Spatial transcriptomics reveals alterations in perivascular macrophage lipid metabolism in the onset of Wooden Breast myopathy in broiler chickens.

This study aims to use spatial transcriptomics to characterize the cell-type-specific expression profile associated with the microscopic features observed in Wooden Breast myopathy. 1 cm3 muscle sample was dissected from the cranial part of the right pectoralis major muscle from three randomly sampled broiler chickens at 23 days post-hatch and processed with Visium Spatial Gene Expression kits (10X Genomics), followed by high-resolution imaging and sequencing on the Illumina Nextseq 2000 system. WB classification was based on histopathologic features identified. Sequence reads were aligned to the chicken reference genome (Galgal6) and mapped to histological images. Unsupervised K-means clustering and Seurat integrative analysis differentiated histologic features and their specific gene expression pattern, including lipid laden macrophages (LLM), unaffected myofibers, myositis and vasculature. In particular, LLM exhibited reprogramming of lipid metabolism with up-regulated lipid transporters and genes in peroxisome proliferator-activated receptors pathway, possibly through P. Moreover, overexpression of fatty acid binding protein 5 could enhance fatty acid uptake in adjacent veins. In myositis regions, increased expression of cathepsins may play a role in muscle homeostasis and repair by mediating lysosomal activity and apoptosis. A better knowledge of different cell-type interactions at early stages of WB is essential in developing a comprehensive understanding.

Integrative transcriptomic and metabolomic analysis reveals alterations in energy metabolism and mitochondrial functionality in broiler chickens with wooden breast.

This integrative study of transcriptomics and metabolomics aimed to improve our understanding of Wooden Breast myopathy (WB). Breast muscle samples from 8 WB affected and 8 unaffected male broiler chickens of 47 days of age were harvested for metabolite profiling. Among these 16 samples, 5 affected and 6 unaffected also underwent gene expression profiling. The Joint Pathway Analysis was applied on 119 metabolites and 3444 genes exhibiting differential abundance or expression between WB affected and unaffected chickens. Mitochondrial dysfunctions in WB was suggested by higher levels of monoacylglycerols and down-regulated genes involved in lipid production, fatty acid beta oxidation, and oxidative phosphorylation. Lower levels of carnosine and anserine, along with down-regulated carnosine synthase 1 suggested decreased carnosine synthesis and hence impaired antioxidant capacity in WB. Additionally, Weighted Gene Co-expression Network Analysis results indicated that abundance of inosine monophosphate, significantly lower in WB muscle, was correlated with mRNA expression levels of numerous genes related to focal adhesion, extracellular matrix and intercellular signaling, implying its function in connecting and possibly regulating multiple key biological pathways. Overall, this study showed not only the consistency between transcript and metabolite profiles, but also the potential in gaining further insights from analyzing multi-omics data.

Identification of circulating metabolites associated with wooden breast and white striping.

Current diagnostic methods for wooden breast and white striping, common breast muscle myopathies of modern commercial broiler chickens, rely on subjective examinations of the pectoralis major muscle, time-consuming microscopy, or expensive imaging technologies. Further research on these disorders would benefit from more quantitative and objective measures of disease severity that can be used in live birds. To this end, we utilized untargeted metabolomics alongside two statistical approaches to evaluate plasma metabolites associated with wooden breast and white striping in 250 male commercial broiler chickens. First, mixed linear modeling was employed to identify metabolites with a significant association with these muscle disorders and found 98 metabolites associated with wooden breast and 44 metabolites associated with white striping (q-value < 0.05). Second, a support vector machine was constructed using stepwise feature selection to determine the smallest subset of metabolites with the highest categorization accuracy for wooden breast. The final support vector machine achieved 94% accuracy using only 6 metabolites. The metabolite 3-methylhistidine, which is often used as an index of myofibrillar breakdown in skeletal muscle, was the top metabolite for both wooden breast and white striping in our mixed linear model and was also the metabolite with highest marginal prediction accuracy (82%) for wooden breast in our support vector machine. Overall, this study identified a candidate set of metabolites for an objective measure of wooden breast or white striping severity in live birds and expanded our understanding of these muscle disorders.

A Student-Led Elective Provides Quality Improvement Feedback for a Required Compounding Course.

Objective. To implement an advanced elective compounding course where pharmacy students conduct investigations to improve compounding-related issues that were subsequently evaluated in a required compounding course. Methods. The elective compounding course required students to engage in self-directed learning, critical thinking, creation and evaluation of laboratory data, and self- and group reflection. Students researched and developed "solutions" to compounded preparation problems, and their solutions were tested in the next iteration of a required compounding course. For example, students in the elective course identified sources of potency variability in a ketoprofen Pluronic organogel (PLO) emulsion preparation. The students identified six variables and executed an investigative action plan. They considered all data collected and proposed a method to reduce potency variation. The recommended solution was implemented in the next offering of a required compounding course and the potency variability results were compared to the previous required course's results. Results. The mean ketoprofen PLO emulsion potency achieved in the required course prior to implementing the elective course recommendation was 129% (SD 21%), n=158. After the recommended change from elective course was implemented, the mean potency was 118% (SD 21%), n=131. Conclusion. The teaching methods and activities conducted in the elective course provided students with a deeper level of learning and understanding of compounding science, while providing practical experience in scientific research methodology. The course also provided a cyclic quality improvement feedback mechanism for the required course.

Blood Gas Disturbances and Disproportionate Body Weight Distribution in Broilers With Wooden Breast.

Wooden breast syndrome is a widespread and economically important myopathy and vasculopathy of fast growing, commercial broiler chickens, primarily affecting birds with high feed efficiency and large breast muscle yield. To investigate potential systemic physiological differences between birds affected and unaffected by wooden breast, a total of 103 market-age Cobb 500 broilers were sampled for 13 blood parameters and the relative weights of the pectoralis major muscle, pectoralis minor muscle, external oblique muscle, wing, heart, lungs, liver, and spleen. Blood analysis was performed on samples taken from the brachial vein of live birds and revealed significant differences in venous blood gases between affected and unaffected chickens. Chickens with wooden breast exhibited significantly higher potassium (K+) and lower partial pressure of oxygen (pO2), oxygen saturation (sO2), and pH. Additionally, affected males had significantly higher partial pressure of carbon dioxide (pCO2) and total carbon dioxide (TCO2) than unaffected males. Wooden breast affected broilers also possessed a significantly heavier pectoralis major muscle and whole feathered wing compared to unaffected broilers. Blood gas disturbances characterized by high pCO2 and low pH are indicative of insufficient respiratory gas exchange, suggesting that wooden breast affected broilers have an elevated metabolic rate that may also be inadequately compensated due to cardiovascular deficiencies such as poor venous return or respiratory insufficiency. Lung tissues from 12 birds with extreme sO2 values were subsequently examined to assess whether lung pathology contributed to the observed blood gas disturbance. Comparison of lung morphology between affected and unaffected birds revealed no apparent differences that could contribute to decreased parabronchial gas exchange. However, an interesting finding was the detection of pulmonary phlebitis in one of the wooden breast-affected samples consistent with vascular changes observed in pectoralis major muscle exhibiting the wooden breast phenotype. Our results suggest that the effects of wooden breast are not limited to the pectoralis major muscle and further indicate the importance of research into metabolic changes associated with the myopathy.

Zika virus infection of pregnant rats and associated neurological consequences in the offspring.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with microcephaly and other neurological disorders in infants born to infected mothers. Despite being declared an international emergency by the World Health Organization, very little is known about the mechanisms of ZIKV pathogenesis or the long-term consequences of maternal ZIKV infection in the affected offspring, largely due to the lack of appropriate rodent models. To address this issue, our lab has developed a working model of prenatal ZIKV infection in rats. In this study, we infected immune competent pregnant female rats with 105-107 PFU of ZIKV (PRVABC59, Puerto Rico/Human/Dec 2015) in order to examine its pathogenesis in the dams and pups. We examined the febrile response and sickness behavior in the dams, in addition to neonatal mortality, microglia morphology, cortical organization, apoptosis, and brain region-specific volumes in the offspring. Here, we demonstrate that pregnant and non-pregnant female rats have a distinct febrile response to ZIKV infection. Moreover, prenatal ZIKV infection increased cell death and reduced tissue volume in the hippocampus and cortex in the neonatal offspring. For the first time, we demonstrate the efficacy and validity of an immunocompetent rat model for maternal ZIKV infection that results in significant brain malformations in the neonatal offspring.

Gene expression profiling of the early pathogenesis of wooden breast disease in commercial broiler chickens using RNA-sequencing.

Wooden Breast Disease (WBD), a myopathy in commercial broiler chickens characterized by abnormally firm consistency of the pectoral muscle, impacts the poultry industry negatively due to severe reduction in meat quality traits. To unravel the molecular profile associated with the onset and early development of WBD in broiler chickens, we compared time-series gene expression profiles of Pectoralis (P.) major muscles between unaffected and affected birds from a high-breast-muscle-yield, purebred broiler line. P. major biopsy samples were collected from the cranial and caudal aspects of the muscle belly in birds that were raised up to 7 weeks of age (i.e. market age). Three subsets of biopsy samples comprising 6 unaffected (U) and 10 affected (A) from week 2 (cranial) and 4 (caudal), and 4U and 11A from week 3 (cranial) were processed for RNA-sequencing analysis. Sequence reads generated were processed using a suite of bioinformatics programs producing differentially expressed (DE) genes for each dataset at fold-change (A/U or U/A) >1.3 and False Discovery Ratio (FDR) <0.05 (week 2: 41 genes; week 3: 618 genes and week 4: 39 genes). Functional analysis of DE genes using literature mining, BioDBnet and IPA revealed several biological processes and pathways associated with onset and progress of WBD. Top among them were dysregulation of energy metabolism, response to inflammation, vascular disease and remodeling of extracellular matrix. This study reveals that presence of molecular perturbations involving the vasculature, extracellular matrix and metabolism are pertinent to the onset and early pathogenesis of WBD in commercial meat-type chickens.

Evidence and role of phlebitis and lipid infiltration in the onset and pathogenesis of Wooden Breast Disease in modern broiler chickens.

Wooden Breast Disease (WBD), a myopathy that frequently affects modern broiler chickens, is a disorder that has been associated with significant economic losses in the poultry industry. To examine tissue changes associated with the onset and early pathogenesis of this disorder, a time-series experiment was conducted using chickens from a high-breast-muscle-yield, purebred commercial broiler line. Birds were raised for up to seven weeks, with a subset of birds sampled weekly. Breast muscle tissues were extracted at necropsy and processed for analysis by light microscopy and transmission electron microscopy. Histologic presentation indicated localized phlebitis with lipogranulomas in Week 1, focal single-myofibril degeneration in Week 2 preceding an inflammatory response that started in Week 3. Lesions in Week 4 were characterized by multifocal to diffuse muscle fibre degeneration, necrosis, interstitial oedema accompanied by increased lipid and inflammatory cell infiltration. Lesions in Weeks 5-7 revealed diffuse muscle degeneration, necrosis, fibrosis and fatty infiltration with lipogranulomas. Ultrastructural examination showed myofibrillar splitting and degeneration, irregular, displaced and degenerated Z-lines, mitochondrial degeneration and interstitial fibrosis with dense regular collagen fibres. This study, therefore, demonstrates that WBD exhibits an earlier onset in modern broilers than when detectable by clinical examination. Further, this study shows that the disease assumes a progressive course with acute vasculitis, lipid deposition and myodegeneration occurring in the earlier stages, followed by a chronic fibrotic phase.

Efficacy of Recombinant HVT-IBD Vaccines Administered to Broiler Chicks from a Single Breeder Flock at 30 and 60 Weeks of Age.

The efficacy of commercially available recombinant herpesvirus of turkeys-infectious bursal disease (rHVT-IBD) virus vaccines was studied in broiler chickens derived from an IBDV-vaccinated breeder flock at 30 wk of age (Trial 1) and 60 wk of age (Trial 2). In parallel, specific-pathogen-free (SPF) white leghorn chickens were used to evaluate vaccine efficacy to control for the effects of maternally derived antibodies (MDA) associated with the broiler chickens. Broilers and SPF leghorns were vaccinated subcutaneously in the neck at 1 day of age with Vaxxitek® HVT+IBD or Vectormune® HVT-IBD vaccines and were placed in isolators. On 10, 14, 18, 22, and 26 days postvaccination (DPV), vaccinated and nonvaccinated broilers and SPF leghorns were bled prior to challenge via the oral-nasal route with infectious bursal disease (IBD) reference strains ST-C, Delaware variant E (Del E), or contemporary field isolates DMV/5038/07 or FF6. Microscopic lesion assessment of the bursa was useful for assessing IBDV challenge in both rHVT-IBD-vaccinated broiler and SPF leghorn chickens. In general, rHVT-IBD vaccines induced greater protection as the time between vaccination and challenge increased. Based on incidence of microscopic lesions (IML) of bursa tissue, Vaxxitek HVT+IBD vaccination of SPF leghorns induced protection by 18 DPV and continued to protect 22 DPV and 26 DPV in Trials 1 and 2. Vectormune HVT-IBD vaccine induced protection of SPF leghorns by 18 or 22 DPV in Trial 1, depending upon the IBDV challenge strain. However, the onset of protection was delayed until 22 or 26 DPV in Trial 2. With either commercial vaccine, rHVT-IBD vaccination of broiler chickens was not as effective as was observed in SPF leghorns, based on IML of bursa tissue. However, Vaxxitek HVT+IBD vaccination protected broilers following challenge with ST-C in both Trial 1 (30-wk-old breeder progeny) and Trial 2 (60-wk-old breeder progeny). Partial protection against FF6 (Trial 1) and DMV/5038/07 (Trial 2) challenges was observed. Vectormune HVT-IBD vaccination protected broilers vs. FF6 challenge in Trial 1. In Trial 2, the vaccine did not offer protection on the basis of IML of bursa tissue. The results indicate that 1) bursa/body weight ratios were not consistently useful as a tool for assessing IBDV challenge in broiler chickens with anti-IBDV MDA compared to assessment by IML of bursa tissue, though were useful for assessing protection in SPF leghorns; and 2) both vaccines may offer some protection to older broilers; however, a window of susceptibility exists between the waning of MDA and the development of vaccine-induced antibodies. The SPF studies showed that some vaccinated chickens were not protected from an IBDV challenge earlier than 14 DPV while broiler studies showed that MDA was not fully protective beyond 10 DPV. Because these vaccines did not protect chickens from an IBDV challenge during this window of susceptibility, our data show that breeder vaccination programs for IBDV must aim to maximize anti-IBDV MDA in progeny to protect against early IBDV challenge.

Characterization of a novel chicken muscle disorder through differential gene expression and pathway analysis using RNA-sequencing.

BACKGROUND: Improvements in poultry production within the past 50 years have led to increased muscle yield and growth rate, which may be contributing to an increased rate and development of new muscle disorders in chickens. Previously reported muscle disorders and conditions are generally associated with poor meat quality traits and have a significant negative economic impact on the poultry industry. Recently, a novel myopathy phenotype has emerged which is characterized by palpably "hard" or tough breast muscle. The objective of this study is to identify the underlying biological mechanisms that contribute to this emerging muscle disorder colloquially referred to as "Wooden Breast", through the use of RNA-sequencing technology. METHODS: We constructed cDNA libraries from five affected and six unaffected breast muscle samples from a line of commercial broiler chickens. After paired-end sequencing of samples using the Illumina Hiseq platform, we used Tophat to align the resulting sequence reads to the chicken reference genome and then used Cufflinks to find significant changes in gene transcript expression between each group. By comparing our gene list to previously published histology findings on this disorder and using Ingenuity Pathways Analysis (IPA®), we aim to develop a characteristic gene expression profile for this novel disorder through analyzing genes, gene families, and predicted biological pathways. RESULTS: Over 1500 genes were differentially expressed between affected and unaffected birds. There was an average of approximately 98 million reads per sample, across all samples. Results from the IPA analysis suggested "Diseases and Disorders" such as connective tissue disorders, "Molecular and Cellular Functions" such as cellular assembly and organization, cellular function and maintenance, and cellular movement, "Physiological System Development and Function" such as tissue development, and embryonic development, and "Top Canonical Pathways" such as, coagulation system, axonal guidance signaling, and acute phase response signaling, are associated with the Wooden Breast disease. CONCLUSIONS: There is convincing evidence by RNA-seq analysis to support localized hypoxia, oxidative stress, increased intracellular calcium, as well as the possible presence of muscle fiber-type switching, as key features of Wooden Breast Disease, which are supported by reported microscopic lesions of the disease.

II. Evaluation of the impact of alternative light technology on male broiler chicken stress.

This study evaluates the impact of light-emitting diodes (LEDs), cold cathode fluorescent lamps (CCFLs), and incandescent lamps on broiler welfare in response to recent interest in the agriculture industry to transition to more energy-efficient lighting technologies. Male Ross 708 broilers (n=672) were raised to 6 wk age in 8 light-tight modified large colony houses under identical intermittent lighting conditions using 4 unique types of lamps, which were gradually dimmed throughout the study. Incandescent lamps served as the control; experimental technologies tested were a CCFL, and 2 different LED lamps. Each technology was tested in duplicate for each of the 4 trials (8 replications total per technology) conducted across the course of one year to account for seasonal variance. Birds were removed from each house at days 7, 14, 35, and 42 to be humanely euthanized and weighed for necropsy evaluation and comparison of body mass. Blood collection via cardiac puncture was performed to obtain heterophil to lymphocyte (H:L) ratios for evaluation of environmental stress. Birds raised under CCFLs had significantly lower body weights (2,871 g±53) than the control (3,000±33 g) by 42 d. Birds raised under CCFLs additionally had significantly higher H:L ratios (0.68±0.06) than the control (0.53±0.03), indicating that these birds may have been chronically stressed (P=0.03). There were not significant differences in the H:L ratio between LED technologies at α=0.05. A significant age and seasonal correlation in H:L ratios was observed across all technologies, along with significant differences among birds raised under the experimental technologies. This study indicates that variation in broiler body weight and stress may be attributed in part to lighting technologies implemented in broiler houses.

I. Evaluation of the impact of alternative light technology on male broiler chicken growth, feed conversion, and allometric characteristics.

This study evaluates the impact of light-emitting diode (LED), cold cathode fluorescent (CCFL), and incandescent lamps on broiler performance. Male Ross 708 broilers (n=672) were raised to 6 wk age in 8 black-out modified large colony houses, under identical intermittent lighting conditions using 4 unique types of lamps, which were gradually dimmed throughout the study. Incandescent lamps served as the control; experimental technologies tested included CCFL and 2 different LED lamps. Each technology was tested in duplicate for each of 4 trials (8 replications total per technology) conducted across the course of one year to account for seasonal variance. Live performance for each technology was evaluated using live broiler body weight (BW), weight gain, feed conversion, and mortality. Birds were removed from each house at 7, 14, 35, and 42 d to be humanely euthanized, weighed, and necropsied for allometric tissue sample analysis. Relative to the technologies tested, results indicate that birds raised under incandescent lamps had significantly higher BW by 42 d, compared to birds raised under CCFL lamps, which had poorer BW performance (P=0.03). Birds raised under both LED technologies grew to final BWs similar to those raised under incandescent light, with significant differences in neither feed conversion nor mortality.

Characterization of nephropathogenic infectious bronchitis virus DMV/1639/11 recovered from Delmarva broiler chickens in 2011.

A limited outbreak of nephropathogenic infectious bronchitis (NIB) occurred in three Delmarva (DMV) commercial broiler chicken flocks in 2011. Isolates of NIB virus (NIBV)--DMV/1639/11, DMV/3432/11, and DMV/3902/11--were characterized by sequence analysis of the N-terminal subunit (S1) of the spike (S) gene. Findings indicated that the isolates were identical to each other and to PA/9579A/10, a 2010 isolate from poultry in Pennsylvania. The 2010 and 2011 isolates appear to have originated from a 1997-2000 NIB outbreak in Pennsylvania. DMV/1639/11 and PA/9579A/10 were determined to be nephropathogenic in susceptible chickens, yielding virus reisolations from kidney and inducing characteristic interstitial nephritis microscopic lesions. In a controlled laboratory study, 40% of chickens vaccinated with a combination live vaccine containing infectious bronchitis virus (IBV) strains Massachusetts (Mass) + Connecticut (Conn) were positive on virus isolation attempts after challenge with DMV/1639/11, compared with only 13% of Mass + Arkansas (Ark) vaccinates. Both combination vaccines gave partial protection against the development of DMV/1639/11-induced renal lesions. Although numerically fewer chickens vaccinated with Mass + Conn had interstitial nephritis compared with those vaccinated with Mass + Ark, neither vaccine combination offered greater protection (P < 0.05) than observed in unvaccinated chickens challenged with DMV/1639/11. Mass + Ark vaccinations, applied under commercial conditions in the hatchery (spray) and on-farm (spray), did not protect the trachea or kidney from DMV/1639/11 challenge. Serologic testing of broiler flocks found < 3% (2 of 69) tested to possess specific antibodies to DMV/1639/11, indicating the virus had not become established in the region.

Influence of submission form characteristics on clinical information received in biopsy accession.

Clinical information supplied to diagnostic laboratories through biopsy submission forms is crucial to accurate, timely diagnosis and to clinicopathologic correlation between microscopic findings and the clinical condition of the patient. The current study attempts to quantify the prevalence of deficient and inadequate submissions in veterinary biopsy service and to determine whether form characteristics, such as the open or closed nature of the form and the presence of specific prompts, influence reporting of essential case information. The hypotheses of this study are, first, that deficient and inadequate biopsy submissions do occur in veterinary medicine and, second, that open-type biopsy submission forms elicit quantitatively and qualitatively more complete case information overall, and in specific content areas, compared to closed-type biopsy submission forms. Three percent of submissions reviewed were information deficient, devoid of information beyond patient signalment, and more than 88% of forms supplied inadequate clinical information in at least 1 key content area. Both form type and specific prompts significantly influenced reporting of important clinical information. This study demonstrates the need and lays the foundation for informational completeness research in veterinary medicine.

The acute cutaneous inflammatory response is attenuated in Slug-knockout mice.

We previously reported ultraviolet radiation (UVR) induction of Slug, a Snail family zinc-finger transcription factor, in the epidermis of mice; we now report that Slug-knockout mice are, unexpectedly, more resistant to sunburn than wild-type mice. There was a marked difference between the cutaneous inflammatory response in the skin of Slug-knockout and wild-type mice from 12 h to 1 week following a single exposure to 3 minimal erythemal doses of UVR. Slug-knockout mice showed a much reduced immediate increase in skin thickness and neutrophil infiltration compared to wild-type mice. However, there were as many or more intraepidermal T cells, dermal mast cells, and dermal blood vessels in the UVR-exposed skin of Slug-knockout mice as in the skin of wild-type mice. Differences in cytokine and chemokine expression following UVR appeared to account for at least some differences between the genotypes in cutaneous inflammatory response. Despite the reported antiapoptotic and antiproliferative role for Slug in some cell types, we observed little difference between the genotypes in UVR-induced keratinocyte apoptosis or proliferation. Our findings indicate an unexpected but important role for Slug in the acute cutaneous inflammatory response to UVR.