Isabel Hennig-Pauka

Infection of Differentiated Porcine Airway Epithelial Cells by Influenza Virus: Differential Susceptibility to Infection by Porcine and Avian Viruses

Background Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. Methodology/Principal Findings To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. Conclusions/Significance Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.

Molecular investigations on the prevalence and viral load of enteric viruses in pigs from five European countries.

Abstract Enteric viral infections in pigs may cause diarrhea resulting in ill-thrift and substantial economic losses. This study reports the enteric infections with porcine astrovirus type 4 (PAstV4), porcine group A rotavirus (GARV), porcine group C rotavirus (GCRV), porcine circovirus type 2 (PCV2) and porcine kobuvirus (PKoV) in 419 pigs, comprising both healthy and diarrheic animals, from 49 farms in five European countries (Austria, Germany, Hungary, Spain and Sweden). Real-time RT-PCR assays were developed to test fecal samples and to compare the prevalence and viral load in relation to health status, farms of origin and age groups. The results showed that PAstV4 (70.4%) was the dominant virus species, followed by PKoV (56.7%), PCV2 (42.2%), GCRV (3%) and GARV (0.9%). Diarrheic pigs had a higher viral load of PAstV4 in the nursery and growing-finishing groups. Rotaviruses were mainly detected in diarrheic pigs, whereas PCV2 was more often detected in clinically healthy than in diarrheic pigs, suggesting that most PCV2 infections were subclinical. PAstV4, PCV2 and PKoV were considered ubiquitous in the European pig livestock and co-infections among them were frequent, independently of the disease status, in contrast to a low prevalence of classical rotavirus infections.

Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses

Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.

PR-39, a porcine host defence peptide, is prominent in mucosa and lymphatic tissue of the respiratory tract in healthy pigs and pigs infected with actinobacillus pleuropneumoniae

BackgroundHost defence peptides are important components of mammalian innate immunity. We have previously shown that PR-39, a cathelicidin host defence peptide, is an important factor in porcine innate immune mechanisms as a first line of defence after infection with Actinobacillus pleuropneumoniae. PR-39 interacts with bacterial and mammalian cells and is involved in a variety of processes such as killing of bacteria and promotion of wound repair. In bronchoalveolar lavage fluid of infected pigs PR-39 concentrations are elevated during the chronic but not during the acute stage of infection when polymorphonuclear neutrophils (known as the major source of PR-39) are highly increased. Thus it was assumed, that the real impact of PR-39 during infection might not be reflected by its concentration in bronchoalveolar lavage fluid.ResultsUsing immunohistochemistry this study demonstrates the actual distribution of PR-39 in tissue of the upper and lower respiratory tract of healthy pigs, and of pigs during the acute and chronic stage of experimental infection with Actinobacillus pleuropneumoniae.During the acute stage of infection PR-39 accumulated adjacent to blood vessels and within bronchi. Immune reactions were mainly localized in the cytoplasm of cells with morphological characteristics of polymorphonuclear neutrophils as well as in extracellular fluids. During the chronic stage of infection pigs lacked clinical signs and lung alterations were characterized by reparation and remodelling processes such as tissue sequestration and fibroblastic pleuritis with a high-grade accumulation of small PR-39-positive cells resembling polymorphonuclear neutrophils. In healthy pigs, PR-39 was homogenously expressed in large single cells within the alveoli resembling alveolar macrophages or type 2 pneumocytes. PR-39 was found in all tissue samples of the upper respiratory tract in healthy and diseased pigs. Within the tracheobronchial lymph nodes, PR-39 dominated in the cytoplasm and nuclei of large cells resembling antigen-presenting cells located in the periphery of secondary follicles.ConclusionsThese immunohistochemical findings indicate that, in addition to polymorphonuclear neutrophils, other cells are involved in the expression, storage, or uptake of PR-39. The presence of PR-39 in healthy lung tissue showed that this antibacterial peptide might be important for the maintenance of health.

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.

Characterization of two Austrian porcine reproductive and respiratory syndrome virus (PRRSV) field isolates reveals relationship to East Asian strains

Porcine reproductive and respiratory syndrome virus (PRRSV) causes major problems for the swine industry worldwide. Due to Austria’s central location in Europe, a large number of animals are transported through the country. However, little is known about current PRRSV strains and epidemiology. We determined full-length genome sequences of two Austrian field isolates (AUT13-883 and AUT14-440) from recent PRRSV outbreaks and of a related German isolate (GER09-613). Phylogenetic analysis revealed that the strains belong to European genotype 1 subtype 1 and form a cluster together with a South Korean strain. Remarkably, AUT14-440 infected the simian cell line MARC-145 without prior adaptation. In addition, this isolate showed exceptional deletions in nonstructural protein 2, in the overlapping region of glycoprotein 3 and 4 and in the 3′ untranslated region. Both Austrian isolates caused similar lung lesions but only pigs infected with AUT14-440 developed clear clinical signs of infection. Taken together, the genetic and biological characterization of two novel Austrian PRRSV field isolates revealed similarities to East Asian strains. This stresses the necessity for a more detailed analysis of current PRRSV strains in Europe beyond the determination of short ORF5 and ORF7 sequences.

Actinobacillus pleuropneumoniae challenge in swine: diagnostic of lung alterations by infrared thermography

BackgroundActinobacillus pleuropneumoniae (A.pp.) is the causative agent of porcine pleuropneumonia leading to high economic losses in the pig industry. Infrared thermography (IRT) of the thorax might offer a new method to select swine with lung alterations for further diagnostics.In this study 50 german landrace pigs were infected with A.pp. in an established model for respiratory tract disease, while 10 healthy pigs served as control animals. To avoid drift errors during IR measurements absolute skin temperatures and temperature differences between a thoracal and an abdominal region were assessed for its diagnostic validity.ResultsIRT findings during the course of experimental A.pp.-infection were verified by computed tomography (CT) before and on days 4 and 21 after infection. Significant correlations were found between clinical scores, CT score and lung lesion score. Ambient temperature, body temperature and abdominal surface temperature were factors influencing the skin surface temperature of the thorax. On day 4 but not on day 21 after infection the right thoracal temperature was significantly higher and the difference between a thoracal region in the height of the left 10th vertebra and an abdominal region was significantly lower in infected pigs than in control pigs. At a cut off of 28°C of right thoracal temperature the specificity of the method was 100% (CI 95%: 69-100%) and the sensitivity 66% (CI 95%: 51-79%).At a cut off of 2°C temperature difference between thoracal and abdominal region on the left body site the specificity of the method was 100% (CI 95%: 69-100%) and the sensitivity 32% (CI 95%: 19-47%) with all control pigs detected negative.Orientation for lung biopsy by IRT resulted in 100% specificity and sensitivity (CI 95%: 69-100%) of bacteriological examination of tissue samples during the acute stage of infection.ConclusionIRT might be a valuable tool for the detection of inflammatory lung alterations in pigs, especially during the acute stage of infection and if ambient temperatures are constant during individual measurements. External and internal factors interfere with this method, so that its application in the field might be restricted to a selection of pigs for further diagnostic with adequate specificity.

Establishment of a quantitative real-time PCR for the detection of Pneumocystis carinii f. sp. suis in bronchoalveolar lavage samples from pigs

Respiratory diseases in pigs are mostly polymicrobial, and the involved pathogens can vary from farm to farm. The impact of Pneumocystis carinii (P. c.) f. sp. suis on respiratory disorders has not been comprehensively appraised because tests were limited to lung tissue samples and, for this reason, it was not possible to detect the fungus in living animals. In the present study, bronchoalveolar lavage fluid (BALF) from 12 pigs and oral fluid samples from 9 pigs were tested for the presence of Pneumocystis by quantitative real-time PCR. The results from these 2 clinical specimens were compared with Pneumocystis quantities in lung tissue samples. Pneumocystis quantities in BALF correlated significantly to those in lung tissue. BALF has proved to be an adequate specimen for detection of various respiratory pathogens in pigs, and the collection procedure directly on farms is also well established. In contrast to the BALF results, all oral fluid samples were negative. Thus, specimens from the lower respiratory tract should generally be preferred for the detection of Pneumocystis. Additionally, under farm conditions, oral fluid is mainly collected in the form of collective samples per pen. In the present study, oral swab sampling of individual pigs was intended but failed in 3 of 12 pigs because they did not salivate sufficiently. As a conclusion, only BALF can be recommended as a useful tool for Pneumocystis herd monitoring in vivo.

Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae

BackgroundActinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia and causes significant losses in the pig industry worldwide. Early host immune response is crucial for further progression of the disease. A. pleuropneumoniae is either rapidly eliminated by the immune system or switches to a long-term persistent form. To gain insight into the host-pathogen interaction during the early stages of infection, pigs were inoculated intratracheally with A. pleuropneumoniae serotype 2 and humanely euthanized eight hours after infection. Gene expression studies of inflammatory cytokines and the acute phase proteins haptoglobin, serum amyloid A and C-reactive protein were carried out by RT-qPCR from the lung, liver, tonsils and salivary gland. In addition, the concentration of cytokines and acute phase proteins were measured by quantitative immunoassays in bronchoalveolar lavage fluid, serum and saliva. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter, Fourier-Transform Infrared (FTIR-) spectroscopy was employed.ResultsSignificant cytokine and acute phase protein gene expression was detected in the lung and the salivary gland however this was not observed in the tonsils. In parallel to the analyses of host response, the impact of the host on the bacterial pathogen was assessed on a metabolic level. For the latter investigations, Fourier-Transform Infrared (FTIR-) spectroscopy was employed. The bacteria isolated from the upper and lower respiratory tract showed distinct IR spectral patterns reflecting the organ-specific acute phase response of the host.ConclusionsIn summary, this study implies a metabolic adaptation of A. pleuropneumoniae to the porcine upper respiratory tract already during early infection, which might indicate a first step towards the persistence of A. pleuropneumoniae. Not only in lung, but also in the salivary gland an increased inflammatory gene expression was detectable during the acute stage of infection.

Identification of a novel bufavirus in domestic pigs by a viral metagenomic approach.

Bufavirus is a single-stranded DNA virus belonging to the genus Protoparvovirus. This study reports the identification and characterization of a porcine bufavirus by a metagenomic approach, and a limited epidemiology investigation of bufavirus in six swine farms. A comparative genome analysis showed a similarity of 93 % to a Hungarian porcine bufavirus. Bayesian and maximum-likelihood analyses of genome sequences showed a close relationship of porcine bufaviruses to human and monkey bufaviruses. Molecular dating of the most recent common ancestors supported a recent introduction of bufaviruses into human and pig populations, respectively. A real-time PCR method was developed to screen 60 faecal samples for the porcine bufavirus DNA, and eight positive samples were found in two neighbouring farms, suggesting a relatively low prevalence (13.3 %). No direct transmission of porcine bufaviruses between two neighbouring farms was found, suggesting that bufaviruses may have spread widely in different geographical regions.