Jan Matiasovic

SPI-1-encoded type III secretion system of Salmonella enterica is required for the suppression of porcine alveolar macrophage cytokine expression

Genes localized at Salmonella pathogenicity island-1 (SPI-1) are involved in Salmonella enterica invasion of host non-professional phagocytes. Interestingly, in macrophages, SPI-1-encoded proteins, in addition to invasion, induce cell death via activation of caspase-1 which also cleaves proIL-1β and proIL-18, precursors of 2 proinflammatory cytokines. In this study we were therefore interested in whether SPI-1-encoded type III secretion system (T3SS) may influence proinflammatory response of macrophages. To test this hypothesis, we infected primary porcine alveolar macrophages with wild-type S. Typhimurium and S. Enteritidis and their isogenic SPI-1 deletion mutants. ΔSPI1 mutants of both serovars invaded approx. 5 times less efficiently than the wild-type strains and despite this, macrophages responded to the infection with ΔSPI1 mutants by increased expression of proinflammatory cytokines IL-1β, IL-8, TNFα, IL-23α and GM-CSF. Identical macrophage responses to that induced by the ΔSPI1 mutants were also observed to the infection with sipB but not the sipA mutant. The hilA mutant exhibited an intermediate phenotype between the ΔSPI1 mutant and the wild-type S. Enteritidis. Our results showed that the SPI-1-encoded T3SS is required not only for cell invasion but in macrophages also for the suppression of early proinflammatory cytokine expression.

Epidemiology and interaction of Salmonella enterica serovar Derby, Infantis and Typhimurium with porcine alveolar macrophages

In this study we were interested in the serovars which are frequently isolated from pigs, i.e. S. Typhimurium, S. Derby and S. Infantis. First we collected different isolates of S. Infantis and S. Derby and compared them by macrorestriction analysis. In the second part of the study we infected porcine alveolar macrophages (PAMs) with representative strains of these serovars and S. Typhimurium and determined intracellular survival, cytotoxicity and cytokine response. In S. Derby, 17 different profiles in 51 isolates have been identified and in S. Infantis, 12 different profiles in 37 isolates have been identified. Four hours post-addition of bacteria to PAMs, higher numbers of intracellular S. Typhimurium than S. Derby or S. Infantis were observed. However, within next 24h, counts of S. Typhimurium did not change while S. Derby and S. Infantis increased their counts 10 and 5 times, respectively. The apparent inability of S. Typhimurium to multiply inside PAMs was caused by its higher cytotoxicity because PAMs infected with S. Typhimurium released LDH 24h post-infection to a significantly higher level than when infected with the other two serovars. The IL-1β, IL-8, IL-12p40, IL-23p19 and TNFα response to S. Derby and S. Infantis was always higher than to S. Typhimurium and the differences among the serovars were more significant at 4 than 24h post-infection. The lower cytokine signaling but higher cytotoxicity of S. Typhimurium for macrophages correlates with the higher virulence for pigs of this serotype when compared with S. Derby or S. Infantis.

Polymorphism and selection in the major histocompatibility complex DRA and DQA genes in the family Equidae

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.

Immunogenomic analysis of insect bite hypersensitivity in a model horse population.

Equine insect bite hypersensitivity (IBH) is a seasonal IgE-mediated dermatosis caused by bites of insects of the genus Culicoides. A familial predisposition for the disease has been shown but, except for the MHC, the genes involved have not been identified so far. An immunogenomic analysis of IBH was performed in a model population of Old Kladruby horses, all living in the same environment. Clinical signs of IBH were used as phenotypic manifestation of IBH. Furthermore, total serum IgE levels were determined in the sera of these horses and used as an independent phenotypic marker for the immunogenetic analysis. Single nucleotide polymorphisms (SNPs) in candidate immunity-related genes were used for association analyses. Genotypes composed of two to five genes encoding interferon gamma -IFNG, transforming growth factor beta 1 -TGFB1, Janus kinase 2 -JAK2, thymic stromal lymphopoietin -TSLP, and involucrin -IVL were associated with IBH, indicating a role of the genes in the pathogenesis of IBH. These findings were supported by analysis of gene expression in skin biopsies of 15 affected and 15 unaffected horses. Two markers associated with IBH, IFNG and TGFB1, showed differences in mRNA expression in skin biopsies from IBH-affected and non-affected horses (p<0.05). Expression of the gene coding for the CD14 receptor molecule -CD14 was different in skin biopsies at p<0.06. When total IgE levels were treated as binary traits, genotypes of IGHE, ELA-DRA, and IL10/b were associated with this trait. When treated as a continuous trait, total IgE levels were associated with genes IGHE, FCER1A, IL4, IL4R, IL10, IL1RA, and JAK2. This first report on non-MHC genes associated with IBH in horses is thus supported by differences in expression of genes known to play a role in allergy and immunity.

Cell-mediated immune response in swine infected with Mycobacterium avium subsp. avium

The zoonotic characteristic of Mycobacterium avium subsp. avium (MAA) represents a veterinary and economic problem in infected pigs. In this study, we analysed cell-mediated immunity six months after experimental infection by measuring interferon-γ (IFN-γ) production and by performing lymphocyte transformation tests after in vitro re-stimulation with the MAA-derived antigen. At the same time, IFN-γ-producing cells were characterised by flow cytometry. In MAA-infected animals, the production of IFN-γ increased in response to the MAA antigen in the blood, spleen and mesenteric lymph nodes. Similarly, a positive antigen-driven response was detected by the proliferation assay. In contrast, IFN-γ production and proliferation was undetectable after stimulation with the MAA antigen in uninfected control animals. These results indicate that both methods can be used for the identification of individual MAA-infected pigs. Using flow cytometry, we found that double-positive CD4(+)CD8(+) lymphocytes were the major T lymphocyte subset producing IFN-γ after in vitro re-stimulation.

Impact of maternally-derived antibodies against Salmonella enterica serovar Typhimurium on the bacterial load in suckling piglets.

The significance of maternal immunity against non-typhoid Salmonella spp. acquired by piglets via colostrum and milk was evaluated in a Salmonella enterica serovar Typhimurium challenge experiment. Piglets from sows vaccinated with an experimental inactivated vaccine exhibited high levels of serum immunoglobulins G and A against S. Typhimurium 4 days after birth, just prior to experimental oral challenge. The S. Typhimurium load in the ileal and caecal wall of piglets 3 days after experimental inoculation was lower by a 2-log magnitude compared to unvaccinated controls. Such a vaccine, delivering colostral/lactogenic immunity to piglets thus has the potential to reduce the prevalence non-typhoid Salmonella spp. infection.

Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) can predispose pigs to secondary respiratory infection with bacteria such as Haemophilus parasuis. Animals infected with both pathogens develop more severe clinical disease. The immune response of porcine alveolar macrophages (PAMs) to simultaneous infection with PRRSV and H. parasuis was analysed in vitro, describing cytokine production, expression of cell surface molecules, and production of reactive oxygen species (ROS). Concurrent infection with PRRSV and H. parasuis increased gene expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-8) in PAMs in comparison with PAMs infected with PRRSV or H. parasuis alone. An additive effect of dual infection on IL-1β production was confirmed at the protein level. PAMs infected with PRRSV showed increased production of ROS compared to controls. Conversely, simultaneous infection of PAMs with PRRSV and H. parasuis decreased production of ROS, indicating the presence of an H. parasuis defence mechanism against respiratory burst. Concurrent infection of PAMs with PRRSV and H. parasuis was shown to elicit a pro-inflammatory immune response represented by significant IL-1β production. Severe multifactorial respiratory disease in natural conditions caused by both pathogens could be the consequence of pro-inflammatory mediated immunopathology.

Interaction of porcine neutrophils with different strains of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is one of the most important causes of post-weaning diarrhea in piglets. Whilst serotype O149:F4 is frequently associated with hemorrhagic gastroenteritis, other serotypes have been found to be associated with mild or moderate enteritis. As neutrophils are recruited to sites of inflammation, the aim of this study was to ascertain whether or not there is any difference in the in vitro interaction between neutrophils and two different ETEC serotypes: O149:F4 and O147:F18. The association of bacteria with neutrophils was evaluated by flow cytometry. The respiratory burst was measured by the fluorescent probe dichlorofluorescein diacetate using flow cytometry and by L012-amplified chemiluminescence. The titers of antibodies against ETEC present in cultivation sera were assessed by agglutination. The viability of E. coli was ascertained by cultivation. It was found that the strains of O149 serotype were more frequently associated with neutrophils and induced a more intensive respiratory burst compared to the strains of O147 serotype. These differences might be due to the presence of different types of fimbriae on the surface of the strains tested and by the presence of anti-fimbrial antibodies in the porcine plasma. However, the intensive interaction between E. coli and the neutrophils and respiratory burst induced by the O149 strain did not lead to more efficient killing of the bacteria. It is suggested that a stronger respiratory burst may be an important factor causing severe clinical signs of post-weaning diarrhea in piglets.

Immune response of pigs to Salmonella enterica serovar Derby and Typhimurium infections

Interaction between pigs and Salmonella enterica serovar Derby (Salmonella Derby) is much less understood in comparison with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). To study interactions of weaned piglets with Salmonella Derby, we compared the course of infections with Salmonella Derby De1 and Salmonella Typhimurium DT104 strains, both isolated from pig herds with a long history of asymptomatic infection. Salmonella Derby strain used was shed during the 28-day experiment period, while Salmonella Typhimurium strain was not found in faeces after day 17 post-infection. When the piglets were co-infected with both strains, Salmonella Derby was present in faeces until the end of the experiment, whilst Salmonella Typhimurium disappeared after day 21 post-infection. At the end of the experiment, Salmonella Derby was present in more tissues when compared with Salmonella Typhimurium. Piglets infected with Salmonella Typhimurium responded earlier with synthesis of anti-lipopolysaccharide IgM and IgG antibodies and with higher antibody levels compared to piglets infected with Salmonella Derby. Cellular immune response to both strains was very low and was detected later than was the onset of IgG antibody production.

Phenotypic characterisation of the monocyte subpopulations in healthy adult pigs and Salmonella-infected piglets by seven-colour flow cytometry

The present study describes the distinct bone marrow (BM) and peripheral blood (PB) monocyte subpopulations detected by seven-colour flow cytometry. Mononuclear phagocytes were identified as viable CD172a(+) SWC8(-) CD203a(-) mononuclear leukocytes. After that, monocyte subpopulations were differentiated by using CD14, CD163 and SLA-DR markers. Four distinct monocyte subpopulations were found in the BM and PB. Based on the discovered populations two possible maturation pathways have been proposed. The first pathway was characterised by release of CD14(hi) CD163(-) SLA-DR(-) BM monocytes into the PB where they matured into CD14(low) CD163(+) SLA-DR(+) monocytes. In the alternative pathway the monocytes finalised their phenotypical maturation in the BM and then they were released into the PB as CD14(low) CD163(+) SLA-DR(+) cells. In Salmonella-infected piglets, the population of CD14(low) CD163(+) SLA-DR(+) monocytes was elevated in the BM and mesenteric lymph nodes (MLN), suggesting the role of this population in pathogenesis of Salmonella infection in pigs.