K. R. B. Sporer

Transcriptional profiling identifies differentially expressed genes in developing turkey skeletal muscle

BackgroundSkeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line (F) selected from the RBC2 line for increased 16wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage.ResultsA total of 3474 genes were differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis was used to group annotated genes into networks, functions, and canonical pathways. The expression of 28 genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR.ConclusionsThe current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products.

Differential gene expression between normal and pale, soft, and exudative turkey meat

In response to high consumer demand, turkeys have been intensively selected for rapid growth rate and breast muscle mass and conformation. The success in breeding selection has coincided with an increasing incidence of pale, soft, and exudative (PSE) meat defect, especially in response to heat stress. We hypothesized that the underlying mechanism responsible for the development of PSE meat arises from differences in expression of several critical genes. The objective of this study was to determine differential gene expression between normal and PSE turkey meat using a 6K turkey skeletal muscle long oligonucleotide microarray. Breast meat samples were collected from Randombred Control Line 2 turkeys at 22 wk of age, and classified as normal or PSE primarily based on marinade uptake (high = normal, low = PSE). Total RNA was isolated from meat samples with the highest (normal, n = 6) and the lowest (PSE, n = 6) marinade uptake. Microarray data confirmation was conducted using quantitative real-time PCR. Selection of differentially expressed genes for pathway analysis was performed using a combination of fold change (FC) ranking (FC < -1.66, FC >1.66) and false discovery rate (<0.35) as criteria. The calcium signaling pathway was highlighted as the top canonical pathway associated with differential gene expression between normal and PSE turkey. Dramatic downregulation of fast-twitch myosin heavy chain coupled with upregulation of slow-twitch myosin and troponin C suggested a switch of skeletal muscle isoforms, which may alter muscle fiber arrangement and formation of actin-myosin complexes. Changes in expression of genes in the actin cytoskeleton signaling pathway also suggest altered structures of actin filaments that may affect cell motility as well as strength and flexibility of muscle cells. Substantial downregulation of pyruvate dehydrogenase kinase, isozyme 4 was observed in PSE samples, suggesting altered regulation of the aerobic metabolic pathway in the birds that developed PSE meat defect.

Differential expression of calcium-regulating genes in heat-stressed turkey breast muscle is associated with meat quality

Aberrant postmortem Ca(2+)-regulation in the early postmortem period is associated with the occurrence of inferior meat quality in turkeys, described as pale, soft, and exudative (PSE). The objective of the current study was to quantify expression of 4 candidate genes responsible for maintaining Ca(2+) homeostasis in turkey skeletal muscle as a function of heat stress: α and β ryanodine receptors (RYR; Ca(2+)-release channels), the sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1), and the sarcoplasmic reticulum, Ca(2+)-storage protein calsequestrin (CASQ1). Two genetic lines of turkeys were used: a growth-selected commercial line and a randombred control line. Market-age birds were subjected to one of 5 heat stress treatments: no heat, 1 d, 3 d, 5 d, or 7 d of heat followed by 7 d of ambient temperature. Breast muscle samples were harvested and classified as normal or PSE using the meat quality parameters percentage of marinade uptake and percentage of cook loss. These parameters differed significantly by line, heat stress treatment, and meat quality status. Expression of candidate genes was measured using TaqMan quantitative PCR. Heat treatment was associated with significantly enhanced expression of αRYR, βRYR, and CASQ1 in normal muscle from both lines. Conversely, mRNA abundance of these genes was reduced in PSE muscle from both lines and recovered or increased by 7 d + 7 d of rest. Genetic line differences were observed at several time points. Expression of SERCA1 in both normal and PSE samples from both lines was unchanged or trended downward with heat stress. Taken together, genetic line and heat-stress treatment affected the expression of important Ca(2+)-regulating genes in association with meat quality status. The data suggest that birds whose meat leads to PSE may fail to respond to heat stress appropriately due to a delay in the upregulation of the important calcium-regulating genes: αRYR, βRYR, and CASQ1.

Characterization of a 6K oligonucleotide turkey skeletal muscle microarray

Consumer demand for lean, inexpensive meat products has driven the domestic turkey (Meleagris gallopavo) industry to unprecedented production; however, this has coincided with an increase in growth-induced myopathies and meat quality defects. With the aim of developing a new tool for the study of turkey growth and development at the muscle transcriptome level, a 6K oligonucleotide microarray was constructed, the Turkey Skeletal Muscle Long Oligo (TSKMLO) microarray. Skeletal muscle samples were collected at three critical stages in muscle development: 18-day embryo (hyperplasia), 1-day post-hatch (hypertrophy), and 16-week (market age) from two genetic lines of turkeys: RBC2, a line maintained without selection pressure, and F, a line selected from the RBC2 line for increased 16-week body weight. Oligonucleotides were designed from sequences obtained from skeletal muscle cDNA libraries from the three developmental stages. Several unique controls, including mismatch and distance controls and scrambled sequences, were designed for 30 genes. Quality control hybridizations were completed, confirming the validity and repeatability of the array. Control features were evaluated across two larger experiments comparing developmental stage within genetic line or genetic line within each developmental stage, totaling 70 arrays. Mismatch and scrambled sequences appeared to be useful controls of specific hybridization for most genes. In addition, quantitative real-time RT-PCR confirmed microarray results. This creation and assessment of the TSKMLO array provides a valuable community resource for the study of gene expression changes related to turkey muscle growth and development.

Versican, matrix Gla protein, and death-associated protein expression affect muscle satellite cell proliferation and differentiation

Our previous transcriptional profiling study using a turkey skeletal muscle-specific oligonucleotide microarray revealed over 3,000 genes that were differentially expressed at 3 critical stages of muscle development: 18 d embryonic, 1 d posthatch, and 16 wk of age. The genes versican, matrix Gla protein (MGP), and death-associated protein (DAP) were selected to study for their potential effects on muscle satellite cell proliferation and differentiation, as their functions in other tissues are suggestive of possible key roles in the regulation of myogenesis and they are differentially expressed throughout muscle development in the turkey. Using small interfering RNA to knockdown the expression of these genes during proliferation and differentiation, each of the genes was found to differentially affect proliferation and differentiation. Versican and MGP predominantly affected proliferation with line effects, but later stages of differentiation were affected by the knockdown of versican and MGP. The underexpression of DAP inhibited myotube formation, which is a necessary stage in the development of muscle fibers. Without myotube development, muscle fiber formation will be inhibited or abolished. This is the first report that these genes with no previously documented functions with regard to muscle development play a critical role in muscle cell proliferation and differentiation.

Reduced humoral immunity and atypical cell-mediated immunity in response to vaccination in cows naturally infected with bovine leukemia virus

Bovine leukemia virus (BLV) is a retrovirus that is widely distributed across US dairy herds: over 83% of herds are BLV-infected and within-herd infection rates can approach 50%. BLV infection reduces both animal longevity and milk production and can interfere with normal immune health. With such a high prevalence of BLV infection in dairy herds, it is essential to understand the circumstances by which BLV negatively affects the immune system of infected cattle. To address this question, BLV- and BLV+ adult, lactating Holstein dairy cows were vaccinated with Bovi-Shield GOLD® FP® 5 L5 HB and their immune response to vaccination was measured over the course of 28days. On day 0 prior to vaccination and days 7, 14 and 28 post-vaccination, fresh PBMCs were characterized for T and B cell ratios in the periphery. Plasma was collected to measure titers of IgM, IgG1 and IgG2 produced against bovine herpesvirus 1 (BHV1), Leptospira hardjo and L. pomona, as well as to characterize neutralizing antibody titers produced against BHV1 and bovine viral diarrhea virus types 1 and 2. On day 18 post-vaccination, PBMCs were cultured in the presence of BHV1 and flow cytometry was used to determine IFNγ production by CD4+, CD8+ and γδ T cells and to investigate CD25 and MHCII expression on B cells. BLV+ cows produced significantly lower titers of IgM against BHV1, L. hardjo and L. pomona and produced lower titers of IgG2 against BHV1. γδ T cells from BLV+ cows displayed a hyper reactive response to stimulation in vitro, although no differences were observed in CD4+ or CD8+ T cell activation. Finally, B cells from BLV+ cows exhibited higher CD25 expression and reduced MHCII expression in response to stimulation in vitro. All together, data from this study support the hypothesis that BLV+ cows fail to respond to vaccination as strongly as BLV- cows and, consequently, may have reduced protective immunity when compared to healthy BLV- cows.

Enhanced production of human influenza virus in PBS-12SF cells with a reduced interferon response

Influenza is one of the most important infectious diseases in humans. The best way to prevent severe illness caused by influenza infection is vaccination. Cell culture-derived influenza vaccines are being considered in addition to the widely used egg-based system in order to support the increasing seasonal demand and to be prepared in case of a pandemic. Cell culture based systems offer increased safety, capacity, and flexibility with reduced downstream processing relative to embryonated eggs. We have previously reported a chick embryo cell line, termed PBS-12SF, that supports replication of human and avian influenza A viruses to high titers (>107 PFU/ml) without the need for exogenous proteases or serum proteins. Viral infections in cells are limited by the Interferon (IFN) response typified by production of type I IFNs that bind to the IFNα/β receptor and activate an antiviral state. In this study, we investigated how neutralizing the interferon (IFN) response in PBS-12SF cells, via shRNA-mediated knock-down of IFNAR1 mRNA expression, affects influenza virus production. We were successful in knocking down ∼90% of IFNAR1 protein expression by this method, resulting in a significant decrease in the response to recombinant chIFNα stimulation in PBS-12SF cells as shown by a reduction in expression of interferon-responsive genes when compared to control cells. Additionally; IFNAR1-knock-down cells displayed enhanced viral HA production and released more virus into cell culture supernatants than parental PBS-12SF cells.

Dairy Cows Naturally Infected with Bovine Leukemia Virus Exhibit Abnormal B- and T-Cell Phenotypes after Primary and Secondary Exposures to Keyhole Limpet Hemocyanin

Bovine leukemia virus (BLV) is a retrovirus that is highly prevalent in US dairy herds: over 83% are BLV infected and the within-herd infection rate can be almost 50% on average. While BLV is known to cause lymphosarcomas, only 5% or fewer infected cattle will develop lymphoma; this low prevalence of cancer has historically not been a concern to dairy producers. However, more recent research has found that BLV+ cows without lymphoma produce less milk and have shorter lifespans than uninfected herdmates. It has been hypothesized that BLV infection interferes with normal immune function in infected cattle, and this could lead to reduced dairy production. To assess how naturally infected BLV+ cows responded to a primary and secondary immune challenge, 10 BLV+ and 10 BLV− cows were injected subcutaneously with keyhole limpet hemocyanin (KLH) and dimethyldioctadecylammonium bromide. B- and T-cell responses were characterized over the following 28 days. A total of 56 days after primary KLH exposure, cows were re-injected with KLH and B- and T-cell responses were characterized again over the following 28 days. BLV+ cows produced less KLH-specific IgM after primary immune stimulation; demonstrated fewer CD45R0+ B cells, altered proportions of CD5+ B cells, altered expression of CD5 on CD5+ B cells, and reduced MHCII surface expression on B cells ex vivo; exhibited reduced B-cell activation in vitro; and displayed an increase in BLV proviral load after KLH exposure. In addition, BLV+ cows had a reduced CD45R0+γδ+ T-cell population in the periphery and demonstrated a greater prevalence of IL4-producing T cells in vitro. All together, our results demonstrate that both B- and T-cell immunities are disrupted in BLV+ cows and that antigen-specific deficiencies can be detected in BLV+ cows even after a primary immune exposure.

T and B cell activation profiles from cows with and without Johne’s disease in response to in vitro stimulation with Mycobacterium avium subspecies paratuberculosis

Johnes disease (JD) is a chronic wasting disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). JD is particularly problematic on US dairy farms: estimates show that over 50% of farms are MAP-contaminated and as many as 91% of dairy herds could be infected. Although estimates vary widely, JD may cost the dairy industry between

The PBS-12SF Cell Line: Development of an Alternative Cell Line for InfluenzaVaccine Production

200 million and