Maria Stadler

Induction of protective immunity by aerosol or oral application of candidate vaccines in a dose-controlled pig aerosol infection model.

In order to outline basic concepts for the design of a bacterial aerosol infection model, the development of a pig model with Actinobacillus pleuropneumoniae is described. First, reproducibility of aerosol parameters should be maintained by optimizing generating and sampling conditions. Survival rates of the chosen strain must be predictable. Secondly, inhalation conditions for the recipients have to be standardized to enable the determination of deposition sites and the dose administered. Subsequently, dose-response relationship should be evaluated to find a suitable challenge dose. Furthermore, it seems necessary to establish methods to obtain local specimens for determination of the local immune responses. The present study demonstrates that after aerosol challenge pigs were completely protected after inhalation and partially protected after oral application of A. pleuropneumoniae vaccines and describes techniques to administer bacteria in a dose-dependent, viable way. Using the infection model several stages of the disease from acute pleuropneumonia to chronic infection can be induced for research purposes.

Bacterial growth kinetic without the influence of the immune system using vacuum-assisted closure dressing with and without negative pressure in an in vitro wound model.

The physical capacity of negative pressure wound therapy (NPWT) dressing on the bacterial growth in an in vitro wound model was investigated. Standardised wounds were contaminated with a clinical Staphylococcus aureus strain and incubated at 35°C for 6 hours. Four wounds were treated with continuous negative pressure (125 mmHg) and four controls without. Bacterial load per gram tissue and per gram polyurethane sponge were measured after 24, 36 and 72 hours. Without negative pressure, the initial mean S. aureus load per gram tissue was 1·42 × 104, with negative pressure 1·84 × 104, P = 0·294. After 24, 36 and 72 hours, both models yielded comparable numbers of organisms (24 hours: P = 0·081; 48 hours: P = 0·455; 72 hours: P = 0·825, respectively). Bacterial load of sponges with or without negative pressure also did not differ. Over a period of 72 hours, sponges with negative pressure yielded 1·60 × 108, those without negative pressure yielded 1·74 × 108 CFU/g sponge (P = 0·876). In non vital tissue without the influence of the immune system, the bacterial load did not decrease in our in vitro model using an NPWT dressing. This observation was independent of the physical effect of continuous negative pressure at 125 mmHg. The reduction in bacteria demonstrated in previous studies appears to be caused by other effects than physical suction alone. However, the results obtained are limited as non viable tissue was used and the effect of suction on dead tissue might be very different from that occurring on perfused tissue, for example, in an animal model or in patients.

Pigs aerogenously immunized with genetically inactivated (ghosts) or irradiated Actinobacillus pleuropneumoniae are protected against a homologous aerosol challenge despite differing in pulmonary cellular and antibody responses

Aerosol immunization is a safe way to induce complete protection against pleuropneumonia in pigs caused by the lung pathogenic bacterium Actinobacillus pleuropneumoniae. In order to determine the local immune responses of vaccinees in concomitant with protection, lung lining fluid before and 3 weeks after immunization from pigs immunized three times with aerosols of either genetically inactivated ghosts which represent whole cell envelope preparations, or irradiated bacteria were examined following an homologous aerosol challenge. Specific antibody isotypes in the bronchoalveolar lavage were assayed by whole cell ELISAs. Total and relative numbers of cells including lymphocyte subsets were determined. In both vaccinated groups a net influx of plasma cells and lymphocytes, as well as a significant increase of specific IgG occurred. Concurrently, the CD4+/CD8+ ratio was found to increase after aerosol immunization. The lymphocyte subsets of IgG+ and IgA+ cells were found significantly higher in the group immunized with irradiated bacteria when compared to pigs immunized with bacterial ghosts. The latter group showed a significant increase of IgA, IgM, and a net influx of lymphoid blasts and granulocytes in the bronchoalveolar lining fluid. Although differences between the local immune responses of both immunized groups occurred, a significant increase of specific IgG and a net influx of plasma cells and lymphocytes were found to be associated with complete protection against a homologous aerosol challenge infection.

Sensitive detection of Foxp3 expression in bovine lymphocytes by flow cytometry

The transcription factor forkhead-box p3 (Foxp3) has been designated as a master regulator for the function of regulatory T cells (Treg). Therefore, the identification of Foxp3 expression in T cells is indispensable for the study of Treg. However, studies on Foxp3 expression in bovine lymphocytes are still sparse, probably due to a lack of Foxp3-specific antibodies with reliable performance in flow cytometry. Our group recently demonstrated that a monoclonal antibody (FJK-16s) developed against murine Foxp3 also binds to porcine Foxp3 and performs well in flow cytometry. A protein sequence alignment of the binding region of the FJK-16s antibody revealed, that within this region the sequences of porcine and bovine Foxp3 are identical. Therefore, we tested this antibody for its suitability in flow cytometry with bovine peripheral blood mononuclear cells (PBMC). By using nonspecific isotype-matched antibodies and competition labeling with non-fluorescent FJK-16s antibodies as negative controls, we readily observed a specific staining of a small subpopulation of CD25(high) lymphocytes within PBMC. Co-staining with monoclonal antibodies against CD3, CD4, CD8β and TCR-γδ revealed that all Foxp3+ cells co-expressed CD3, and were in their vast majority CD4+. However, minor populations of Foxp3+CD8β+ and Foxp3+TCR-γδ+ lymphocytes could also be identified. In summary, our data demonstrate that the FJK-16s antibody is a valuable tool to promote the study of Foxp3+ T cells and their biological relevance in cattle.

Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs

The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F 2 descendants of F 1 Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA-1 , SLA-2 , SLA-3 ) and class II ( SLA-DRB1 , SLA-DQB1 , SLA-DQA ) genes of 27 purebred Pietrain pigs using a combination of the high-resolution sequence-based typing (SBT) method and a low-resolution (Lr) PCR-based method using allele-group, sequence-specific primers (PCR-SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr-43.0 ( SLA-1 *11XX- SLA-3 *04XX- SLA-2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr-0.14 [ SLA-DRB1 *0901- SLA-DQB1 *0801- SLA-DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr-Pie-0.1 [ SLA-DRB1 *01XX/be01/ha04- SLA-DQB1 *05XX- SLA - DQA*blank] and Lr-Pie-0.2 [ SLA-DRB1 *06XX- SLA-DQB1 *03XX- SLA-DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR-SSP-based assays represents a rapid and cost-effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease-resistant pigs in the context of SLA antigens to improve overall swine health.

Magnitude and kinetics of multifunctional CD4+ and CD8β+ T cells in pigs infected with swine influenza A virus

Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4+ and CD8β+ T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ+TNF-α+IL-2+ multifunctional CD4+ T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4+ T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4+ T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4+ T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4+ T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67+perforin+ CD8β+ T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8β+ T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.

Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung

ABSTRACT Pigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67+) CD8+ T cells with an early effector phenotype (perforin+ CD27+) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4+ and CD8+ T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4+ and CD8+ T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4+ and CD8+ memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs. IMPORTANCE Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.

PCV2 vaccination induces IFN-γ/TNF-α co-producing T cells with a potential role in protection

Porcine circovirus type 2 (PCV2) is one of the economically most important pathogens for swine production worldwide. Vaccination is a powerful tool to control porcine circovirus diseases (PCVD). However, it is not fully understood how PCV2 vaccination interacts with the porcine immune system. Especially knowledge on the cellular immune response against PCV2 is sparse. In this study we analysed antigen-specific T cell responses against PCV2 in a controlled vaccination and infection experiment. We focused on the ability of CD4+ T cells to produce cytokines using multicolour flow cytometry (FCM). Vaccination with a PCV2 subunit vaccine (Ingelvac CircoFLEX®) induced PCV2-specific antibodies only in five out of 12 animals. Conversely, vaccine-antigen specific CD4+ T cells which simultaneously produced IFN-γ and TNF-α and had a phenotype of central and effector memory T cells were detected in all vaccinated piglets. After challenge, seroconversion occurred earlier in vaccinated and infected pigs compared to the non-vaccinated, infected group. Vaccinated pigs were fully protected against viremia after subsequent challenge. Therefore, our data suggests that the induction of IFN-γ/TNF-α co-producing T cells by PCV2 vaccination may serve as a potential correlate of protection for this type of vaccine.

Porcine CD3(+)NKp46(+) Lymphocytes Have NK-Cell Characteristics and Are Present in Increased Frequencies in the Lungs of Influenza-Infected Animals.

The CD3−NKp46+ phenotype is frequently used for the identification of natural killer (NK) cells in various mammalian species. Recently, NKp46 expression was analyzed in more detail in swine. It could be shown that besides CD3−NKp46+ lymphocytes, a small but distinct population of CD3+NKp46+ cells exists. In this study, we report low frequencies of CD3+NKp46+ lymphocytes in blood, lymph nodes, and spleen, but increased frequencies in non-lymphatic organs, like liver and lung. Phenotypic analyses showed that the majority of CD3+NKp46+ cells coexpressed the CD8αβ heterodimer, while a minor subset expressed the TCR-γδ, which was associated with a CD8αα+ phenotype. Despite these T-cell associated receptors, the majority of CD3+NKp46+ lymphocytes displayed a NK-related phenotype (CD2+CD5−CD6−CD16+perforin+) and expressed mRNA of NKp30, NKp44, and NKG2D at similar levels as NK cells. Functional tests showed that CD3+NKp46+ lymphocytes produced IFN-γ and proliferated upon cytokine stimulation to a similar extent as NK cells, but did not respond to the T-cell mitogen, ConA. Likewise, CD3+NKp46+ cells killed K562 cells with an efficiency comparable to NK cells. Cross-linking of NKp46 and CD3 led to degranulation of CD3+NKp46+ cells, indicating functional signaling pathways for both receptors. Additionally, influenza A(H1N1)pdm09-infected pigs had reduced frequencies of CD3+NKp46+ lymphocytes in blood, but increased frequencies in the lung in the early phase of infection. Thus, CD3+NKp46+ cells appear to be involved in the early phase of influenza infections. In summary, we describe a lymphocyte population in swine with a mixed phenotype of NK and T cells, with results so far indicating that this cell population functionally resembles NK cells.

Influence of Selective Bowel Decontamination on the Organisms Recovered During Bacteremia in Neutropenic Patients

OBJECTIVE To assess the influence of prophylactic selective bowel decontamination (SBD) on the spectrum of microbes causing bloodstream infection (BSI). DESIGN The microbes causing BSI in neutropenic patients of a hematologic ward (HW) and a bone marrow transplantation unit (BMTU), respectively, were compared by retrospective analysis of blood culture results from January 1996 to June 2003. SETTING A 30-bed HW (no SBD) and a BMTU including a 7-bed normal care ward and an 8-bed intensive care unit (SBD used) of a 2,200-bed university teaching hospital. RESULTS The overall incidences of bacteremia in the HW and the BMTU were similar (72.6 vs 70.6 episodes per 1,000 admissions; P = .8). Two hundred twenty episodes of BSI were recorded in 164 neutropenic patients of the HW and 153 episodes in 127 neutropenic patients of the BMTU. Enterobacteriaceae (OR, 3.14; CI95, 1.67-5.97; P= .0002) and Streptococcus species (OR, 2.04; CI95, 1.14-3.70; P = .015) were observed more frequently in HW patients and coagulase-negative staphylococci more frequently in BMTU patients (OR, 0.15; CI95, 0.09-0.26; P < .00001). No statistically significant differences were found for gram-negative nonfermentative bacilli (P = .53), Staphylococcus aureus (P = .21), Enterococcus species (P = .48), anaerobic bacteria (P = .1), or fungi (P = .50). CONCLUSIONS SBD did not lead to a significant reduction in the incidence of bacteremia, but significant changes in microbes recovered from blood cultures were observed. SBD should be considered when empiric antimicrobial therapy is prescribed for suspected BSI.