Phenotypic Characterization of Recombinant Marek's Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence.

Marek's disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek's disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

Type 2 polysaccharide storage myopathy in Quarter Horses is a novel glycogen storage disease causing exertional rhabdomyolysis.

BACKGROUND: Both type 1 (PSSM1) and type 2 polysaccharide storage myopathy (PSSM2) are characterised by aggregates of abnormal polysaccharide in skeletal muscle. Whereas the genetic basis for PSSM1 is known (R309H GYS1), the cause of PSSM2 in Quarter Horses (PSSM2-QH) is unknown and glycogen concentrations not defined. OBJECTIVES: To characterise the histopathological and biochemical features of PSSM2-QH and determine if an associated monogenic variant exists in genes known to cause glycogenosis. STUDY DESIGN: Retrospective case control. METHODS: Sixty-four PSSM2-QH, 30 PSSM1-QH and 185 control-QH were identified from a biopsy repository and clinical data, histopathology scores (0-3), glycogen concentrations and selected glycolytic enzyme activities compared. Coding sequences of 12 genes associated with muscle glycogenoses were identified from whole genome sequences and compared between seven PSSM2-QH and five control-QH. RESULTS: Exertional rhabdomyolysis in PSSM2-QH occurred predominantly in barrel racing and working cow/roping performance types and improved with regular exercise and a low starch/fat-supplemented diet. Histopathological scores, including the amount of amylase-resistant polysaccharide (PSSM2-QH 1.4 ± 0.6, PSSM1-QH 2.1 ± 0.3, control-QH 0 ± 0, p < 0.001), and glycogen concentrations (PSSM2-QH 129 ± 62, PSSM1-QH 175 ± 9, control-QH 80 ± 27 mmol/kg, p < 0.0001) were intermediate in PSSM2-QH with significant differences among groups. In PSSM2-QH, abnormal polysaccharide had a less filamentous ultrastructure than PSSM1-QH and phosphorylase and phosphofructokinase activities were normal. Seventeen of 30 PSSM2-QH with available pedigrees descended from one of three stallions within four generations. Of the 29 predicted high or moderate impact genetic variants identified in candidate genes, none were present in only PSSM2-QH and absent in control-QH. MAIN LIMITATIONS: Analyses of PSSM2-QH and PSSM1-QH were performed on shipped samples, controls on frozen samples. CONCLUSIONS: PSSM2-QH is a novel glycogen storage disorder that is not the result of a mutation in genes currently known to cause muscle glycogenoses in other species.

CONTEXTO: Ambos os tipos 1 e 2 de miopatia por acúmulo de polissacarídeo (PSSM) são caracterizados por agregados de polissacarídeos anormais no músculo esquelético. Enquanto a base genética do PSSM 1 é conhecida (R309H GYS1), a causa do PSSM2 em cavalos Quarto de Milha (PSSM2-QH) é desconhecida, e a concentração de glicogênio não é definida. OBJETIVOS: Identificar as características histopatológicas e bioquímicas do PSSM-QH e determinar se há uma variante monogênica em genes conhecidos por causar glicogenose. DELINEAMENTO DO ESTUDO: Caso controlado retrospectivo. METODOLOGIA: 64 PSSM2-QH, 30 PSSM1-QH e 185 QH controles foram identificados em um arquivo de dados. Informação clínica, achados histológicos (escala 0-3), concentração de glicogênio e atividade enzimática de algumas enzimas glicolíticas foram comparadas. Sequências codificadas de 12 genes associados com glicogenose muscular foram identificados nas sequências genômicas completas, e comparadas entre 7 PSSM2-QH e 5 QH controles. RESULTADOS: Rabdomiólise por exercício em PSSM2-QH ocorreu predominantemente em cavalos de corrida de tambor e cavalos de team roping/trabalho com gado, e melhorou com exercício regular e uma dieta com baixo amido e alta gordura. A escala histopatológica, incluindo a quantidade de polissacarídeos resistentes à amilase (PSSM2-QH 1.4 ± 0.6, PSSM1-QH 2.1 ± 0.3, controle-QH 0 ± 0, P < 0.001), e concentrações de glicogênio (PSSM2-QH 129 ± 62, PSSM1-QH 175 ± 9, controle-QH 80 ± 27 mmol/kg, P < 0.0001) foram intermediárias em PSSM2-QH com diferença significante entre grupos. Em PSSM2-QH, polissacarídeo anormal teve uma ultraestrutura menos filamentosa do que PSSM1-QH e as atividades de fosforilase e fosfofrutoquinase foram normais. Dezessete dos 30 PSSM2-QH com pedigree disponível descendiam de 1 de 3 garanhões dentro de 4 gerações. Das 29 variações genéticas preditas a terem impacto moderado ou alto como genes candidatos, nenhuma estava presente apenas em PSSM2-QH e ausente no grupo controle-QH. PRINCIPAIS LIMITAÇÕES: As análises feitas nas amostras de PSSM2-QH e PSSM1-QH foram realizadas em amostras enviadas por correio, e as amostras dos animais controles eram amostras congeladas. CONCLUSÕES: PSSM2-QH é uma nova doença por acúmulo de glicogênio que não é o resultado de uma mutação nos genes conhecidos por causarem glicogenose muscular em outras espécies.

Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis.

Certain Standardbred racehorses develop recurrent exertional rhabdomyolysis (RER-STD) for unknown reasons. We compared gluteal muscle histopathology and gene/protein expression between Standardbreds with a history of, but not currently experiencing rhabdomyolysis (N = 9), and race-trained controls (N = 7). Eight RER-STD had a few mature fibers with small internalized myonuclei, one out of nine had histologic evidence of regeneration and zero out of nine degeneration. However, RER-STD versus controls had 791/13,531 differentially expressed genes (DEG). The top three gene ontology (GO) enriched pathways for upregulated DEG (N = 433) were inflammation/immune response (62 GO terms), cell proliferation (31 GO terms), and hypoxia/oxidative stress (31 GO terms). Calcium ion regulation (39 GO terms), purine nucleotide metabolism (32 GO terms), and electron transport (29 GO terms) were the top three enriched GO pathways for down-regulated DEG (N = 305). DEG regulated RYR1 and sarcoplasmic reticulum calcium stores. Differentially expressed proteins (DEP ↑N = 50, ↓N = 12) involved the sarcomere (24% of DEP), electron transport (23%), metabolism (20%), inflammation (6%), cell/oxidative stress (7%), and other (17%). DEP included ↑superoxide dismutase, ↑catalase, and DEP/DEG included several cysteine-based antioxidants. In conclusion, gluteal muscle of RER-susceptible Standardbreds is characterized by perturbation of pathways for calcium regulation, cellular/oxidative stress, inflammation, and cellular regeneration weeks after an episode of rhabdomyolysis that could represent therapeutic targets.

The Impact of N-Acetyl Cysteine and Coenzyme Q10 Supplementation on Skeletal Muscle Antioxidants and Proteome in Fit Thoroughbred Horses.

Horses have one of the highest skeletal muscle oxidative capacities amongst mammals, which, combined with a high glycolytic capacity, could perturb redox status during maximal exercise. We determined the effect of 30 d of oral coenzyme Q10 and N-acetyl-cysteine supplementation (NACQ) on muscle glutathione (GSH), cysteine, ROS, and coenzyme Q10 concentrations, and the muscle proteome, in seven maximally exercising Thoroughbred horses using a placebo and randomized cross-over design. Gluteal muscle biopsies were obtained the day before and 1 h after maximal exercise. Concentrations of GSH, cysteine, coenzyme Q10, and ROS were measured, and citrate synthase, glutathione peroxidase, and superoxide dismutase activities analyzed. GSH increased significantly 1 h post-exercise in the NACQ group (p = 0.022), whereas other antioxidant concentrations/activities were unchanged. TMT proteomic analysis revealed 40 differentially expressed proteins with NACQ out of 387 identified, including upregulation of 13 mitochondrial proteins (TCA cycle and NADPH production), 4 Z-disc proteins, and down regulation of 9 glycolytic proteins. NACQ supplementation significantly impacted muscle redox capacity after intense exercise by enhancing muscle glutathione concentrations and increasing expression of proteins involved in the uptake of glutathione into mitochondria and the NAPDH-associated reduction of oxidized glutathione, without any evident detrimental effects on performance.