Cross-species transmission of canine distemper virus—an update

AbstractCanine distemper virus (CDV) is a pantropic morbillivirus with a worldwide distribution, which causes fatal disease in dogs. Affected animals develop dyspnea, diarrhea, neurological signs and profound immunosuppression. Systemic CDV infection, resembling distemper in domestic dogs, can be found also in wild canids (e.g. wolves, foxes), procyonids (e.g. raccoons, kinkajous), ailurids (e.g. red pandas), ursids (e.g. black bears, giant pandas), mustelids (e.g. ferrets, minks), viverrids (e.g. civets, genets), hyaenids (e.g. spotted hyenas), and large felids (e.g. lions, tigers). Furthermore, besides infection with the closely related phocine distemper virus, seals can become infected by CDV. In some CDV outbreaks including the mass mortalities among Baikal and Caspian seals and large felids in the Serengeti Park, terrestrial carnivores including dogs and wolves have been suspected as vectors for the infectious agent. In addition, lethal infections have been described in non-carnivore species such as peccaries and non-human primates demonstrating the remarkable ability of the pathogen to cross species barriers. Mutations affecting the CDV H protein required for virus attachment to host-cell receptors are associated with virulence and disease emergence in novel host species. The broad and expanding host range of CDV and its maintenance within wildlife reservoir hosts considerably hampers disease eradication

Beneficial and detrimental effects of regulatory T cells in neurotropic virus infections

Neurotropic viruses infect the central nervous system (CNS) and cause acute or chronic neurologic disabilities. Regulatory T cells (Treg) play a critical role for immune homeostasis, but may inhibit pathogen-specific immunity in infectious disorders. The present review summarizes the current knowledge about Treg in human CNS infections and their animal models. Besides dampening pathogen-induced immunopathology, Treg have the ability to facilitate protective responses by supporting effector T cell trafficking to the infection site and the development of resident memory T cells. Moreover, Treg can reduce virus replication by inducing apoptosis of infected macrophages and attenuate neurotoxic astrogliosis and pro-inflammatory microglial responses. By contrast, detrimental effects of Treg are caused by suppression of antiviral immunity, allowing for virus persistence and latency. Opposing disease outcomes following Treg manipulation in different models might be attributed to differences in technique and timing of intervention, infection route, genetic background, and the host’s age. In addition, mouse models of virus-induced demyelination revealed that Treg are able to reduce autoimmunity and immune-mediated CNS damage in a disease phase-dependent manner. Understanding the unique properties of Treg and their complex interplay with effector cells represents a prerequisite for the development of new therapeutic approaches in neurotropic virus infections

Untersuchung des möglichen Einflusses von Umweltchemikalien auf die lymphatischen Organe von Schweinswalen aus der Nord- und Ostsee sowie dem Atlantik unter besonderer Berücksichtigung der Zytokin-Expression

1. Neben hohen Beifangzahlen und einem verminderten Nahrungsangebot sind die Schweinswale der Deutschen Nord- und Ostsee durch die zunehmende Häufigkeit bakterieller und parasitärer Erkrankungen gefährdet. Weiterhin akkumulieren die Wale große Mengen an organischen und anorganischen Schadstoffen. Ziel der vorliegenden Arbeit ist es, den möglichen Zusammenhang zwischen der Schadstoffbelastung und Veränderungen im Immunsystem der Schweinswale zu untersuchen. 2. Im Literaturteil wird die Taxonomie und Ökologie sowie die aktuelle Situation der Schweinswale in der Nord- und Ostsee dargestellt. Weiterhin werden Studien zur zellulären und humoralen Immunantwort sowie zur Immuntoxizität bei marinen Säugetieren beschrieben. Neben morphologischen Aspekten werden pathologische Veränderungen der lymphatischen Organe verschiedener Wal- und Robbenarten berücksichtigt. Abschließend wird der gegenwärtige Stand der Forschung auf dem Gebiet der Immunphänotypisierung lymphatischer Zellen und der Zytokine bei Meeressäugetieren erläutert. 3. Im ersten Teil der vorliegenden Arbeit wurden 26 monoklonale (mAK) und ein polyklonaler Antikörper (mAK) unterschiedlicher Spezies auf ihre immunhistologische Kreuzreaktivität mit gefrorenen und Formalin-fixierten, Paraffin-eingebetteten lymphatischen Geweben des Schweinswales getestet. T-Lymphozyten wurden durch einen mAK und pAK Anti-CD3-Antikörper sowie durch zwei monoklonale walspezifische Anti-CD2-Antikörper markiert. Ein walspezifischer CD45R-Marker zeigte eine starke Reaktion auf B-Lymphozyten, während T-Lymphozyten eine schwache Reaktion aufwiesen. MHC Klasse II-Antigen, welches durch einen bovinen und walspezifischen mAK erkannt wurde, fand sich auf T- und B-Lymphozyten. Ein kaniner MHC Klasse II-spezifischer mAK reagierte mit Oberflächenantigenen von Antigen-präsentierenden Zellen und B-Lymphozyten. Ein equiner Pan-Leukozytenmarker erkannte die meisten Zellen der T- und B-Zellkompartimente. 4. Zur Untersuchung der zellulären Immunantwort wurden periphere Blutlymphozyten von vier Schweinswalen isoliert. Nach Inkubation mit Concanavalin A (Con A), Phytohämagglutinin (PHA) und „pokeweed mitogen“ (PWM) wurde die Zellproliferation durch BrdU-Inkorporation photometrisch gemessen. Con A und PHA stellten die effektivsten Mitogene dar, während PWM eine vergleichsweise geringe Proliferation der Lymphozyten induzierte. 5. Mittels RT-PCR wurde die Genexpression ausgewählter Zytokine und des Haushaltsgens GAPDH im Lymphozytenstimulationstest untersucht. Durch die selektierten Primer konnte die mRNS von IL-2, IL-4, IL-6, IL-10, TGF-beta und TNF-alpha in Con A und PWM-stimulierten Zellen detektiert werden. In PHA-stimulierten Zellen konnte keine IL-2-mRNS nachgewiesen werden. IL-10 und GAPDH fanden sich in unstimulierten und stimulierten Zellen. Die Spezifität der Amplifikate wurde durch die DNS-Analyse bestätigt. 6. Gegenstand der Hauptuntersuchungen waren 61 gestrandete und beigefangene Schweinswale aus der Nord- und Ostsee sowie aus isländischen und norwegischen Gewässern. Für die histologische Befunderhebung wurden Formalin-fixierte, Paraffin-eingebettete, HE-gefärbte Gewebsschnitte verwendet. Die Gradierung der Thymusatrophie und Milzdepletion erfolgte anhand morphologischer Kriterien. Weiterhin wurde der Gesundheitsstatus der Tiere aufgrund des Ernährungszustandes und der pathologischen Veränderungen ermittelt. Durch toxikologische Analysen wurden die PCB-, PBDE-, Toxaphen-, DDE- und DDT-Konzentrationen im Fettgewebe quantifiziert. 7. Der Respirationstrakt wies die häufigsten, pathologischen Befunde auf. Gestrandete Schweinswale zeigten einen schlechteren Ernährungs- und Gesundheitszustand als beigefangene Tiere. Weiterhin fand sich eine positive Korrelation zwischen der Thymusatrophie bzw. Milzdepletion und erhöhten PCB- und PBDE-Konzentrationen. Zusätzlich bestand ein signifikanter Zusammenhang zwischen der Depletion beider Organe und dem Gesundheits- bzw. Ernährungszustand. 8. Mittels Immunhistologie wurden unterschiedlich stark depletierte Thymi und Milzen von 29 Tieren phänotypisch charakterisiert. Zusätzlich wurde mittels semiquantitativer Echtzeit-RT-PCR die Zytokin-Expression in diesen Organen und dem Blut der Tiere gemessen. 9. Die Thymusatrophie war mit einer Depletion der CD2+/CD3epsilon+-Zellen vergesellschaftet. In hochgradig atrophischen Thymi fand sich mit dem bovinen Antikörper eine kortikale MHC Klasse II-Aufregulation. Weiterhin war die Thymusatrophie mit einem Verlust der medullären CD45R- und MHC Klasse II-exprimierenden Zellen verbunden. Die Milzdepletion war durch einen Zellschwund in den Follikeln und der Marginalzone, ohne phänotypische Veränderungen charakterisiert. Jedoch fand sich in der PALS ein selektiver Verlust der CD2+- und CDepsilon+-Lymphozyten. 10. Mittels Echtzeit-RT-PCR konnte mit zunehmender Atrophie eine verminderte Expression von IL-2, IL-4, IL-6, IL-10 und TNF-alpha im Thymus nachgewiesen werden, jedoch zeigte lediglich die Reduktion von IL-6 einen signifikanten Zusammenhang. Die Zytokin-Expression in der Milz wies keine eindeutige Verbindung mit der Depletion auf. In einzelnen intakten und hochgradig depletierten Organen konnte jedoch eine Aufregulation von IL-6, TNF-alpha und TGF-beta festgestellt werden. 11. Die mRNS-Menge von IL-10 im Blut wies einen signifikanten Zusammenhang mit der Milzdepletion auf. Es konnte keine eindeutige Korrelation zwischen der Thymusatrophie und den Zytokinen im Blut festgestellt werden. Jedoch wiesen drei Schweinswale mit hochgradiger Thymusatrophie eine deutliche IL-6-Aufregulation auf. 12. Mit zunehmender Thymusatrophie wird ein T-Zellverlust beobachtet. Die immunphänotypischen Veränderungen sprechen für eine Depletion der unreifen, kortikalen Thymozyten. Weiterhin ist die Reduktion medullärer B-Lymphozyten möglicherweise mit einer gestörten Thymopoese vergesellschaftet. Die Depletion der T- und B-Zellkompartimente in der Milz sowie der selektive Verlust von T-Lymphozyten in der PALS kann als eine Suppression ortständiger Zellen oder eine gestörten Migration aus primären lymphatischen Organen interpretiert werden. 13. Die semiquantitative Echtzeit-RT-PCR stellt ein sensitives Verfahren für den Zytokin-Nachweis dar. Die ausgewählten Zytokine spielen eine Rolle in der Thymopoese und Immunantwort der Schweinswale und der mögliche Zusammenhang mit der Thymusatrophie bzw. Milzdepletion wird diskutiert. Möglicherweise induziert die IL-10-Aufregulation im Blut eine lymphozytäre Suppression in der Milz der untersuchten Wale. 14. Die Untersuchungen unterstützen die Hypothese eines negativen Einflusses von PCB und PBDE auf das Immunsystem der Schweinswale. Jedoch müssen bei der Interpretation weitere immunsuppressive Faktoren wie chronische Erkrankungen und Kachexie der Tiere, welche ebenfalls zu einer lymphozytären Depletion führen können, berücksichtigt werden.1. The harbor porpoises in the German North and Baltic Seas represents an endangered species due to high by-catch rates, depletion of food sources, and a growing incidence of bacterial and parasitic diseases. As a top predator, whales accumulate environmental contaminants. The aim of the present study was to investigate the possible influence of xenobiotics on the immune system of harbor porpoises. 2. Taxonomy, ecology, and the current status of harbor porpoises in the North and Baltic Seas is reviewed in the literature section. Additionally, investigations upon the cellular and humoral immune response, as well as upon the immunotoxicity in marine mammals are described. Special emphasis is given to the morphology and pathological changes in lymphoid organs of different cetacean and pinniped species. Finally, techniques for immunophenotyping of lymphoid cells and detection of cytokines in marine mammals are described. 3. In the first part of the study 26 monoclonal antibodies (mAb) and one polyclonal antibody (mAb) of different species were tested for immunohistochemical cross-reactivity on frozen or formalin-fixed, paraffin-embedded lymphatic tissues of the harbor porpoise. T-lymphocytes were labeled by a mAb and a pAb directed against the CD3 complex and by two cetacean mAb specific for CD2 antigen. CD45R, labeled by a cetacean mAb, was strongly expressed on B and weakly on T cells. MHC class II antigen, recognized by cetacean and bovine-specific mAbs, was expressed on T and B cells. A canine MHC class II-specific mAb recognized an epitope on the surface of antigen-presenting cells and B lymphocytes. An equine-pan-leukocyte marker labeled the majority of cells in B- and T-cell compartments. 4. To investigate the cellular immune response of peripheral lymphocytes, blood cells were isolated from four harbor porpoises. After concanavalin A (Con A)-, phytohemagglutinin (PHA)-, and pokeweed mitogen (PWM)-stimulation cell proliferation was measured photometrically using BrdU-assay. Con A and PHA were the most effective mitogens, while PWA induced only a comparatively low proliferation. 5. Gene expression of selected cytokines and the housekeeping gene GAPDH in the lymphocyte transformation test was investigated by RT-PCR. Using specific primers IL-2-, IL-4-, IL-6-, IL-10-, TGFbeta-and TNFalpha-cDNA were amplified in Con A- and PWM-stimulated cells. IL-2 was not detected in PHA-activated cells. The mRNA of IL-10 and GAPDH was expressed in non-stimulated and stimulated cells. Additionally, specificity of PCR-products was confirmed by sequence analysis. 6. Sixty one by-caught and stranded harbor porpoises from the North and Baltic Seas, as well as from Icelandic and Norwegian waters were investigated. Histological examination was performed on formalin-fixed, paraffin-embedded and HE-stained tissue sections. Grading of thymic atrophy and splenic depletion was based on morphological criteria. Additionally, the nutritional state and health status of harbor porpoises was determined. Body burden of PCB, PBDE, toxaphen, DDE and DDT was quantified by toxicological analysis. 7. The most severe pathological findings were demonstrated in the respiratory tract. In comparison to by-caught harbor porpoises, most stranded animals showed a poor nutritional state and health status. Furthermore, thymic atrophy and splenic depletion was positively correlated to increased PCB- and PBDE-concentrations. In both organs lymphoid depletion was correlated with a poor nutritional state and health status. 8. Phenotypical changes in thymus and spleen of 29 harbor porpoises were investigated using immunohistochemistry. Furthermore, cytokine expression in these organs and the blood was measured using semiquantitative real-time RT-PCR. 9. Thymic atrophy was associated with a depletion of CD2+/CD3epsilon+-cells. Using the bovine mAb a cortical MHC class II-up-regulation was found in severely atrophic thymuses. Furthermore, thymic atrophy was correlated to a decrease of medullary CD45R- and MHC class II-expressing cells. Splenic depletion was characterized by cellular loss in follicles and the marginal zone, without phenotypical changes. However, in the PALS a selective loss of CD2+- and CD3epsilon+-lymphocytes was demonstrated. 10. Using real-time RT-PCR a loss of IL-2, IL-4, IL-6, Il-10 and TNF-alpha expression was demonstrated in atrophic thymuses. However, only down-regulation of IL-6-mRNA was statistically significant. Cytokine expression in the spleen showed no association with lymphoid depletion, and in few normal and severely depleted organs a marked up-regulation of IL-6, TNF-alpha and TGF-beta was found. 11. Splenic depletion was significantly associated with an up-regulation of IL-10 in the blood. No definite correlation was found between cytokine expression and thymic atrophy. However, in three animals with severe thymic atrophy high levels of IL-6 were found in the blood samples. 12. Thymic atrophy of the harbor porpoise is associated with a loss of T cells predominantly in the cortex. Furthermore, immunophenotypical changes demonstrated a depletion of immature cortical thymocytes. Reduction of medullary B cells may be associated with diminished thymopoiesis. Additionally depletion of splenic T and B cell compartments can be interpreted due to suppression of splenic stem cells or a failure of immigration from primary lymphoid organs. 13. Semiquantitative real time RT-PCR is a sensitive method for the detection of cytokines. The possible role of selected cytokines in the thymopoiesis and the immune response as well as the possible interaction with the observed lymphoid depletion in harbor porpoises is discussed. Up-regulation of IL-10 in the blood may induce a splenic suppression in the investigated harbor porpoises. 14. The results of the present investigation indicate a potential negative influence of PCB and PBDE upon the immune system of harbor porpoises. However, other immunosuppressive factors, such as chronic illness and emaciation can lead to similar changes in lymphoid organs of the animals

Polyadenine insertion disrupting the G6PC1 gene in German Pinschers with glycogen storage disease type Ia (GSD1A).

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Study of congenital Morgagnian cataracts in Holstein calves

Cataracts are focal to diffuse opacities of the eye lens causing impaired vision or complete blindness. For bilateral congenital cataracts in Red Holsteins a perfectly cosegregating mutation within the CPAMD8 gene (CPAMD8:g.5995966C>T) has been reported. We genotyped the CPAMD8:g.5995966C>T variant in Holstein calves affected by congenital bilateral congenital cataracts, their unaffected relatives and randomly selected herd mates. Ophthalmological examinations were performed in all affected individuals to confirm a congenital cataract. Whole genome sequencing was employed to screen variants in candidate genes for the Morgagnian cataract phenotype. In the present study, 3/35 cases were confirmed as homozygous mutated and 6/14 obligate carriers. Further 7/46 unaffected animals related with these cases were heterozygous mutated for the CPAMD8:g.5995966C>T variant. However 32 cases with a congenital cataract showed the wild type for the CPAMD8 variant. We did not identify variants in the candidate genes CPAMD8 and NID1 or in their close neighborhood as strongly associated with the congenital cataract phenotype in Holstein calves with the CPAMD8 wild type. In conclusion, the CPAMD8:g.5995966C>T variant is insufficient to explain the majority of Morgagnian congenital cataract phenotypes in Holsteins. It is very likely that congenital bilateral cataracts may be genetically heterogeneous and not yet known variants in genes other than CPAMD8 and NID1 are involved

Harbour porpoise thyroids: Histological investigations and potential interactions with environmental factors

The thyroid plays an important role in development and is of primary importance in metabolism and heat loss for cetaceans, including the harbor porpoise (Phocoena phocoena). Several studies have demonstrated that environmental contaminants can alter various aspects of thyroid function in mammals and may contribute to various histologic changes. The present study completes the data set of a 2006 study by Das et al., by performing histological and immunohistologic investigations on thyroids of 36 harbor porpoises from Belgian and United Kingdom waters. The number and mean diameter of follicles (µm) and the relative proportion of follicular, connective, and vascular tissue (%) were quantified in the thyroid gland of each individual. Interfollicular fibrosis has been observed in these thyroid glands, and the collective findings support the hypothesis of an endocrine disruption of thyroid function through organochlorinated compounds. Our study aimed also to reveal potential relationships between thyroid morphometric data and metal levels (Cd, Fe, Zn, Cu, Se, and Hg) using multivariate statistical analysis. The multiple regressions revealed statistically significant relationships between trace elements (cadmium, selenium, and copper) and thyroid fibrosis. The largely negative relationships are interesting findings but do not support the hypothesis that these elements have an adverse effect on thyroid morphometry. Further research is needed to understand the nature of any relationship between organochlorine and trace element exposure and thyroid gland morphology and function in harbor porpoises.Peer reviewe

Standardisation of canine meningioma grading: Inter‐observer agreement and recommendations for reproducible histopathologic criteria

The human grading system is currently applied to canine meningioma, although it has not been validated in dogs. The present study focused on standardising the human grading system applied to canine meningioma. Four veterinary neuropathologists graded 186 canine meningiomas as follows: Grade I tumour, with 50% of the tumour; (6) Report necrosis, small cells, hypercellularity and macronucleoli, even when focal; (7) Report anaplasia if multifocal

A Comparative Transcriptome Analysis of Human and Porcine Choroid Plexus Cells in Response to Streptococcus suis Serotype 2 Infection Points to a Role of Hypoxia

Streptococcus suis (S. suis) is an important opportunistic pathogen, which can cause septicemia and meningitis in pigs and humans. Previous in vivo observations in S. suisinfected pigs revealed lesions at the choroid plexus (CP). In vitro experiments with primary porcine CP epithelial cells (PCPEC) and human CP epithelial papilloma (HIBCPP) cells demonstrated that S. suis can invade and traverse the CP epithelium, and that the CP contributes to the inflammatory response via cytokine expression. Here, next generation sequencing (RNA-seq) was used to compare global transcriptome profiles of PCPEC and HIBCPP cells challenged with S. suis serotype (ST) 2 infected in vitro, and of pigs infected in vivo. Identified differentially expressed genes (DEGs) were, amongst others, involved in inflammatory responses and hypoxia. The RNA-seq data were validated via quantitative PCR of selected DEGs. Employing Gene Set Enrichment Analysis (GSEA), 18, 28, and 21 enriched hallmark gene sets (GSs) were identified for infected HIBCPP cells, PCPEC, and in the CP of pigs suffering from S. suis ST2 meningitis, respectively, of which eight GSs overlapped between the three different sample sets. The majority of these GSs are involved in cellular signaling and pathways, immune response, and development, including inflammatory response and hypoxia. In contrast, suppressed GSs observed during in vitro and in vivo S. suis ST2 infections included those, which were involved in cellular proliferation and metabolic processes. This study suggests that similar cellular processes occur in infected human and porcine CP epithelial cells, especially in terms of inflammatory response

Interfollicular fibrosis and organohalogens in the thyroid of the harbour porpoise (phocoena phocoena) of the British and Belgian coasts

Previous studies have described high levels of polychlorobiphenyls (PCB), p,p`-dichlordiphenyl-dichlorethen (DDT), p,p`-dichlordiphenyl-trichlorethan (DDE) and polybrominated diphenylether (PBDE) in the blubber of the harbour porpoise (Phocoena phocoena) from the North Sea raising the question of a potential endocrine disruption in this species. In the present study, the thyroids of 36 harbour porpoises from the British and the Belgian coast have been collected for histological and immunohistological investigations. The number and mean diameter of follicles and the relative distribution of follicular, connective and vascular tissues (%) were quantified in the thyroid of each individual. Then, the relationship between the thyroid morphometry data and previously described organic compounds (namely PCB, DDT, DDE and PBDE) was investigated using factor analysis and multiple regressions. The results of this work were compared to the previous study of harbour porpoises from the German (North and Baltic Seas), Norwegian and Icelandic coasts. Thyroid morphology was similar between the harbour porpoises from the British and Belgian coast. Compared to the previous study, the thyroids differed strongly between sampling sites. Porpoises from the German (North and Baltic Seas) and Norwegian coasts displayed a high percentage of connective tissues between 30 and 38% revealing severe inter-follicular fibrosis. The thyroids of Icelandic coasts displayed only a light inter-follicular fibrosis (percentage of connective tissue of 3%). The thyroids of the porpoises from the British and Belgian coasts occupied an intermediary place with a moderate fibrosis (percentage of connective tissue of 10%). A relation between mean diameter of follicles and the body size and mass suggesting a downward trend of activity of the thyroid with increasing body size and mass and thus, the age of the porpoises. A correlation-based principal component analysis (PCA) revealed one principal components explaining 69% of the total variance. The variables PCB, PBDE, DDT, and DDE compounds loaded highest on PC1. Our results pointed out a relationship between PCBs, PBDE, DDE and DDT (PC1) compounds and interfollicular fibrosis in the thyroids of harbour porpoises from the German coast of the Baltic Sea. The present report supports the hypothesis of a contaminant-induced thyroid fibrosis in harbour porpoises raising the question of the long term-viability in highly polluted area