Redundancy of p75NTR neurotrophin receptor function in development, growth and fertility in the rat.

The p75NTR neurotrophin receptor has positive and negative roles regulating cell survival in the nervous system. Unambiguous interpretation of p75NTR function in vivo has been complicated, however, by residual expression of alternate forms of p75NTR protein in initial p75NTR knock-out mouse models. As rats are the preferred rodent for studying brain and behaviour, and to simplify interpretation of the knock-out phenotype, we report here the generation of a mutant rat devoid of the p75NTR protein. TALEN-mediated recombination in embryonic stem cells (ESCs) was used to flank exon 2 of p75NTR with Lox P sites and produce transgenic rats carrying either un-recombined floxed p75NTREx2-fl, or recombined, exon-2 deleted p75NTREx2-Δ alleles. Crossing p75NTREx2-fl rats with a Cre-deleter strain efficiently removed exon 2 in vivo. Excision of exon 2 causes a frameshift after p75NTR Gly23 and eliminated p75NTR protein expression. Rats lacking p75NTR were healthy, fertile, and histological analysis did not reveal significant changes in cellular density or overall structure in their brains. p75NTR function is therefore largely dispensable for normal development, growth and basal homeostasis in the rat. However, the availability of constitutive and conditional p75NTREx2-Δ rats provides new opportunities to investigate specific roles of p75NTR upon injury and during tissue repair.

Balancing selection at a premature stop mutation in the myostatin gene underlies a recessive leg weakness syndrome in pigs.

Balancing selection provides a plausible explanation for the maintenance of deleterious alleles at moderate frequency in livestock, including lethal recessives exhibiting heterozygous advantage in carriers. In the current study, a leg weakness syndrome causing mortality of piglets in a commercial line showed monogenic recessive inheritance, and a region on chromosome 15 associated with the syndrome was identified by homozygosity mapping. Whole genome resequencing of cases and controls identified a mutation causing a premature stop codon within exon 3 of the porcine Myostatin (MSTN) gene, similar to those causing a double-muscling phenotype observed in several mammalian species. The MSTN mutation was in Hardy-Weinberg equilibrium in the population at birth, but significantly distorted amongst animals still in the herd at 110 kg, due to an absence of homozygous mutant genotypes. In heterozygous form, the MSTN mutation was associated with a major increase in muscle depth and decrease in fat depth, suggesting that the deleterious allele was maintained at moderate frequency due to heterozygous advantage (allele frequency, q = 0.22). Knockout of the porcine MSTN by gene editing has previously been linked to problems of low piglet survival and lameness. This MSTN mutation is an example of putative balancing selection in livestock, providing a plausible explanation for the lack of disrupting MSTN mutations in pigs despite many generations of selection for lean growth.

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus.

Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (>99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.

Evaluation of a Harmonized Undergraduate Catalog for Veterinary Public Health and Food Hygiene Pedagogy in Europe.

Current and emerging veterinary public health (VPH) challenges raised by globalization, climate change, and industrialization of food production require the veterinarian's role to evolve in parallel and veterinary education to adapt to reflect these changes. The European Food Hygiene catalog was developed to provide a list of topics relevant to Day One Competencies in VPH. A study was undertaken to ensure that the catalog and teaching practices were pertinent to the work of public health veterinarians. Relevant stakeholders were consulted using questionnaires and semi-structured interviews. A long questionnaire was distributed to 49 academics teaching VPH in European veterinary schools to review topics listed in the catalog. Eighteen responses were received (36.7%), representing 12 European countries. There was general agreement that most topics were appropriate for the undergraduate VPH curriculum. A short questionnaire was distributed to 348 European veterinarians working in the industry. Twenty-four questionnaires (6.7%) were received, representing eight European countries. Despite the low participation rate, topics needing greater emphasis in the undergraduate curriculum included Hazard Analysis Critical Control Points (HACCP), food microbiology, and audits. Seven semi-structured interviews with public health veterinarians working in the UK identified the need for curricular changes including greater practical experience and a shift from a focus on meat inspection to risk management. This may be partly achieved by replacing traditional lectures with authentic case-based scenarios. The study findings can be used to inform the future direction to VPH education for veterinary students across Europe.

Investigating mechanisms underlying genetic resistance to Salmon Rickettsial Syndrome in Atlantic salmon using RNA sequencing.

BACKGROUND: Salmon Rickettsial Syndrome (SRS), caused by Piscirickettsia salmonis, is one of the primary causes of morbidity and mortality in Atlantic salmon aquaculture, particularly in Chile. Host resistance is a heritable trait, and functional genomic studies have highlighted genes and pathways important in the response of salmon to the bacteria. However, the functional mechanisms underpinning genetic resistance are not yet well understood. In the current study, a large population of salmon pre-smolts were challenged with P. salmonis, with mortality levels recorded and samples taken for genotyping. In parallel, head kidney and liver samples were taken from animals of the same population with high and low genomic breeding values for resistance, and used for RNA-Sequencing to compare their transcriptome profile both pre and post infection. RESULTS: A significant and moderate heritability (h2 = 0.43) was shown for the trait of binary survival. Genome-wide association analyses using 38 K imputed SNP genotypes across 2265 animals highlighted that resistance is a polygenic trait. Several thousand genes were identified as differentially expressed between controls and infected samples, and enriched pathways related to the host immune response were highlighted. In addition, several networks with significant correlation with SRS resistance breeding values were identified, suggesting their involvement in mediating genetic resistance. These included apoptosis, cytoskeletal organisation, and the inflammasome. CONCLUSIONS: While resistance to SRS is a polygenic trait, this study has highlighted several relevant networks and genes that are likely to play a role in mediating genetic resistance. These genes may be future targets for functional studies, including genome editing, to further elucidate their role underpinning genetic variation in host resistance.

Ultrastructural insights into the replication cycle of salmon pancreas disease virus (SPDV) using salmon cardiac primary cultures (SCPCs).

Salmon pancreas disease virus (SPDV) has been affecting the salmon farming industry for over 30 years, but despite the substantial amount of studies, there are still a number of recognized knowledge gaps, for example in the transmission of the virus. In this work, an ultrastructural morphological approach was used to describe observations after infection by SPDV of an ex vivo cardiac model generated from Atlantic salmon embryos. The observations in this study and those available on previous ultrastructural work on SPDV are compared and contrasted with the current knowledge on terrestrial mammalian and insect alphaviral replication cycles, which is deeper than that of SPDV both morphologically and mechanistically. Despite their limitations, morphological descriptions remain an excellent way to generate novel hypotheses, and this has been the aim of this work. This study has used a target host, ex vivo model and resulted in some previously undescribed features, including filopodial membrane projections, cytoplasmic stress granules or putative intracytoplasmic budding. The latter suggests a new hypothesis that warrants further mechanistic research: SPDV in salmon may have retained the capacity for non-cytolytic (persistent) infections by intracellular budding, similar to that noted in arthropod vectors of other alphaviruses. In the notable absence of a known intermediate host for SPDV, the presence of this pattern suggests that both cytopathic and persistent infections may coexist in the same host. It is our hope that the ultrastructural comparison presented here stimulates new research that brings the knowledge on SPDV replication cycle up to a similar level to that of terrestrial alphaviruses.

Proteomic characterization of serum proteins from Atlantic salmon (Salmo salar L.) from an outbreak with cardiomyopathy syndrome.

Cardiomyopathy syndrome (CMS), caused by piscine myocarditis virus (PMCV), is a serious challenge to Atlantic salmon (Salmo salar L.) aquaculture. Regrettably, husbandry techniques are the only tool to manage CMS outbreaks, and no prophylactic measures are available at present. Early diagnosis of CMS is therefore desirable, preferably with non-lethal diagnostic methods, such as serum biomarkers. To identify candidate biomarkers for CMS, the protein content of pools of sera (4 fish/pool) from salmon with a CMS outbreak (3 pools) and from clinically healthy salmon (3 pools) was compared using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Overall, seven proteins were uniquely identified in the sera of clinically healthy fish, while 27 proteins were unique to the sera of CMS fish. Of the latter, 24 have been associated with cardiac disease in humans. These were grouped as leakage enzymes (creatine kinase, lactate dehydrogenase, glycogen phosphorylase and carbonic anhydrase); host reaction proteins (acute-phase response proteins-haptoglobin, fibrinogen, α2-macroglobulin and ceruloplasmin; and complement-related proteins); and regeneration/remodelling proteins (fibronectin, lumican and retinol). Clinical evaluation of the suitability of these proteins as biomarkers of CMS, either individually or as part of a panel, is a logical next step for the development of early diagnostic tools for CMS.

Efficacy of heat-killed and formalin-killed vaccines against Tilapia tilapinevirus in juvenile Nile tilapia (Oreochromis niloticus).

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is considered to be a new threat to the global tilapia industry. The objective of this study was to develop simple cell culture-based heat-killed (HKV) and formalin-killed (FKV) vaccines for the prevention of disease caused by TiLV. The fish were immunized with 100 µl of either HKV or FKV by intraperitoneal injection with each vaccine containing 1.8 × 106 TCID50- inactivated virus. A booster vaccination was carried out at 21-day post-vaccination (dpv) using the same protocol. The fish were then challenged with a lethal dose of TiLV at 28 dpv. The expression of five immune genes (IgM, IgD, IgT, CD4 and CD8) in the head kidney and spleen of experimental fish was assessed at 14 and 21 dpv and again after the booster vaccination at 28 dpv. TiLV-specific IgM responses were measured by ELISA at the same time points. The results showed that both vaccines conferred significant protection, with relative percentage survival of 71.3% and 79.6% for HKV and FKV, respectively. Significant up-regulation of IgM and IgT was observed in the head kidney of fish vaccinated with HKV at 21 dpv, while IgM, IgD and CD4 expression increased in the head kidney of fish receiving FKV at the same time point. After booster vaccination, IgT and CD8 transcripts were significantly increased in the spleen of fish vaccinated with the HKV, but not with FKV. Both vaccines induced a specific IgM response in both serum and mucus. In summary, this study showed that both HKV and FKV are promising injectable vaccines for the prevention of disease caused by TiLV in Nile tilapia.

Simultaneous detection of scale drop disease virus and Flavobacterium columnare from diseased freshwater-reared barramundi Lates calcarifer.

Freshwater farming of barramundi Lates calcarifer in Thailand is a growing sector in aquaculture, but mortalities due to infectious diseases are still a major threat to this industry. In 2018, an episode of severe mortality in juvenile barramundi was noted in a freshwater earth pond site. Fish presented with severe gill necrosis, as well as severe cutaneous hemorrhages, scale loss, and discoloration at the base of dorsal fin (saddleback lesions). Histopathology revealed extensive necrosis of skeletal muscle and gill filaments, as well as basophilic inclusion bodies and megalocytosis in muscle, gill, liver, and kidney. Scale drop disease virus (SDDV) infection was subsequently confirmed by virus-specific semi-nested PCR. Further, DNA sequences of the viral major capsid protein (MCP) and ATPase genes had a respective homology of 99.85 and 99.92% with sequences of SDDV infecting barramundi in saltwater culture. Gill necrosis and saddleback lesions are not typical lesions associated with scale drop syndrome. Their presence was explained by Flavobacterium columnare isolation from the gill, followed by positive F. columnare-specific PCR. To our knowledge, this is the first report of SDDV-associated mortality in freshwater-farmed barramundi. Furthermore, this mortality presented as a concurrent infection with SDDV and F. columnare, with typical lesions of both infections.

Changes in distribution, morphology and ultrastructure of chloride cell in Atlantic salmon during an AGD infection.

Amoebic gill disease (AGD) is emerging as one of the most significant health challenges affecting farmed Atlantic salmon in the marine environment. It is caused by the amphizoic amoeba Neoparamoeba perurans, with infestation of gills causing severe hyperplastic lesions, compromising overall gill integrity and function. This study used histology, transmission electron microscopy (TEM), immunohistochemistry and transcript expression to relate AGD-associated pathological changes to changes in the morphology and distribution of chloride cells (CCs) in the gills of Atlantic salmon (Salmo salar L.) showing the progression of an AGD infection. A marked reduction in numbers of immunolabelled CCs was detected, and a changing pattern in distribution and morphology was closely linked with the level of basal epithelial hyperplasia in the gill. In addition, acute degenerative ultrastructural changes to CCs at the lesion site were observed with TEM. These findings were supported by the early-onset downregulation of Na+ /K+ -ATPase transcript expression. This study provides supportive evidence that histological AGD lesion assessment was a good qualitative tool for AGD scoring and corresponded well with qPCR genomic Paramoeba perurans quantification. Ultrastructural changes induced in salmon CCs as a result of AGD are reported here for the first time.

Canine and feline emphysematous gastritis may be differentiated from gastric emphysema based on clinical and imaging characteristics: Five cases.

Gastric pneumatosis is an imaging finding defined as the presence of gas foci in the gastric wall. In humans, this imaging feature can result from one of two separate clinical entities: life-threatening emphysematous gastritis or clinically benign gastric emphysema. This retrospective case series study describes the clinical and imaging features in five animals diagnosed with spontaneous gastric pneumatosis without gastric dilatation-volvulus. Three canine and two feline cases of spontaneous gastric pneumatosis were identified on radiographic and ultrasonographic examinations. In addition to gastric pneumatosis, one dog and two cats presented concomitant systemic signs such as lethargy, hematemesis, anemia, or leukocytosis. Two dogs remained asymptomatic or presented mild gastrointestinal signs. Portal gas was described in two dogs and one cat, and pneumoperitoneum in one dog. These features were not considered clinically significant. The dog and two cats with systemic signs were euthanized due to clinical deterioration and diagnosed with emphysematous gastritis. The gastric pneumatosis of both dogs without systemic signs resolved while on medical management without antibiotic therapy. These latter cases were interpreted as consistent with gastric emphysema. Findings from the current study indicated that gastric pneumatosis can occur without gastric dilatation-volvulus in cats and dogs and that a combination of clinical and imaging characteristics may help to differentiate between potentially life-threatening emphysematous gastritis and relatively benign gastric emphysema. More studies are needed to determine the etiology and risk factors associated with these conditions.

Use of Salmon Cardiac Primary Cultures (SCPCs) of different genotypes for comparative kinetics of mx expression, viral load and ultrastructure pathology, after infection with Salmon Pancreas Disease Virus (SPDV).

In vitro fish based models have been extensively applied in human biomedical research but, paradoxically, less frequently in the research of fish health issues. Farmed Atlantic salmon can suffer from several viral conditions affecting the heart. Therefore, species-specific, cardiac in vitro models may represent a useful tool to help further understanding and management of these diseases. The mechanisms underlying genotype based resistance are complex and usually rely on a combined effect of elements from both the innate and adaptive immune response, which are further complicated by external environmental factors. Here we propose that Salmon Cardiac Primary Cultures (SCPCs) are a useful tool to investigate these mechanisms as the basis for genotypic differences between Atlantic salmon families in susceptibility to cardiotropic viral disease. Using SCPCs produced from two different commercially available Atlantic salmon embryonated ova (Atlantic Ova IPN sensitive" (S) and "Atlantic QTL-innOva® IPN/PD" (R)), the influence of host genotype on the viral load and mx expression following Salmon Pancreas Disease Virus infection was assessed over a 15 day period. Both R and S SCPCs groups were successfully infected. A measurable difference between groups of viral nsP1 and host antiviral mx gene expression was observed (i.e. a later, but larger onset of mx expression in the R group). Mx expression peaks were followed by a decrease in viral nsP1 in both groups. Additionally, ultrastructural examination of infected SCPCs allowed the description of degenerative changes at the individual cell level. The SCPC model presents some advantages, over current fish cell culture monolayers and in vivo material, such as the presence of different cell components normally present in the target organ, as well as the removal of a layer of functional complexity (acquired immunity), making it possible to focus on tissue specific, early innate immune mechanisms. These preliminary results highlight the importance of considering genetic origin when selecting the fish source for the production of SCPCs, as well as their usefulness as screening tools for assessment of genotypic differences in disease resistance.

Ultrasound and computed tomography of sacculitis and appendicitis in a rabbit.

A 9-month-old neutered male rabbit was referred for lethargy, anorexia, and gastrointestinal stasis. Routine hematology, serum biochemistry, and diagnostic imaging were performed. Computed tomography revealed a wall thickening of the sacculus rotundus and appendix, which was further confirmed on abdominal ultrasound. Full thickness biopsies were collected with histopathology diagnosing a chronic multifocal heterophilic granulomatous sacculitis and appendicitis. The patient was treated medically and at 6 weeks follow-up, clinical signs and intestinal changes had completely regressed. Inflammation of the sacculus rotundus and appendix should be considered as a cause of gastrointestinal stasis in rabbits.

Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Quantitative imaging of tissue sections using infrared scanning technology.

Quantification of immunohistochemically (IHC) labelled tissue sections typically yields semi-quantitative results. Visualising infrared (IR) 'tags', with an appropriate scanner, provides an alternative system where the linear nature of the IR fluorophore emittance enables realistic quantitative fluorescence IHC (QFIHC). Importantly, this new technology enables entire tissue sections to be scanned, allowing accurate area and protein abundance measurements to be calculated from rapidly acquired images. Here, some of the potential benefits of using IR-based tissue imaging are examined, and the following are demonstrated. Firstly, image capture and analysis using IR-based scanning technology yields comparable area-based quantification to those obtained from a modern high-resolution digital slide scanner. Secondly, IR-based dual target visualisation and expression-based quantification is rapid and simple. Thirdly, IR-based relative protein abundance QIHC measurements are an accurate reflection of tissue sample protein abundance, as demonstrated by comparison with quantitative fluorescent Western blotting data. In summary, it is proposed that IR-based QFIHC provides an alternative method of rapid whole-tissue section low-resolution imaging for the production of reliable and accurate quantitative data.

Zebrafish as model organisms for studying drug-induced liver injury.

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high-throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers.

Preliminary study of proton magnetic resonance spectroscopy to assess bone marrow adiposity in the third metacarpus or metatarsus in Thoroughbred racehorses.

BACKGROUND: Magnetic resonance spectroscopy (MRS) has been used to investigate metabolic changes within human bone. It may be possible to use MRS to investigate bone metabolism and fracture risk in the distal third metacarpal/tarsal bone (MC/MTIII) in racehorses. OBJECTIVES: To determine the feasibility of using MRS as a quantitative imaging technique in equine bone by using the 1H spectra for the MC/MTIII to calculate fat content (FC). STUDY DESIGN: Observational cross-sectional study. METHODS: Limbs from Thoroughbred racehorses were collected from horses that died or were subjected to euthanasia on racecourses. Each limb underwent magnetic resonance imaging (MRI) at 3 T followed by single-voxel MRS at three regions of interest (ROI) within MC/MTIII (lateral condyle, medial condyle, proximal bone marrow [PBM]). Percentage FC was calculated at each ROI. Each limb underwent computed tomography (CT) and bone mineral density (BMD) was calculated for the same ROIs. All MR and CT images were graded for sclerosis. Histology slides were graded for sclerosis and proximal marrow space was calculated. Pearson or Spearman correlations were used to assess the relationship between BMD, FC and marrow space. Kruskal-Wallis tests were used to check for differences between sclerosis groups for BMD or FC. RESULTS: Eighteen limbs from 10 horses were included. A negative correlation was identified for mean BMD and FC for the lateral condyle (correlation coefficient = -0.60, p = 0.01) and PBM (correlation coefficient = -0.5, p = 0.04). There was a significant difference between median BMD for different sclerosis grades in the condyles on both MRI and CT. A significant difference in FC was identified between sclerosis groups in the lateral condyle on MRI and CT. MAIN LIMITATIONS: Small sample size. CONCLUSIONS: 1H Proton MRS is feasible in the equine MC/MTIII. Further work is required to evaluate the use of this technique to predict fracture risk in racehorses.

Developing an Injury-Free 15 Hour Preservation Protocol of Donor Porcine Kidneys Using Normothermic Machine Perfusion.

Normothermic machine perfusion (NMP) offers a superior alternative to hypothermic preservation but is currently time limited. Extending this time could electivise transplantation and enable physiologic assessments of functionality. Porcine kidneys were retrieved, stored on ice for 3.5 hours before being placed onto a NMP circuit for 12 hours. Hemodynamics, biochemistry, and urine output were assessed. After 12 hours, kidneys were scored using the clinical assessment score. Biopsies were collected for histological assessment. Kidneys demonstrated continual improvements in hemodynamics. Perfusate sodium concentrations remained within physiologic parameters. Sodium bicarbonate increased over-time with corresponding decreases in lactate, demonstrating active renal gluconeogenesis and Cori cycle processes. Urine production began immediately and was sustained, indicating renal functionality. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury free. Our NMP protocol safely preserves kidneys for over 15 hours. Successful perfusion was achieved with stable hemodynamics and biochemistry, with maintained urination. Importantly, kidneys remained in optimal health, with no evidence of injury. This may enable electivisation of transplantation, while reducing hypothermic injury.