Karl-Heinz Waldmann

Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep.

Background Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep. Methodology/Principal Findings The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation. Conclusions/Significance This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.

A novel Respiratory Health Score (RHS) supports a role of acute lung damage and pig breed in the course of an Actinobacillus pleuropneumoniae infection

BackgroundBacterial lung infections are a major cause of economic losses in the pig industry; they are responsible for approximately 50% of the antibiotics used in pigs and, therefore, also present an increasing concern to consumer protection agencies. In response to this changing market we investigated the feasibility of an old approach aimed at the breeding selection of more resistant pigs. As a first step in this direction we applied a new respiratory health score system to study the susceptibility of four different pig breeding lines (German Landrace, Piétrain, Hampshire, Large White) towards the respiratory tract pathogen Actinobacillus (A.) pleuropneumoniae.ResultsA controlled experimental aerosol infection with an A. pleuropneumoniae serotype 7 isolate was performed using 106 weaning pigs of defined breeding lines from the breeds German Landrace, Piétrain, Hamphire, and Large White. Pigs were clinically assessed on days 4 and 20 post infection following a novel scoring system, the Respiratory Health Score (RHS), which combines clinical, sonographic and radiographic examination results. The ranking on day 4 was significantly correlated with the ranking based on the pathomorphological Lung Lesion Score (LLS; Spearman Rank Correlation Coefficient of 0.86 [p < 0.0001]). Based on their RHS pigs were assigned to the different quartiles independent of the breeding line. The RHS-based rankings of pigs on day 4 and on day 20 were highly correlated (Spearman Rank Correlation Coefficient of 0.82 [p < 0.0001]) independent of the breeding line. Pigs of the Hampshire line were predominantly found in the lowest scoring quartile (47.6%) and absent in the highest scoring quartile. In contrast, pigs of the German Landrace and Piétrain breeding lines were predominantly found in the highest scoring quartile (32.3% and 35.7%, respectively).ConclusionThese results demonstrate that the RHS obtained from live pigs shows a highly significant correlation to the lung lesion score considered as a gold standard. The correlation of the ranking at days 4 and 20 post infection implies that the course of disease is highly dependent on the acute lung damage. The different severity of signs among the tested pig breeding lines clearly suggests a genetic difference in the susceptibility of pigs to A. pleuropneumoniae infection.

Herd Factors Associated with the Serological Yersinia Prevalence in Fattening Pig Herds

Recent epidemiological evidence has demonstrated that pork is an important source of yersiniosis in humans. Identifying risk factors and potential interventions in swine production that may decrease the risk of pork production contamination during harvest and processing is an important step before controlling Yersinia spp. Therefore, management strategies and production processes that might be associated with fattening pigs testing seropositive for pathogenic Yersinia spp. were investigated in 80 fattening pig farms. Although >70 farm characteristics were included in the risk assessment, there were only a few that seemed to be connected with serological prevalence: housing on a fully slatted floor and the use of municipal water were observed more often in herds with low serological Yersinia prevalence, whereas recurring health problems and a low daily weight gain compared with the mean of the herds included in the study were found in herds with a high prevalence. Besides, the Yersinia prevalence seemed to be inversely proportional to the herds serological Salmonella status collected in accordance with German legislation. Additionally, the development of the serological Yersinia status of selected herds was assessed over a period of a year to gain knowledge of the dynamics of Yersinia infections in fattening pig herds. Three out of four serological negative herds maintained a low level of Yersinia prevalence, whereas one herd shifted between negative status and a prevalence of 100%. The reason for these considerable fluctuations could not be explained, and there was no direct association with the analyzed risk factors. Further research should be carried out to prove the given risk factors, especially the possible relation to the Salmonella prevalence before implementing a combined zoonoses surveillance and control program.

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.

In vivo degradation of magnesium alloy LA63 scaffolds for temporary stabilization of biological myocardial grafts in a swine model

Abstract Synthetic or biological patch materials used for surgical myocardial reconstruction are often fragile. Therefore, a transient support by degradable magnesium scaffolds can reduce the risk of dilation or rupture of the patch until physiological remodeling has led to a sufficient mechanical durability. However, there is evidence that magnesium implants can influence the growth and physiological behavior of the host’s cells and tissue. Hence, we epicardially implanted scaffolds of the magnesium fluoride-coated magnesium alloy LA63 in a swine model to assess biocompatibility and degradation kinetics. Chemical analysis of the pigs’ organs revealed no toxic accumulation of magnesium ions in the skeletal muscle, myocardium, liver, kidney, and bone of the pigs 1, 3, and 6 months postimplantation. The implants were surrounded by a fibrous granulation tissue, but no signs of necrosis were histologically evaluable. A sufficiently slow degradation rate of the magnesium alloy scaffold can be demonstrated via micro-computed tomography investigation. We conclude that stabilizing scaffolds of the magnesium fluoride-coated magnesium alloy LA63 can be used for epicardial application because no significant adverse effects to myocardial tissue were noted. Thus, degradable stabilizing scaffolds of this magnesium alloy with a slow degradation rate can extend the indication of innovative biological and synthetic patch materials.

Campylobacter spp. - prevalence on pig livers and antimicrobial susceptibility.

The objective of the study was to determine the prevalence of Campylobacter spp. on surfaces of slaughtered pig livers. Multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter coli isolates. Additionally, C. coli and Campylobacter jejuni isolates were tested for antimicrobial susceptibility by the broth dilution method. The minimal inhibitory concentrations were determined for erythromycin, gentamicin, ampicillin, ampicillin/sulbactam, nalidixic acid, ciprofloxacin, tetracycline and trimethoprim/sulphamethoxazole. Samples were taken during the slaughtering process in a slaughterhouse in Lower Saxony, Germany. Altogether, 10% of 1500 surfaces of pig livers from 50 fattening herds was found to be Campylobacter positive, with C. coli as the predominant species (76%) followed by C. jejuni (21%). Resistance to erythromycin and tetracycline was higher in C. jejuni compared to C. coli, whereas C. coli were more resistant to quinolone compared to C. jejuni. Fluoroquinolone resistance is usually associated with cross-resistance to quinolone, but in the presented investigation C. coli as well as C. jejuni showed a higher resistance to ciprofloxacin (28.6% and 20.0%, respectively) than to nalidixic acid (9.5% and 0%, respectively). A high genetic diversity of the C. coli isolates was demonstrated by MLST. Differences in STs and antimicrobial resistance pattern indicate that the Campylobacter strains originated from the pig itself and not from the slaughterhouse. A comparison of the STs with those reported in the C. jejuni/coli PubMLST database showed an overlap of porcine and human isolates, indicating that C. coli isolates from pigs should be considered as potential sources of human infection.

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.

Antibiotic Susceptibility of Salmonella, Campylobacter coli, and Campylobacter jejuni Isolated from Northern German Fattening Pigs

This study was conducted to assess the antimicrobial susceptibility rate of Salmonella and Campylobacter spp. isolated from Northern German fattening pigs. From 540 lymph node samples, 16 Salmonella Typhimurium, 1 Salmonella Brandenburg, 37 Campylobacter coli, and 11 Campylobacter jejuni strains were isolated. Antibiotic susceptibility testing was carried out by the broth dilution method. The 14 tested antibiotics for Salmonella were ampicillin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. The eight tested antibiotics for Campylobacter spp. were ampicillin, ampicillin-sulbactam (2:1), ciprofloxacin, erythromycin, gentamicin, nalidixic acid, sulfamethoxazole-trimethoprim (1:19), and tetracycline. In total, 93.7% (n = 16) of Salmonella Typhimurium, 75.7% (n = 37) of C. coli, and 54.5% (n = 11) of C. jejuni isolates were resistant to at least one of the tested antibiotics. Multiresistance to three antibiotics was observed in 75% of Salmonella Typhimurium, 16.2% of C. coli, and 0% of C. jejuni isolates. Pansusceptibility was detected in 6.3% of Salmonella Typhimurium, 24.3% of C. coli, and 45.5% of C. jejuni isolates. Multiresistance is defined as resistance to three or more antibiotics, and pansusceptibility is defined as not having resistance to any antibiotic. Regarding drugs of last resort--cefotaxime, ciprofloxacin, and nalidixic acid--resistance was not common among Salmonella (6.3%). The resistance rate of Campylobacter spp. to last-resort drugs--erythromycin, ciprofloxacin, and nalidixic acid--varied between species. The observed trend was not statistically significant. No C. coli isolates and few C. jejuni isolates (9.1%) were resistant to erythromycin. In contrast to C. jejuni, the C. coli isolates were more likely to be resistant to ciprofloxacin (9.1 and 18.9%, respectively) and nalidixic acid (0 and 13.5%, respectively). The same phenomenon was detected for tetracycline (27.3 and 62.2%, respectively), sulfamethoxazole (9.1 and 43.2%, respectively), and ampicillin (9.1 and 21.6%, respectively).

Prevalence of Pathogenic Yersinia enterocolitica Strains on Liver Surfaces of Pigs and Their Antimicrobial Susceptibility

A study to determine the occurrence of pathogenic Yersinia enterocolitica on surfaces of slaughtered pig livers and the antimicrobial resistant pattern of the isolates was carried out in a slaughterhouse in Lower Saxony, Germany. During the slaughtering process, 1,500 surfaces of pig livers from 50 fattening herds were swabbed in order to isolate and characterize Y. enterocolitica isolates by serotyping, detecting the virulence plasmid coding the yopT gene, and resistance testing. Of the livers tested, 4.7% were positive for Y. enterocolitica O:3, which was the only identified serotype. The virulence gene yopT was found in 90.0% of these isolates. Antimicrobial susceptibility was tested by the broth dilution method, and the MICs were determined for 13 antimicrobials. All isolates were resistant to ampicillin and sulfamethoxazole but were susceptible to ciprofloxacin, nalidixic acid, gentamicin, ceftiofur, tetracycline, kanamycin, cefotaxime, and chlorphenicol. Up to now, resistance to florfenicol has always been described in combination with resistance to chloramphenicol. In the present study, 15.3% of the isolates were resistant to florfenicol, while no chloramphenicol-resistant strains could be identified. Multiresistance to three or more antimicrobials was detected in 22 strains (27.3%). Nevertheless, third-generation cephalosporines or fluoroquinolones, which were recommended for extraintestinal Y. enterocolitica infection in humans, were not affected.

Pegivirus Infection in Domestic Pigs, Germany

To the Editor: The family Flaviviridae includes many human and animal virus pathogens. Recently, in addition to the genera Flavivirus, Hepacivirus, and Pestivirus, a fourth genus, Pegivirus, has been identified (1). In addition to human pegiviruses, a range of phylogenetic, highly divergent pegiviral sequences have been identified in various animal species, including primates, bats, rodents, and horses (2). We report the detection of a porcine pegivirus (PPgV) in serum samples from pigs. n nInitially, we investigated pooled serum samples by using high-throughput sequencing methods and isolated RNA from individual porcine serum samples by using the QIAmp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). We prepared libraries compatible with Illumina (San Diego, CA, USA) sequencing from pooled samples and individual serum samples by using the ScriptSeq version 2 RNA-Seq Library Preparation Kit (Epicenter, Madison, WI, USA) and sequenced them by using a HiSeq 2500 (2 × 150 cycles paired-end; Illumina) for pooled samples and MiSeq (2 × 250 cycles paired-end; Illumina) for individual samples (3). n nWe conducted quantitative reverse transcription PCR (RT-PCR) by using a Quantitect-SYBR Green Assay (QIAGEN) and primers PPgV_fwd: 5′-CTGTCTATGCTGGTCACGGA-3′ and PPgV_rev: 5′-GCCATAGAACGGGAAGTCGC-3′. By using high-throughput sequencing of the pooled serum sample library (23,167,090 reads), we identified 1 contig (4,582 bp) that had distant nucleotide sequence simi-larity to bat pegivirus (69% and 4% sequence coverage) and 2 contigs (2,683 bp and 665 bp) that had 73% sequence coverage, thereby covering 8% and 37% of the identified sequence. RT-PCR with primers designed on basis of recovered sequences identified the sample containing pegivirus sequences. Subsequent MiSeq analysis (7,085,595 reads) of an RNA library prepared from a sample from 1 animal identified 1 contig (9,145 nt) with sequence similarity to pe-givirus sequences. n nWe performed 3′ end completion of the viral genome by rapid amplification of cDNA ends and identified the entire open reading frame of PPgV_903 encoding 2,972 aa (GenBank accession no. KU351669). Analysis of the pegivirus 5′-untranslated region identified a highly structured internal ribosome entry site motif (Technical Appendix), which was similar in structure to previously described 5′ untranslated region structures of other pegiviruses (4,5). n nPegiviruses do not encode a protein homologous to the capsid protein of other viruses of the family Flaviviridae, another common feature of pegiviruses (6). The presence of cleavage sites for cellular signal peptidases and viral proteases indicates that, similar to polyproteins of other pegiviruses and members of the genus Hepacivirus, the pegivirus polyprotein NH2-E1- E2-Px-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (E [envelope], NS [nonstructural], and Px [protein X]) is cleaved co-translationally and posttranslationally. n nWe tested 3 additional animals from the same breeding cohort for virus RNA at irregular intervals for 22 months. One animal was positive for pegivirus RNA for 7 months, and the other 2 animals had pegivirus RNA in serum for 16 and 22 months. None of these animals showed obvious clinical signs attributable to virus infection. Follow-up investigation of 455 serum samples from 37 swine holdings from Germany identified 10 (2.2%) samples from 6 pig holdings that contained pegivirus RNA. We obtained 2 additional near full-length genomic sequences (PPgV_80F and PPgV_S8-7) from 2 animals in different herds by high-throughput sequencing, RT-PCR, and Sanger sequencing (GenBank accession nos. KU351670 and KU351671). n nPhylogenetic analyses of complete coding regions showed the close relationship of the 3 pegivirus sequences from Germany. These 3 sequences formed a separate clade within the genus Pegivirus (Figure). Pairwise comparison between PPgV_903 and the other 2 pegivirus sequences showed strong nucleotide identities (96.0%–98.4%). A distance scan over the entire polyprotein showed genetic distance to other pegiviruses and demonstrated that NS3 and NS5B contain the most conserved regions among pegivirus polyproteins (Technical Appendix). n n n nFigure n nPhylogenetic analysis of human and animal pegiviruses. We constructed a maximum-likelihood tree on the basis of the complete coding region and used the general time reversible model for modeling of substitutions. Bootstrap analysis was performed with ... n n n nIn horses, 2 distinct pegiviruses that had different potentials to cause clinical disease in infected animals have been described (4,7). No obvious clinical effects were observed in pegivirus-infected animals during our study. However, potential consequences of viral infection for animal health and food production need to be explored more closely under field and experimental conditions. Pegiviruses can interact with the immune system of the host. Co-infection with human pegivirus and HIV can have beneficial effects, which result in decreased retroviral loads and delayed disease progression (8). n nIt will be useful to investigate whether co-infections with pegiviruses can influence clinical manifestations of infectious diseases of swine, including multifactorial diseases such as postweaning multisystemic wasting syndrome, in which unknown immune modulating virus infections have been suggested to influence the degree of clinical illness (9). RNA viruses have considerable potential to adapt to new environmental conditions and to overcome host restrictions (10). Until now, the host tropism of PPgV has not been investigated in detail. Therefore, additional studies will be required to elucidate whether the spectrum of potential hosts might include other farm or companion animals, and whether the virus might be able to infect humans. n nTechnical Appendix: nAdditional information on pegivirus infection in domestic pigs, Germany. n nClick here to view.(302K, pdf)