Search

Abstract Borna disease virus 1 (BoDV-1) is the causative agent of Borna disease, a fatal neurologic disorder of domestic mammals and humans, resulting from spill-over infection from its natural reservoir host, the bicolored white-toothed shrew (Crocidura leucodon). The known BoDV-1-endemic area is remarkably restricted to parts of Germany, Austria, Switzerland and Liechtenstein. To gain comprehensive data on its occurrence, we analysed diagnostic material from suspected BoDV-1-induced encephalitis cases based on clinical and/or histopathological diagnosis. BoDV-1 infection was confirmed by RT-qPCR in 207 domestic mammals, 28 humans and seven wild shrews. Thereby, this study markedly raises the number of published laboratory-confirmed human BoDV-1 infections and provides a first comprehensive summary. Generation of 136 new BoDV-1 genome sequences from animals and humans facilitated an in-depth phylogeographic analysis, allowing for the definition of risk areas for zoonotic BoDV-1 transmission and facilitating the assessment of geographical infection sources. Consistent with the low mobility of its reservoir host, BoDV-1 sequences showed a remarkable geographic association, with individual phylogenetic clades occupying distinct areas. The closest genetic relatives of most human-derived BoDV-1 sequences were located at distances of less than 40 km, indicating that spill-over transmission from the natural reservoir usually occurs in the patient´s home region.

The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid “aa” in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

Abstract Jamaican fruit bats (Artibeus jamaicensis) naturally harbor a wide range of viruses of human relevance. These infections are typically mild in bats, suggesting unique features of their immune system. To better understand the immune response to viral infections in bats, we infected male Jamaican fruit bats with the bat-derived influenza A virus (IAV) H18N11. Using comparative single-cell RNA sequencing, we generated single-cell atlases of the Jamaican fruit bat intestine and mesentery. Gene expression profiling showed that H18N11 infection resulted in a moderate induction of interferon-stimulated genes and transcriptional activation of immune cells. H18N11 infection was predominant in various leukocytes, including macrophages, B cells, and NK/T cells. Confirming these findings, human leukocytes, particularly macrophages, were also susceptible to H18N11, highlighting the zoonotic potential of this bat-derived IAV. Our study provides insight into a natural virus-host relationship and thus serves as a fundamental resource for future in-depth characterization of bat immunology.

Abstract As immunohistochemistry is valuable for determining tissue and cell tropism of avian influenza viruses (AIV), but time-consuming, an artificial intelligence-based workflow was developed to automate the AIV antigen quantification. Organ samples from experimental AIV infections including brain, heart, lung and spleen on one slide, and liver and kidney on another slide were stained for influenza A-matrixprotein and analyzed with QuPath: Random trees algorithms were trained to identify the organs on each slide, followed by threshold-based quantification of the immunoreactive area. The algorithms were trained and tested on two different slide sets, then retrained on both and validated on a third set. Except for the kidney, the best algorithms for organ selection correctly identified the largest proportion of the organ area. For most organs, the immunoreactive area assessed following organ selection was significantly and positively correlated to a manually assessed semiquantitative score. In the validation set, intravenously infected chickens showed a generally higher percentage of immunoreactive area than chickens infected oculonasally. Variability between the slide sets and a similar tissue texture of some organs limited the ability of the algorithms to select certain organs. Generally, suitable correlations of the immunoreactivity data results were achieved, facilitating high-throughput analysis of AIV tissue tropism.

Abstract Background Brachyspira (B.) pilosicoli is a zoonotic pathogen, able to infect different animal species such as pigs, poultry, and rodents, causing intestinal spirochetosis. An association of gastrointestinal clinical signs, such as diarrhea, with the isolation of B. pilosicoli from fecal samples or rectal swabs has not been proven in dogs. Other Brachyspira species commonly isolated from dogs, such as “B. canis” and “B. pulli”, are considered commensals. This study investigated the occurrence of different Brachyspira species in rectal swabs and fecal samples in an independent canine cohort in central Germany. These included samples from shelter dogs, hunting dogs, and dogs presenting at regional small animal practices with various clinical signs. Data about the dogs, including potential risk factors for Brachyspira isolation, were obtained using a standardized questionnaire. The study also longitudinally investigated a colony of Beagle dogs for Brachyspira over 5 years. Results The rate of Brachyspira spp. isolation was 11% and included different Brachyspira species (“B. canis”, “B. pulli”, and B. pilosicoli). “B. canis” was detected in 18 dogs, whereas B. pilosicoli was only isolated from 1 dog in the independent cohort (not including the Beagle colony). Risk factors for shedding Brachyspira and “B. canis” were being less than 1 year of age and shelter origin. Gastrointestinal signs were not associated with the shedding of Brachyspira. B. pilosicoli and “B. canis” were isolated from several dogs of the same Beagle colony in 2017 and again in 2022, while Brachyspira was not isolated at multiple sampling time points in 2021. Conclusions Shedding of B. pilosicoli in dogs appears to be uncommon in central Germany, suggesting a low risk of zoonotic transmission from dogs. Commensal status of “B. canis” and “B. pulli” is supported by the results of this study. Findings from the longitudinal investigation of the Beagle colony agree with an asymptomatic long-term colonization of dogs with “B. canis” and B. pilosicoli and suggest that introducing new animals in a pack can trigger an increased shedding of B. pilosicoli.

IntroductionCrested chickens show abnormalities in their anatomy of the skull, endocranium, and brain (including cerebral elongation) and can be appropriate model systems for neuroanatomical evolution, brain–skull integration, and skull and brain deformities. Here, we give a detailed comprehensive description of the skull of crested chickens using the example of the Padovana chicken, including ontogenetic aspects and an allometric analysis of their brain size.MethodsIn total, 109 chickens of two different strains of the Padovana chicken were hatched together. All animals were X-rayed weekly during growth. Nine juvenile (ready for hatch) and 22 adult skulls were processed for histology and morphological descriptions, and a further 20 individuals were processed for brain analysis.ResultsAt hatching, all chicks were already crested, and a distinctive bony protuberance was first observed at the age of 4 weeks. Juvenile chickens exhibit either an open neurocranium or a protuberance. In the adult skull, foramina of different sizes can be found in the frontal bone, but no completely open neurocrania are observed in juveniles. Particularly in Padovana with cranial protuberances, several peculiarities can be observed in the os mesethmoidale, os nasale, os praemaxillare, orbit, and cranial fossae. Additionally, the brain of Padovana with cranial protuberances looks drawn in length with the shape of an hourglass and showed significantly larger encephalization indices than plain-headed breeds, topped only by another crested chicken breed.DiscussionInvestigations on chickens with cerebral elongation may facilitate the understanding of skull and brain dysplasia and may provide meaningful insights into cerebral hernia development. Additionally, crested breeds, combined with standard chickens, form a promising comparative system for investigating the emergence of novel brain and skull morphologies.

To decipher the evolution of the hominoid brain and its functions, it is essential to conduct comparative studies in primates, including our closest living relatives. However, strong ethical concerns preclude in vivo neuroimaging of great apes. We propose a responsible and multidisciplinary alternative approach that links behavior to brain anatomy in non-human primates from diverse ecological backgrounds. The brains of primates observed in the wild or in captivity are extracted and fixed shortly after natural death, and then studied using advanced MRI neuroimaging and histology to reveal macro- and microstructures. By linking detailed neuroanatomy with observed behavior within and across primate species, our approach provides new perspectives on brain evolution. Combined with endocranial brain imprints extracted from computed tomographic scans of the skulls these data provide a framework for decoding evolutionary changes in hominin fossils. This approach is poised to become a key resource for investigating the evolution and functional differentiation of hominoid brains.

One of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum (C. parvum). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies.

Abstract Cryptosporidiosis is one of the main causes of diarrhea in children and young livestock. The interaction of the parasite with the intestinal host cells has not been characterized thoroughly yet but may be affected by the nutritional demand of the parasite. Hence, we aimed to investigate the impact of C. parvum infection on glucose metabolism in neonatal calves. Therefore, N = 5 neonatal calves were infected with C. parvum on the first day of life, whereas a control group was not (N = 5). The calves were monitored clinically for one week, and glucose absorption, turnover and oxidation were assessed using stable isotope labelled glucose. The transepithelial transport of glucose was measured using the Ussing chamber technique. Glucose transporters were quantified on gene and protein expression level using RT-qPCR and Western blot in the jejunum epithelium and brush border membrane preparations. Plasma glucose concentration and oral glucose absorption were decreased despite an increased electrogenic phlorizin sensitive transepithelial transport of glucose in infected calves. No difference in the gene or protein abundance of glucose transporters, but an enrichment of glucose transporter 2 in the brush border was observed in the infected calves. Furthermore, the mRNA for enzymes of the glycolysis pathway was increased indicating enhanced glucose oxidation in the infected gut. In summary, C. parvum infection modulates intestinal epithelial glucose absorption and metabolism. We assume that the metabolic competition of the parasite for glucose causes the host cells to upregulate their uptake mechanisms and metabolic machinery to compensate for the energy losses.

Abstract Background Streptoccocus suis (S. suis) is a major porcine pathogen causing meningitis, septicemia, arthritis and endocarditis. These diseases severely impair welfare of pigs. Experimental studies in pigs are important to better understand the pathogenesis and to identify protective antigens, as so far there is no vaccine available protecting against various serotypes (cps). Due to the severity of disease, application of appropriate refinement strategies in experimental S. suis infections is essential to reduce distress imposed on the piglets without jeopardizing the scientific output. The objectives of this study were to evaluate buprenorphine treatment as a refinement measure and serum cortisol levels as a distress read out parameter in a new S. suis cps3 infection model in pigs. Results Intravenous application of 2 × 108 CFU of S. suis cps3 (sly +, mrp +) to 6-week-old piglets led to severe morbidity in approximately 50% of the animals. Main pathological findings included suppurative meningoencephalitis and arthritis as well as fibrinosuppurative endocarditis. Buprenorphine treatment (0.05 mg/kg every 8 h) did not prevent signs of severe pain, high clinical scores, moderate to severe pathologies or high levels of serum cortisol in single severely affected piglets. Significant differences in the course of leukocytosis, induction of specific antibodies and bactericidal immunity were not recorded between groups with or w/o buprenorphine treatment. Of note, clinically unobtrusive piglets showed serum cortisol levels at 2 and 5 days post infectionem (dpi) comparable to the levels prior to infection with cps3. Cortisol levels in serum were significantly increased in piglets euthanized due to severe disease in comparison to clinically unobtrusive pigs. Conclusions Different clinical courses and pathologies are induced after intravenous challenge of piglets with 2 × 108 CFU of this S. suis cps3 strain. The chosen protocol of buprenorphine application does not prevent severe distress in this infection model. Important parameters of the humoral immune response, such as the level of IgM binding to S. suis cps3, do not appear to be affected by buprenorphine treatment. Serum cortisol is a meaningful parameter to measure distress in piglets experimentally infected with S. suis and to evaluate refinement strategies. In this intravenous model, which includes close clinical monitoring and different humane endpoints, clinics and cortisol levels suggest convalescence in surviving piglets within 5 days following experimental infection.

Bacterial endometritis is one of the major problems in equine reproduction and usually treated with antimicrobial drugs. The study aimed to compare the effects of intrauterine ozone application and systemic antibiotic treatment (trimethoprim-sulfadimethoxine) on intrauterine bacterial growth and possible side effects on the endometrium in a clinical setting. Mares (n = 30) with signs of endometritis (positive uterine bacterial culture and cytological findings) were assigned randomly to different treatments: intrauterine insufflation of an ozone-air-mix (240 ml, 80 μg ozone/ml) twice at a 48 h-interval (Ozone; n = 10), systemic antibiotic therapy with trimethoprim-sulfadimethoxine (30 mg/kg, p.o., twice daily) for 5 days (TMS; n = 10), or intrauterine insufflation of air (240 ml, sterile-filtered) twice at a 48 h-interval (air; n = 10). Endometrial biopsy for histological examination was obtained before the treatment. Histological examination revealed no differences among groups. A control examination, including transrectal ultrasound, bacterial culture, cytological evaluation, and biopsy, was performed 7 days after the last treatment. Overall bacterial growth was reduced in every group after the treatment (p < 0.05), irrespective of the therapy [Ozone: 4/9 (positive culture after treatment/number of mares), TMS: 3/10 and Air: 6/10; p > 0.05]. However, Ozone and TMS (p < 0.05) were more effective in reducing growth of gram-negative bacteria as compared to Air (p > 0.05). No effects on the number of polymorphonuclear granulocytes (cytology) were observed (p > 0.05). In conclusion, trimethoprim-sulfadimethoxine and intrauterine ozone insufflation are safe treatment options for bacterial endometritis in mares but the efficacy of both treatments in reducing bacterial growth did not result in a complete absence of intrauterine bacterial growth.

Abstract Background Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection in humans and animals may lead to severe symptoms in the offspring, especially in the brain. A suitable animal model for human congenital toxoplasmosis is currently lacking. The aim of this study is to establish and validate the guinea pig as a model for human congenital toxoplasmosis by investigating the impact of the T. gondii infection dose, the duration of infection and the gestational stage at infection on the seroconversion, survival rate of dams, fate of the offspring, T. gondii DNA loads in various offspring tissues and organs and the integrity of the offspring brain. Methods Pregnant guinea pigs were infected with three different doses (10, 100, 500 oocysts) of T. gondii strain ME49 at three different time points during gestation (15, 30, 48 days post-conception). Serum of dams was tested for the presence of T. gondii antibodies using immunoblotting. T. gondii DNA levels in the dam and offspring were determined by qPCR. Offspring brains were examined histologically. Results We found the survival rate of dams and fate of the offspring to be highly dependent on the T. gondii infection dose with an inoculation of 500 oocysts ending lethally for all respective offspring. Moreover, both parameters differ depending on the gestational stage at infection with infection in the first and third trimester of gestation resulting in a high offspring mortality rate. The duration of infection was found to substantially impact the seroconversion rate of dams with the probability of seroconversion exceeding 50% after day 20 post-infection. Furthermore, the infection duration of dams influenced the T. gondii DNA loads in the offspring and the integrity of offspring brain. Highest DNA levels were found in the offspring brain of dams infected for ≥ 34 days. Conclusion This study contributes to establishing the guinea pig as a suitable model for human congenital toxoplasmosis and thus lays the foundation for using the guinea pig as a suitable animal model to study scientific questions of high topicality and clinical significance, which address the pathogenesis, diagnosis, therapy and prognosis of congenital toxoplasmosis. Graphical abstract

Bovine tuberculosis (bTB) not only poses a zoonotic threat to humans but also has a significant economic impact on livestock production in many areas of the world. Effective vaccines for humans, livestock, and wildlife are highly desirable to control tuberculosis. Suitable large animal models are indispensable for meaningful assessment of vaccine candidates. Here, we describe the refinement of an animal model for bTB in goats. Intrabronchial inoculation procedure via video-guided endoscopy in anesthetized animals, collection of lungs after intratracheal fixation in situ, and imaging of lungs by computed tomography (CT) were established in three goats using barium sulfate as surrogate inoculum. For subsequent infection experiments, four goats were infected with 4.7 × 102 colony-forming units of M. bovis by intrabronchial inoculation using video-guided endoscopy with spray catheters. Defined amounts of inoculum were deposited at five sites per lung. Four age-matched goats were mock-inoculated. None of the goats developed clinical signs until they were euthanized 5 months post infection, but simultaneous skin testing confirmed bTB infection in all goats inoculated with M. bovis. In tissues collected at necropsy, M. bovis was consistently re-isolated from granulomas in lymph nodes, draining the lungs of all the goats infected with M. bovis. Further dissemination was observed in one goat only. Pulmonary lesions were quantified by CT and digital 2D radiography (DR). CT revealed mineralized lesions in all the infected goats ranging from 10 mm in diameter. Small lesions

The current article served to provide the most up-to-date information regarding the causes of keel bone fracture. Although elevated and sustained egg production is likely a major contributing factor toward fractures, new information resulting from the development of novel methodologies suggests complementary causes that should be investigated. We identified 4 broad areas that could explain variation and increased fractures independent of or complementing elevated and sustained egg production: the age at first egg, late ossification of the keel, predisposing bone diseases, and inactivity leading to poor bone health. We also specified several topics that future research should target, which include continued efforts to link egg production and bone health, examination of noncommercial aves and traditional breeds, manipulating of age at first egg, a detailed histological and structural analysis of the keel, assessment of prefracture bone condition, and the relationship between individual activity patterns and bone health.

Abstract An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

ABSTRACTIn 2016/2017, a severe epidemic of HPAIV H5N8 clade 2.3.4.4 group B (H5N8B) affected Europe. To analyse the role of mallards in the spatiotemporal dynamics of global HPAIV H5N8B dispersal, mallards (Anas platyrhynchos), naturally exposed to various AIV and therefore seropositive, were challenged with H5N8B. All experiments were controlled by infection and co-housing of seronegative juvenile Pekin ducklings. All ducks that survived the first infection were re-challenged 21 dpi with the homologous H5N8B strain. After the first H5N8B infection, seropositive mallards showed only mild clinical symptoms. Moderate to low viral shedding, occurring particularly from the oropharynx and lasting for 7 days maximum, led to severe clinical disease of all contact ducklings. All challenged seronegative Pekin ducks and contact ducklings died or had to be euthanized. H5-specific antibodies were detected in surviving birds within 2 weeks. Virus and viral RNA could be isolated from several water samples until 6 and 9 dpi, respectively. Conversely, upon re-infection with homologous H5N8B neither inoculated nor contact ducklings showed any clinical symptoms, nor was an antibody titer increase of seropositive mallards or any seroconversion of contact ducklings observed. Mallard ducks naturally pre-exposed to LPAIV can play a role as a clinically unsuspicious virus reservoir for H5N8B effective in virus transmission. Mallards with homologous immunity did not contribute to virus transmission.

Rift Valley fever phlebovirus (RVFV) causes Rift Valley fever (RVF), an emerging zoonotic disease that causes abortion storms and high mortality rates in young ruminants as well as severe or even lethal complications in a subset of human patients. This study investigates the pathomechanism of intranuclear inclusion body formation in severe RVF in a mouse model. Liver samples from immunocompetent mice infected with virulent RVFV 35/74, and immunodeficient knockout mice that lack interferon type I receptor expression and were infected with attenuated RVFV MP12 were compared to livers from uninfected controls using histopathology and immunohistochemistry for RVFV nucleoprotein, non-structural protein S (NSs) and pro-apoptotic active caspase-3. Histopathology of the livers showed virus-induced, severe hepatic necrosis in both mouse strains. However, immunohistochemistry and immunofluorescence revealed eosinophilic, comma-shaped, intranuclear inclusions and an intranuclear (co-)localization of RVFV NSs and active caspase-3 only in 35/74-infected immunocompetent mice, but not in MP12-infected immunodeficient mice. These results suggest that intranuclear accumulation of RVFV 35/74 NSs is involved in nuclear translocation of active caspase-3, and that nuclear NSs and active caspase-3 are involved in the formation of the light microscopically visible inclusion bodies.

We genetically characterized highly pathogenic avian influenza virus A(H5N6) clade 2.3.4.4b isolates found in Germany in 2017–2018 and assessed pathogenicity of representative H5N5 and H5N6 viruses in ferrets. These viruses had low pathogenicity; however, continued characterization of related isolates is warranted because of their high potential for reassortment.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus that causes severe disease in humans and ruminants. The infection is characterized by abortions in pregnant animals, high mortality in neonates as well as febrile illness in humans that develop in 1% of cases encephalitis or hemorrhagic fever. There is presently no specific antiviral treatment for RVFV infection available. In this study, two monoclonal antibodies (mAbs), raised against glycoprotein Gn, were applied in a therapeutic study. Treatment of RVFV infected mice with neutralizing mAb Gn3 alone at two different time points (30 minutes before or 30 minutes after virus challenge) showed only moderate efficacy of about 58.3% survival in both applications. However, a combination therapy together with non-neutralizing mAb Gn32 demonstrated complete protection (100% survival) when applied 30 minutes after the lethal challenge dose. The increase of mAb efficacy is probably based on cooperative neutralization effects. These data suggest that a combination therapy with mAbs Gn3 and Gn32 could be an effective treatment option against RVFV infection.

Rift Valley fever (RVF) is a zoonotic disease caused by RVF Phlebovirus (RVFV). The RVFV MP-12 vaccine strain is known to exhibit residual virulence in the case of a deficient interferon type 1 response. The hypothesis of this study is that virus replication and severity of lesions induced by the MP-12 strain in immunocompromised mice depend on the specific function of the disturbed pathway. Therefore, 10 strains of mice with deficient innate immunity (B6-IFNARtmAgt, C.129S7(B6)-Ifngtm1Ts/J, B6-TLR3tm1Flv, B6-TLR7tm1Aki, NOD/ShiLtJ), helper T-cell- (CD4tm1Mak), cytotoxic T-cell- (CD8atm1Mak), B-cell- (Igh-Jtm1DhuN?+N2), combined T- and B-cell- (NU/J) and combined T-, B-, natural killer (NK) cell- and macrophage-mediated immunity (NOD.Cg-PrkdcscidIl2rgtm1WjI/SzJ (NSG) mice) were subcutaneously infected with RVFV MP-12. B6-IFNARtmAgt mice were the only strain to develop fatal disease due to RVFV-induced severe hepatocellular necrosis and apoptosis. Notably, no clinical disease and only mild multifocal hepatocellular necrosis and apoptosis were observed in NSG mice, while immunohistochemistry detected the RVFV antigen in the liver and the brain. No or low virus expression and no lesions were observed in the other mouse strains. Conclusively, the interferon type 1 response is essential for early control of RVFV replication and disease, whereas functional NK cells, macrophages and lymphocytes are essential for virus clearance.

Abstract Highly pathogenic avian influenza virus (HPAIV) infection in poultry caused devastating mortality and economic losses. HPAIV of subtypes H5 and H7 emerge from precursor viruses of low pathogenicity (LP) by spontaneous mutation associated with a shift in the susceptibility of the endoproteolytic cleavage site of the viral hemagglutinin protein from trypsin- to furin-like proteases. A recently described natural pair of LP/HP H7N7 viruses derived from two spatio-temporally linked outbreaks in layer chickens was used to study how a minority of mutated HP virions after de novo generation in a single host might gain primacy. Co-infection experiments in embryonated eggs and in chickens were conducted to investigate amplification, spread and transmissionof HPAIV within a poultry population that experiences concurrent infection by an antigenically identical LP precursor virus. Simultaneous LPAIV co-infection (inoculum dose of 106 egg-infectious dose 50% endpoint (EID50)/0.5 mL) withincreasing titers of HPAIV from 101 to 105.7 EID50/0.5 mL) had a significant impeding impact on HP H7 replication, viral excretion kinetics, clinical signs and histopathological lesions (in vivo) and on embryo mortality (in ovo). LP/HP co-infected chickens required a hundredfold higher virus dose (HPAIV inoculum of 105 EID50) compared to HPAIV mono-infection (HPAIV inoculum of 103 EID50) to develop overt clinical signs, mortality and virus spread to uninfected sentinels. Escape and spread of HP phenotypes after de novo generation in an index host may therefore be highly precarious due to significant competition with co-circulating LP precursor virus.

Abstract In classical bovine spongiform encephalopathy (C-BSE), an orally acquired prion disease of cattle, the ileal Peyer’s patch (IPP) represents the main entry port for the BSE agent. In earlier C-BSE pathogenesis studies, cattle at 4–6 months of age were orally challenged, while there are strong indications that the risk of infection is highest in young animals. In the present study, unweaned calves aged 4–6 weeks were orally challenged to determine the earliest time point at which newly formed PrPBSE and BSE infectivity are detectable in the IPP. For this purpose, calves were culled 1 week as well as 2, 4, 6 and 8 months post-infection (mpi) and IPPs were examined for BSE infectivity using a bovine PrP transgenic mouse bioassay, and for PrPBSE by immunohistochemistry (IHC) and protein misfolding cyclic amplification (PMCA) assays. For the first time, BSE prions were detected in the IPP as early as 2 mpi by transgenic mouse bioassay and PMCA and 4 mpi by IHC in the follicular dendritic cells (FDCs) of the IPP follicles. These data indicate that BSE prions propagate in the IPP of unweaned calves within 2 months of oral uptake of the agent.

Pigs are anatomically, genetically and physiologically comparable to humans and represent a natural host for influenza A virus (IAV) infections. Thus, pigs may represent a relevant biomedical model for human IAV infections. We set out to investigate the systemic as well as the local immune response in pigs upon two subsequent intranasal infections with IAV H1N1pdm09. We detected decreasing numbers of peripheral blood lymphocytes after the first infection. The simultaneous increase in the frequencies of proliferating cells correlated with an increase in infiltrating leukocytes in the lung. Enhanced perforin expression in αβ and γδ T cells in the respiratory tract indicated a cytotoxic T cell response restricted to the route of virus entry such as the nose, the lung and the bronchoalveolar lavage. Simultaneously, increasing frequencies of CD8αα expressing αβ T cells were observed rapidly after the first infection, which may have inhibited uncontrolled inflammation in the respiratory tract. Taking together, the results of this study demonstrate that experimental IAV infection in pigs mimics major characteristics of human seasonal IAV infections.

The main findings of the post-mortem examination of poultry infected with highly pathogenic avian influenza viruses (HPAIV) include necrotizing inflammation and viral antigen in multiple organs. The lesion profile displays marked variability, depending on viral subtype, strain, and host species. Therefore, in this study, a semiquantitative scoring system was developed to compare histopathological findings across a wide range of study conditions. Briefly, the severity of necrotizing lesions in brain, heart, lung, liver, kidney, pancreas, and/or lymphocytic depletion in the spleen is scored on an ordinal four-step scale (0 = unchanged, 1 = mild, 2 = moderate, 3 = severe), and the distribution of the viral antigen in parenchymal and endothelial cells is evaluated on a four-step scale (0 = none, 1 = focal, 2 = multifocal, 3 = diffuse). These scores are used for a meta-analysis of experimental infections with H7N7 and H5N8 (clade 2.3.4.4b) HPAIV in chickens, turkeys, and ducks. The meta-analysis highlights the rather unique endotheliotropism of these HPAIV in chickens and a more severe necrotizing encephalitis in H7N7-HPAIV-infected turkeys. In conclusion, the proposed scoring system can be used to condensate HPAIV-typical pathohistological findings into semiquantitative data, thus enabling systematic phenotyping of virus strains and their tissue tropism.

Rift Valley fever phlebovirus (RVFV) is an arthropod-borne zoonotic pathogen, which is endemic in Africa, causing large epidemics, characterized by severe diseases in ruminants but also in humans. As in vitro and field investigations proposed amphibians and reptiles to potentially play a role in the enzootic amplification of the virus, we experimentally infected African common toads and common agamas with two RVFV strains. Lymph or sera, as well as oral, cutaneous and anal swabs were collected from the challenged animals to investigate seroconversion, viremia and virus shedding. Furthermore, groups of animals were euthanized 3, 10 and 21 days post-infection (dpi) to examine viral loads in different tissues during the infection. Our data show for the first time that toads are refractory to RVFV infection, showing neither seroconversion, viremia, shedding nor tissue manifestation. In contrast, all agamas challenged with the RVFV strain ZH501 carried virus genomes in the spleens at 3 dpi, but the animals displayed neither viremia nor virus shedding. In conclusion, the results of this study indicate that amphibians are not susceptible and reptiles are only susceptible to a low extent to RVFV, indicating that both species play, if at all, rather a subordinate role in the RVF virus ecology.

In 2012 and 2013, the genomic sequences of two novel influenza A virus (IAV) subtypes, designated H17N10 and H18N11, were identified via next-generation sequencing in the feces of the little yellow-shouldered fruit bat (Sturnira lilium) and the flat-faced fruit-eating bat (Artibeus planirostris), respectively. The pathogenesis caused by these viruses in their respective host species is currently insufficiently understood, which is primarily due to the inability to obtain and keep these bat species under appropriate environmental and biosafety conditions. Seba’s short-tailed bats (Carollia perspicillata), in contrast, are close relatives and a natural H18N11 reservoir species, with the advantage of established animal husbandry conditions in academic research. To study viral pathogenesis in more detail, we here oro-nasally inoculated Seba’s short-tailed bats with the bat IAV H18N11 subtype. Following inoculation, bats appeared clinically healthy, but the histologic examination of tissues revealed a mild necrotizing rhinitis. Consistently, IAV-matrix protein and H18-RNA positive cells were seen in lesioned respiratory and olfactory nasal epithelia, as well as in intestinal tissues. A RT-qPCR analysis confirmed viral replication in the conchae and intestines as well as the presence of viral RNA in the excreted feces, without horizontal transmission to naïve contact animals. Moreover, all inoculated animals seroconverted with low titers of neutralizing antibodies.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 is endemic in poultry in Egypt where the highest number of human infections worldwide was reported. During the last 12 years the Egyptian A/H5N1 evolved into several genotypes. In 2007-2014 vaccinated poultry suffered from antigenic drift variants of clade 2.2.1.1 and in 2014/2015 an unprecedented upsurge of A/H5N1 clade 2.2.1.2 occurred in poultry and humans. Factors contributing to the endemicity or re-emergence of A/H5N1 in poultry in Egypt remain unclear. Here, three potential factors were studied: climatic factors (temperature, relative humidity, and wind speed), biological fitness in vitro, and pathogenicity in domestic Pekin and Muscovy ducks. Statistical analyses using negative binomial regression models indicated that ambient temperature in winter months influenced the spread of A/H5N1 in different geographic areas analyzed in this study. In vitro, at 4 and 56°C 2.2.1.1 and recent 2.2.1.2 viruses were more stable than other viruses used in this study. Further, Pekin ducks were more resistant than Muscovy ducks and the viruses were excreted for up to 2 weeks post-infection assuming a strong role as a reservoir. Taken together, ambient temperature in winter months potentially contributes to increasing outbreaks in some regions in Egypt. Heat stability of clade 2.2.1.1 and recent 2.2.1.2 viruses probably favors their persistence at elevated temperatures. Importantly, asymptomatically infected Pekin ducks may play an important role in the spread of avian and human-like A/H5N1 in Egypt. Therefore, control measures including targeted surveillance and culling of silently infected Pekin ducks should be considered.

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research.

Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread.

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease.

Rift Valley fever phlebovirus (RVFV), the causative agent of an emerging zoonotic disease in Africa and Arabia, can infect a variety of species, predominantly ruminants, camelids, and humans. While clinical symptoms are mostly absent in adult ruminants and camelids, RVFV infection may lead to a serious, sometimes fatal disease in humans. Virus transmissions between individuals and between species mainly occur through mosquito bites, but direct or even indirect contact with infectious materials may also result in infection. Although the main reservoir of the virus is not yet identified, small mammals such as rodents and bats may act as amplifying hosts. We therefore inoculated Rousettus aegyptiacus fruit bats that are abundant in northern Africa with the vaccine strain MP-12, in order to elucidate the general competence of this species for virus propagation and transmission. We were able to detect the RVFV genome in the spleen of each of these animals, and re-isolated the virus from the spleen and liver of some animals. Moreover, we were able to identify the Gc RVFV surface antigen in mild subacute multifocal necrotizing hepatic lesions of one bat which was sacrificed 7 days post exposure. These findings demonstrate that Rousettus aegyptiacus fruit bats can propagate RVFV.

In contrast to previous incursions of highly pathogenic avian influenza (HPAIV) H5 viruses, H5N8 clade 2.3.4.4b viruses caused numerous cases of lethal infections in white-tailed sea eagles (Haliaeetus albicilla) affecting mainly young eagles (younger than five years of age) in Germany during winter 2016/2017. Until April 2017, 17 HPAIV H5N8-positive white-tailed sea eagles had been detected (three found alive and 14 carcasses) by real-time RT-PCR and partial nucleotide sequence analyses. Severe neurological clinical signs were noticed which were corroborated by immunohistopathology revealing mild to moderate, oligo- to multifocal necrotizing virus-induced polioencephalitis. Lethal lead (Pb) concentrations, a main factor of mortality in sea eagles in previous years, could be ruled out by atomic absorption spectrometry. HPAIV H5 clade 2.3.4.4b reportedly is the first highly pathogenic influenza virus known to induce fatal disease in European white-tailed see eagles. This virus strain may become a new health threat to a highly protected species across its distribution range in Eurasia. Positive cloacal swabs suggest that eagles can spread the virus with their faeces.

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms "viral replication" and "humoral immune response" as well as down-regulated genes functionally related to "metabolite and energy generation".

BACKGROUND: Multiple microarray analyses of multiple sclerosis (MS) and its experimental models have been published in the last years. OBJECTIVE: Meta-analyses integrate the information from multiple studies and are suggested to be a powerful approach in detecting highly relevant and commonly affected pathways. DATA SOURCES: ArrayExpress, Gene Expression Omnibus and PubMed databases were screened for microarray gene expression profiling studies of MS and its experimental animal models. STUDY ELIGIBILITY CRITERIA: Studies comparing central nervous system (CNS) samples of diseased versus healthy individuals with n >1 per group and publically available raw data were selected. MATERIAL AND METHODS: Included conditions for re-analysis of differentially expressed genes (DEGs) were MS, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in rats, proteolipid protein-induced EAE in mice, Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), and a transgenic tumor necrosis factor-overexpressing mouse model (TNFtg). Since solely a single MS raw data set fulfilled the inclusion criteria, a merged list containing the DEGs from two MS-studies was additionally included. Cross-study analysis was performed employing list comparisons of DEGs and alternatively Gene Set Enrichment Analysis (GSEA). RESULTS: The intersection of DEGs in MS, EAE, TMEV-IDD, and TNFtg contained 12 genes related to macrophage functions. The intersection of EAE, TMEV-IDD and TNFtg comprised 40 DEGs, functionally related to positive regulation of immune response. Over and above, GSEA identified substantially more differentially regulated pathways including coagulation and JAK/STAT-signaling. CONCLUSION: A meta-analysis based on a simple comparison of DEGs is over-conservative. In contrast, the more experimental GSEA approach identified both, a priori anticipated as well as promising new candidate pathways.

Understanding the basic mechanisms that control CNS remyelination is of direct clinical relevance. Suitable model systems include the analysis of naturally occurring and genetically generated mouse mutants and the transplantation of oligodendrocyte precursor cells (OPCs) following experimental demyelination. However, aforementioned studies were exclusively carried out in rats and little is known about the in vivo behavior of transplanted murine OPCs. Therefore in the present study, we (i) established a model of ethidium bromide-induced demyelination of the caudal cerebellar peduncle (CCP) in the adult mouse and (ii) studied the distribution and marker expression of the murine OPC line BO-1 expressing the enhanced green fluorescent protein (eGFP) 10 and 17 days after stereotaxic implantation. Injection of ethidium bromide (0.025%) in the CCP resulted in a severe loss of myelin, marked astrogliosis, and mild to moderate axonal alterations. Transplanted cells formed an invasive and liquorogenic metastasizing tumor, classified as murine giant cell glioblastoma. Transplanted BO-1 cells displayed substantially reduced CNPase expression as compared to their in vitro phenotype, low levels of MBP and GFAP, prominent upregulation of NG2, PDGFRα, nuclear p53, and an unaltered expression of signal transducer and activator of transcription (STAT)-3. Summarized environmental signaling in the brain stem was not sufficient to trigger oligodendrocytic differentiation of BO-1 cells and seemed to block CNPase expression. Moreover, the lack of the remyelinating capacity was associated with tumor formation indicating that BO-1 cells may serve as a versatile experimental model to study tumorigenesis of glial tumors.