. Calves were orally challenged with the live, calf-virulent S. typhimurium SVA44 strain: either 10(6) bacteria (equivalent to 100 25% lethal doses [LD25S]) or 10(9) bacteria (100,000 LD25S doses). The live oral vaccine gave significantly better protection than the heat-vaccinated, subcutaneously injected vaccine since (i) only control calves and calves given the killed vaccine developed profuse diarrhea, (ii) clinically, the mild fever responses seen after challenge in calves given the live vaccine were significantly lower (P less than 0.0005), (iii) autopsies performed 21 days after the challenge infection revealed normal findings in calves given the live vaccine, whereas calves given the killed vaccine showed signs of acute enteritis and chronic salmonellosis, (iv) all 12 calves given either 100 X or 100,000 X the LD25 survived the 21-day observation period; the mean survival time in nonvaccinated calves was 8.0 days; in calves given heat-inactivated vaccine and 100 X the LD25 it was 21.0 days, and in calves given 100,000 X the LD25 it was 11.5 days, (v) the fecal bacterial counts of the challenge S. typhimurium SVA44 strain were significantly lower (P less than 0.0005) in both groups given the live vaccine, and (vi) upon autopsy followed by culture, the qualitative recovery of the challenge strain from the alimentary canals and tissues of calves given the live vaccine was significantly lower (P less than 0.005).

Groups of six calves, 4 to 5 weeks old, were vaccinated either orally with a live auxotrophic Salmonella typhimurium (O-antigen 1,4,12) SL1479 vaccine (10(8) bacteria on day zero, 10(10) bacteria on days 7 and 14) or subcutaneously with a heat-inactivated (56 degrees C, 30 min) S. typhimurium SVA1232 vaccine (10(10) bacteria suspended in 30% [vol/vol] aluminum hydroxide on days zero, 7, and 14). The calves were then orally challenged with either 10(6) (approximately 100 X the 25% lethal dose) or 10(9) (approximately 100,000 X the 25% lethal dose) live bacteria of the calf-virulent S. typhimurium SVA44 strain. The immune reactivity of these calves and of nonvaccinated control calves was followed before and after the challenge infection up to 42 days by (i) intradermal injection of S. typhimurium crude extract, outer membrane protein preparation (porins), and lipopolysaccharide (LPS), (ii) in vitro stimulation of peripheral blood lymphocytes estimated by using uptake of [3H]thymidine, with S. typhimurium crude extract, porins, LPS, and polysaccharide (O-antigenic polysaccharide chain free of lipid A), and Salmonella sp. serotype thompson (O-antigen 6,7) strain IS40 LPS and polysaccharide, and (iii) estimation of the class-specific immunoglobulin G (IgG) and IgM antibody responses against S. typhimurium LPS and porins, and Salmonella sp. serotype thompson LPS. The immune studies showed that in calves given the live vaccine orally, the skin test reactivity and lymphocyte stimulation indices were significantly higher (P values ranging from less than 0.025 to less than 0.0005) against homologous, but not heterologous, antigens than those seen in calves given the heat-inactivated vaccine subcutaneously. In contrast, the IgG and IgM antibody titers against homologous LPS and porins were significantly higher (P less than 0.0005) in sera collected on day 21 from calves given the heat-inactivated vaccine than in calves given the live vaccine. After the oral challenge, calves given the live vaccine showed reduced cell-mediated immune reactions, in agreement with the observation that the host defense could eradicate the challenge organism, whereas calves given the heat-inactivated vaccine showed significantly increased cell-mediated immune reactions (P values ranging from less than 0.025 to less than 0.005), in agreement with the observation that in these calves, the challenge strain caused enteritis as well as systemic invasion. The increased cell-mediated immune reactivity in calves given the live vaccine correlated well with the excellent protection against challenge infection seen in these animals.

Specific delayed skin reactions developed after intradermal injection of Salmonella typhimurium lipopolysaccharide in 12-week-old calves which had been orally infected with S. typhimurium 5 weeks earlier. Uninfected calves showed no delayed skin reactions. Skin biopsies from skin swellings showed a massive infiltration of mononuclear cells in the skin of infected calves but not in those of uninfected calves. Persistence of infection in infected calves was confirmed by isolation of S. typhimurium from fecal specimens. The delayed skin reactions could be shown to be specific and directed against the O-polysaccharide chain of the lipopolysaccharide since none of the lipopolysaccharide chain of the lipopolysaccharide since none of the lipopolysaccharide preparations from a rough mutant of S. typhimurium, two strains with different O-polysaccharide chains, or lipid A elicited skin reactivity. To cause a reaction, the O-polysaccharide had to be in a macromolecular complex, since skin swellings were seen only after injections of either O-polysaccharide chains cross-linked by 1,2,3,4-diepoxybutane or octasaccharides from the O chain covalently linked to a straight 12-carbon aliphatic chain forming an artificial glycolipid. Injection of the pure octasaccharides of O-polysaccharide chains failed to elicit delayed skin reactions.

Postweaning multisystemic wasting syndrome (PMWS) is causally associated with porcine circovirus type 2 (PCV2) infection of pigs. PCV2 has been present in the Swedish pig population for at least ten years. Despite this, no signs of PMWS were observed in pigs of Sweden until 2003. Since then the disease has spread slowly in Sweden. The pig population of Sweden is geographically isolated, the density of pigs and the pathogen load in the country is low and the trade of animals is organised in a restricted way. To date, there has been little information placed in the peer-reviewed literature on the study of the spread of PMWS in a country during the early phase of the disease outbreak, mainly because diagnosis of PMWS has only occurred after the disease has been recognised on a number of farms. This manuscript documents the studies to date on the PMWS outbreak in Sweden which has been closely monitored since the initial outbreak of diseases on a single farm in 2003 was diagnosed. In Sweden PMWS was officially reported for the first time at a progeny test station during December 2003. One year later sixteen herds have been diagnosed and reported to the authorities: one progeny test station, one nucleus herd, thirteen piglet producers or integrated herds and one specialised fattening herd. Fifteen suspected cases have been deemed negative for PMWS with disease scenarios associated with Lawsonia intracellularis, Brachyspira pilosicoli, Mycoplasma hyopneumoniae, shortcomings in feed composition etc. To date, one year after the index case was reported ten other herds suspected for PMWS are under observation. The PMWS cases are concentrated into the southern part of Sweden. However, the disease is spreading slowly, and in general there are no obvious physical links between affected herds. The data generated to date in Sweden suggest that introduction of a new contagious microbe into the country that is responsible for the PMWS outbreaks appears unlikely, as does the spread of PMWS via semen.

Peripheral blood lymphocytes collected from calves infected experimentally with Salmonella typhimurium (O antigens 4,5,12) or Salmonella sp. serotype dublin (O 9,12) were stimulated with various bacterial cell envelope components, and their [3H]thymidine incorporation was measured. It was found that peripheral blood lymphocytes from infected calves incorporated significantly more [3H]thymidine than peripheral blood lymphocytes from uninfected controls (P values ranged from less than 0.05 to less than 0.0005). The responder cell type was found in a B-cell-depleted and T-cell-enriched population. The Salmonella infections elicited T-cell responses against at least two cell envelope components: (i) a specific response against the O-antigenic polysaccharide chain of the lipopolysaccharide (This was evident in that a polysaccharide from S. enteritidis [O 9,12] which shares a trisaccharide structure [O antigen 12 determinant] with S. typhimurium stimulated [3H]thymidine uptake, which, although lower than in the homologous system, was significantly higher than that seen after incubation with unrelated Salmonella sp serotype thompson polysaccharide.) and (ii) a response against outer membrane proteins (porins), which are present in both S. typhimurium and Salmonella sp. serotype dublin. The experiments with peripheral blood lymphocytes from Salmonella sp. serotype dublin-infected calves gave results in excellent agreement with those obtained in S. typhimurium-infected calves.