Robert Klopfleisch

Commensal Akkermansia muciniphila Exacerbates Gut Inflammation in Salmonella Typhimurium-Infected Gnotobiotic Mice

Excessive mucin degradation by intestinal bacteria may contribute to inflammatory bowel diseases because access of luminal antigens to the intestinal immune system is facilitated. This study investigated how the presence of a mucin degrading commensal bacterium affects the severity of an intestinal Salmonella enterica Typhimurium-induced gut inflammation. Using a gnotobiotic C3H mouse model with a background microbiota of eight bacterial species (SIHUMI) the impact of the mucin-degrading commensal bacterium Akkermansia muciniphila (SIHUMI-A) on inflammatory and infectious symptoms caused by S. Typhimurium was investigated. Presence of A. muciniphila in S. Typhimurium-infected SIHUMI mice caused significantly increased histopathology scores and elevated mRNA levels of IFN-γ, IP-10, TNF-α, IL-12, IL-17 and IL-6 in cecal and colonic tissue. The increase in pro-inflammatory cytokines was accompanied by 10-fold higher S. Typhimurium cell numbers in mesenteric lymph nodes of SIHUMI mice associated with A. muciniphila and S. Typhimurium (SIHUMI-AS) compared to SIHUMI mice with S. Typhimurium only (SIHUMI-S). The number of mucin filled goblet cells was 2- to 3- fold lower in cecal tissue of SIHUMI-AS mice compared to SIHUMI-S, SIHUMI-A or SIHUMI mice. Reduced goblet cell numbers significantly correlated with increased IFN-γ mRNA levels (r2 = −0.86, ***P<0.001) in all infected mice. In addition, loss of cecal mucin sulphation was observed in SIHUMI mice containing both A. muciniphila and S. Typhimurium compared to other mouse groups. Concomitant presence of A. muciniphila and S. Typhimurium resulted in a drastic change in microbiota composition of SIHUMI mice: the proportion of B. thetaiotaomicron in SIHUMI-AS mice was 0.02% of total bacteria compared to 78% – 88% in the other mouse groups and the proportion of S. Typhimurium was 94% in SIHUMI-AS mice but only 2.2% in the SIHUMI-S mice. These results indicate that A. muciniphila exacerbates S. Typhimurium-induced intestinal inflammation by its ability to disturb host mucus homeostasis.

Pathology of Natural Infections by H5N1 Highly Pathogenic Avian Influenza Virus in Mute (Cygnus olor) and Whooper (Cygnus cygnus) Swans

Mortality in wild aquatic birds due to infection with highly pathogenic avian influenza viruses (HPAIV) is a rare event. During the recent outbreak of highly pathogenic avian influenza in Germany, mortality due to H5N1 HPAIV was observed among mute and whooper swans as part of a rapid spread of this virus. In contrast to earlier reports, swans appeared to be highly susceptible and represented the mainly affected species. We report gross and histopathology and distribution of influenza virus antigen in mute and whooper swans that died after natural infection with H5N1 HPAIV. At necropsy, the most reliable lesions were multifocal hemorrhagic necrosis in the pancreas, pulmonary congestion and edema, and subepicardial hemorrhages. Major histologic lesions were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasmacytic encephalitis with neuronal necrosis. Adrenals displayed consistently scattered cortical and medullary necrosis. In spleen and Peyers patches, mild lymphocyte necrosis was present. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas, adrenals, liver, and brain was strongly consistent with histologic lesions. In the brain, a large number of neurons and glial cells, especially Purkinje cells, showed immunostaining. Occasionally, ependymal cells of the spinal cord were also positive. In the lungs, influenza virus antigen was identified in a few endothelial cells but not within pneumocytes. The infection of the central nervous system supports the view that the neurotropism of H5N1 HPAIV leads to nervous disturbances with loss of orientation. More investigations are necessary to clarify the mechanisms of the final circulatory failure, lung edema, and rapid death of the swans.

Neurotropism of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) in experimentally infected pigeons (Columbia livia f. domestica)

This investigation assessed the susceptibility of experimentally infected pigeons to the highly pathogenic avian influenza virus (HPAIV) H5N1 that caused recent outbreaks of avian influenza in birds and humans in several countries of Asia. For this purpose 14 pigeons were infected ocularly and nasally with 108 EID50 and clinical signs were recorded and compared with five chickens infected simultaneously as positive controls. The chickens demonstrated anorexia, depression, and 100% mortality within 2 days postinoculation. Three of the pigeons died after a history of depression and severe neurological signs consisting of paresis to paralysis, mild enteric hemorrhage, resulting in a mortality of 21%. Gross lesions in these pigeons were mild and inconsistent. Occasionally subcutaneous hyperemia and hemorrhage and cerebral malacia were observed. Microscopic lesions and detection of viral antigen were confined to the central nervous system of these pigeons. In the cerebrum and to a minor extent in the brain stem a lymphohistiocytic meningoencephalitis with disseminated neuronal and glial cell necrosis, perivascular cuffing, glial nodules, and in one bird focally extensive liquefactive necrosis could be observed. The remaining nine pigeons showed neither clinical signs nor gross or histological lesions associated with avian influenza, although seroconversion against H5 indicated that they had been infected. These results confirm that pigeons are susceptible to HPAIV A/chicken/Indonesia/2003 (H5N1) and that the disease is associated with the neurotropism of this virus. Although sentinel chickens and most pigeons did not develop disease, further experiments have to elucidate whether or not Columbiformes are involved in transmission and spread of highly pathogenic avian influenza.

Metastatic canine mammary carcinomas can be identified by a gene expression profile that partly overlaps with human breast cancer profiles

BackgroundSimilar to human breast cancer mammary tumors of the female dog are commonly associated with a fatal outcome due to the development of distant metastases. However, the molecular defects leading to metastasis are largely unknown and the value of canine mammary carcinoma as a model for human breast cancer is unclear. In this study, we analyzed the gene expression signatures associated with mammary tumor metastasis and asked for parallels with the human equivalent.MethodsMessenger RNA expression profiles of twenty-seven lymph node metastasis positive or negative canine mammary carcinomas were established by microarray analysis. Differentially expressed genes were functionally characterized and associated with molecular pathways. The findings were also correlated with published data on human breast cancer.ResultsMetastatic canine mammary carcinomas had 1,011 significantly differentially expressed genes when compared to non-metastatic carcinomas. Metastatic carcinomas had a significant up-regulation of genes associated with cell cycle regulation, matrix modulation, protein folding and proteasomal degradation whereas cell differentiation genes, growth factor pathway genes and regulators of actin organization were significantly down-regulated. Interestingly, 265 of the 1,011 differentially expressed canine genes are also related to human breast cancer and, vice versa, parts of a human prognostic gene signature were identified in the expression profiles of the metastatic canine tumors.ConclusionsMetastatic canine mammary carcinomas can be discriminated from non-metastatic carcinomas by their gene expression profiles. More than one third of the differentially expressed genes are also described of relevance for human breast cancer. Many of the differentially expressed genes are linked to functions and pathways which appear to be relevant for the induction and maintenance of metastatic progression and may represent new therapeutic targets. Furthermore, dogs are in some aspects suitable as a translational model for human breast tumors in order to identify prognostic molecular signatures and potential therapeutic targets.

Avian Malaria Deaths in Parrots, Europe

To the Editor: Avian malaria is an insect-borne disease induced by a so far unknown number of protozoan blood parasites of the genera Plasmodium and Haemoproteus (hematozoa) (1,2). The unintentional introduction of P. relictum to the Hawaiian Islands, USA, has had fatal effects for the native bird fauna (3). In Europe, asymptomatic blood infections by hematozoa have been regularly observed, with an especially high prevalence in songbirds (4). However, numerous outbreaks of fatal protozoan infections have been reported over the past 40 years, mainly among psittacines of Australia that have been kept in aviaries (5,6). Diagnosis in all these cases was based on histopathologic detection of protozoan cyst-like structures of unexplained origin in the heart and skeletal muscles and, to a lesser extent, in other organs. In most cases, the protozoans were identified as members of the genus Leucocytozoon because of their morphologic features. Recent studies suggest that these cases may, in fact, have been infections of Besnoitia spp. (Sarcocystidae) or other unknown hematozoa (5); however, genetic evidence is lacking. In August 2010, sudden deaths of parrots were noticed in 2 separate aviaries in northern Germany and Switzerland (Technical AppendixTable). Nine yellow-crowned parakeets (Cyanoramphus auriceps), 3 barred parakeets (Bolborhynchus lineola), and 2 budgerigars (Melopsittacus undulatus) died within 2–5 days after a history of reduced general condition and reduced activity and food intake before death. In addition, 2 budgerigars and 1 barred parakeet in the aviary in Germany showed lethargy and reduced food intake for 2 weeks but fully recovered. About half of the birds were juvenile. No new birds had been introduced into the aviaries during the previous 24 months. Necropsy and histologic examination of 7 animals with fatal disease showed numerous large cyst-like protozoan structures (size up to 800 µm in diameter; Technical Appendix Figure) in myocardial and skeletal muscles and, to a lesser extent, in the lung and the smooth muscles of the intestinal tract without obvious signs of inflammation. The cyst-like structures had a thick eosinophilic outer wall, were partly compartmented by internal septae, and were filled with many merozoites. Surrounding muscle fibers were degenerated or necrotic and, in some cases, associated with hemorrhage. Blood smears of clinically affected animals screened for ≈5 × 105 cells each did not show parasites. To further characterize the parasites, we carried out a nested PCR and subsequent DNA sequencing as described (7). Notably, phylogenetic comparison of 479 bp of the mitochondrial cytochrome b gene derived from protozoan cyst-like structures with known sequences of avian hematozoa found 99%–100% homology of parasites from both outbreaks with the avian malaria parasites (Haemoproteus spp.) of European songbirds (Figure). Identical cytochrome b sequences were detected in a yellow-crowned parakeet from Switzerland (CYAUR1), a budgerigar from Germany (MEUND1), and a Haemoproteus sp. (TUPHI1) previously found in the blood of a song thrush (Turdus philomelos) in Bulgaria. The sequence derived from the barred parakeet (BOLIN1) of the German outbreak was identical with H. minutus of the common blackbird (T. merula). In fact, different psittacine species of the German outbreak were infected with different Haemoproteus spp. Because all affected parrots had been bred in Europe and had no contact to imported birds, these results suggest that infection was the result of previously unknown cross-species transmission of Haemoproteus spp. between birds of only distantly related orders (8,9). Figure Phylogenetic relationships based on alignment of 479 bp of the cytochrome b gene of Haemoproteus spp. isolated from megalomeronts (m) of infected muscles and blood (b) of parrots with related hematozoan parasites in GenBank and the database MalAvi (http://mbio-serv4.mbioekol.lu.se/avianmalaria ... Blood samples from surviving, asymptomatic animals from the German outbreak were tested cytologically and by nested PCR for the presence of Haemoproteus spp. PCR identified Haemoproteus sequences in the blood of 3 of 26 psittacines, although parasitic structures were not identifiable in blood smears. Retrieved sequences were identical with that of MEUND1, except for a single-nucleotide polymorphism in 1 sequence (MEUND3; Figure). A latent infection of these animals therefore seems possible and may constitute a potential risk for further horizontal transmission in aviaries by blood-sucking insects such as biting midges (Culicoides), the vectors for Haemoproteus spp. of passerine birds in Europe (2). In conclusion, we identified the cause of a previously unexplained lethal disease of captive parrots in Europe, induced by numerous large cyst-like megalomeronts in several organs, including the heart. Morphologically, the parasitic structures were strikingly similar to yet undetermined parasites of numerous previous outbreaks (5,6). Genetically, the parasites had 99%–100% homology to known Haemoproteus spp. from wild European songbirds. The avian malaria parasites identified are highly prevalent in the native songbird population but generally do not cause overt disease or death in their natural hosts. In contrast, the cases reported here suggest that these parasites that have adapted to European songbirds may cause fatal outbreaks in native psittacines of Australia, New Zealand, and South America that are raised in captivity. These findings also show that preexisting pathogens may be a potential hazard for invading species. Avian malaria should therefore be considered a threat for exotic parrots in Europe until results of further epidemiologic and experimental studies are available. Because many European bird species have been introduced to the native range of the psittacines studied here, a concern has been expressed that these parasites already have become established in these areas and are affecting the natural populations.

Experimental Infection and Natural Contact Exposure of Dogs with Avian Influenza Virus (H5N1)

Experiments that exposed influenza virus (H5N1)–infected cats to susceptible dogs did not result in intraspecies or interspecies transmission. Infected dogs showed increased body temperatures, viral RNA in pharyngeal swabs, and seroconversion but not fatal disease.

Distribution of Lesions and Antigen of Highly Pathogenic Avian Influenza Virus A/Swan/Germany/R65/06 (H5N1) in Domestic Cats after Presumptive Infection by Wild Birds

In early 2006, the highly pathogenic avian influenza virus (HPAIV) H5N1 of the Asian lineage caused the death of wild aquatic birds in Northern Germany. In the mainly affected areas, a trans-species transmission of HPAIV H5N1 to mammals occurred between birds and domestic cats and 1 Stone Marten (Martes foina), respectively. Here, we report lesions and distribution of influenza virus antigen in 3 cats infected naturally with HPAIV H5N1 A/swan/Germany/R65/06. The hemagglutinin partial nucleotide sequences of the viruses were genetically closely related to a H5N1 HPAIV obtained from a dead Whooper Swan (Cygnus cygnus) of the same area. At necropsy, within the patchy dark-red and consolidated lungs, there was granulomatous pneumonia caused by Aelurostrongylus sp. Histologically, the main findings associated with influenza in all cats were bronchointerstitial pneumonia and marked random hepatic necrosis. In addition, all animals displayed lymphoid necrosis in the spleen and Peyers patches and necrosis of the adrenal cortex. Immunohistochemically, nucleoprotein of HPAIV was present intralesionally in the lungs, liver, adrenal glands, and lymphoid tissues. Oropharyngeal swabs were shown to be suited to detect HPAIV by quantitative real-time polymerase chain reaction (RT-PCR) in these cats, despite the paucity of influenza virus antigen in the upper respiratory tract by means of immunohistochemistry. The results show that outdoor cats in areas affected by HPAIV in wild birds are at risk for lethal infection. In conclusion, hepatic necrosis was, besides bronchointerstitial pneumonia, the primary lesion, suggesting that in naturally infected cats, damage to the liver plays an important role in the pathogenesis of H5N1 influenza.

Molecular Carcinogenesis of Canine Mammary Tumors News From an Old Disease

Studies focusing on the molecular basis of canine mammary tumors (CMT) have long been hampered by limited numbers of molecular tools specific to the canine species. The lack of molecular information for CMT has impeded the identification of clinically relevant tumor markers beyond histopathology and the introduction of new therapeutic concepts. Additionally, the potential use for the dog as a model for human breast cancer is debatable until questions are answered regarding cellular origin, mechanisms, and cellular pathways. During the past years, increasing numbers of canine molecular tools have been developed on the genomic, RNA, and protein levels, and an increasing number of studies have shed light on specific aspects of canine carcinogenesis, particularly of the mammary gland. This review summarizes current knowledge on the molecular carcinogenesis of CMT, including the role of specific oncogenes, tumor suppressors, regulators of apoptosis and DNA repair, proliferation indices, adhesion molecules, circulating tumor cells, and mediators of angiogenesis in CMT progression and clinical behavior. Whereas the data available are far from complete, knowledge of molecular pathways has a significant potential to complement and refine the current diagnostic and therapeutic approach to this tumor type. Furthermore, current data show that significant similarities and differences exist between canine and human mammary tumors at the molecular level. Clearly, this is only the beginning of an understanding of the molecular mechanisms of CMT and their application in clinical patient management.

Proteome of metastatic canine mammary carcinomas: similarities to and differences from human breast cancer.

Mammary tumors are a major health threat to women and female dogs. In both, metastasis of the primary tumor to distant organs is the most common cause of tumor-related death. Nevertheless, the molecular mechanisms of tumor metastasis are far from being understood, and it is still unknown why some human and canine carcinomas metastasize and others do not. Using 2D-DIGE and MALDI-TOF-MS we identified 21 proteins with significant changes (fold change >1.5; p < 0.05) in protein expression between metastasizing (n = 6) and nonmetastasizing (n = 6) canine mammary carcinomas. Quantitative RT-PCR was used to identify transcriptional or post-transcriptional regulation of protein expression. Up-regulated proteins in metastatic carcinomas included proliferating cell nuclear antigen, ferritin light chain, bomapin, tropomyosin 3, thioredoxin-containing domain C5, adenosin, ornithine aminotransferase, coronin 1A, RAN-binding protein 1,3-phosphoglycerate dehydrogenase, and eukaryotic translation elongation factor 1. Down-regulated proteins in metastatic carcinomas included calretinin, myosin, light chain 2, peroxiredoxin 6, maspin, ibrinogen beta chain, vinculin, isocitrate dehydrogenase 1, tropomyosin 1, annexin A5, and Rho GTPase activating protein 1. Interestingly, 19 of these 21 proteins have been described with a malignancy-associated expression in human breast cancer and other human cancer types before. Further investigations are now necessary to test whether these markers are of prognostic value for canine mammary carcinomas and whether their expression is directly involved in canine mammary carcinogenesis or represent solely a secondary reactive phenotype.

Multiparametric and semiquantitative scoring systems for the evaluation of mouse model histopathology - a systematic review

BackgroundHistopathology has initially been and is still used to diagnose infectious, degenerative or neoplastic diseases in humans or animals. In addition to qualitative diagnoses semiquantitative scoring of a lesion`s magnitude on an ordinal scale is a commonly demanded task for histopathologists. Multiparametric, semiquantitative scoring systems for mouse models histopathology are a common approach to handle these questions and to include histopathologic information in biomedical research.ResultsInclusion criteria for scoring systems were a first description of a multiparametric, semiquantiative scoring systems which comprehensibly describe an approach to evaluate morphologic lesion. A comprehensive literature search using these criteria identified 153 originally designed semiquantitative scoring systems for the analysis of morphologic changes in mouse models covering almost all organs systems and a wide variety of disease models. Of these, colitis, experimental autoimmune encephalitis, lupus nephritis and collagen induced osteoarthritis colitis were the disease models with the largest number of different scoring systems. Closer analysis of the identified scoring systems revealed a lack of a rationale for the selection of the scoring parameters or a correlation between scoring parameter value and the magnitude of the clinical symptoms in most studies.ConclusionAlthough a decision for a particular scoring system is clearly dependent on the respective scientific question this review gives an overview on currently available systems and may therefore allow for a better choice for the respective project.