Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates.

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

Infection of porcine enteroids and 2D differentiated intestinal epithelial cells with rotavirus A to study cell tropism and polarized immune response.

Intestinal epithelial cell interactions with enteric pathogens have been incompletely elucidated owing to the lack of model systems that recapitulate the cellular diversity, architecture and functionality of the intestine. To analyze rotavirus (RV) infection and the subsequent innate immune response, we established cultures of differentiated porcine intestinal epithelial cells in three different variations: basolateral-out enteroids, apical-out enteroids and two-dimensional (2D) filter-grown intestinal epithelial cells. Application of specific antibodies for fluorescent staining indicated that enteroids and enteroid-derived cell cultures contain multiple intestinal epithelial cell types. Infection studies indicated that both apical-out enteroids and 2D intestinal epithelial cells are susceptible to porcine RV infection. However, 2D intestinal epithelial cells are more useful for a detailed characterization and comparison of apical and basolateral infection than apical-out enteroids. Virus-induced apoptosis was observed in apical-out enteroids at 24 h post infection but not at earlier time points after infection. RV infected not only enterocytes but also goblet cells and Paneth cells in apical-out enteroids and 2D intestinal epithelial cells. Interestingly, despite the lack of significant differences in the efficiency of infection after apical and basolateral infection of 2D intestinal epithelial cells, stronger innate immune and inflammatory responses were observed after basolateral infection as compared to infection via the apical route. Therefore, apical-out enteroids and 2D intestinal epithelial cells provide useful primary cell culture models that can be extended to analyze invasion and replication strategies of agents implicated in enteric diseases or to study immune and inflammatory responses of the host induced by enteric pathogens.

Case report: Urolithiasis, nephrolithiasis and a urinary bladder malformation in a seven-month-old alpaca cria.

Urolithiasis is a common condition in male small ruminants where predisposing factors have been identified. Occasionally, urolithiasis is diagnosed in South American camelids (SACs). However, nephrolithiasis is rarely diagnosed in ruminants. To our knowledge, this is the first report focusing on a combined appearance of nephrolithiasis and urolithiasis in an alpaca cria. A 7-month-old alpaca cria suffering from impaired urinary flow was presented for examination. On admission, the alpaca had a wet prepuce and showed a standing posture with a wide-based stance. Ultrasonographic examination of the abdomen showed a distended bladder. Clinical chemistry revealed azotemia and hypophosphatemia. After the first examination, repeated urination was observed. Conservative therapy using antibiotics, anti-inflammatory and spasmolytic drugs was started with the suspected diagnosis of urinary calculus. During the first 24 h, plasma concentrations of creatinine and urea decreased, but increased again during the following days. During the second day after admission, urination was not observed for 16 h while the concentration of urea and creatinine further increased. Therefore, the animal was euthanized due to financial concerns of the owner. Necropsy revealed that calculi were located in the left kidney as well as in the urethra. In addition, the animal exhibited uroperitoneum. The urinary bladder was intact, moderately distended with urine and showed a malformation, which was covered with a translucent mucosal membrane. Histologic examination revealed that this malformation was a bladder diverticulum. The extent to which the unilateral nephroliths affected the general condition and renal function of the animal is unclear, since the uroliths also cause azotemia, and abdominal pain. Further studies are needed for a better understanding of obstructive urinary disease in SACs.

First report about a cerebrospinal nematode infection in an alpaca (Vicugna pacos).

The number of New World camelids in European farms is rising and thus, the need for veterinary care towards these animals arises. However, veterinary care requires sophisticated knowledge on disease and pathogen occurrence within New World camelids. Here, an alpaca cria with neurological signs was admitted to the veterinary clinic. Although the animal was treated with antibiotics, vitamins and dexamethason, it refused to drink milk and the clinical status worsened. After euthanasia, necropsy and histopathological examination were carried out and revealed intracerebral nematode larvae. The morphology of these larvae strongly suggests them to be Baylisascaris procyonis, a parasite of raccoons. The extended history revealed that a fully grown raccoon was living within farm enclosures, suggesting an infection of the alpaca and the development of a cerebrospinal larva migrans. This zoonotic disease is characterized by aberrant larval migration that typically shows extraintestinal migration in dead-end hosts. The aim of this report is to sensitize practical colleagues towards this rare, but occasionally fatal infection in New World camelids while familiarizing diagnostic pathologists with the morphological characteristics of this disease.

Sero- and apx-typing of German Actinobacillus pleuropneumoniae field isolates from 2010 to 2019 reveals a predominance of serovar 2 with regular apx-profile.

Serotyping is the most common method to characterize field isolates of Actinobacillus (A.) pleuropneumoniae, the etiological agent of porcine pleuropneumonia. Based on serology, many farms seem to be infected and antibodies against a wide variety of serovars are detectable, but, so far it is unknown to what degree respective serovars contribute to outbreaks of clinical manifest disease. In this study, 213 German A. pleuropneumoniae field isolates retrieved for diagnostic purposes from outbreaks of porcine pleuropneumonia between 2010 and 2019 were genetically serotyped and analyzed regarding their apx-toxin gene profile using molecular methods. Serotyping revealed a prominent role of serovar 2 in clinical cases (64% of all isolates) and an increase in the detection of this serovar since 2010 in German isolates. Serovar 9/11 followed as the second most frequent serovar with about 15% of the isolates. Furthermore, very recently described serovars 16 (n = 2) and 18 (n = 8) were detected. Most isolates (93.4%) showed apx-profiles typical for the respective serovar. However, this does not hold true for isolates of serovar 18, as 75% (n = 6) of all isolates of this serovar deviated uniformly from the "typical" apx-gene profile of the reference strain 7311555. Notably, isolates from systemic lesions such as joints or meninges did not harbor the complete apxICABD operon which is considered typical for highly virulent strains. Furthermore, the extremely low occurrence (n = 1) of NAD independent (biovar II) isolates in German A. pleuropneumoniae was evident in our collection of clinical isolates.

Serotyping and pathotyping of Glaesserella parasuis isolated 2012-2019 in Germany comparing different PCR-based methods.

Glaesserella parasuis is an important pathogen in swine production. It acts as a primary pathogen in systemic Glässer´s disease and as a secondary pathogen in Porcine Respiratory Disease Complex. In this study, a collection of 308 isolates from carrier animals and individuals with respiratory or Glässer´s disease isolated 2012-2019 in Germany was analysed. Isolates were characterized for serovar implementing two different PCR methods. Additionally, two different PCR methods for pathotyping isolates were applied to the collection and results compared. Serovar 6 (p < 0.0001) and 9 (p = 0.0007) were correlated with carrier isolates and serovar 4 was associated with isolates from animals with respiratory disease (p = 0.015). In systemic isolates, serovar 13 was most frequently detected (18.9%). Various other serovars were isolated from all sites and the ratio of serovar 5 to serovar 12 was approximately 1:2. These two serovars together represented 14.3% of the isolates; only serovar 4 was isolated more frequently (24.7%). The pathotyping method based on the leader sequence (LS = ESPR of vta) was easy to perform and corresponded well to the clinical background information. Of the carrier isolates 72% were identified as non-virulent while 91% of the systemic isolates were classified as virulent (p < 0.0001). Results of the pathotyping PCR based on 10 different marker genes overall were in good agreement with clinical metadata as well as with results of the LS-PCR. However, the pathotyping PCR was more complicated to perform and analyze. In conclusion, a combination of the serotyping multiplex-PCR and the LS-PCR could improve identification of clinically relevant G. parasuis isolates, especially from respiratory samples.

Link between Heterotrophic Carbon Fixation and Virulence in the Porcine Lung Pathogen Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniae instead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae Isotope mass spectrometry revealed heterotrophic fixation of carbon from stable isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed by nano-scale secondary ion mass spectrometry at a single-cell level. By gas chromatography-combustion-isotope ratio mass spectrometry we could further show that the labeled carbon atom is mainly incorporated into the amino acids aspartate and lysine, which are derived from the TCA metabolite oxaloacetate. We therefore suggest that carbon fixation occurs at the interface of glycolysis and the reductive branch of the TCA cycle. The heme precursor δ-aminolevulinic acid supported growth of A. pleuropneumoniae, similar to bicarbonate, implying that anaplerotic carbon fixation is needed for heme synthesis. However, deletion of potential carbon-fixing enzymes, including PEP-carboxylase (PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate decarboxylase, as well as various combinations thereof, did not affect carbon fixation. Interestingly, generation of a deletion mutant lacking all four enzymes was not possible, suggesting that carbon fixation in A. pleuropneumoniae is an essential metabolic pathway controlled by a redundant set of enzymes. A double deletion mutant lacking PEPC and PEPCK was not impaired in carbon fixation in vitro but showed reduction of virulence in a pig infection model.

Genetic variability of porcine pegivirus in pigs from Europe and China and insights into tissue tropism.

Pegiviruses belong to the family Flaviviridae and have been found in humans and other mammalian species. To date eleven different pegivirus species (Pegivirus A-K) have been described. However, little is known about the tissue tropism and replication of pegiviruses. In 2016, a so far unknown porcine pegivirus (PPgV, Pegivirus K) was described and persistent infection in the host, similar to human pegivirus, was reported. In this study, qRT-PCR, phylogenetic analyses and fluorescence in situ hybridization (FISH) were implemented to detect and quantify PPgV genome content in serum samples from domestic pigs from Europe and Asia, in tissue and peripheral blood mononuclear cell (PBMC) samples and wild boar serum samples from Germany. PPgV was detectable in 2.7% of investigated domestic pigs from Europe and China (viral genome load 2.4 × 102 to 2.0 × 106 PPgV copies/ml), while all wild boar samples were tested negative. Phylogenetic analyses revealed pairwise nucleotide identities >90% among PPgVs. Finally, PPgV was detected in liver, thymus and PBMCs by qRT-PCR and FISH, suggesting liver- and lymphotropism. Taken together, this study provides first insights into the tissue tropism of PPgV and shows its distribution and genetic variability in Europe and China.

Multi-organ spreading of Actinobacillus pleuropneumoniae serovar 7 in weaned pigs during the first week after experimental infection.

Actinobacillus (A.) pleuropneumoniae is normally considered strictly adapted to the respiratory tract of swine. Despite this, scattered case reports of arthritis, osteomyelitis, hepatitis, meningitis or nephritis exist, in which A. pleuropneumoniae remained the only detectable pathogen. Therefore, the aim of this study was to investigate whether spreading to other organs than the lungs is incidental or may occur more frequently. For this, organ samples (blood, liver, spleen, kidney, tarsal and carpal joints, meninges, pleural and pericardial fluids) from weaners (n = 47) infected experimentally with A. pleuropneumoniae serovar 7 by aerosol infection (infection dose: 10.9 × 103 cfu/animal) were examined by culture during the first week after infection. In addition, tissue samples of eight weaners were examined by histology and immunohistochemistry (IHC). A. pleuropneumoniae was isolated in all examined sample sites (86.7% pleural fluids, 73.3% pericardial fluids, 50.0% blood, 61.7% liver, 51.1% spleen, 55.3% kidney, 14.9% tarsal joints, 12.8% carpal joints, 27.7% meninges). These results were also obtained from animals with only mild clinical symptoms. IHC detection confirmed these findings in all locations except carpal joints. Histological examination revealed purulent hepatitis (n = 2), nephritis (n = 1) and beginning meningitis (n = 2). Isolation results were significantly correlated (p < 0.001) with the degree of lung colonization and, to a lower extent, with the severity of disease. Detection of A. pleuropneumoniae in peripheral tissues was significantly correlated to spleen colonization. In conclusion, multi-organ spreading of A. pleuropneumoniae serovar 7 strain AP 76 seems to occur more frequently during acute infection following effective lung colonization than previously thought.

Efficacy of a one-shot marbofloxacin treatment on acute pleuropneumonia after experimental aerosol inoculation of nursery pigs.

BACKGROUND: Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is a bacterial respiratory disease of swine. Acute outbreaks of the disease are often accompanied by high mortality and economic losses. As severe cases of the disease frequently require parenteral antibiotic treatment of the animals, the efficacy of a single, high dose of marbofloxacin was compared to a three-time application of a dose of enrofloxacin under experimental conditions. METHODS: A blinded, controlled, randomized and blocked dose confirmation study was conducted to test the efficacy and safety of a single dose of 8 mg/kg marbofloxacin (160 mg/ml, Forcyl® Swine, Vetoquinol SA, France) to treat acute porcine pleuropneumonia after experimental aerosol inoculation of pigs with A. pleuropneumoniae serotype 2. The results were compared to a three consecutive day treatment of 2.5 mg/kg enrofloxacin and a mock (saline) treatment. Criteria for the assessment of efficacy were severity of lung lesions, bacteriological cure and the course of clinical disease after treatment. RESULTS: Thirty six nursery pigs were divided into three treatment groups: marbofloxacin (T1), enrofloxacin (T2) and mock (T3). Statistically significant superiority (p < 0.05) of marbofloxacin and enrofloxacin compared to the mock-treated group was demonstrated for all efficacy criteria. The need of rescue euthanasia due to severity of symptoms was significantly reduced in both treatment groups (T1: 1 pig; T2: 0 pigs; vs. T3: 8 pigs). On day 6 after treatment initiation, clinical cure was observed in 10 (T1), 10 (T2) but only 1 of the piglets in T3. Extent of lung lesions (mean of lung lesion score T1: 3.9, T2: 6.0, T3: 21.1) and bacteriological isolation from lung tissue (on day 6 after treatment initiation: T1 = 0 pigs; T2 = 1 pig; T3 = all pigs) were also significantly reduced within both treatment groups. There were no adverse events linked to the drug administration and no injection site reactions were observed. CONCLUSIONS: Both applied antimicrobial treatments were proven safe and efficacious for the treatment of acute porcine pleuropneumonia. No statistically significant differences were detected between the antibiotic treatments.

Ciliostasis of airway epithelial cells facilitates influenza A virus infection.

Porcine precision-cut lung slices (PCLS) were used to analyze the effect of the ciliary activity on infection of airway epithelial cells by influenza viruses. Treatment of slices with 2% NaCl for 30 min resulted in reversible ciliostasis. When PCLS were infected by a swine influenza virus of the H3N2 subtype under ciliostatic conditions, the viral yield was about twofold or threefold higher at 24 or 48 h post-infection, respectively, as compared to slices with ciliary activity. Therefore, the cilia beating not only transports the mucus out of the airways, it also impedes virus infection.

Evaluation of the predictive value of tonsil examination by bacteriological culture for detecting positive lung colonization status of nursery pigs exposed to Actinobacillus pleuropneumoniae by experimental aerosol infection.

BACKGROUND: Actinobacillus (A.) pleuropneumoniae is the causative agent of porcine pleuropneumonia. For control of the disease the detection of sub-clinically infected pigs is of major importance to avoid transmitting of subclinical infections. One method recommended is the testing of tonsillar samples for the presence of A. pleuropneumoniae. This is routinely done by PCR techniques. However, based upon PCR susceptibility testing and monitoring of resistance development is impossible. Therefore, in this study the informative values of bacteriological culture of tonsilar samples for the colonisation status of pigs were tested. In total, 163 German Landrace nursery pigs were experimentally exposed to A. pleuropneumoniae serotype 7 by aerosol and the rate of isolation from lung tissue and tonsils and the corresponding degree of lung lesions were investigated. RESULTS: Overall a significant correlation (p < 0.001) between degree of clinical disease, degree of lung alterations and degree of A. pleuropneumoniae isolation from tonsillar and lung tissue after exposure was detected. Of these animals tested, 74.8% were tested positive in tonsillar and lung samples, 7.4% remained completely negative and in 4.3% the tonsils were tested positive despite negative isolation results from lung tissue. In 13.5% of the pigs A. pleuropneumoniae could be isolated in lung tissue but not in tonsillar samples. In 36.4% of these animals a heavy colonization of the lungs and in 40.9% moderate to severe lung alterations were proven. Hence, the diagnostic sensitivity for the detection of a positive colonization status of the pigs by bacterial culture examination of tonsillar samples was 84.7%, the diagnostic specificity was 66.7% and the predictive values were 94.6% (positive) and 35.3% (negative). The overall sensitivity for A. pleuropneumoniae exposure was 78.2% (tonsils) and 88.0% (lung tissue). CONCLUSIONS: In conclusion, tonsil examination alone for the detection of a positive colonization status of pigs performed might lead to false negative results as lungs might be heavily colonized despite negative tonsillar isolation results. Therefore culture of tonsillar samples should not be the sole test for the confirmation of a pigs' status but used in combination with methods also evaluating the colonization status of the lower respiratory tract.

Treatment of infected lungs by ex vivo perfusion with high dose antibiotics and autotransplantation: A pilot study in pigs.

The emergence of multi-drug resistant bacteria threatens to end the era of antibiotics. Drug resistant bacteria have evolved mechanisms to overcome antibiotics at therapeutic doses and further dose increases are not possible due to systemic toxicity. Here we present a pilot study of ex vivo lung perfusion (EVLP) with high dose antibiotic therapy followed by autotransplantation as a new therapy of last resort for otherwise incurable multidrug resistant lung infections. Severe Pseudomonas aeruginosa pneumonia was induced in the lower left lungs (LLL) of 18 Mini-Lewe pigs. Animals in the control group (n = 6) did not receive colistin. Animals in the conventional treatment group (n = 6) received intravenous application of 2 mg/kg body weight colistin daily. Animals in the EVLP group (n = 6) had their LLL explanted and perfused ex vivo with a perfusion solution containing 200 µg/ml colistin. After two hours of ex vivo treatment, autotransplantation of the LLL was performed. All animals were followed for 4 days following the initiation of treatment. In the control and conventional treatment groups, the infection-related mortality rate after five days was 66.7%. In the EVLP group, there was one infection-related mortality and one procedure-related mortality, for an overall mortality rate of 33.3%. Moreover, the clinical symptoms of infection were less severe in the EVLP group than the other groups. Ex vivo lung perfusion with very high dose antibiotics presents a new therapeutic option of last resort for otherwise incurable multidrug resistant pneumonia without toxic side effects on other organs.

Porcine pulmonary auto-transplantation for ex vivo therapy as a model for new treatment strategies.

OBJECTIVES: Lung auto-transplantation is the surgical key step in experiments involving ex vivo therapy of severe or end-stage lung diseases. Ex vivo therapy has become a clinical reality because of systems such as the Organ Care System (OCS) Lung, which is the only commercially available portable lung perfusion system. However, survival experiments involving porcine lung auto-transplantation pose special surgical and anaesthesiological challenges. This current study was designed to describe the development of surgical techniques and aneasthesiological management strategies that facilitate lung auto-transplantation survival surgery including a follow-up period of 4 days. METHODS: Left pneumonectomy was performed in 12 Mini-Lewe miniature pigs. After ex vivo treatment of the harvested lungs within the OCS Lung for 2 h, the lungs were retransplanted into the same animal (auto-transplantation). Four animals were used to develop the optimal techniques and establish an experimental protocol. According to the final protocol, eight additional animals were operated. The follow-up period was 4 days. RESULTS: There were four severe intraoperative surgical complications [anatomical variant of the superior vena cava (two times), a complication related to the bronchial anastomosis and a complication related to the pulmonary arterial anastomosis]. The major postoperative problems were hyperkalaemia, prolonged recovery from anaesthesia and pulmonary oedema after reperfusion. Establishment of the surgical technique showed that using a pericardial tube to facilitate the anastomosis of the thin left superior pulmonary vein should be considered to prevent thrombosis. However, routine use of the patch technique to construct venous and arterial anastomoses is not necessary. Furthermore, traction on the venous anastomoses can be avoided by performing the bronchial anastomosis first. CONCLUSIONS: Lung auto-transplantation is a feasible experimental model for ex vivo therapy of lung diseases and is applicable for experimental questions concerning human lung transplantation.

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.

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.

Pathway deregulation and expression QTLs in response to Actinobacillus pleuropneumoniae infection in swine.

Actinobacillus (A.) pleuropneumoniae is among the most important pathogens in pig. The agent causes severe economic losses due to decreased performance, the occurrence of acute or chronic pleuropneumonia, and an increase in death incidence. Since therapeutics cannot be used in a sustainable manner, and vaccination is not always available, new prophylactic measures are urgently needed. Recent research has provided evidence for a genetic predisposition in susceptibility to A. pleuropneumoniae in a Hampshire × German Landrace F2 family with 170 animals. The aim of the present study is to characterize the expression response in this family in order to unravel resistance and susceptibility mechanisms and to prioritize candidate genes for future fine mapping approaches. F2 pigs differed distinctly in clinical, pathological, and microbiological parameters after challenge with A. pleuropneumoniae. We monitored genome-wide gene expression from the 50 most and 50 least susceptible F2 pigs and identified 171 genes differentially expressed between these extreme phenotypes. We combined expression QTL analyses with network analyses and functional characterization using gene set enrichment analysis and identified a functional hotspot on SSC13, including 55 eQTL. The integration of the different results provides a resource for candidate prioritization for fine mapping strategies, such as TF, TFRC, RUNX1, TCN1, HP, CD14, among others.

Identification of QTL affecting resistance/susceptibility to acute Actinobacillus pleuropneumoniae infection in swine.

Actinobacillus pleuropneumoniae is among the most important pathogens worldwide in pig production. The agent can cause severe economic losses due to decreased performance, acute or chronic pleuropneumonia and an increased incidence of death. Therapeutics cannot be used in a sustainable manner, and vaccination is not always available, but discovering more about host defence and disease mechanisms might lead to new methods of prophylaxis. The aim of the present study was to detect quantitative trait loci (QTL) associated with resistance/susceptibility to A. pleuropneumoniae. Under controlled conditions, 170 F2 animals of a Hampshire/Landrace family, with known differences in founder populations regarding A. pleuropneumoniae resistance, were challenged with an A. pleuropneumoniae serotype 7 aerosol followed by a detailed clinical, radiographic, ultrasonographic, pathological and bacteriological examination. F2 pigs were genotyped with 159 microsatellite markers. Significant QTL were identified on Sus scrofa chromosomes (SSC) 2, 6, 12, 13, 16, 17 and 18. They explained 6-22% of phenotypic variance. One QTL on SSC2 reached significance on a genome-wide level for five associated phenotypic traits. A multiple regression analysis revealed a combinatory effect of markers SWR345 (SSC2) and S0143 (SSC12) on Respiratory Health Score, Clinical Score and the occurrence of death. The results indicate the genetic background of A. pleuropneumoniae resistance in swine and provide new insights into the genetic architecture of resistance/susceptibility to porcine pleuropneumonia. The results will be helpful in identifying the underlying genes and mechanisms.

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.

BACKGROUND: Host 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. RESULTS: Using 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. CONCLUSIONS: These 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.