Überprüfung der Verträglichkeit des Salmonella Typhimurium – Lebendimpfstoffes Salmoporc® bei oraler Anwendung für drei Tage alte Saugferkel unter Berücksichtigung der Ausscheidung, Persistenz und Immunogenität des Impfstamms

Safety of Salmonella Typhimurium-live vaccine Salmoporc® in oral application in 3 day old piglets with consideration of elimination, persistence and immunogenicity of the vaccine strain The aim of the present study was to examine the Salmonella Typhimurium live vaccine Salmoporc® for its safety for 3 days old piglets applicated by the oral route. Therefore Salmonella negative piglets were vaccinated orally twice with a 10fold overdose of the vaccine when the piglets were 3 and day 21 days old. In a second assay investigations about the immunological response to this early vaccination were made and spread and persistence of the vaccine strain in different tissues were investigated. The piglets in this group were vaccinated with a single dose of the vaccine also at day 3 and 21. In both cases negative control piglets within the same age were given an equivalent amount of sterile physiological saline solution orally. In both investigations, the faeces consistence, body temperature, suckling frequency and general health were evaluated in a period of time comprising 8 hours after vaccination. Control of body weight and bacteriological examinations concerning the shedding of the vaccine strain, were done weekly. Furthermore, serological surveys concerning the immunological response to this early vaccination were made. As a result of the 10fold overdosed vaccination at day 3 and 21, an increase of the mean body temperature was seen lasting 8 hours after vaccination. The number of piglets showing a body temperature higher than 39, 8°C was significant higher for the overdosed pigs at both time points. No significance concerning clinical signs and suckling frequency were observed between vaccinated piglets and the control group after the first overdose. In a consequence of the 10fold overdosed vaccination at day 21, a significant increase in respiration frequency and lethargic behaviour as well a decreasing frequency of suckling was seen within the piglets of the vaccination group. From the fourth day after the overdosed vaccination until the end of the tenth living week of the vaccinated piglets, a significant lower mean body weight was measured in comparison to the control group. After the single dosed vaccination on day 3 and 21 the piglets of the vaccinated group had a significant higher mean body weight at the time of weaning at the age of 4 weeks. A significant increase in the occurrence of diarrhoea could be observed in both assays in time course of 14 days after the first vaccination, as well as in time course of 14 days after the second vaccination with one fold dose of vaccine. After the ten fold overdosed vaccination, the vaccination strain was detected in the faeces at latest at day 49 after the second vaccine application. In case of the one fold vaccinated piglets, the vaccine strain was detected in faeces at least until day 7 after the second vaccination. The colonization of the internal tissues with the vaccine strain was timely restricted until six weeks after the second immunization, as a sample of the ileum and the colon have been found to be positive. The vaccine strain was isolated in decreasing amounts from samples of the ileoceacal lymph nodes, the ileum, ceacum content, colon, lung as well as in samples of the liver and skeletal- and heart muscle. Samples from the kidney were not found to be positive for the vaccine strain at any time. Salmonella field strains could be detected neither in the control nor in the vaccinated group in any time during this study. A serological response in answer to the single dosed vaccination on day 3 and 21 could be seen from day 7 after the second vaccination. A significant higher mean antibody concentration in the serum of the piglets of the vaccinated group could be detected from the third week after the second vaccination until the end of the study after ten weeks. The maximum of the mean antibody concentration was measured 49 days after the second vaccination.In der vorliegenden Untersuchung sollte der Salmonella Typhimurium-Lebendimpfstoff Salmoporc® auf seine Verträglichkeit nach oraler Applikation für Ferkel mit einem Erstimmunisierungsalter von 3 Tagen, sowie einer Revakzination am 21. Lebenstag untersucht werden. Zu diesem Zweck wurden salmonellenfreie Ferkel an ihrem 3. sowie 21. Lebenstag mit der 10fachen der normalen, sowie in einem weiteren Versuch mit der Vom Hersteller empfohlenen Dosis oral geimpft. Als Negativkontrolle dienten in beiden Versuchen Ferkel, die eine äquivalente Menge steriler, physiologischer Kochsalzlösung oral verabreicht bekamen. Bis 8 Stunden nach der Impfung wurde bei allen Ferkeln kontinuierlich die Kotkonsistenz, Körpertemperatur, Säugehäufigkeit und das Allgemeinbefinden bewertet. Des weiteren erfolgten bis zum Ende des Versuchszeitraums in der 10. Lebenswoche der Ferkel neben der täglichen Kontrolle der Körpertemperatur und Kotkonsistenz eine wöchentliche Gewichtskontrolle, sowie bakteriologische Untersuchungen über die Dauer der Impfstammausscheidung. Zusätzlich zu den oben erwähnten Parametern wurde die Verteilung und Peristenz des Impfstamms in verschiedenen Geweben der Ferkel nach zweimaliger Applikation der einfachen Dosis am 3. und 21. Lebenstag. Die Immunantwort nach der einfach dosierten Impfung wurde mittels serologischer Untersuchung auf Salmonella-Antikörper überprüft. Die 10fache normale Dosis des Impfstoffs führte zu beiden Impfzeitpunkten bei den geimpften Ferkeln zu einem signifikanten Ansteigen der Körpertemperatur. Die Anzahl der Ferkel mit einer Körpertemperatur über 39,8°C war im Vergleich zur Kontrollgruppe signifikant größer. Nach der Überdosierung am 3. Lebenstag traten bei je drei Ferkeln der Impfgruppe Lethargie, Atemfrequenzerhöhung und milder Durchfall auf. Eine signifikante Häufung im Auftreten von Ferkeln mit Atemfrequenzerhöhung und Lethargie sowie eine geringere Säugehäufigkeit bei den Ferkeln der Impfgruppe war nach der erneuten Überdosierung am 21. Lebenstag zu beobachten. Die 10fach überdosierten Ferkel wiesen bereits 4 Tage nach der ersten Überdosierung ein signifikant geringeres Gewicht auf. Dieser Status blieb bis zum Versuchsende in der zehnten Lebenswoche der Ferkel erhalten. Nach der einfach dosierten Impfung waren die Ferkel der Impfgruppe am Tag des Absetzens signifikant schwerer. Ein signifikant häufigeres Auftreten von Durchfall konnte sowohl nach Verabreichung der einfachen als auch der zehnfachen Dosis des Impfstoffs innerhalb von 14 Tagen nach der ersten Impfung, sowie im Zeitraum von 14 Tagen nach der zweiten Impfung bei einfacher Dosierung beobachtet werden. Nach Verabreichung der 10fachen Dosis des Impfstoffs gelang der letzte kulturelle Nachweis des Impfstamms im Kot der geimpften Ferkel am 49. Tag nach der zweiten Impfstoffapplikation (70. Lebenstag der Ferkel). Nach der Applikation der einfachen Dosis wurde der Impfstamm letztmalig 7 Tage nach der zweiten Impfung (28. Lebenstag der Ferkel) im Kot nachgewiesen. Die Besiedlung der untersuchten Gewebe durch den Impfstamm nach der einfach dosierten Impfung war zeitlich begrenzt. Der letzte Nachweis des Impfstamms gelang 6 Wochen nach der zweiten Impfung aus Proben des Dünndarms und Dickdarms. Der Impfstamm wurde in absteigender Häufigkeit aus Proben der Ileozäkallymphknoten, des Dünndarms, Blinddarminhalts, Dickdarms, der Lunge sowie aus Proben der Leber und Skelett- sowie Herzmuskulatur nachgewiesen. Aus Proben der Nieren konnte zu keinem Untersuchungszeitpunkt der Impfstamm isoliert werden. Feldstämme wurden weder aus Kot- noch aus den Organproben von Impf- oder Kontrollgruppe isoliert. Die Kontrolltiere blieben bakteriologisch in beiden Versuchen bis zum Ende des Versuchszeitraums Impf- und Feldstamm negativ. Eine Serokonversion wurde ab dem 7. Tag nach der zweiten Impfung beobachtet. Die mittlere Antikörperkonzentration der geimpften Ferkel war ab der 3. Woche nach der zweiten Immunisierung signifikant höher als die der Kontrolltiere. Die maximale mittlere Antikörperkonzentration im Serum der Ferkel wurde 49 Tage nach der zweiten Impfung gemessen

Examination on the Occurrence of Coinfections in Diagnostic Transmittals in Cases of Stillbirth, Mummification, Embryonic Death, and Infertility (SMEDI) Syndrome in Germany

The stillbirth, mummification, embryonic death, and infertility (SMEDI) syndrome is most commonly associated with porcine parvovirus 1 (PPV1) infections. Little is known about the occurrence of coinfections with SMEDI-associated pathogens and the associations among these pathogens. In our study, we included 40 SMEDI-affected litters from 18 different farms. In total, 158 out of 358 available fetuses from diagnostic transmittals were selected by systematic random sampling and examined for PCV2, PCV3, PPV1, and Leptospira spp. by q-PCR. Results from diagnostic materials showed the following results: in eleven farms, PCV2 was present; in nine farms, PPV1 was present; in five farms, PCV3 was present; and in two farms, Leptospira spp. was present. The detection of Leptospira spp. was significantly associated with a PCV2 coinfection (OR: 26.3; p < 0.001). PCV3 positivity resulted in a reduced probability of detecting PCV2 in the corresponding fetus (OR: 0.078; p = 0.008). Fetal maceration was associated with Leptospira spp. detection (OR: 8.6; p = 0.003), whereas mummification (p = 0.047), reduced crown-rump length (p < 0.001), and bodyweight (p = 0.001) of fetuses were significantly associated with PPV1 and PCV2 coinfection and thus, presumably, a shorter time to death after infection, indicating an enhanced negative effect on the development of fetuses with PCV2 + PPV1 coinfection

Bone regeneration of minipig mandibular defect by adipose derived mesenchymal stem cells seeded tri-calcium phosphate- poly(D,L-lactide-co-glycolide) scaffolds

Reconstruction of bone defects represents a serious issue for orthopaedic and maxillofacial surgeons, especially in extensive bone loss. Adipose-derived mesenchymal stem cells (ADSCs) with tri-calcium phosphates (TCP) are widely used for bone regeneration facilitating the formation of bone extracellular matrix to promote reparative osteogenesis. The present study assessed the potential of cell-scaffold constructs for the regeneration of extensive mandibular bone defects in a minipig model. Sixteen skeletally mature miniature pigs were divided into two groups: Control group and scaffolds seeded with osteogenic differentiated pADSCs (n=8/group). TCP-PLGA scaffolds with or without cells were integrated in the mandibular critical size defects and fixed by titanium osteosynthesis plates. After 12 weeks, ADSCs seeded scaffolds (n=7) demonstrated significantly higher bone volume (34.8%+/- 4.80%) than scaffolds implanted without cells (n=6, 22.4%+/- 9.85%) in the micro-CT (p < 0.05). Moreover, an increased amount of osteocalcin deposition was found in the test group in comparison to the control group (27.98 +/- 2.81% vs 17.10 +/- 3.57%, p < 0.001). In conclusion, ADSCs seeding on ceramic/polymer scaffolds improves bone regeneration in large mandibular defects. However, further improvement with regard to the osteogenic capacity is necessary to transfer this concept into clinical use

Explorative Field Study on the Use of Oral Fluids for the Surveillance of Actinobacillus pleuropneumoniae Infections in Fattening Farms by an Apx-Real-Time PCR

Simple Summary Oral fluid sampling (OFS) is an animal friendly and easy way for surveillance purposes in domestic swine populations, especially concerning respiratory diseases. In case of Actinobacillus (A.) pleuropneumoniae surveillance, measures are usually combined with burdensome sampling for animals and humans. In the present study, we evaluated the suitability of oral fluids (OFs) for surveillance purposes of A. pleuropneumoniae infections in fattening pigs using an Apx-toxin real-time PCR. We were able to demonstrate that the examination of OFs by an Apx-toxin real-time PCR is suitable for A. pleuropneumoniae surveillance in the field in an animal friendly and easy way. These results might contribute to an increased compliance of laboratory diagnostic measures on pig farms and thereby to increased animal welfare due to less burdensome sampling and improved animal health. Oral fluids (OFs) represent a cost effective and reliable tool for surveillance purposes, mostly regarding viruses. In the present study, we evaluated the suitability of OFs for surveillance purposes concerning Actinobacillus (A.) pleuropneumoniae infections in fattening pigs under field conditions. OFs were examined with an Apx-toxin real-time PCR that detects the genes encoding for Apx I-, Apx III-, and Apx IV-toxin. For this purpose, we conducted a pen-wise collection of OFs over one fattening period from fattening pigs of two farms (farm A and B) with a known history of A. pleuropneumoniae infection. Lung lesions were determined at slaughter to estimate the extend of pulmonary lesions and pleural affection. Apx III- and Apx IV-toxin DNA were present in the OFs of both farms whereas Apx I-toxin DNA was present on farm A only. We were able to detect Apx I-, Apx III-, and Apx IV-toxin DNA in different patterns directly after introduction of the new pigs in the farms and over the entire study period. In summary, or results indicate the suitability of OFS for the early detection and surveillance of A. pleuropneumoniae in fattening farms

Update on shedding and transmission routes of porcine haemotrophic mycoplasmas in naturally and experimentally infected pigs

Horizontal transmission of Mycoplasma suis via parenteral exposure during standard practices or through bites during fightings have been identified as key epidemiological routes. However, as knowledge gaps on other potential shedding and transmission routes exist, the present study combines both laboratory experiments and field surveys to gain new insights into the epidemiology of porcine haemotrophic mycoplasmas. Splenectomised pigs were orally inoculated with a M. suis field strain and investigated for clinical signs related to infectious anaemia of pigs (IAP) and the presence of M. suis in blood, urine and saliva samples by qPCR. All blood samples were negative for M. suis and animals did not show obvious clinical signs of IAP throughout the entire study period. Additionally, urine, nasal and saliva samples from sows of conventional piglet producing farms and semen samples from a boar stud revealed no detection of M. suis and ‘Candidatus Mycoplasma haemosuis’ by qPCR. Thus, the results indicate that blood-independent transmission routes might be of minor relevance under field conditions

Repair of Oronasal Fistulae by Interposition of Multilayered Amniotic Membrane Allograft

Background: Oronasal fistulas are a frequent complication after cleft palate surgery. Numerous repair methods have been described, but wound-healing problems occur often. The authors investigated, for the first time, the suitability of multilayered amniotic membrane allograft for fistula repair in a laboratory experiment (part A), a swine model (part B), and an initial patient series (part C). Methods: In part A, one-, two-, and four-layer porcine and human amniotic membranes (n = 20 each) were fixed in a digital towing device and the force needed for rupture was determined. In part B, iatrogenic oronasal fistulas in 18 piglets were repaired with amniotic membrane allograft, autofetal amniotic membrane, or small intestinal submucosa (n = 6 each). Healing was evaluated by probing and visual inflammation control (no/moderate/strong) on postoperative days 3, 7, 10, and 76. Histological analysis was performed to visualize tissue architecture. In part C, four patients (two women and two men, ages 21 to 51 years) were treated with multilayered amniotic membrane allograft. Results: In part A, forces needed for amniotic membrane rupture increased with additional layers (p < 0.001). Human amniotic membrane was stronger than porcine membrane (p < 0.001). In part B, fistula closure succeeded in all animals treated with amniotic membrane with less inflammation than in the small intestinal submucosa group. One fistula remained persistent in the small intestinal submucosa group. In part C, all fistulas healed completely without inflammation. Conclusions: Amniotic membrane is an easily available biomaterial and can be used successfully for oronasal fistula repair. The multilayer technique and protective plates should be utilized to prevent membrane ruptures

Detection of Mycoplasma suis in pre-suckling piglets indicates a vertical transmission

Background Transmission of Mycoplasma (M.) suis mainly occurs via iatrogenic or zootechnical manipulations or due to ranking fights. Other transmission routes including ingestion of secretes/excretes; blood-sucking arthropods and intra-uterine transmission have thought to play an epidemiological role without being experimentally proven. To investigate a vertical transmission of M. suis under field conditions blood samples from pre-suckling piglets and their corresponding dam were examined for M. suis by quantitative polymerase chain reaction (qPCR) in 21 farms in Southern Germany. Results A total of 14.35% of the 474 blood samples from pre-suckling piglets reacted qPCR positive. Additionally, M. suis was detected in 65 (31.25%) of the 208 sows at farrowing. On farm level, 16 (76.2%) of the 21 farms had at least one M. suis positive animal. M. suis positive farms had an average of 0.41 more stillborn piglets per litter than M. suis negative farms (p = 0.007). Conclusion The present study provides further insights into M. suis infection dynamics as it is the first detection of M. suis in piglets immediately after birth prior to colostrum intake and the first large scale investigation of M. suis in sows at farrowing

Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains

Background: The occurrence of the novel porcine circovirus type 3 (PCV3) was reported from the Americas, Asia arid Europe. Although this virus was detected in association with various clinical syndromes in pigs, its role as possible swine pathogen remains unclear. PCV3 was detected with high prevalence in Polish farms, but to date no genome sequences were available from European PCV3 strains. Methods: We collected 1060 serum samples from piglets at the age of 20-24 weeks from 53 farms distributed all over Germany. PCV3 DNA was detected using a real-time PCR and subsequently complete PCV3 genome sequences were obtained after multiply primed rolling circle amplification and sequencing of overlapping PCR products. Phylogenetic analysis was performed by neighbor-joining method and maximum likelihood method. Results: We obtained 15 complete PCV3 genome sequences as well as nine partial sequences including the putative ORFs 1, 2 and 3 from PCV3 viremic animals in German pig farms. Phylogenetic analysis of these German as well as 30 full genome sequences received from GenBank divided the PCV3 strains into two main groups and several subclusters. Furthermore, we were able to define group specific amino acid patterns in open reading frame 1 and 2. Conclusion: PCV3 is distributed with high prevalence in German pig industry. Phylogenetic analysis revealed two clearly separated groups of PCV3 strains, which might be considered as PCV3 genotypes. Specific nucleotide and amino acid marker positions may serve for easy and fast intraspecies classification and genotyping of PCV3 strains. No correlation between PCV3 variants with their geographical origin was evident We found the same diversity of PCV3 strains in Germany as in other countries. We hypothesize that PCV3 is not a newly emerging virus in the German pig population. Future studies will have to show, if PCV3 genotype specific biological properties are evident

Effects of the glucagon-like peptide-1 receptor agonist liraglutide in juvenile transgenic pigs modeling a pre-diabetic condition

Background: The glucagon-like peptide-1 receptor (GLP1R) agonist liraglutide improves glycemic control and reduces body weight of adult type 2 diabetic patients. However, efficacy and safety of liraglutide in adolescents has not been systematically investigated. Furthermore, possible pro-proliferative effects of GLP1R agonists on the endocrine and exocrine pancreas need to be further evaluated. We studied effects of liraglutide in adolescent pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) in the beta-cells, leading to a pre-diabetic condition including disturbed glucose tolerance, reduced insulin secretion and progressive reduction of functional beta-cell mass. Methods: Two-month-old GIPRdn transgenic pigs were treated daily with liraglutide (0.6-1.2 mg per day) or placebo for 90 days. Glucose homeostasis was evaluated prior to and at the end of the treatment period by performing mixed meal and intravenous glucose tolerance tests (MMGTT and IVGTT). Finally animals were subjected to necropsy and quantitative-stereological analyses were performed for evaluation of alpha-and beta-cell mass, beta-cell proliferation as well as acinus-cell proliferation. Results: MMGTT at the end of the study revealed 23% smaller area under the curve (AUC) for glucose, a 36% smaller AUC insulin, and improved insulin sensitivity, while IVGTT showed a 15% smaller AUC glucose but unchanged AUC insulin in liraglutide-vs. placebo-treated animals. Liraglutide led to marked reductions in body weight gain (-31%) and food intake (-30%) compared to placebo treatment, associated with reduced phosphorylation of insulin receptor beta (INSRB)/insulin-like growth factor-1 receptor beta (IGF1RB) and protein kinase B (AKT) in skeletal muscle. Absolute alpha-and beta-cell mass was reduced in liraglutide-treated animals, but alpha-and beta-cell mass-to-body weight ratios were unchanged. Liraglutide neither stimulated beta-cell proliferation in the endocrine pancreas nor acinus-cell proliferation in the exocrine pancreas, excluding both beneficial and detrimental effects on the pig pancreas. Conclusions: Although plasma liraglutide levels of adolescent transgenic pigs treated in our study were higher compared to human trials, pro-proliferative effects on the endocrine or exocrine pancreas or other liraglutide-related side-effects were not observed

Ear tagging in piglets: the cortisol response with and without analgesia in comparison with castration and tail docking

The objectives of the present study were to compare the cortisol response caused by ear tagging piglets with the distress caused by other known painful husbandry procedures (e.g. castration and tail docking) and to evaluate the effectiveness of analgesia with meloxicam to reduce the cortisol response caused by these procedures. In total, 210 male piglets were randomised to equal numbers (n=30) into one of seven groups: a control group which was only handled (H), an ear tagged group that received no analgesia (ET), an ear tagged group with analgesia (ETM), a castration group with no analgesia (C), a castration group with analgesia (CM), a tail-docked group with no analgesia (TD) and a tail-docked group with analgesia (TDM). The procedures were carried out on day 3 or 4 after farrowing. Five blood samples were taken from each piglet: 30 min before the respective procedure (baseline value), and 30, 60 min, 4 and 7 h after processing, to assess cortisol concentrations. Means as well as the area under the curve (AUC) value were analysed and the effective sizes of the procedures were established. At 7 h after the experimental treatment, cortisol concentrations had returned to base values in all groups. ET evoked a greater cortisol response than H piglets at 30 min (P<0.001) and 60 min (P=0.001). The cortisol response to ET was lower than C at 30 min (P=0.001) but did not differ significantly at the other sample times. The mean cortisol response was similar between ET and TD piglets over all sample times. Taking both intensity and duration of the cortisol response into account (AUC), ET evoked a greater response than TD. Analgesia (ETM) resulted in significantly lower cortisol levels than ET at 30 and 60 min post-procedure. Castration (C) provoked the highest cortisol response of all procedures; a significant analgesic effect (CM) was shown only at 4 h post-procedure. TD resulted in significantly higher cortisol levels than H piglets only at 30 min; analgesia (TDM) significantly reduced the cortisol response at 30 min. We conclude that ear tagging causes a dramatic increase in cortisol levels compared with handling alone in piglets, which suggests that this procedure causes substantial distress. However, further research is needed to confirm these results