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Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most significant diseases of swine. IDEXX HerdChek PRRS, a commercially available enzyme-linked immunosorbent assay (ELISA), has become the industry standard for the detection of antibodies against PRRS virus (PRRSV). The need to accurately determine the PRRSV serostatus of herds and individual animals has prompted the development of several follow-up assay methods. A highly specific and repeatable blocking ELISA (bELISA) was developed on the basis of the use of an expressed PRRSV nucleocapsid (N) protein as the antigen and a biotinylated monoclonal antibody. Validation of the bELISA used sera from 316 animals experimentally and naturally infected with North American PRRSV and sera from 370 uninfected animals. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 97.8% and a diagnostic specificity of 100%. The between-run coefficient of variation of an internal quality control serum was 4.24%. The bELISA was able to detect seroconversion as well as the IDEXX ELISA and the indirect fluorescent antibody (IFA) assay; kappa values were 0.94 and 0.96, respectively. A collection of 133 serum samples with unexpected positive IDEXX ELISA results was obtained from 4,038 diagnostic samples submitted from farms from which PRRS-negative results were expected. The bELISA identified 97% of the samples as PRRS seronegative, while the IFA identified 100% as seronegative. The anticipated use of the bELISA is as a follow-up test to the IDEXX ELISA for determining the PRRSV serostatus of individual animals with unexpected positive test results from swine herds from which negative results are expected.

Skeletal muscle fibers are surrounded by an extracellular matrix. The extracellular matrix is composed of glycoproteins, collagen, and proteoglycans. Proteoglycans have been suggested to play an important functional role in tissue differentiation. However, an understanding of how the extracellular matrix affects skeletal muscle development and function is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), a late embryonic increase in the expression of decorin is followed by a subsequent increase in collagen crosslinking. The sarcomere organization, collagen fibril diameter and organization were investigated using transmission electron microscopy. Measurements were made at 20 days of embryonic development and 6 weeks posthatch. These studies showed changes in sarcomere organization and deterioration of muscle fibril structure in the LSN pectoral muscle. In vitro satellite cell cultures were developed and assayed for mitochondrial activity, and protein synthesis and degradation. In these analyses, mitochondrial activity from LSN satellite cells was significantly higher than those from normal pectoral muscle satellite cells. Protein synthesis rates between the normal and LSN satellite cell-derived myotubes were similar, but protein degradation rates were higher in the LSN cultures. Based on the reported functions of decorin as a regulator of cell proliferation and collagen fibril organization, it is possible that the late embryonic increase in decorin may be influencing the alterations in LSN sarcomere and collagen organization.

Variation in response to growth factor stimuli in myogenic satellite cell populations was investigated using a clonal-derived satellite cell culture system. Satellite cell clones were established from one muscle from one individual animal. One clone ("Early") which reached confluence on day 19 and one clone ("Late"), which reached confluence on day 29, were chosen for further examination. In previous studies, these two clones were found to differ in their growth rates in serum-containing medium. In the present study, the influence of growth factors on the proliferation of the two clones was compared in serum-free defined medium. Although basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), insulin, and platelet-derived growth factor-BB (PDGF-BB) stimulated proliferation of both clones, the Early clone was more responsive to all growth factors tested than the Late clone (P < or = 0.05). The Early clone was also more responsive to the proliferative and differentiative depressing effects of administered transforming growth factor-beta (P < or = 0.05). Examination of properties of the PDGF, FGF, and IGF-I receptors on these two clones revealed no differences in either dissociation constants or receptor numbers (P > or = 0.05). The results suggest that there is heterogeneity in satellite cell response to growth factors.

Myogenic satellite cells were isolated from the turkey pectoralis major (PM), a muscle composed largely of white fibers, and the biceps femoris (BF), a muscle composed largely of red fibers, and their properties were compared in culture. Satellite cells derived from the PM and BF muscles exhibited differences in metabolic parameters, growth factor receptor characteristics, and mitogenic responses. PM satellite cells exhibited greater responsiveness to platelet-derived growth factor (PDGF), and the PDGF receptor on these cells had a higher affinity toward ligand compared to BF cells (P < 0.05). Protein synthesis, protein degradation, and glucose uptake rates were higher in BF satellite cell cultures (P < 0.05), correlating with previously reported in vivo measurements using red and white muscle fibers.

Satellite cells were isolated from the pectoralis major (PM) and biceps femoris (BF) muscles of 5-week-old broiler chickens to compare growth and differentiation characteristics in vitro. BF cells proliferated at greater rates in the growth medium and were more responsive to the mitogenic effects of chicken serum than PM cells at all levels tested (p < or = 0.05). When low serum-containing medium was administered, the levels of creatine kinase, a marker of differentiation, increased at greater rates in PM cultures than in BF cultures (p < or = 0.05). Administration of increasing levels of fibroblast growth factor in serum-free medium resulted in similar responsiveness of the two lines to this mitogen. No differences were detected in rates of protein synthesis or degradation in myotube cultures from the two muscle sources. The results suggest that satellite cells derived from PM and BF muscles of the chicken have different responsiveness to serum mitogens.

Myogenic satellite cell clones were established from the pectoralis major muscle of chickens with the low score normal (LSN) muscle weakness and controls. The percentage of cells which attached to substrata and began to proliferate was higher for the control line than the LSN line (55% vs 30%). Furthermore, of those clones which initiated growth, 63% of the control cells and only 32% of the LSN cells proliferated to confluence in 25 cm2 tissue culture flasks. Proliferation rates were significantly lower with LSN satellite cells than with controls (p < 0.05). LSN satellite cells were less responsive to the mitogenic effects of chicken serum (p < 0.05) and differentiation rates were lower compared with controls (p < 0.05). There was a greater (p < 0.05) number of insulin-like growth factor receptors on LSN satellite cells compared with controls. The IGF receptor binding affinities (Kds) between the two cell lines were similar (p > 0.05). The results suggest that a defect in satellite cell physiology may contribute to the skeletal muscle weakness seen in the LSN line.

Satellite cells were isolated from the skeletal muscles of turkey varieties which grow at different rates to explore cellular mechanisms that may influence the rate of muscular growth. Satellite cells from the fast growing variety (Nicholas) were more responsive to insulin-like growth factors (IGFs) and insulin, and less responsive to fibroblast growth factor than were cells derived from the slow growing (Merriam's) turkey. IGF receptor affinities were similar between the Nicholas and Merriam's turkey satellite cells. The results suggest that differences in the responses of satellite cells to growth factors may influence rates of skeletal muscle accretion and whole body growth.

Protein synthesis, protein degradation, and glucose uptake were compared in clonal-derived turkey myogenic satellite cells (clone D5-SC) and embryonic myoblasts (EM) and between satellite cell cultures from Nicholas (DN) and Merriam's (WM) turkeys. Protein synthesis rates were higher and degradation rates were lower in myotube cultures of D5-SC compared with EM (P < or = 0.05). Protein synthesis and degradation rates did not differ between cultures of DN and WM (P > or = 0.05). Glucose transport rates were significantly higher in EM than D5-SC cultures and did not differ between DN and WM cultures. Insulin-like growth factors and insulin stimulated protein synthesis, decreased protein degradation, and increased glucose uptake in all cell lines.

1. The distribution and relative insulin-like growth factor (IGF) binding capacities of membranes derived from 14 tissues of the turkey were examined. 2. Affinity cross-linking analyses using [125I]IGF-I and [125I]IGF-II with membranes derived from the liver, pectoralis major muscle, gizzard, heart and brain indicated that both IGFs interact with only type-I IGF receptors on these tissues. 3. There was no evidence for the existence of a type-II IGF receptor in any tissue. 4. Although considerable variation was detected in the molecular weights of the IGF receptor alpha subunits between tissues (112.2-132.9 kDa), these differences did not appear to influence receptor-ligand affinities.

1. The interaction of insulin-like growth factors (IGFs) with receptors on clonal-derived turkey satellite cells and embryonic myoblasts was compared using competitive binding assays and affinity cross-linking analysis. 2. Although [125I]IGF-I and [125I]IGF-II were displaced similarly by IGF-I and IGF-II within cell lines (P greater than 0.05), displacement, and therefore dissociation constants, differed between cell lines (P less than 0.0001). 3. Receptor cross-linking analysis using iodinated IGFs suggests that both IGF-I and IGF-II interact with the type I receptor on turkey embryonic and posthatch myogenic cells.

A cDNA-encoding turkey prolactin (PRL) has been isolated from a turkey pituitary library. The 953-base pair cDNA clone contains a 229-amino acid open reading frame which consists of a 30-amino acid signal peptide followed by a 199-amino acid mature PRL. The deduced amino acid sequence of turkey PRL shows greater than 90% homology to chicken PRL and 54-78% homology to other mammalian prolactins. A mRNA of 1100 nucleotides was detected in total RNA extracted from turkey pituitaries. Levels of PRL mRNA increased approximately 10-, 20-, and 100-fold in photostimulated, laying, and incubating hens, respectively, relative to that found in nonphotostimulated hens. The corresponding increases in plasma PRL levels were 2-, 5.5-, and 50-fold and in pituitary PRL content were 2-, 4-, and 13.4-fold, respectively. The transition from incubation to the photorefractory phase resulted in a 10-fold reduction in PRL mRNA, a 3.7-fold decrease in pituitary PRL, and a dramatic 50-fold decrease in plasma PRL. The changes in the abundance of pituitary PRL mRNA appear to be related to the changes in PRL-releasing activity observed at each of the reproductive stages. This study provides the first characterization of pituitary PRL mRNA and its comparison with plasma and pituitary PRL levels during the avian reproductive cycle.