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Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

An 11-year-old, English cocker spaniel was presented with subacute progressive signs of vestibular ataxia, tetraparesis, left-sided proprioceptive deficits, positional ventrolateral strabismus of the right eye and a right-sided menace deficit. Magnetic resonance imaging of the cranium and cranial cervical spinal cord revealed multifocal T2 hyper-/T1-hypointense intradural lesions with dural tail signs and intra-axial and intramedullary extension. Medical treatment resulted in initial improvement before deterioration was noticed. Cytological examination results of computed tomography-guided fine-needle aspiration biopsy of the C1–C2 lesion were consistent with mesenchymal neoplasia. Three days later, after progressive clinical deterioration, euthanasia was performed. Postmortem examination and subsequent histological examination of the brainstem and spinal cord revealed multifocal, strongly infiltrative growth of neoplastic cells with areas of pseudo-rosette formation by cylindrical neoplastic cells with moderately large, oval nuclei in addition to areas of spindle-shaped neoplastic cells with meningothelial whorls. The final diagnosis was a papillary (grade III) meningioma with multifocal leptomeningeal spread.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV), a coronavirus of domestic fowl. IB is a major concern in the poultry industry, causing worldwide economic losses through decreased egg production and quality and by increasing the chicken's susceptibility for secondary bacterial infections, particularly Escherichia coli. In this study, the anti-IBV activity of cathelicidins, small antimicrobial peptides of the innate immune system was investigated. The cell culture adapted (nonvirulent) IBV strain Beaudette was effectively inhibited by the human cathelicidin LL-37 in bovine hamster kidney-21 cells at nontoxic concentrations. The peptide needed to be present during virus inoculation to effectively inhibit the infection of IBV-Beaudette, indicating that LL-37 likely bound viral particles. However, no clear morphological changes in the IBV virion upon binding were observed by electron microscopy. In this cell culture model, chicken cathelicidins (CATH1-3) were inactive against IBV-Beaudette. In contrast, in multicellular infection models using the virulent IBV-M41 strain the activities of human and chicken cathelicidins were different. In particular, upon inoculation of 10-day-old embryonic eggs with IBV-M41, CATH-2 reduced the viral load to a higher extend than LL-37. Similarly, viral infection of chicken tracheal organ cultures with IBV-M41 was significantly reduced in the presence of CATH-2 but not LL-37. These results indicate a potential antiviral role for CATH-2 upon IBV infection in vivo.

Humans impact natural systems at an unprecedented rate. The North Sea is one of the regions in the world with the highest levels of anthropogenic activity. Here, the harbor porpoise (Phocoena phocoena) is an abundant species and is often regarded as an ecosystem sentinel. A post-mortem surveillance program was established in the Netherlands aimed at increasing knowledge of the effects of human activities on harbor porpoises. In this study, we describe the pathological findings related to anthropogenic and natural causes of death categories in 612 harbor porpoises that stranded between 2008 and 2019, and assess their relations to age, sex, season, and location. The largest anthropogenic category was bycatch (17%), with mainly juveniles affected and peak periods in March and September–October. Other, infrequently diagnosed anthropogenic causes of death were trauma (4%), largely most likely due to ship collisions, and marine debris ingestion and entanglement (0.3%). The risk of dying from anthropogenic causes was highest for juveniles. Lesions compatible with noise-induced hearing loss were investigated in carcasses which were fresh enough to do so (n = 50), with lesions apparent in two porpoises. Non-direct human-induced threats included infectious diseases, which were by far the largest cause of death category (32%), and affected mainly adults. Also, gray seal (Halichoerus grypus) attacks were a frequently assigned cause of death category (24%). There were more acute predation cases in the earlier study years, while porpoises with lesions that suggested escape from gray seal attacks were diagnosed more recently, which could suggest that porpoises adapted to this threat. Our study contributes to understanding porpoise health in response to persisting, new, emerging, and cumulative threats. Building up such knowledge is crucial for conservation management of this protected species.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the disease with the highest economic loss for the broiler industry. However, studies focusing on the prevalence and population structure of APEC in the broiler production pyramid are scarce. Here, we used genotyping and serotyping data to elucidate the APEC population structure and its changes in different broiler production stages along with whole-genome sequencing (WGS) in a subset of APEC isolates to determine transmission patterns amongst dominant APEC sequence types (STs) and characterize them in detail. Comparison of genotypes encountered in both APEC and avian fecal E. coli (AFEC) provided further insights. Overall, APEC-related mortality, as the proportion of the total sampled mortality in the broiler production, was high (35%), while phylogroup C and serogroup O78 were predominant amongst APEC isolates. We found a low (34.0%) and high (53.3%) incidence of colibacillosis in chicks and end-cycle broilers, respectively, which may be related to a shift in APEC genotypes, suggesting a trend from commensalism to pathogenicity across different broiler production stages. Despite considerable APEC genotypic diversity, there was substantial genotype overlap (40.9%, overall) over the production stages and convergence of STs to the four clusters. Within these clusters, WGS data provided evidence of clonal transmission events and revealed an enriched virulence and resistance APEC repertoire. More specifically, sequenced APEC were assigned to defined pathotypes based on their virulence gene content while the majority (86%) was genotypically multi-drug resistant. Interestingly, WGS-based phylogeny showed that a subset of APEC, which are cephalosporin-resistant, may originate directly from cephalosporin-resistant AFEC. Finally, exploration of the APEC plasmidome indicated that the small fraction of the APEC virulome carried by IncF plasmids is pivotal for the manifestation of the APEC pathotype; thus, plasmid exchange can promote pathogenicity in strains that are at the edge of the commensal and pathogenic states.

Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (Pwald = 4.83x10-9) and 19 (Pwald = 2.25x10-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility., Author summary We have identified two regions of the canine genome that explain a striking 35% of risk for developing histiocytic sarcoma in FCRs. The disease is uniformly lethal, affects 20% of FCRs, and parallels a cancer of the same name in humans. Both regions harbor genes involved in cell migration and cancer-related pathways. The first includes variants in regulatory regions at the tumor suppressor PIK3R6 locus that are strongly associated with histiocytic sarcoma and likely confer risk for other hematopoietic cancers. FCRs with risk alleles at the second locus demonstrate increased expression of TNFAIP6, which correlates with poor prognosis in multiple human cancers. In identifying genomic differences between affected and unaffected dogs, we advance our understanding of both canine and human health biology and set the stage for the development of diagnostic and therapeutic strategies.

Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding. Ten single Asn–to–Ala substitutions at the predicted N-glycosylation sites of the M41–RBD were evaluated along with two control Val–to–Ala substitutions. CD analysis revealed that the secondary structure of all variants was retained compared with the unmodified M41–RBD construct. Six of the 10 glycosylation variants lost binding to chicken trachea tissue and an ELISA-presented α2,3-linked sialic acid oligosaccharide ligand. LC/MSE glycomics analysis revealed that glycosylation sites have specific proportions of N-glycan subtypes. Overall, the glycosylation patterns of most variant RBDs were highly similar to those of the unmodified M41–RBD construct. In silico docking experiments with the recently published cryo-EM structure of the M41 IBV spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. Combined with the results of previous array studies, the glycosylation and mutational analyses presented here suggest a unique glycosylation-dependent binding modality for the M41 spike protein.

BACKGROUND: Chronic hepatitis (CH) in dogs is common and has the tendency to progress to liver cirrhosis (LC). Circulating microRNAs might have the potential as markers for disease progression. OBJECTIVES: To investigate whether concentration of specific microRNAs in serum correlate with the stage and grade of CH in Labrador Retrievers. ANIMALS: Twenty-two Labrador Retrievers with histological CH (n = 8), LC (n = 7), and normal liver (NL, n = 7). METHODS: In this retrospective study, serum concentrations of miR-122, miR-29a, miR-133a, miR-181b, and miR-17-5p were measured by quantitative real-time PCR and evaluated using univariate linear regression in dogs. A multivariate model was fit including the grade of hepatitis and the stage of fibrosis. RESULTS: Of the 5 microRNAs, only circulating miR-122 and miR-29a were significantly associated with the grade of hepatitis and the stage of fibrosis. A positive correlation was identified between the grade of hepatitis with miR-122 (rs = 0.79, P

Antimicrobials are commonly used in conventional livestock production and manure is widely applied to agricultural lands as fertilizer. This practice raises questions regarding the effects of fertilization on (i) soil microbiota composition and (ii) spread of antimicrobials and antimicrobial resistance (AMR) in the environment. This study was conducted in a high-density farming area of Northern Italy and aimed at assessing the impact of (dairy cattle, chickens and swine) manure application on soil microbiome, antimicrobial concentrations and antimicrobial resistance gene (ARG) abundance. We found the microbial community composition in manure to be different and less diverse than in soil, with manure application altering only marginally the soil microbiome. Exceptions were the phyla Firmicutes, Tenericutes and Cloacimonetes, which significantly enriched in fertilized soil. Of the antimicrobials investigated, only flumequine concentrations increased after manure application, albeit non-significantly. ARGs were more abundant in manure, with ermA, ermB, blaOXA-1 and oqxA being significantly enriched in fertilized soil. Positive correlations between oqxA and qnrS abundances and flumequine concentrations were observed, together with the co-occurrence of some ARGs and microbial taxa (e.g. oqxA correlated with Acidobacteria and Gemmatimonadetes). This study showed that manure application has little effect on soil microbiome but may contribute to the dissemination of specific ARGs into the environment. Moreover, flumequine residues seem to enhance the emergence of oqxA and qnrS in soil.

Objectives To describe the signalment, ophthalmic examination findings, and follow-up of dogs affected with a previously unreported retrocorneal pigmentary lesion. Materials and methods Retrospective record evaluation spanning 2009-2019. Results Retrocorneal pigmentary lesions were described in 34 patients (46 eyes). German Shepherds (n = 7), Jack Russel terriers (n = 5), and terrier crosses (n = 4) made up 16/34 (47.1%) of the cases. The mean age was 13.5 years (range 1.4-14.2 years), and 16/30 (53.3%) dogs were female. Most dogs were affected unilaterally (22/34 (64.7%)), the others bilaterally, and 5/34 (14.7%) were referred for it while the others were incidentally diagnosed. The lesions affected the ventral, peripheral, inner cornea and had a round/undulated leading edge. The number of corneal clock hours affected was known for 41/46 (89.1%) eyes and involved 1-3 clock hours in 32/41 (78.1%) eyes, 4-6 in 6/41 (14.6%), 7-9 in 2/41 (4.9%), and 10 in 1/41 (2.4%). The central cornea was affected in 9/46 (19.6%) eyes, and in 5/9 (55.6%), the median corneal clarity score was G2 (scale: G0-G4). The commonest additional findings included free-floating uveal cysts (11/34 dogs, 32.4%), cataracts (6/34 dogs, 17.6%), and primary glaucoma (5/34 dogs, 14.7%). Gonioscopy was available in 16/34 (47.1%) dogs and was normal except in primary glaucoma cases. Follow-up was documented in 13/34 (38.2%) dogs with a mean follow-up of 17 months (range: 5-26 months). Lesion progression was documented in 6/13 (46.2%) dogs. Conclusions Retrocorneal pigmentation occurs as a slowly progressive lesion of older dogs that could impact vision. Histological studies of affected eyes are warranted.

Avian infectious bronchitis virus (IBV) is a coronavirus with great economic impact on the poultry industry, causing an acute and highly contagious disease in chickens that primarily affects the respiratory and reproductive systems. The cellular regulation of IBV pathogenesis and the host immune responses involved remain to be fully elucidated. MicroRNAs (miRNAs) have emerged as a class of crucial regulators of numerous cellular processes, including responses to viral infections. Here, we employed a high-throughput sequencing approach to analyze the miRNA composition of the spleen and the lungs of chicken embryos upon IBV infection. Compared to healthy chicken embryos, 13 and six miRNAs were upregulated in the spleen and the lungs, respectively, all predicted to influence viral transcription, cytokine production, and lymphocyte functioning. Subsequent downregulation of NFATC3, NFAT5, SPPL3, and TGFB2 genes in particular was observed only in the spleen, demonstrating the biological functionality of the miRNAs in this lymphoid organ. This is the first study that describes the modulation of miRNAs and the related host immune factors by IBV in chicken embryos. Our data provide novel insight into complex virus-host interactions and specifically highlight components that could affect the host's immune response to IBV infection.

Highlights • IBV-QX vaccine and virulent progenitor have comparable RNA titers in the trachea. • IBV-QX vaccine shows delayed induction of lesions in the respiratory tract. • IBV-QX vaccine has reduced ability to disseminate from the site of inoculation to other target tissues. • IBV-QX vaccine displays no nephropathogenicity in SPF broilers. • Ascending virus transport seems to contribute to kidney infection., Infectious bronchitis (IB) is a highly contagious respiratory disease of poultry, caused by the avian coronavirus infectious bronchitis virus (IBV). Currently, one of the most relevant genotypes circulating worldwide is IBV-QX (GI-19), for which vaccines have been developed by passaging virulent QX strains in embryonated chicken eggs. Here we explored the attenuated phenotype of a commercially available QX live vaccine, IB Primo QX, in specific pathogens free broilers. At hatch, birds were inoculated with QX vaccine or its virulent progenitor IBV-D388, and postmortem swabs and tissues were collected each day up to eight days post infection to assess viral replication and morphological changes. In the trachea, viral RNA replication and protein expression were comparable in both groups. Both viruses induced morphologically comparable lesions in the trachea, albeit with a short delay in the vaccinated birds. In contrast, in the kidney, QX vaccine viral RNA was nearly absent, which coincided with the lack of any morphological changes in this organ. This was in contrast to high viral RNA titers and abundant lesions in the kidney after IBV D388 infection. Furthermore, QX vaccine showed reduced ability to reach and replicate in conjunctivae and intestines including cloaca, resulting in significantly lower titers and delayed protein expression, respectively. Nephropathogenic IBVs might reach the kidney also via an ascending route from the cloaca, based on our observation that viral RNA was detected in the cloaca one day before detection in the kidney. In the kidney distal tubular segments, collecting ducts and ureter were positive for viral antigen. Taken together, the attenuated phenotype of QX vaccine seems to rely on slower dissemination and lower replication in target tissues other than the site of inoculation.

Finch trichomonosis in Europe is caused by a Trichomonas gallinae subtype A1 strain, considered to be clonal because lacking genetic heterogeneity in partial genotyping. The disease recently emerged and has been associated with a 66% reduction of the British breeding greenfinch (Chloris chloris) population. In contrast, in the Netherlands, where trichomonosis was detected in 2009, the breeding greenfinch population continued to grow in subsequent years. This study aimed to elucidate whether this discrepancy in population trends is because Trichomonas infection in Dutch greenfinches is associated with less severe disease, i.e., disease being less fatal. Therefore, it characterized and quantified trichomonosis in a convenience sample of greenfinches found dead and examined post-mortem between 2009 and 2017 and compared results to published data from Great Britain. Trichomonads were detected by cytology, histology, or culture in 95/101 greenfinches. The birds with trichomonads all had microscopic lesions in the upper digestive tract consistent with trichomonosis, indicating the trichomonads caused disease. The occurrence of significant lesions due to other causes was low. Some greenfinches with trichomonosis showed no macroscopic lesions. These birds showed significantly less ulceration of the mucosa and less extensive heterophil infiltration, but extent of macrophage infiltration and presence of bacteria was similar to that of birds with macroscopic lesions, and significant lesions due to other causes were equally rare. Therefore, trichomonosis was considered similarly fatal in both groups. The frequency of fatal trichomonosis in the Dutch greenfinches did not differ significantly from that reported from Great Britain. Partial genotyping of the ITS1-5,8S-ITS2 and Fe-hydrogenase regions of T. gallinae was performed to detect genetic heterogeneity, that could indicate the presence of other, possibly less virulent, strains. In 60/63 samples there was full alignment of sequences with the clonal strain of T. gallinae subtype A1. The remaining three samples had the same single synonymous nucleotide difference in the Fe-hydrogenase region; however, pathology is these three was identical to the others. Collectively, the results provide no clear evidence for less severe disease as explanation for the discrepancy in census data trends. We conclude that trichomonosis is a threat concealed in Dutch breeding greenfinch census data.

Highlights • Accessory genes 3 and 5 are not essential for IBV’s replication in ovo and in vivo. • IBVs lacking 3ab and/or 5ab reduce ciliostasis and are thus attenuated. • The attenuated IBV strains can protect against homologues challenge. • Targeted RNA recombination enables the development of live attenuated IBV vaccines., Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens, causing severe economic losses in poultry industry worldwide. Live attenuated viruses are widely used in both the broiler and layer industry because of their efficacy and ability to be mass applied. Recently, we established a novel reverse genetics system based on targeted RNA recombination to manipulate the genome of IBV strain H52. Here we explore the possibilities to attenuate IBV in a rational way in order to generate safe and effective vaccines against virulent IBV (van Beurden et al., 2017). To this end, we deleted the nonessential group-specific accessory genes 3 and/or 5 in the IBV genome by targeted RNA recombination and selected the recombinant viruses in embryonated eggs. The resulting recombinant (r) rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab could be rescued and grew to the same virus titer as recombinant and wild type IBV strain H52. Thus, genes 3ab and 5ab are not essential for replication in ovo. When administered to one-day-old chickens, rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab showed reduced ciliostasis as compared to rIBV H52 and wild type H52, indicating that the accessory genes contribute to the pathogenicity of IBV. After homologous challenge with the virulent IBV strain M41, all vaccinated chickens were protected against disease based on reduced loss of ciliary movement in the trachea compared to the non-vaccinated but challenged controls. Taken together, deletion of accessory genes 3ab and/or 5ab in IBV resulted in mutant viruses with an attenuated phenotype and the ability to induce protection in chickens. Hence, targeted RNA recombination based on virulent IBV provides opportunities for the development of a next generation of rationally designed live attenuated IBV vaccines.

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAc alpha 2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAc alpha 2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding

Summary Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research., Highlights • Feline organoids can be cultured from fresh and frozen liver and from needle biopsy • A feline-specific liver culture medium allows organoid expansion up to 32 passages • When fed fatty acids, feline liver organoids store more lipids than human organoids • Feline liver organoids can be used to model hepatic steatosis, In this study Kruitwagen and colleagues establish and characterize a feline liver organoid culture, which has adult stem cell properties and can be differentiated toward hepatocyte-like cells. They propose liver organoids as a tool to model hepatic steatosis and show that feline liver organoids accumulate more lipids than human organoids when provided with excess fatty acids.

International audience; Harbour porpoises (Phocoena phocoena) are the most prevalent cetaceans in the North Sea. The fecal viral flora of 21 harbour porpoises stranded along the Dutch coastline was analyzed by a metagenomics approach. Sequences of a novel cetacean mastadenovirus, designated harbour porpoise adenovirus 1 (HpAdV-1), were detected. The sequence of a 23-kbp genomic region, spanning the conserved late region, was determined using primer walking. Phylogenetic analysis indicated that HpAdV-1 is most closely related to bottlenose dolphin adenovirus and clusters with Cetartiodactyla adenoviruses. The prevalence of HpAdV-1 was low (2.6%) based on targeted PCR-screening of the intestinal contents of 151 harbour porpoises stranded between 2010 and 2013.

Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls., Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here, we studied GfCoV diversity and evaluated its phenotypic consequences. Over the period of 2014 to 2016, affected guinea fowl flocks were sampled in France, and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and that of the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that, in addition to the previously elucidated biantennary di-N-acetyllactosamine (diLacNAc) receptor, viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014 S1 has an increased affinity for these glycans compared to that of GfCoV/2011 S1, which was in agreement with the increased avidity of GfCoV/2014 S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties. IMPORTANCE Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls.

8658860258318000Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D (SDHB and SDHD) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL, together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases, SDHB p.Arg38Gln (n = 1) and SDHD p.Lys122Arg (n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways.

Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA–receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA–sfGFP proteins was studied using glycan arrays and tissue staining. The HA–sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA–sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.

Ranaviruses are the second deadliest pathogens for amphibian populations throughout the world. Despite their wide distribution in America, these viruses have never been reported in Mexico, the country with the fifth highest amphibian diversity in the world. This paper is the first to address an outbreak of ranavirus in captive American bullfrogs (Lithobates catesbeianus) from Sinaloa, Mexico. The farm experienced high mortality in an undetermined number of juveniles and sub-adult bullfrogs. Affected animals displayed clinical signs and gross lesions such as lethargy, edema, skin ulcers, and hemorrhages consistent with ranavirus infection. The main microscopic lesions included mild renal tubular necrosis and moderate congestion in several organs. Immunohistochemical analyses revealed scant infected hepatocytes and renal tubular epithelial cells. Phylogenetic analysis of five partial ranavirus genes showed that the causative agent clustered within the Frog virus 3 clade. Risk assessment with the Pandora+ protocol demonstrated a high risk for the pathogen to affect amphibians from neighboring regions (overall Pandora risk score: 0.619). Given the risk of American bullfrogs escaping and spreading the disease to wild amphibians, efforts should focus on implementing effective containment strategies and surveillance programs for ranavirus at facilities undertaking intensive farming of amphibians.

Sequence-based typing of Francisella tularensis has led to insights in the evolutionary developments of tularemia. In Europe, two major basal clades of F. tularensis subsp. holarctica exist, with a distinct geographical distribution. Basal clade B.6 is primarily found in Western Europe, while basal clade B.12 occurs predominantly in the central and eastern parts of Europe. There are indications that tularemia is geographically expanding and that strains from the two clades might differ in pathogenicity, with basal clade B.6 strains being potentially more virulent than basal clade B.12. This study provides information on genotypes detected in the Netherlands during 2011–2017. Data are presented for seven autochthonous human cases and for 29 European brown hares (Lepus europaeus) with laboratory confirmed tularemia. Associated disease patterns are described for 25 European brown hares which underwent post-mortem examination. The basal clades B.6 and B.12 are present both in humans and in European brown hares in the Netherlands, with a patchy geographical distribution. For both genotypes the main pathological findings in hares associated with tularemia were severe (sub)acute necrotizing hepatitis and splenitis as well as necrotizing lesions and hemorrhages in several other organs. Pneumonia was significantly more common in the B.6 than in the B.12 cases. In conclusion, the two major basal clades present in different parts in Europe are both present in the Netherlands. In hares found dead, both genotypes were associated with severe acute disease affecting multiple organs. Hepatitis and splenitis were common pathological findings in hares infected with either genotype, but pneumonia occurred significantly more frequently in hares infected with the B.6 genotype compared to hares infected with the B.12 genotype.

Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic (example, IBV-M41) and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field., Infectious bronchitis virus (IBV) infects ciliated epithelial cells in the chicken respiratory tract. While some IBV strains replicate locally, others can disseminate to various organs, including the kidney. Here, we elucidate the determinants for kidney tropism by studying interactions between the receptor-binding domain (RBD) of the viral attachment protein spike from two IBV strains with different tropisms. Recombinantly produced RBDs from the nephropathogenic IBV strain QX and from the nonnephropathogenic strain M41 bound to the epithelial cells of the trachea. In contrast, only QX-RBD binds more extensively to cells of the digestive tract, urogenital tract, and kidneys. While removal of sialic acids from tissues prevented binding of all proteins to all tissues, binding of QX-RBD to trachea and kidney could not be blocked by preincubation with synthetic alpha-2,3-linked sialic acids. The lack of binding of QX-RBD to a previously identified IBV-M41 receptor was confirmed by enzyme-linked immunosorbent assay (ELISA), demonstrating that tissue binding of QX-RBD is dependent on a different sialylated glycan receptor. Using chimeric RBD proteins, we discovered that the region encompassing amino acids 99 to 159 of QX-RBD was required to establish kidney binding. In particular, QX-RBD amino acids 110 to 112 (KIP) were sufficient to render IBV-M41 with the ability to bind to kidney, while the reciprocal mutations in IBV-QX abolished kidney binding completely. Structural analysis of both RBDs suggests that the receptor-binding site for QX is located at a different location on the spike than that of M41. IMPORTANCE Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic (example, IBV-M41) and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field.

Introduction: Infectious bronchitis (IB) is a gammacoronavirus-induced respiratory disease in chickens. IB virus strain QX is additionally known for its detrimental nephropathogenicity. In this study we compared the phenotypic differences between an IB QX vaccine virus and its field progenitor with focus on viral replication and dissemination through the host. Materials and Methods: Specific pathogen free chickens were inoc-ulated intratracheally with either 10 3 EID 50 vaccine strain Nobilis IB Primo QX (MSD Animal Health) or field strain IBV-D388 and killed at 1 day intervals over an 8-day period. Histopathology was studied over time in the trachea, kidney, cloaca and gastrointestinal tract. Immunohistochemistry was used to demonstrate viral protein expression in these tissues. qPCR was performed on tracheal and cloacal swabs and kidney to reveal viral RNA loads. Results: IB Primo QX and IBV-D388 induced comparable tracheal le-sions, characterized by epithelial cell death and desquamation and heter-ophilic and lymphohistiocytic infiltration. Changes were earlier and more severe with IBV-D388, while tracheal viral RNA loads were comparable over time for both viruses. In contrast, degenerative and inflammatory le-sions and viral protein and RNA were only found after IBV-D388 infection in the kidneys. This renal viral presence was preceded by viral RNA detection in the cloaca and viral protein expression in the gastrointestinal tract, albeit without associated lesions in these tissues. Discussion: Dissemination to the kidneys by IBV-D388 might result from cloacal ascending infection. In contrast to its field progenitor, IB Primo QX was unable to cause renal infection, which likely considerably contributes to attenuation of its phenotype.

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research—histopathologic scoring, immunohistochemistry, and polymerase chain reaction—to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.

Between the 8 th January and the 25 th February 2016, the largest sperm whale Physeter macrocephalus mortality event ever recorded in the North Sea occurred with 30 sperm whales stranding in five countries within six weeks. All sperm whales were immature males. Groups were stratified by size, with the smaller animals stranding in the Netherlands, and the largest in England. The majority (n = 27) of the stranded animals were necropsied and/ or sampled, allowing for an international and comprehensive investigation into this mortality event. The animals were in fair to good nutritional condition and, aside from the pathologies caused by stranding, did not exhibit significant evidence of disease or trauma. Infectious agents were found, including various parasite species, several bacterial and fungal pathogens and a novel alphaherpesvirus. In nine of the sperm whales a variety of marine litter was found. However, none of these findings were considered to have been the primary cause of the stranding event. Potential anthropogenic and environmental factors that may have caused the sperm whales to enter the North Sea were assessed. Once sperm whales enter the North Sea and head south, the water becomes progressively shallower (

Avian coronavirus infectious bronchitis virus (IBV) infects domestic fowl, resulting in respiratory disease and causing serious losses in unprotected birds. Its control is mainly achieved by using live attenuated vaccines. Here we explored the possibilities for rationally attenuating IBV to improve our knowledge regarding the function of IBV accessory proteins and for the development of next-generation vaccines with the recently established reverse genetic system for IBV H52 based on targeted RNA recombination and selection of recombinant viruses in embryonated eggs. To this aim, we selectively removed accessory genes 3a, 3b, 5a and 5b individually, and rescued the resulting recombinant (r) rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b. In vitro inoculation of chicken embryo kidney cells with recombinant and wild-type viruses demonstrated that the accessory protein 5b is involved in the delayed activation of the interferon response of the host after IBV infection. Embryo mortality after the inoculation of 8-day-old embryonated chicken eggs with recombinant and wild-type viruses showed that rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b had an attenuated phenotype in ovo, with reduced titres at 6 h p.i. and 12 h p.i. for all viruses, while growing to the same titre as wild-type rIBV at 48 h p.i. When administered to 1-day-old chickens, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b showed reduced ciliostasis in comparison to the wild-type viruses. In conclusion, individual deletion of accessory genes in IBV H52 resulted in mutant viruses with an attenuated phenotype.

Knowledge about reproductive parameters in male harbour porpoises such as testicular histology and germ cell maturation as well as seasonal changes in spermatogenesis is scarce. Thus, the aim of the present study was to report changes in the histological appearance of the testicular morphology of neonatal and juvenile harbour porpoises during maturation, to identify stages of spermatogenesis in adult males and to detect seasonal modifications. The identification of these stages can be used to assess the developmental profile of gene expression during spermatogenesis and to identify defects in spermatogenesis arising in pathological conditions. Testes of adult male harbour porpoises from the North and Baltic Sea that became stranded or by-caught in the years 1998-2016 were histologically examined using Haematoxylin and Eosin - staining. The Periodic Acid Schiff (PAS) staining was used for spermatogenic staging and the evaluation of the development of the acrosomic cap. For the identification of changes in testes morphology and morphometry during the course of the year, histological characteristics like germ cell associations and diameter of the convoluted seminiferous tubules were noted for each month. The analysis showed that in adult males more than one stage of spermatogenesis could be found per cross section of the convoluted seminiferous tubules similar to findings in men and some ape species. This rare phenomenon is called multi-stage-arrangement. In sexually active males from the peak breeding season (June and July) eight stages of spermatogenesis were identified and all stages occurred simultaneously, while during the low breeding season (August to May) only residual spermatogenesis or constituent germ cell populations were found. Missing germ cell generations were recorded in specimens from July to September. Our investigations provide a detailed staging of spermatogenesis and give new insight into the reproductive biology of male harbour porpoises. With these new basic parameters, indicators for endocrine disruptors can be developed in the future, aiming to detect how environmental factors could affect male fertility in wildlife.

Ranaviruses are pathogenic viruses for poikilothermic vertebrates worldwide. The identification of a common midwife toad virus (CMTV) associated with massive die-offs in water frogs (Pelophylax spp.) in the Netherlands has increased awareness for emerging viruses in amphibians in the country. Complete genome sequencing of 13 ranavirus isolates collected from ten different sites in the period 2011-2016 revealed three CMTV groups present in distinct geographical areas in the Netherlands. Phylogenetic analysis showed that emerging viruses from the northern part of the Netherlands belonged to CMTV-NL group I. Group II and III viruses were derived from the animals located in the center-east and south of the country, and shared a more recent common ancestor to CMTV-amphibian associated ranaviruses reported in China, Italy, Denmark, and Switzerland. Field monitoring revealed differences in water frog host abundance at sites where distinct ranavirus groups occur; with ranavirus-associated deaths, host counts decreasing progressively, and few juveniles found in the north where CMTV-NL group I occurs but not in the south with CMTV-NL group III. Investigation of tandem repeats of coding genes gave no conclusive information about phylo-geographical clustering, while genetic analysis of the genomes revealed truncations in 17 genes across CMTV-NL groups II and III compared to group I. Further studies are needed to elucidate the contribution of these genes as well as environmental variables to explain the observed differences in host abundance.

Effective management of natural resources involves a multidisciplinary perspective to address complex issues in data poor-environments. With mobile species that do not conform to human-defined borders a cross-boundary approach is essential. There is a continuing concern of ecological sustainability of marine environments, which demands monitoring of ecosystem indicators. Such indicators are increasingly derived from monitoring sentinel species. Harbour porpoises (Phocoena phocoena) are included as indicator species in several national and international agreements. Increasing exposure to anthropogenic stressors may impact harbour porpoise populations. To investigate these risks, a better understanding of threats and their effect is required. This study aimed to identify current knowledge gaps, to predict future pressures or threats, and to define useful conservation indicators to facilitate future research on harbour porpoises in the North Sea, through expert elicitation gained in a two-round Delphi approach. The three most important knowledge gaps addressed were bycatch, population dynamics, and the cumulative effects of multiple stressors. Bycatch was predicted as the highest concern for porpoises in the next 20 years, followed by chemical and noise pollution, respectively. A list of essential indicators aiming to increase understanding of harbour porpoises’ health status was established and studying causes of death, distribution, abundance, habitat use and diet composition were scored as most relevant. These results should guide research focus and management objectives of harbour porpoise populations and the study design could be translated to serve managers in other geographical areas aiming to identify knowledge gaps and defining research priorities for other wildlife species.

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.

Harbour porpoises are often found to be infected by endoparasites in several organs including the lungs and stomach as well as the heart, liver and ears. Nevertheless there is still little knowledge about the impact, ecology, transmission, and virulence of these parasitic infections. Here, we profile the presence of parasites in 4 frequently infected organs (lungs, stomach, liver and ears) in relation to biological parameters of harbour porpoises stranded along the Dutch coastline between December 2008 and December 2013. We found that parasites were common, with prevalence of 68% in lungs, 74.4% in ears, 26% in stomach and 23.5% in liver. We used generalised linear models to further quantify parasite presence in relation to biological data gathered during necropsy (sex, body length and nutritive condition). Body length (used as a proxy for age) was significant in explaining parasite presence for all organs with increasing probability of having the parasite with increasing body length. For the parasitic infections in the ears and stomach the nutritive condition was an additional significant factor, with a higher probability of parasite presence in porpoises in a poorer nutritive condition. The results of this study can be used as a baseline for assessing parasite presence in harbour porpoises and are a first step towards linking parasite infections to basic biological data gathered during necropsy.

In addition to transporting ions, the multisubunit Na + ,K + -ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na + ,K + -ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na + ,K + -ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections. IMPORTANCE Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na + ,K + -ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na + ,K + -ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

Viruses exploit molecules on the target membrane as receptors for attachment and entry into host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host to a particular virus. Previously, others and we have shown that respiratory pathogens of the genus Gammacoronavirus , including chicken infectious bronchitis virus (IBV), require specific α2,3-linked sialylated glycans for attachment and entry. Here, we studied determinants of binding of enterotropic avian gammacoronaviruses, including turkey coronavirus (TCoV), guineafowl coronavirus (GfCoV), and quail coronavirus (QCoV), which are evolutionarily distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV, and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 proteins of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo . Glycan array analyses revealed that, in contrast to the S1 protein of IBV, S1 proteins of enteric gammacoronaviruses recognize a unique set of nonsialylated type 2 poly- N -acetyl-lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrate not only that enteric gammacoronaviruses recognize a novel glycan receptor but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host. IMPORTANCE Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus Gammacoronavirus , including those causing enteric disease, are hardly studied. In turkey, guineafowl, and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe, and Australia for several decades. Recently, a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the tropism and pathogenicity of these viruses explained by their receptor specificity and receptor expression on tissues was therefore needed. Here, we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines.

Highlights • This review gives an overview of the literature on avian coronavirus spike proteins. • Avian gammacoronaviruses are represented by chicken infectious bronchitis virus. • The review summarizes the spike's contribution to host and tissue tropism. • The role of the spike in pathogenesis and interventions is included. • Focus is on recent results on binding of soluble spike proteins to chicken tissues., Avian coronaviruses of the genus Gammacoronavirus are represented by infectious bronchitis virus (IBV), the coronavirus of chicken. IBV causes a highly contagious disease affecting the respiratory tract and, depending on the strain, other tissues including the reproductive and urogenital tract. The control of IBV in the field is hampered by the many different strains circulating worldwide and the limited protection across strains due to serotype diversity. This diversity is believed to be due to the amino acid variation in the S1 domain of the major viral attachment protein spike. In the last years, much effort has been undertaken to address the role of the avian coronavirus spike protein in the various steps of the virus’ live cycle. Various models have successfully been developed to elucidate the contribution of the spike in binding of the virus to cells, entry of cell culture cells and organ explants, and the in vivo tropism and pathogenesis. This review will give an overview of the literature on avian coronavirus spike proteins with particular focus on our recent studies on binding of recombinant soluble spike protein to chicken tissues. With this, we aim to summarize the current understanding on the avian coronavirus spike's contribution to host and tissue predilections, pathogenesis, as well as its role in therapeutic and protective interventions.

Frog virus 3 was isolated from a strawberry poison frog ( Oophaga pumilio ) imported from Nicaragua via Germany to the Netherlands, and its complete genome sequence was determined. Frog virus 3 isolate Op /2015/Netherlands/UU3150324001 is 107,183 bp long and has a nucleotide similarity of 98.26% to the reference Frog virus 3 isolate.

A 15-year old draft horse mare was presented to the University Clinic for evaluation of lethargy, anorexia, ptyalism, weight loss and ventral oedema. Clinical examination and rectal palpation revealed generalised lymphadenopathy and numerous firm subcutaneous and abdominal masses of various sizes. Transcutaneous ultrasonography revealed significant bilateral pleural fluid accumulation and a single hypoechoic structure in the abdomen lateral to the liver. Blood analysis showed several abnormalities including a marked leucocytosis with an increased number of segmented neutrophils, atypical lymphocytes and monocytosis, suggestive of leukaemic lymphoma. A significant hyperproteinaemia with a hypoalbuminemia and a monoclonal gammopathy was identified. At necropsy myriad masses presented through the whole body. Histology confirmed the suspicion of lymphoma, which was classified as a T-cell rich B-cell multicentric lymphoma. This article describes the clinical and pathologic findings of this case of leukaemic lymphoma.

Aortic rupture in horses is a rare condition. Although it is relatively common in the Friesian breed, only limited histopathologic information is available. Twenty Friesian horses (1–10 years old) were diagnosed with aortic rupture by postmortem examination. Ruptured aortic walls were analyzed with histology and immunohistochemistry. Based on the histologic and immunohistochemical findings, these cases were divided into 3 groups: acute ( n = 4, 20%), subacute ( n = 8, 40%), and chronic ( n = 8, 40%). Features common to samples from horses in all groups included accumulation of mucoid material; disorganization and fragmentation of the elastic laminae; aortic medial smooth muscle hypertrophy; and medial necrosis of varying degrees, ranging from mild and patchy in the acute cases to severe midzonal necrosis in the chronic cases. Inflammation, most likely secondary to medial necrosis, varied from predominantly neutrophilic infiltrates in the media and periadventitial tissue in the acute group to the presence of mainly hemosiderophages in the periadventitial tissue in the chronic group. Medial fibrosis with aberrant collagen morphology was seen in the subacute group and, more commonly, in the chronic group. Only minimal changes were seen in the aortic vasa vasorum. Smooth muscle hypertrophy and accumulation of mucoid material were not related to the age of the lesions. The findings of this study suggest that a connective tissue disorder affecting elastin or collagen in the aortic media is potentially the underlying cause of aortic rupture in Friesian horses.

The infection of the avian coronavirus infectious bronchitis virus (IBV) is initiated by the binding of the spike glycoprotein S to sialic acids on the chicken host cell. In this study we identified the receptor-binding domain (RBD) of the spike of the prototype IBV strain M41. By analyzing the ability of recombinantly expressed chimeric and truncated spike proteins to bind to chicken tissues, we demonstrate that the N-terminal 253 amino acids of the spike are both required and sufficient for binding to chicken respiratory tract in an α-2,3-sialic acid-dependent manner. Critical amino acids for attachment of M41 spike are present within the N-terminal residues 19–69, which overlap with a hypervariable region in the S1 gene. Our results may help to understand the differences between IBV S1 genotypes and the ultimate pathogenesis of IBV in chickens., Highlights • The RBD of the IBV M41 spike maps to the N-terminal 253 residues. • The NTD is both required and sufficient for α-2,3-sialic acid binding. • Critical residues for attachment of the M41 spike include N38, H43, P63 and T69.

A formulation of an antibody with antibacterial properties for topical use on Staphylococcal skin infections was developed and characterized. The best formulation was obtained with 1.5% (w/v) sodium carboxymethylcellulose containing 10 mg/ml immunoglobulin. Spraying forces and rheological behavior were measured in order to characterize the hydrogel formulation. The percentage of antibody aggregates in gel as well as the antibody release, folding and target binding properties of the released antibody were analyzed to proof an acceptable shelf life and no significant changes in the activity of the antibody over time. No microbial contamination was observed in the chosen non-airless application container. Functional testing of the topical skin formulation was performed with an ex vivo biopsy culture model of dog skin. Histological analysis indicated efficacy in protection from Staphylococcus mediated skin damage and antibody delivery restricted to the epidermal surface. The results demonstrate that this hydrogel is suitable for cutaneous antibody applications in the medical field.

SUMMARY A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection., Graphical Abstract, In Brief Broszeit and colleagues demonstrate that influenza A viruses recognize either N-acetyl or N-glycolyl neuraminic acid, and they explain these specificities using X-ray structures. NeuGc-binding viruses are perfectly viable, and neuraminidases can cleave NeuGc-containing receptor structures. There is an apparent selection now for NeuAc, as no known NeuGc-binding virus currently circulates.

With global increases in anthropogenic pressures on wildlife populations comes a responsibility to manage them effectively. The assessment of marine ecosystem health is challenging and often relies on monitoring indicator species, such as cetaceans. Most cetaceans are however highly mobile and spend the majority of their time hidden from direct view, resulting in uncertainty on even the most basic population metrics. Here, we discuss the value of long-term and internationally combined stranding records as a valuable source of information on the demographic and mortality trends of the harbour porpoise (Phocoena phocoena) in the North Sea. We analysed stranding records (n = 16,181) from 1990 to 2017 and demonstrate a strong heterogeneous seasonal pattern of strandings throughout the North Sea, indicative of season-specific distribution or habitat use, and season-specific mortality. The annual incidence of strandings has increased since 1990, with a notable steeper rise particularly in the southern North Sea since 2005. A high density of neonatal strandings occurred specifically in the eastern North Sea, indicative of areas important for calving, and large numbers of juvenile males stranded in the southern parts, indicative of a population sink or reflecting higher male dispersion. These findings highlight the power of stranding records to detect potentially vulnerable population groups in time and space. This knowledge is vital for managers and can guide, for example, conservation measures such as the establishment of time-area -specific limits to potentially harmful human activities, aiming to reduce the number and intensity of human -wildlife conflicts.

Equine intervertebral disc degeneration is thought to be rare and of limited clinical relevance, although research is lacking. To objectively assess pathological changes of the equine intervertebral disc and their clinical relevance, description of the normal morphology and a practical, biologically credible grading scheme are needed. The objectives of this study are to describe the gross and histological appearance of the equine intervertebral discs and to propose a grading scheme for macroscopic degeneration. Spinal units from 33 warmblood horses were grossly analyzed and scored. Of the 286 intervertebral discs analyzed, 107 (37%) were assigned grade 1 and grade 2 (considered normal) and were analyzed histologically. A nucleus pulposus and an annulus fibrosus could be identified macroscopically and histologically. Histologically, the nucleus pulposus was composed of a cartilaginous matrix and the annulus fibrosus of parallel collagenous bands. A transition zone was also histologically visible. Intra- and inter-observer reliability scores were high for all observers. Higher grades were associated with greater age. Gross changes associated with equine intervertebral disc degeneration (grades 3–5)—that is, yellow discoloration, cleft formation (tearing), and changes in consistency of the nucleus pulposus—were largely similar to those in humans and dogs and were most prevalent in the caudal cervical spine. Equine intervertebral disc degeneration was not associated with osteophyte formation. Changes of the vertebral bone were most common in the thoracolumbar spine but were not correlated with higher grades of intervertebral disc degeneration. Thus, changes of the vertebral bone should be excluded from grading for equine intervertebral disc degeneration.

The white-beaked dolphin (Lagenorhynchus albirostris) is an endemic species in the North Sea with an estimated population of around 36,000 individuals. Recently, concerns have been raised among conservationists regarding increasing water temperatures as a result of climate change, which could result in a decline in population numbers in certain areas of the white-beaked dolphin’s range. Here we use stranding frequencies of white-beaked dolphins as an indicator of distribution and investigate whether there have been spatiotemporal patterns and changes in stranding frequencies in the south western North Sea in the last 27 years (1991-2017). A total of 407 strandings was recorded and the distribution of stranded animals throughout this period revealed a higher density of animals in the southern countries in earlier years, with slightly increased densities in the north western area more recently. This could be a first indication of a change in habitat use and population distribution from southern to northern regions. A potential explanation for the observed shift is climate change and its effect on prey distribution and availability. This study highlights the potential of using stranding records as a way to collect high resolution spatiotemporal data, making this a valuable addition to surveys of live animals assessing species distribution and abundance. Additional research into metrics such as causes of mortality, life history and diet parameters (all of which are currently largely unknown for this species) would provide a welcome contribution to assess more detailed measures of the status of the population.

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk.