Chondrozytärer Kohlenhydratmetabolismus und Zellvitalität boviner artikulärer Knorpelexplantate in statischer Kultur zur Analyse der Bedeutung von FCS und des nutritiven Potentials von Fructose für Knorpel in vitro

Unter Beleuchtung des chondrozytären Kohlenhydratstoffwechsels und Zellüberlebens in vitro sollten im Rahmen dieser Studie folgende Fragen beantwortet werden: 1. Ist FCS als Nährmediumsupplement zusätzlich zu ITS vorteilhaft? 2. Kann Fructose Glucose im Medium als chondrozytäres Energiesubstrat mit Minimierung von Lactatproduktion und pH-Wert-Verschiebung ersetzen? 3. Wie hoch ist der Energiebedarf des kultivierten Knorpels und hat die Höhe des nutritiven Angebots Auswirkungen auf Zellmetabolismus bzw. -vitalität? Dazu wurden 96 Knorpelchips aus den Schultergelenken eines eineinhalb Jahre alten Rindes 16 Versuchsgruppen zugeordnet und bei 37° C sowie atmosphärischem O2- und CO2-Gehalt unter statischen Bedingungen in Multiwell-Platten inkubiert. Das Nährmedium (DMEM) wurde alle 48 h gewechselt und in 4 Varianten eingesetzt: M1A - 1 g/L Fructose; M1B - 1 g/L Fruct, 2% (V/V) FCS; M2A - 1 g/L Glucose; M2B - 1 g/L Gluc, 2 % (V/V) FCS. Alle enthielten 2 mM Glutamin und 1 % (V/V) ITS. Für jeden Mediumtyp gab es eine mit 2 mL versorgte Gruppe und eine weitere mit 5 mL. Jede dieser Bedingungen fand sich in einer 8- und einer 16-tägig geführten Gruppe wieder. Als Kontrollen wurden 10 Chips direkt nach der Biopsie ohne Kultivierung in Formaldehyd-Lösung fixiert und in Paraffin eingebettet. Auch die kultivierten Knorpel wurden bei Versuchsende so aufgearbeitet. Anhand der an Gewebeschnitten durchgeführten HE-Färbung und immunhistochemischen Methoden TUNEL bzw. M30 CytoDEATH® wurde der Vitalitätszustand der Chondrozyten bestimmt und am Lichtmikroskop quantifiziert. In Mediumüberständen wurden Glucose- bzw. Fructose- sowie Lactat- und LDH-Gehalt nach photometrischem Prinzip bestimmt und der pH-Wert gemessen. Die statistische Auswertung erfolgte nicht-parametrisch mit dem Kruskal-Wallis-(H-)Test und post hoc einem Bonferroni-korrigierten Mann-Whitney-U-Test. Dabei wurden i. R. des U-Tests Paare verglichen, die sich in nur einer Versuchsbedingung unterschieden. Der Anteil histomorphologisch vitaler Zellen der nicht kultivierten Kontrollen lag bei durchschnittlich 90,8 ± 5,3 %, in den Versuchsgruppen global bei 66,4 ± 18,9 %. Während die Vitalität der Kontrollen das Zellüberleben des kultivierten Knorpels gesamt überstieg (geschätzter Lageunterschied ~Δ: 20,5 %; p-Wert α’) in den Glucosegruppen höher. Sie korrelierte negativ mit dem pH-Wert (Pearson-Koeffizient: -0,62; p-Wert < 0,05), der in den Fructosemedien im Versuchsverlauf ausnahmslos signifikant größer - i. e. näher an den physiologischen 7,4 (van den Berg, 2003) - als in den Glucosemedien war (~Δ: 0,11-0,39). Als Fazit bleibt unter Berücksichtigung der eingangs gestellten Fragen dies festzuhalten: 1. FCS im Nährmedium bringt Chondrozyten keinen Überlebensvorteil und erhöht ihren Kohlenhydratstoffwechsel nicht. Somit kann es in dieser Hinsicht als Supplement nicht empfohlen werden. 2. Fructose kann Glucose im Medium ersetzen. Im Rahmen dieser Studie wurde meines Wissens erstmalig gezeigt, dass Knorpelzellen Fructose zur Energiegewinnung verstoffwechseln können. Ohne Auswirkung auf die Zellvitalität bewirkt Fructose im Vergleich zu Glucose eine niedrigere Kohlenhydratausschöpfung, Lactatproduktion und pH-Wert-Verschiebung in den Kulturmedien. 3. Der Nährstoffbedarf von Knorpel lag weit unter der angebotenen Menge und könnte in Zukunft auf ca. ein Drittel (~o,33 mg/L) reduziert werden. Bei höherem Zuckerangebot waren Kohlenhydratstoffwechsel, Lactatanfall und pH-Wert-Verschiebung ebenfalls größer, die Zellvitalität blieb unbeeinflusst

In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes

A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3·9 kb (M39). Expression of the reporter gene {beta}-glucuronidase (GUS) (2–8 µg per 106 cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1–2 µg per 106 cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production by in situ hybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development

Migratory Status Is Not Related to the Susceptibility to HPAIV H5N1 in an Insectivorous Passerine Species

Migratory birds have evolved elaborate physiological adaptations to travelling, the implications for their susceptibility to avian influenza are however unknown. Three groups of stonechats (Saxicola torquata) from (I) strongly migrating, (II) weakly migrating and (III) non-migrating populations were experimentally infected with HPAIV H5N1. The different bird groups of this insectivorous passerine species were infected in autumn, when the migrating populations clearly exhibit migratory restlessness. Following infection, all animals succumbed to the disease from 3 through 7 days post inoculation. Viral shedding, antigen distribution in tissues, and survival time did not differ between the three populations. However, notably, endothelial tropism of the HPAIV infection was exclusively seen in the group of resident birds. In conclusion, our data document for the first time the high susceptibility of an insectivorous passerine species to H5N1 infection, and the epidemiological role of these passerine birds is probably limited due to their high sensitivity to HPAIV H5N1 infection. Despite pronounced inherited differences in migratory status, the groups were generally indistinguishable in their susceptibility, survival time, clinical symptoms and viral shedding. Nevertheless, the migratory status partly influenced pathogenesis in the way of viral tropism

Immunohistochemistry for detection of avian infectious bronchitis virus strain M41 in the proventriculus and nervous system of experimentally infected chicken embryos

<p>Abstract</p> <p>Background</p> <p>Infectious bronchitis virus primarily induces a disease of the respiratory system, different IBV strains may show variable tissue tropisms and also affect the oviduct and the kidneys. Proventriculitis was also associated with some new IBV strains. Aim of this study was to investigate by immunohistochemistry (IHC) the tissue tropism of avian infectious bronchitis virus (IBV) strain M41 in experimentally infected chicken embryos.</p> <p>Results</p> <p>To this end chicken embryos were inoculated in the allantoic sac with 10³EID₅₀of IBV M41 at 10 days of age. At 48, 72, and 120 h postinoculation (PI), embryos and chorioallantoic membranes (CAM) were sampled, fixed, and paraffin-wax embedded. Allantoic fluid was also collected and titrated in chicken embryo kidney cells (CEK). The sensitivity of IHC in detecting IBV antigens in the CAM of inoculated eggs matched the virus reisolation and detection in CEK. Using IHC, antigens of IBV were detected in nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons of the inoculated embryos. These results were consistent with virus isolation and denote the wide tissue tropism of IBV M41 in the chicken embryo. Most importantly, we found infection of vasculature and smooth muscle of the proventriculus which has not seen before with IBV strain M41.</p> <p>Conclusion</p> <p>IHC can be an additional useful tool for diagnosis of IBV infection in chickens and allows further studies to foster a deeper understanding of the pathogenesis of infections with IBV strains of different virulence. Moreover, these results underline that embryonic tissues in addition to CAM could be also used as possible source to generate IBV antigens for diagnostic purposes.</p

Tropism of Puumala orthohantavirus and endoparasite coinfection in the bank vole reservoir

In Europe, most cases of human hantavirus disease are caused by Puumala orthohantavirus (PUUV) transmitted by bank voles (Clethrionomys glareolus, syn. Myodes glareolus), in which PUUV causes inconspicuous infection. Little is known about tropism and endoparasite coinfections in PUUV-infected reservoir and spillover-infected rodents. Here, we characterized PUUV tropism, pathological changes and endoparasite coinfections. The voles and some non-reservoir rodents were examined histologically, immunohistochemically, by in situ hybridization, indirect IgG enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. PUUV RNA and anti-PUUV antibodies were detected simultaneously in a large proportion of the bank voles, indicating persistent infection. Although PUUV RNA was not detected in non-reservoir rodents, the detection of PUUV-reactive antibodies suggests virus contact. No specific gross and histological findings were detected in the infected bank voles. A broad organ tropism of PUUV was observed: kidney and stomach were most frequently infected. Remarkably, PUUV was detected in cells lacking the typical secretory capacity, which may contribute to the maintenance of virus persistence. PUUV-infected wild bank voles were found to be frequently coinfected with Hepatozoon spp. and Sarcocystis (Frenkelia) spp., possibly causing immune modulation that may influence susceptibility to PUUV infection or vice versa. The results are a prerequisite for a deeper understanding of virus–host interactions in natural hantavirus reservoirs

Immunogenicity Studies in Carnivores Using a Rabies Virus Construct with a Site-Directed Deletion in the Phosphoprotein

Different approaches have been applied to develop highly attenuated rabies virus vaccines for oral vaccination of mesocarnivores. One prototype vaccine construct is SAD dIND1, which contains a deletion in the P-gene severely limiting the inhibition of type-1 interferon induction. Immunogenicity studies in foxes and skunks were undertaken to investigate whether this highly attenuated vaccine would be more immunogenic than the parental SAD B19 vaccine strain. In foxes, it was demonstrated that SAD dIND1 protected the animals against a rabies infection after a single oral dose, although virus neutralizing antibody titres were lower than in foxes orally vaccinated with the SAD B19 virus as observed in previous experiments. In contrast, skunks receiving 107.5 FFU SAD dIND1 did not develop virus neutralizing antibodies and were not protected against a subsequent rabies infection

Formalin-fixed and paraffin-embedded tissues of chickens are useful for retrospective studies on pathology of H5N1 Highly Pathogenic Avian Influenza Virus (HPAI) outbreaks in Nigeria

In a retrospective study, histopathology and immunohistochemistry (IHC) were performed on formalin-fixed paraffin embedded (FFPE) archival tissues from chickens obtained during outbreaks of highly pathogenic avian influenza (HPAI) H5N1 that occurred in Nigeria in 2006 and 2007. Ten samples as representative of 10 outbreaks were selected, and following the detection of HPAI viral antigen in different chicken tissues using IHC, RNA was extracted from each sample and molecular analysis was performed using real-time reverse transcription-polymerase chain reaction (rRT-PCR) targeting matrix protein. Seven rRT-PCR positive samples were then subjected to conventional and rRT-PCR assays for the amplification of hemagglutinin (HA) gene. Four of them were further characterized by sequence analysis of a short HA2-part of the H5 gene. Along the 154 nucleotides sequenced, differences at 4 positions were detected in one sample. One of these mutations led to an amino acid exchange at position 544 (Ala&gt;Thr) whereas the others were silent. The study suggests the potential application for retrospective IHC and PCR analysis of FFPE tissues from chickens involved in the AI outbreaks for pathologic studies and providing short fragment sequences which may help in the characterization of viral strains and tracing the outbreaks. This is important as archived poultry tissues can be re-examined for possibility of earlier introduction of the virus.Keywords: Avian influenza; FFPE; H5N1; Nigeria; Immunohistochemistry; real-time RT-PC

Experimental Infection of Swans and Geese with Highly Pathogenic Avian Influenza Virus (H5N1) of Asian Lineage

Susceptibility to infection, duration of illness, and concentration of asymptomatic viral shedding vary between species of swans and geese

Protection and Virus Shedding of Falcons Vaccinated against Highly Pathogenic Avian Influenza A Virus (H5N1)

Virus shedding by vaccinated birds was markedly reduced

Pathogenicity of Highly Pathogenic Avian Influenza Virus (H5N1) in Adult Mute Swans

Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/Cygnus cygnus/Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus–specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity