Neuropathological lesions in intravenous BCG-stimulated K18-hACE2 mice challenged with SARS-CoV-2.

In the wake of the COVID-19 pandemic caused by SARS-CoV-2, questions emerged about the potential effects of Bacillus Calmette-Guérin (BCG) vaccine on the immune response to SARS-CoV-2 infection, including the neurodegenerative diseases it may contribute to. To explore this, an experimental study was carried out in BCG-stimulated and non-stimulated k18-hACE2 mice challenged with SARS-CoV-2. Viral loads in tissues determined by RT-qPCR, histopathology in brain and lungs, immunohistochemical study in brain (IHC) as well as mortality rates, clinical signs and plasma inflammatory and coagulation biomarkers were assessed. Our results showed BCG-SARS-CoV-2 challenged mice presented higher viral loads in the brain and an increased frequency of neuroinvasion, with the greatest differences observed between groups at 3-4 days post-infection (dpi). Histopathological examination showed a higher severity of brain lesions in BCG-SARS-CoV-2 challenged mice, mainly consisting of neuroinflammation, increased glial cell population and neuronal degeneration, from 5 dpi onwards. This group also presented higher interstitial pneumonia and vascular thrombosis in lungs (3-4 dpi), BCG-SARS-CoV-2 mice showed higher values for TNF-α and D-dimer values, while iNOS values were higher in SARS-CoV-2 mice at 3-4 dpi. Results presented in this study indicate that BCG stimulation could have intensified the inflammatory and neurodegenerative lesions promoting virus neuroinvasion and dissemination in this experimental model. Although k18-hACE2 mice show higher hACE2 expression and neurodissemination, this study suggests that, although the benefits of BCG on enhancing heterologous protection against pathogens and tumour cells have been broadly demonstrated, potential adverse outcomes due to the non-specific effects of BCG should be considered.

Intrapancreatic accessory spleens in African swine fever infection of wild boar (Sus scrofa).

Intrapancreatic accessory spleen (IPAS) is one of the most frequent congenital splenic anomalies in humans; however, studies in veterinary medicine are scarce. This study aimed to describe the macroscopic, histopathological and immunohistochemical features of 11 suspected cases of IPAS in wild boar piglets of 3-4 months old. Seven of the 11 animals were immunised with a low virulence isolate of African swine fever virus (ASFV) and subsequently challenged with a highly virulent ASFV isolate (LVI-HVI group). The remaining four animals were exclusively infected with a highly virulent isolate of ASFV (HVI group). Grossly, lesions comprised focal or multifocal reddish areas of variable shape, located on the surface of the pancreatic tail or within the parenchyma. Histological and immunohistochemical studies (anti-CD79 and CD3) confirmed the presence of IPAS in eight of the 11 cases. IPAS shared the same histological structure and alterations as those observed in the original spleen. The immunohistochemical study against ASFV revealed the presence of VP72+ cells in both the spleen and IPAS of seven of the eight piglets. The results of this study describe for the first time the presence of IPAS in ASFV infection of wild boar (Sus scrofa) regardless the isolate and suggest that the infection may induce the development of ectopic splenic tissue due to an increased demand for phagocytic cells from the reticuloendothelial system. However, further studies are needed to understand the immunological mechanisms that trigger the formation of these accessory organs.

Forensic investigations of religious rituals involving poultry: a case report.

Animal victims of human cruelty are receiving increasing attention from the press and society. Veterinary pathologists and civic authorities have a duty not only to elucidate the cause, method and manner of death but also to address the motivation behind a case. Poultry are commonly used as offering to gods in Santería rituals (ebós). Only a few cases have been reported in scientific journals, in contrast with the number of cases described in the media. Here, a hen and a cockerel (Gallus domesticus) were submitted to the VISAVET Health Surveillance Centre for autopsy, which comprised macroscopic and histological studies and microbiological testing for Chlamydia psittaci, avian orthoavulavirus 1 (Newcastle disease virus), West Nile virus and avian influenza virus. The carcasses were in an active decay stage of decomposition, containing larvae of Lucilia eximia. Both animals had been decapitated and haemorrhages in the cutting region were observed histologically. Post-mortem burns were also observed. To the best of the authors' knowledge, this is the first post-mortem description of lesions in a domestic avian species due to religious rituals. European Union and Spanish forensic practitioners should be aware that these ritual sacrifices occur in some European countries. Moreover, future European and national legislation updates should consider this type of animal abuse.

An Outbreak of Aeromonas salmonicida in Juvenile Siberian Sturgeons (Acipenser baerii).

Aeromonas salmonicida is one of the major threats to world aquaculture, causing fish furunculosis and high mortality rates in cultured fish, particularly salmonids. Although Aeromonas spp. is a thoroughly studied pathogen, little is known regarding aeromoniasis in sturgeons. After a mortality outbreak, four juvenile sturgeons (Acipenser baerii) were submitted for autopsy and tissue samples were collected for histopathological and microbiological studies. The external examination revealed size heterogenicity, skin hyperpigmentation and reduced body condition of sturgeons. Within the abdominal cavity, mild hepatomegaly and splenomegaly were observed, as well as generalized organic congestion. Histology revealed severe multifocal haemorrhagic and ulcerative dermatitis, mainly localized in the dorsal and latero-ventral areas of fish. The histological study also showed moderate to severe inflammation of gills and organic lesions compatible with septicaemia. Bacterial isolates were identified as Aeromonas salmonicida subsp. salmonicida using MALDI-TOF MS and PCR. Overall, the lesions first described here are consistent with those previously reported in other cultured fish species and contribute to a better understanding of the pathogenesis of Aeromonas salmonicida subsp. salmonicida in the Siberian sturgeon, aside from providing new diagnostic tools for bacterial diseases impacting the fast-growing industry of caviar.

Immunopathology of early and advanced epididymis lesions caused by Brucella ovis in rams.

Ovine brucellosis is an infectious disease that causes alterations in the reproductive tract in ram and abortion in ewes. Their negative economic impact in ovine production warrants a thorough understanding the interactions between B. ovis and the host. Here, epididymis lesions of rams infected by B. ovis were histopathologically staged into early and advanced. Expression by immunohistochemistry of Brucella antigens, inflammatory cell markers (CD3, CD79αcy) and cytokines (IFN-γ, TNF-α, TGF-ß1) was assessed in both stages. Early lesions were characterized by epithelial changes, interstitial inflammation, and mild fibrosis; whereas advanced lesions displayed caseous granulomas containing numerous macrophages, multinucleated giant cells, lymphocytes, and plasma cells. Expression of Brucella antigens were observed in both stages. The cellular response in B. ovis lesions were predominantly of T-cells (CD3+) whereas low numbers of B-cells and plasma cells (CD79αcy+) were present in both early and advanced lesions. IFN-γ was expressed by lymphocytes in early lesions suggesting that the adaptive immune response against B. ovis is initiated by Th1 cells, this response was also preserved in advanced stages. Expression of TNF-α was observed in neutrophils of epithelial microabscesses and intraepithelial T-cells of early lesions suggesting a promotion of neutrophil phagocytosis triggered by TNF-α. On the other hand, advanced lesions showed a reduction of TNF-α expression which may permit B. ovis persistence in granulomas. Lastly, TGF-ß1 expression (fibroblast, macrophages and less in lymphocytes) were increased with time, suggesting that B. ovis promotes TGF-ß1 secretion promoting chronicity of the lesions.

First detection of Flavobacterium psychrophilum in juvenile Siberian sturgeons (Acipenser baerii) and description of the pathological findings.

Flavobacterium psychrophilum affects many cultured fish species and is considered one of the most important bacterial pathogens causing substantial economic losses in salmonid aquaculture worldwide. Here, F. psychrophilum was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nested PCR as the aetiological agent causing mortality in diseased juvenile Siberian sturgeons (Acipenser baerii) reared on a freshwater fish farm. Diseased sturgeons were lethargic and displayed dark skin pigmentation, increased mucus production and the presence of skin ulcerations and haemorrhages specially on the ventral side and the base of fins. The histological examination of fish revealed proliferative branchitis, ulcerative and necrotizing dermatitis and myositis, lymphoid tissue atrophy, liver and kidney degeneration and thrombosis. To the best of our knowledge, this is the first report describing the infection of Siberian sturgeons by F. psychrophilum. The detection of F. psychrophilum in diseased Siberian sturgeons and the description of the pathological findings observed during the outbreak may contribute to a better understanding of the bacterium pathogenicity and the range of fish species susceptible to infection.

Genome Editing of Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes to Model Genetic Ocular Diseases.

Genome editing with the use of CRISPR/Cas9 ribonucleoprotein complexes of induced pluripotent stem cells can be used to model many diseases. The combination of stem cells and gene editing technologies is a valuable tool to study ocular disorders, as many have been identified to be caused by specific genetic mutations. This protocol provides experimentally derived guidelines for genome editing of human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 ribonucleoprotein complexes to generate iPSCs harboring single nucleotide variants from ocular disorders. Edited iPSC can be further differentiated into retinal cells in order to study disease mechanisms as well as screen potential therapies.

AMD-Like Substrate Causes Epithelial Mesenchymal Transition in iPSC-Derived Retinal Pigment Epithelial Cells Wild Type but Not C3-Knockout.

The Bruch's membrane (BrM) is a five-layered extracellular matrix (ECM) that supports the retinal pigment epithelium (RPE). Normal age-related changes in the BrM may lead to RPE cell damage and ultimately to the onset and progression of age-related macular degeneration (AMD), which is the most common cause of visual loss among the elderly. A role for the complement system in AMD pathology has been established, but the disease mechanisms are poorly understood, which hampers the design of efficient therapies to treat millions of patients. In an effort to identify the mechanisms that lead from normal aging to pathology, we have developed a cell-based model using complement deficient human induced pluripotent stem cell (iPSC)-derived RPE cells cultured on an AMD-like ECM that mimics BrM. The data present evidence that changes in the ECM result in loss of differentiation and promote epithelial mesenchymal transition (EMT) of healthy RPE cells. This pathological process is mediated by complement activation and involves the formation of a randomly oriented collagen meshwork that drives the dedifferentiation of the RPE monolayer. Genetic ablation of complement component 3 has a protective effect against EMT but does not prevent the abnormal deposition of collagens. These findings offer new insights into the sequence of events that initiate AMD and may guide the design of efficient therapies to treat this disease with unmet medical needs.

Tick-over-mediated complement activation is sufficient to cause basal deposit formation in cell-based models of macular degeneration.

Despite numerous unsuccessful clinical trials for anti-complement drugs to treat age-related macular degeneration (AMD), the complement system has not been fully explored as a target to stop drusen growth in patients with dry AMD. We propose that the resilient autoactivation of C3 by hydrolysis of its internal thioester (tick-over), which cannot be prevented by existing drugs, plays a critical role in the formation of drusenoid deposits underneath the retinal pigment epithelium (RPE). We have combined gene editing tools with stem cell technology to generate cell-based models that allow the role of the tick-over in sub-RPE deposit formation to be studied. The results demonstrate that structurally or genetically driven pathological events affecting the RPE and Bruch's membrane can lead to dysregulation of the tick-over, which is sufficient to stimulate the formation of sub-RPE deposits. This can be prevented with therapies that downregulate C3 expression. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

Differential Immune Transcriptome and Modulated Signalling Pathways in Rainbow Trout Infected with Viral Haemorrhagic Septicaemia Virus (VHSV) and Its Derivative Non-Virion (NV) Gene Deleted.

Viral haemorrhagic septicaemia virus (VHSV) is one of the worst viral threats to fish farming. Non-virion (NV) gene-deleted VHSV (dNV-VHSV) has been postulated as an attenuated virus, because the absence of the NV gene leads to lower induced pathogenicity. However, little is known about the immune responses driven by dNV-VHSV and the wild-type (wt)-VHSV in the context of infection. Here, we obtained the immune transcriptome profiling in trout infected with dNV-VHSV and wt-VHSV and the pathways involved in immune responses. As general results, dNV-VHSV upregulated more trout immune genes than wt-VHSV (65.6% vs 45.7%, respectively), whereas wt-VHSV maintained more non-regulated genes than dNV-VHSV (45.7% vs 14.6%, respectively). The modulated pathways analysis (Gene-Set Enrichment Analysis, GSEA) showed that, when compared to wt-VHSV infected trout, the dNV-VHSV infected trout upregulated signalling pathways (n = 19) such as RIG-I (retinoic acid-inducible gene-I) like receptor signalling, Toll-like receptor signalling, type II interferon signalling, and nuclear factor kappa B (NF-kappa B) signalling, among others. The results from individual genes and GSEA demonstrated that wt-VHSV impaired the activation at short stages of infection of pro-inflammatory, antiviral, proliferation, and apoptosis pathways, delaying innate humoral response and cellular crosstalk, whereas dNV-VHSV promoted the opposite effects. Therefore, these results might support future studies on using dNV-VHSV as a potential live vaccine.

Identification of the functional regions of the viral haemorrhagic septicaemia virus (VHSV) NV protein: Variants that improve function.

Non-virion (NV) protein is essential for an efficient replication increasing the pathogenicity of the Salmonid novirhabdovirus (formerly IHNV), Piscine novirhabdovirus (formerly VHSV), and Hirame novirhabdovirus (HIRV). The interferon system, apoptosis, and other immune-related genes are modulated by NV to finally induce a deficient antiviral state in the cell. However, little is known about the VHSV NV regions involved in function and location. Here, eight different NV 07.71 fragments and eleven NV 07.71 mutants derived from the region between the two first α-helices have been studied in order to establish the mx and il8 transcript levels in ZF4 cells and the subcellular location. As a result, we determined that the N-terminal part of NV preserves the same ability as the wild-type (wt) NV in mx/il8 modulation and it also shares the subcellular location. Among NV mutants, some induced mx upregulation (N34A, C35A, D38A, and S40A) but maintained the il8 levels stable when compared to wt-NV in ZF4. Four NV mutants (D28A, N31A, L33A, and F37A) were not affected by the mutation and showed mx and il8 transcript levels similar to wt-NV. Surprisingly, mutants D36A, R39A, and D41A induced a stronger downregulation of both mx and il8 transcript levels than wt-NV, suggesting that a more stable structure and an improved interaction with ligands could be achieved through these mutations. Amino acids at positions 36 and 39 are conserved among known VHSV NV proteins whereas at position 41 two different amino acids have been described. To date, no natural NV proteins with alanine at positions 36, 39, and 41 have been found. In addition, wt-NV, all NV mutants, and one N-terminal NV fragment were located at cytoplasm with a characteristic pattern, which might support that cytoplasm is the site for interaction with candidate ligands such as PPM1Bb. Taken together, the data presented in this work indicated that NV function relies on the first part of the molecule and is dependent on tertiary structure rather than on the linear one. This study could lead to a better knowledge of VHSV escape from fish antiviral mechanisms as well as to future studies on immune targets.

Transcriptome analysis of rainbow trout in response to non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV).

The non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV), an economically important fish novirhabdovirus, has been implicated in the interference of some host innate mechanisms (i.e. apoptosis) in vitro. This work aimed to characterise the immune-related transcriptome changes in rainbow trout induced by NV protein that have not yet been established in vivo. For that purpose, immune-targeted microarrays were used to analyse the transcriptomes from head kidney and spleen of rainbow trout (Oncorhynchus mykiss) after injection of recombinant NV (rNV). Results showed the extensive downregulation (and in some cases upregulation) of many innate and adaptive immune response genes not related previously to VHSV infection. The newly identified genes belonged to VHSV-induced genes (vigs), tumour necrosis factors, Toll-like receptors, antigen processing and presentation, immune co-stimulatory molecules, interleukins, macrophage chemotaxis, transcription factors, etc. Classification of differentially downregulated genes into rainbow trout immune pathways identified stat1 and jun/atf1 transcription factor genes as the most representative of the multipath gene targets of rNV. Altogether, these results contribute to define the role and effects of NV in trout by orchestrating an immunosuppression of the innate immune responses for favouring viral replication upon VHSV infection. Finally, these transcriptome results open up the possibility to find out new strategies against VHSV and better understand the interrelationships between some immune pathways in trout.

Identification of multipath genes differentially expressed in pathway-targeted microarrays in zebrafish infected and surviving spring viremia carp virus (SVCV) suggest preventive drug candidates.

Spring viremia carp virus (SVCV) is a rhabdovirus seasonally affecting warm-water cyprinid fish farming causing high impacts in worldwide economy. Because of the lack of effective preventive treatments, the identification of multipath genes involved in SVCV infection might be an alternative to explore the possibilities of using drugs for seasonal prevention of this fish disease. Because the zebrafish (Danio rerio) is a cyprinid susceptible to SVCV and their genetics and genome sequence are well advanced, it has been chosen as a model for SVCV infections. We have used newly designed pathway-targeted microarrays 3-4-fold enriched for immune/infection functional-relevant probes by using zebrafish orthologous to human genes from selected pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG). The comparative analysis of differential expression of genes through 20 pathways in 2-day exposed or 30-day survivors of SVCV infection allowed the identification of 16 multipath genes common to more than 6 pathways. In addition, receptors (Toll-like, B-cell, T-cell, RIG1-like) as well as viral RNA infection pathways were identified as the most important human-like pathways targeted by SVCV infection. Furthermore, by using bioinformatic tools to compare the promoter sequences corresponding to up and downregulated multipath gene groups, we identified putative common transcription factors which might be controlling such responses in a coordinated manner. Possible drug candidates to be tested in fish, can be identified now through search of data bases among those associated with the human orthologous to the zebrafish multipath genes. With the use of pathway-targeted microarrays, we identified some of the most important genes and transcription factors which might be implicated in viral shutoff and/or host survival responses after SVCV infection. These results could contribute to develop novel drug-based prevention methods and consolidate the zebrafish/SVCV as a model for vertebrate viral diseases.

Optimization of fixed-permeabilized cell monolayers for high throughput micro-neutralizing antibody assays: application to the zebrafish/viral hemorrhagic septicemia virus (vhsv) model.

A new high throughput centrifugation-free method to estimate viral neutralizing antibody levels in low volumes and large numbers of plasma blood samples is described. Cell monolayers were, (i) plated on poly-d-Lys coated 96-wells, (ii) infected with viruses previously incubated with fish plasma containing antibodies, (iii) fixed with formaldehyde to increase cell recovery and avoid centrifugation steps, (iv) permeabilized with Saponin, (v) immunostained in the presence of Saponin by using a monoclonal antibody (MAb) to viral protein, (vi) digested with trypsin to detach cells from the monolayer, in the absence of Saponin to reduce damage of intracellular MAb-antigen complexes, and (vii) gated by flow cytometry using automatic 96-well batch analysis. The method was applied to the determination of plasma neutralizing antibodies from zebrafish (Danio rerio) surviving infections with viral hemorrhagic septicemia virus (VHSV) (an important rhabdovirus of salmonids). This semi-automatic, rapid and practical assay detected anti-VHSV neutralizing antibodies in the plasma (∼3 µl per fish) of 95.1% of the zebrafish surviving VHSV infections. The fixed-permeabilized monolayer (FIXPERM) micro-neutralization method might help to analyze sera/plasma from small fish under standarized high throughput conditions.

In vitro neutralization of viral hemorrhagic septicemia virus by plasma from immunized zebrafish.

We studied humoral long-term adaptive viral neutralization responses in zebrafish (Danio rerio), an increasingly useful vertebrate model for viral diseases actually limited by the absence of standardized anti-zebrafish immunoglobulin M (IgM) antibodies. We established an alternative method, similar to those used in other fish, to achieve a first estimation of zebrafish anti-viral antibody-like responses. We used the viral hemorrhagic septicemia virus (VHSV) model because, although protection after this non-natural infection was demonstrated in cold-acclimatized zebrafish, little is known about their induced anti-VHSV antibody-like responses. Therefore, we first optimized a micro-neutralization method based on immunostaining VHSV-infected fish cell monolayers to detect zebrafish neutralizing activity in plasma samples in one day. We then used the method to measure the specific anti-VHSV neutralization in plasma obtained from individual zebrafish under various VHSV challenges or immunization protocols. The neutralizing activity was inhibited by protein A-sepharose and rabbit anti-zebrafish IgM antibodies, suggesting the implication of IgM zebrafish antibodies in such responses. To our knowledge, this is the first report to demonstrate detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections. This micro-method might be useful, not only for the follow-up of infection/vaccine development in the zebrafish/VHSV model in particular, but also for similar work involving other in vitro neutralizable zebrafish pathogens. This technique might also further the development of alternative ELISA assay methods to measure specific immunoglobulins in zebrafish.