Proteomic and immunoproteomic insights into the exoproteome of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, a thorough analysis of the A. pleuropneumoniae exoproteome is still missing. Using a complementary approach of quantitative proteomics and immunoproteomics we gained an in-depth insight into the A. pleuropneumoniae serotype 2 exoproteome, which provides the basis for future functional studies. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 593 exoproteins, of which 104 were predicted to be virulence factors. The RTX toxins ApxIIA and ApxIIIA -were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome. Furthermore, the ApxIVA toxin was one of the proteins showing the highest abundance, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Our study revealed several antigens, including proteins with moonlight functions, such as the elongation factor (EF)-Tu, and proteins linked to specific metabolic traits, such as the maltodextrin-binding protein MalE, that warrant future functional characterization and might present potential targets for novel therapeutics and vaccines. Our Ig-classes specific serological proteome analysis (SERPA) approach allowed us to explore the development of the host humoral immune response over the course of the infection. These SERPAs pinpointed proteins that might play a key role in virulence and persistence and showed that the immune response to the different Apx toxins is distinct. For instance, our results indicate that the ApxIIIA toxin has properties of a thymus-independent antigen, which should be studied in more detail.

Development of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella abortisuis isolated from pigs.

The first description of Trueperella (T.) abortisuis was presented in Japan in 2009 by Azuma and colleagues. In the current study, eight T. abortisuis strains were identified by a newly developed loop-mediated isothermal amplification (LAMP) assay based on the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) encoding gene gap. Two T. abortisuis strains were obtained from prepuce of a seven-month-old boar and pooled foetal stomach contents in the United Kingdom, while the other six T. abortisuis strains were recovered from aborted foetal material of six pigs from a single farm in Germany. The developed LAMP assay showed an analytical sensitivity of 22 pg µL-1T. abortisuis DNA. T. abortisuis DSM 19515T and field strain T. abortisuis P504054/19/1 were directly detectable in artificially contaminated vaginal swabs up to concentrations of 980 CFU and 770 CFU per swab, respectively. There was no cross reactivity with control strains representing six species of genus Trueperella and six species of the closely related genus Arcanobacterium and Schaalia (Actinomyces) hyovaginalis. Further field research is required to determine the usefulness of the designed LAMP assay for identifying T. abortisuis isolated from pigs of various origins and from test samples directly obtained at farm level.

New Insights into Neutrophil Extracellular Trap (NETs) Formation from Porcine Neutrophils in Response to Bacterial Infections.

Actinobacillus pleuropneumoniae (A.pp, Gram negative) and Streptococcus (S.) suis (Gram positive) can cause severe diseases in pigs. During infection, neutrophils infiltrate to counteract these pathogens with phagocytosis and/or neutrophil extracellular traps (NETs). NETs consist of a DNA-backbone spiked with antimicrobial components. The NET formation mechanisms in porcine neutrophils as a response to both of the pathogens are not entirely clear. The aim of this study was to investigate whether A.pp (serotype 2, C3656/0271/11) and S. suis (serotype 2, strain 10) induce NETs by NADPH oxidase- or CD18-dependent mechanisms and to characterize phenotypes of NETs in porcine neutrophils. Therefore, we investigated NET induction in porcine neutrophils in the presence and absence of NET inhibitors and quantified NETs after 3 h. Furthermore, NETosis and phagocytosis were investigated by transmission electron microscopy after 30 min to characterize different phenotypes. A.pp and S. suis induce NETs that are mainly ROS-dependent. A.pp induces NETs that are partially CD18-dependent. Thirty minutes after infection, both of the pathogens induced a vesicular NET formation with only slight differences. Interestingly, some neutrophils showed only NET-marker positive phagolysosomes, but no NET-marker positive vesicles. Other neutrophils showed vesicular NETs and only NET-marker negative phagolysosomes. In conclusion, both of the pathogens induce ROS-dependent NETs. Vesicular NETosis and phagocytosis occur in parallel in porcine neutrophils in response to S. suis serotype 2 and A.pp serotype 2.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

Impacts of global warming on confined livestock systems for growing-fattening pigs: simulation of heat stress for 1981 to 2017 in Central Europe.

In the mid-latitudes, pigs and poultry are kept predominantly in confined livestock buildings with a mechanical ventilation system. In the last decades, global warming has already been a challenge which causes hat stress for animals in such systems. Heat stress inside livestock buildings was assessed by a simulation model for the indoor climate, which is driven by meteorological parameters. Besides the meteorological conditions, the thermal environment inside the building depends on the sensible and latent energy release of the animals, the thermal properties of the building and the ventilation system and its control unit. For a site in Austria in the north of the Alpine Ridge, which is representative for confined livestock buildings for growing-fattening pigs in Central Europe, meteorological data between 1981 and 2017 were used for the model calculations of heat stress measures. This business-as-usual simulation over these 37 years resulted in an increase of the mean relative annual heat stress parameters in the range between 0.9 and 6.4% per year since 1981. In order to minimise the negative economic impact as the consequence of this positive trend of heat stress, adaptation measures are needed. The calculations for growing-fattening pigs show that such a simulation model for the indoor climate is an appropriate tool to determine the level of heat stress of livestock inside confined livestock buildings.

Neutrophil extracellular trap formation in the Streptococcus suis-infected cerebrospinal fluid compartment.

Streptococcus suis is an important meningitis-causing pathogen in pigs and humans. Neutrophil extracellular traps (NETs) have been identified as host defense mechanism against different pathogens. Here, NETs were detected in the cerebrospinal fluid (CSF) of S. suis-infected piglets despite the presence of active nucleases. To study NET-formation and NET-degradation after transmigration of S. suis and neutrophils through the choroid plexus epithelial cell barrier, a previously described model of the human blood-CSF barrier was used. NETs and respective entrapment of streptococci were recorded in the "CSF compartment" despite the presence of active nucleases. Comparative analysis of S. suis wildtype and different S. suis nuclease mutants did not reveal significant differences in NET-formation or bacterial survival. Interestingly, transcript expression of the human cathelicidin LL-37, a NET-stabilizing factor, increased after transmigration of neutrophils through the choroid plexus epithelial cell barrier. In good accordance, the porcine cathelicidin PR-39 was significantly increased in CSF of piglets with meningitis. Furthermore, we confirmed that PR-39 is associated with NETs in infected CSF and inhibits neutrophil DNA degradation by bacterial nucleases. In conclusion, neutrophils form NETs after breaching the infected choroid plexus epithelium, and those NETs may be protected by antimicrobial peptides against bacterial nucleases.

Frequency of Th17 cells correlates with the presence of lung lesions in pigs chronically infected with Actinobacillus pleuropneumoniae.

Porcine contagious pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) remains one of the major causes of poor growth performance and respiratory disease in pig herds. While the role of antibodies against APP has been intensely studied, the porcine T cell response remains poorly characterized. To address this, pigs were intranasally infected with APP serotype 2 and euthanized during the acute phase [6-10 days post-infection (dpi)] or the chronic phase of APP infection (27-31 dpi). Lymphocytes isolated from blood, tonsils, lung tissue and tracheobronchial lymph nodes were analyzed by intracellular cytokine staining (ICS) for IL-17A, IL-10 and TNF-α production after in vitro stimulation with crude capsular extract (CCE) of the APP inoculation strain. This was combined with cell surface staining for the expression of CD4, CD8α and TCR-γδ. Clinical records, microbiological investigations and pathological findings confirmed the induction of a subclinical APP infection. ICS-assays revealed the presence of APP-CCE specific CD4+CD8αdim IL-17A-producing T cells in blood and lung tissue in most infected animals during the acute and chronic phase of infection and a minor fraction of these cells co-produced TNF-α. APP-CCE specific IL-17A-producing γδ T cells could not be found and APP-CCE specific IL-10-producing CD4+ T cells were present in various organs but only in a few infected animals. The frequency of identified putative Th17 cells (CD4+CD8αdimIL-17A+) in lung and blood correlated positively with lung lesion scores and APP-specific antibody titers during the chronic phase. These results suggest a potential role of Th17 cells in the immune pathogenesis of APP infection.

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

BACKGROUND: Actinobacillus (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. RESULTS: Significant 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. CONCLUSIONS: In 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.

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.

Characterization of surfactant alterations in pigs infected with Actinobacillus pleuropneumoniae.

PURPOSE/AIM OF THE STUDY: Surfactant, a surface active complex of phospholipids and proteins located at the inner surface of alveoli and small conducting airways is necessary for breathing. Bacterial respiratory tract infections frequently lead to surfactant alterations and to an increase in surface tension. Pigs, often used in experimental lung research, could suffer from severe pleuropneumonia, a highly contagious disease often characterized by sudden onset, short clinical course, high morbidity, and high mortality. It is caused by the gram-negative bacterium Actinobacillus pleuropneumoniae (A.pp.). This study tests the hypothesis that also in the subacute stage pathomorphological lung alterations are accompanied with increased inactive surfactant components. Clinical lung scores, functional and ultrastructural analysis of porcine surfactant were performed in pigs before infection and in the subacute state of infection. Clinical signs were determined using inter alia different subscores. Surfactant was isolated from the BALF for functional and quantitative ultrastructural studies. RESULTS: In the subacute stage clinical, ultrosonographic and radiographic scores as well as the overall Respiratory Health Score showed significantly higher values than before infection. However, surfactant surprisingly contained more active surfactant subtypes and significantly less inactive subtypes such as unilamellar vesicles. The quantity of multilamellar vesicles with unclear function did not differ. The minimal surface tension of surfactant before and after infection was comparable. CONCLUSIONS: Thus, in spite of continued severe lung tissue alterations the surfactant system show signs of recovery. This may be the result of an effective adaption to inflammatory lung disorders caused by swine-specific pathogens.

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.

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

BACKGROUND: Actinobacillus 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. RESULTS: IRT 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. CONCLUSION: IRT 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.

Spontaneous Lethal Outbreak of Influenza A Virus Infection in Vaccinated Sows on Two Farms Suggesting the Occurrence of Vaccine-Associated Enhanced Respiratory Disease with Eosinophilic Lung Pathology.

Influenza A virus (IAV) infections in swine are usually subclinical, but they can reach high morbidity rates. The mortality rate is normally low. In this study, six vaccinated, spontaneously deceased sows revealed IAV infection and enhanced neutrophilic bronchopneumonia with unexpectedly large numbers of infiltrating eosinophils. The purpose of this study was to characterize these lung lesions with special emphasis on the phenotypes of inflammatory cells, the presence of eosinophilic peroxidase (EPO), and neutrophil extracellular traps (NETs). The number of Sirius red-stained eosinophils was significantly higher in the lungs of IAV-infected sows compared to healthy pigs, indicating a migration of eosinophils from blood vessels into the lung tissue stimulated by IAV infection. The detection of intra- and extracellular EPO in the lungs suggests its contribution to pulmonary damage. The presence of CD3+ T lymphocytes, CD20+ B lymphocytes, and Iba-1+ macrophages indicates the involvement of cell-mediated immune responses in disease progression. Furthermore, high numbers of myeloperoxidase-positive cells were detected. However, DNA-histone-1 complexes were reduced in IAV-infected sows, leading to the hypothesis that NETs are not formed in the IAV-infected sows. In conclusion, our findings in the lungs of IAV-infected vaccinated sows suggest the presence of so far unreported field cases of vaccine-associated enhanced respiratory disease.

Multilocus sequence typing schemes for the emerging swine pathogen Mycoplasma hyosynoviae.

Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson's ID (0.992) than conventional MLST (Simpson's ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates.

Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria.

Introduction: Influenza A viruses (IAVs) infect the respiratory tract of mainly humans, poultry, and pigs. Co-infections with pathogenic lung bacteria are a common event and contribute to the severity of disease progression. Neutrophils are a major cell type of the innate immune system and are rapidly recruited to the site of infection. They have several effector functions to fight invading pathogens such as the secretion of reactive oxygen species (ROS) or the release of neutrophil extracellular traps (NETs). NETs are known to promote the growth of Pasteurellaceae bacteria, especially if degraded by nucleases. Methods: In this study, bronchoalveolar lavage fluid (BALF) from 45 field-infected pigs was analyzed for 1) NET markers, 2) influence on growth of lung bacteria, and 3) impact on neutrophil functions. BALF samples from 21 IAV-positive pigs and 24 lung diseased but IAV-negative pigs were compared. Results: Here, we show that neutrophils in the lungs of IAV-positive pigs release vesicular NETs. Several NET markers were increased in the BALF of IAV-positive pigs compared with the BALF from IAV-negative pigs. The amount of NET markers positively correlated with the viral load of the IAV infection. Interestingly, the BALF of IAV-positive pigs enhanced the growth of bacteria belonging to the family of Pasteurellaceae as potential coinfecting bacteria. These effects were weaker with the BALF derived from IAV-negative pigs with other lung infections. The intensity of oxidative burst in neutrophils was significantly decreased by BALF from IAVpositive pigs, indicating impaired antimicrobial activity of neutrophils. Finally, the lung milieu reflected by IAV-positive BALF does not enable neutrophils to kill Actinobacillus pleuropneumoniae but rather enhances its growth. Discussion: In summary, our data show that an IAV infection is affecting neutrophil functions, in particular the release of NETs and ROS. Furthermore, IAV infection seems to provide growth-enhancing factors for especially coinfecting Pasteurellaceae and reduces the killing efficiency of neutrophils.

Influences on the Decision to Euthanize a Compromised Pig.

The decision to euthanize a compromised pig can be challenging for pig farmers and veterinarians. To understand more about the challenges in Germany, a cross-sectional online survey was conducted. Based on a hybrid design, the responses of 39 veterinarians and 62 pig farmers were analyzed to generate a list of common clinical signs associated with the euthanasia of sows, fatteners, and piglets. Moreover, a set of influences on the farm, due to economic and personal considerations, were found to shape the decision-making process. The two most salient reasons outlined for the delay of timely euthanasia were uncertainty and misinterpretation of the chance for healing. The lack of valid clinical signs or a sound justification was most frequently mentioned as a challenge to the general decision-making process. In summation, this study highlights the need to generate a valid taxonomy for clinical signs that includes their development in a compromised pig over time. Future studies should elaborate on the justification of euthanasia decisions to facilitate the resolution of ethical dilemmas among the involved pig farmers and veterinarians. Lastly, the results suggest that clinical reasoning and consultation skills should be included when decision-making behavior is to be trained.

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.

Rapid Diagnostic of Streptococcus suis in Necropsy Samples of Pigs by thrA-Based Loop-Mediated Isothermal Amplification Assay.

Streptococcus (S.) suis presents a serious threat to the pig industry as well as food safety and public health. Although several LAMP assays have been developed for the identification of S. suis, no universal assay is so far available for the field-suitable examination of clinical pig specimens. Based on the thrA housekeeping gene, a new loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of S. suis in the brain and joints of pigs. For this LAMP assay, two different methods for the extraction of DNA from brain and joint swabs were compared. Using the LPTV boiling method, the detection limit of LAMP was 1.08 CFU/reaction, while the detection limit was 53.8 CFU/reaction using a commercial DNA extraction kit. The detection limits of thrA-LAMP in combination with the LPTV boiling method were 104-105 CFU/swab in the presence of brain tissue and 103-104 CFU/swab in the presence of joint tissue. The diagnostic quality criteria of LAMP were determined by the examination of 49 brain swabs and 34 joint swabs obtained during routine diagnostic necropsies. Applying the LPTV boiling method to brain swabs, the sensitivity, specificity, and positive and negative predictive values of thrA-LAMP were 88.0, 95.8, 95.7, and 88.5% using cultural investigation as a reference method, and 76.7, 100, 100, and 73.1% using real-time PCR as a reference method. Based on these results, the thrA-LAMP assay combined with the LPTV boiling method is suitable for rapid detection of S. suis from brain swabs.

Streptococcus equi subspecies zooepidemicus - a case report of sudden death in a German sow farm.

A farm in North-West Germany experienced a high morbidity and mortality in their sow herd. Sows showed fever, lethargy, oedema, mucosal discharge and dyspnoea. Necropsy revealed a severe fibrinous and purulent polyserositis. Haematological and histological examinations confirmed septicaemia. Streptococcus equi subspecies zooepidemicus was isolated in high yields from major organs. Sequence typing of this isolate (21/455) revealed a new sequence type showing a significantly higher proliferation rate in comparison to two other isolates. Other infectious agents (influenza A virus, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus 2, african swine fever virus, classical swine fever virus, Actinobacillus pleuropneumoniae) were excluded by routine diagnostic examinations. A climate check revealed an insufficient air supply in the area for the gestating sows. This case describes the first disease outbreak in swine due to S. zooepidemicus in Germany.