Expression of miRNAs in turkey muscle satellite cells and differential response to thermal challenge.

Thermal stress alters the transcriptome and subsequent tissue physiology of poultry; thus, it can negatively impact poultry production through reduced meat quality, egg production, and health and wellbeing. The modulation of gene expression is critical to embryonic development and cell proliferation, and growing evidence suggests the role of non-coding RNAs (RNA:RNA interaction) in response to thermal stress in animals. MicroRNAs (miRNAs) comprise a class of small regulatory RNAs that modulate gene expression through posttranscriptional interactions and regulate mRNAs, potentially altering numerous cellular processes. This study was designed to identify and characterize the differential expression of miRNAs in satellite cells (SCs) from the turkey pectoralis major muscle and predict important miRNA:mRNA interactions in these developing SCs under a thermal challenge. Small RNA sequencing was performed on RNA libraries prepared from SCs cultured from 1-week-old male Nicholas commercial turkeys (NCTs) and non-selected Randombred Control Line 2 turkeys during proliferation and differentiation at the control temperature (38°C) or under a thermal challenge (33°C or 43°C). A total of 353 miRNAs (161 known and 192 novel) were detected across the sequenced libraries. Expression analysis found fewer differentially expressed miRNAs in the SCs of NCT birds, suggesting that the miRNA response to heat stress has been altered in birds selected for their modern commercial growth traits. Differentially expressed miRNAs, including those with described roles in muscle development, were detected both among temperature treatments and between genetic lines. A prominent differential expression of miR-206 was found in proliferating turkey SCs with a significant response to thermal challenges in both lines. In differentiating SCs, isoforms of miR-1 had significant differential responses, with the expression of miR-206 being mainly affected only by cold treatment. Target gene predictions and Gene Ontology analysis suggest that the differential expression of miRNAs during thermal stress could significantly affect cellular proliferation and differentiation.

Transcriptome Response of Differentiating Muscle Satellite Cells to Thermal Challenge in Commercial Turkey.

Early muscle development involves the proliferation and differentiation of stem cells (satellite cells, SCs) in the mesoderm to form multinucleated myotubes that mature into muscle fibers and fiber bundles. Proliferation of SCs increases the number of cells available for muscle formation while simultaneously maintaining a population of cells for future response. Differentiation dramatically changes properties of the SCs and environmental stressors can have long lasting effects on muscle growth and physiology. This study was designed to characterize transcriptional changes induced in turkey SCs undergoing differentiation under thermal challenge. Satellite cells from the pectoralis major (p. major) muscle of 1-wk old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (Randombred Control Line 2, RBC2) were proliferated for 72 h at 38 °C and then differentiated for 48 h at 33 °C (cold), 43 °C (hot) or 38 °C (control). Gene expression among thermal treatments and between turkey lines was examined by RNAseq to detect significant differentially expressed genes (DEGs). Cold treatment resulted in significant gene expression changes in the SCs from both turkey lines, with the primary effect being down regulation of the DEGs with overrepresentation of genes involved in regulation of skeletal muscle tissue regeneration and sarcomere organization. Heat stress increased expression of genes reported to regulate myoblast differentiation and survival and to promote cell adhesion particularly in the NCT line. Results suggest that growth selection in turkeys has altered the developmental potential of SCs in commercial birds to increase hypertrophic potential of the p. major muscle and sarcomere assembly. The biology of SCs may account for the distinctly different outcomes in response to thermal challenge on breast muscle growth, development, and structure of the turkey.

Transcriptome response of proliferating muscle satellite cells to thermal challenge in commercial turkey.

Thermal stress poses a threat to agricultural systems through increased risk to animal growth, health, and production. Exposure of poultry, especially hatchlings, to extreme temperatures can seriously affect muscle development and thus compromise subsequent meat quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells (SCs) cultured from commercial birds under thermal challenge to determine the applicability of previous results obtained for select research lines. Satellite cells isolated from the pectoralis major muscle of 1-week old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (RBC2) were proliferated in culture at 38°C or 43°C for 72 h. RNAseq analysis found statistically significant differences in gene expression among treatments and between turkey lines with a greater number of genes altered in the NCT SCs suggesting early myogenesis. Pathway analysis identified cell signaling and regulation of Ca2+ as important responses. Expression of the intercellular signaling Wnt genes, particularly Wnt5a and 7a was significantly altered by temperature with differential response between lines. The peripheral calcium channel RYR3 gene was among the genes most highly upregulated by heat stress. Increased expression of RYR3 would likely result in higher resting cytosolic calcium levels and increased overall gene transcription. Although responses in the calcium signaling pathway were similar among the RBC2 and NCT lines, the magnitude of expression changes was greater in the commercially selected birds. These results provide evidence into how SC activity, cellular fate, and ultimately muscle development are altered by heat stress and commercial selection.

Data Mining Identifies Differentially Expressed Circular RNAs in Skeletal Muscle of Thermally Challenged Turkey Poults.

Precise regulation of gene expression is critical for normal muscle growth and development. Changes in gene expression patterns caused by external stressors such as temperature can have dramatic effects including altered cellular structure and function. Understanding the cellular mechanisms that underlie muscle growth and development and how these are altered by external stressors are crucial in maintaining and improving meat quality. This study investigated circular RNAs (circRNAs) as an emerging aspect of gene regulation. We used data mining to identify circRNAs and characterize their expression profiles within RNAseq data collected from thermally challenged turkey poults of the RBC2 and F-lines. From sequences of 28 paired-end libraries, 8924 unique circRNAs were predicted of which 1629 were common to all treatment groups. Expression analysis identified significant differentially expressed circRNAs (DECs) in comparisons between thermal treatments (41 DECs) and between genetic lines (117 DECs). No intersection was observed between the DECs and differentially expressed gene transcripts indicating that the DECs are not simply the result of expression changes in the parental genes. Comparative analyses based on the chicken microRNA (miRNA) database suggest potential interactions between turkey circRNAs and miRNAs. Additional studies are needed to reveal the functional significance of the predicted circRNAs and their role in muscle development in response to thermal challenge. The DECs identified in this study provide an important framework for future investigation.

The hepatic transcriptome of the turkey poult (Meleagris gallopavo) is minimally altered by high inorganic dietary selenium.

There is interest in supplementing animals and humans with selenium (Se) above Se-adequate levels, but the only good biomarker for toxicity is tissue Se. We targeted liver because turkeys fed 5 µg Se/g have hepatic Se concentrations 6-fold above Se-adequate (0.4 µg Se/g) levels without effects on growth or health. Our objectives were (i) to identify transcript biomarkers for high Se status, which in turn would (ii) suggest proteins and pathways used by animals to adapt to high Se. Turkey poults were fed 0, 0.025, 0.4, 0.75 and 1.0 µg Se/g diet in experiment 1, and fed 0.4, 2.0 and 5.0 µg Se/g in experiment 2, as selenite, and the full liver transcriptome determined by RNA-Seq. The major effect of Se-deficiency was to down-regulate expression of a subset of selenoprotein transcripts, with little significant effect on general transcript expression. In response to high Se intake (2 and 5 µg Se/g) relative to Se-adequate turkeys, there were only a limited number of significant differentially expressed transcripts, all with only relatively small fold-changes. No transcript showed a consistent pattern of altered expression in response to high Se intakes across the 1, 2 and 5 µg Se/g treatments, and there were no associated metabolic pathways and biological functions that were significant and consistently found with high Se supplementation. Gene set enrichment analysis also found no gene sets that were consistently altered by high-Se and supernutritional-Se. A comparison of differentially expressed transcript sets with high Se transcript sets identified in mice provided high Se (~3 µg Se/g) also failed to identify common differentially expressed transcript sets between these two species. Collectively, this study indicates that turkeys do not alter gene expression in the liver as a homeostatic mechanism to adapt to high Se.

Transcriptional Profiling and Molecular Characterization of the yccT Mutant Link: A Novel STY1099 Protein with the Peroxide Stress Response and Cell Division of Salmonella enterica Serovar Enteritidis.

Uncharacterized protein STY1099, encoded by the yccT gene, was previously identified as the most altered (i.e., upregulated) protein among the ZnO nanoparticle (NP) stimulon of Salmonella enterica serovar Enteritidis. Here we combined various stress response-related assays with functional genetics, global transcriptomic and proteomic analyses to characterize the yccT gene and its STY1099 product. Exposure of S. enterica Enteritidis to H2O2 (i.e., hydrogen peroxide) resulted in a significant (p < 0.0001) upregulation of the yccT gene, whereas exposure to paraquat (i.e., superoxide) did not alter the expression of the yccT gene. The ∆yccT mutant of S. enterica Enteritidis exposed to 0.75 mM H2O2, showed significantly reduced (p < 0.05) viability compared to the wild type strain. Further, comparative transcriptome analyses supported by Co-immunoprecipitation (Co-IP) assay revealed that STY1099 protein plays a role in redox homeostasis during the peroxide stress assault via involvement in the processes of respiratory nitrate reductase, oxidoreductase activities, cellular uptake and stress response. In addition, we found that the STY1099 protein has the monopolar subcellular location and that it interacts with key cell division proteins, MinD, and FtsH, as well as with a rod shape-determining protein MerB.

Antibiotics and Host-Tailored Probiotics Similarly Modulate Effects on the Developing Avian Microbiome, Mycobiome, and Host Gene Expression.

The microbiome is important to all animals, including poultry, playing a critical role in health and performance. Low-dose antibiotics have historically been used to modulate food production animals and their microbiome. Identifying alternatives to antibiotics conferring similar modulatory properties has been elusive. The purpose of this study was to determine if a host-tailored probiotic could recapitulate effects of a low-dose antibiotic on host response and the developing microbiome. Over 13 days of life, turkey poults were supplemented continuously with a low-dose antibiotic or oral supplementation of a prebiotic with or without two different probiotics (8 cage units, n = 80 per group). Gastrointestinal bacterial and fungal communities of poults were characterized by 16S rRNA gene and ITS2 amplicon sequencing. Localized and systemic host gene expression was assessed using transcriptome sequencing (RNA-Seq), kinase activity was assessed by avian-specific kinome peptide arrays, and performance parameters were assessed. We found that development of the early-life microbiome of turkey poults was tightly ordered in a tissue- and time-specific manner. Low-dose antibiotic and turkey-tailored probiotic supplementation, but not nontailored probiotic supplementation, elicited similar shifts in overall microbiome composition during development compared to controls. Treatment-induced bacterial changes were accompanied by parallel shifts in the fungal community and host gene expression and enhanced performance metrics. These results were validated in pen trials that identified further additive effects of the turkey-tailored probiotic combined with different prebiotics. Alternative approaches to low-dose antibiotic use in poultry are feasible and can be optimized utilizing the indigenous poultry microbiome. Similar approaches may also be beneficial for humans.IMPORTANCE Alternative approaches are greatly needed to reduce the need for antibiotic use in food animal production. This study utilized a pipeline for the development of a host-tailored probiotic to enhance performance in commercial turkeys and modulate their microbiota, similar to the effects of low-dose antibiotic administration. We determined that a host-tailored probiotic, developed in the context of the commercial turkey gut microbiome, was more effective at modulating these parameters than a nontailored probiotic cocktail. Furthermore, the host-tailored probiotic mimicked many of the effects of a low-dose antibiotic growth promoter. Surprisingly, the effects of the antibiotic growth promoter and host-tailored probiotic were observed across kingdoms, illustrating the coordinated interkingdom effects of these approaches. This work suggests that tailored approaches to probiotic development hold promise for modulating the avian host and its microbiota.

Altered Gene Response to Aflatoxin B1 in the Spleens of Susceptible and Resistant Turkeys.

Susceptibility and/or resistance to aflatoxin B1 (AFB1) is a threshold trait governed principally by glutathione S transferase (GST)-mediated detoxification. In poultry, domesticated turkeys are highly sensitive to AFB1, most likely due to dysfunction in hepatic GSTs. In contrast, wild turkeys are comparatively resistant to aflatoxicosis due to the presence of functional hepatic GSTAs and other possible physiological and immunological interactions. The underlying genetic basis for the disparate GST function in turkeys is unknown as are the broader molecular interactions that control the systemic response. This study quantifies the effects of dietary AFB1 on gene expression in the turkey spleen, specifically contrasting genetically distinct domesticated (DT, susceptible) and Eastern wild (EW, resistant) birds. Male turkey poults were subjected to a short-term AFB1 treatment protocol with feed supplemented with 320 ppb AFB1 beginning on day 15 of age and continuing for 14 days. Spleen tissues were harvested and subjected to deep RNA sequencing and transcriptome analysis. Analysis of differential gene expression found the effects of AFB1 treatment on the spleen transcriptomes considerably more prominent in the DT birds compared to EW. However, expression of the differentially expressed genes (DEGs) was directionally biased, with the majority showing higher expression in EW (i.e., down-regulation in DT). Significantly altered pathways included FXR/RXR and LXR/RXR activation, coagulation system, prothrombin activation, acute phase response, and atherosclerosis signaling. Differential extra-hepatic expression of acute phase protein genes was confirmed by quantitative real time PCR (qRT-PCR) in the original experiment and additional turkey lines. Results demonstrate that wild turkeys possess a capacity to more effectively respond to AFB1 exposure.

Differential Transcriptome Responses to Aflatoxin B1 in the Cecal Tonsil of Susceptible and Resistant Turkeys.

The nearly-ubiquitous food and feed-borne mycotoxin aflatoxin B1 (AFB1) is carcinogenic and mutagenic, posing a food safety threat to humans and animals. One of the most susceptible animal species known and thus a good model for characterizing toxicological pathways, is the domesticated turkey (DT), a condition likely due, at least in part, to deficient hepatic AFB1-detoxifying alpha-class glutathione S-transferases (GSTAs). Conversely, wild turkeys (Eastern wild, EW) are relatively resistant to the hepatotoxic, hepatocarcinogenic and immunosuppressive effects of AFB1 owing to functional gene expression and presence of functional hepatic GSTAs. This study was designed to compare the responses in gene expression in the gastrointestinal tract between DT (susceptible phenotype) and EW (resistant phenotype) following dietary AFB1 challenge (320 ppb for 14 days); specifically in cecal tonsil which functions in both nutrient absorption and gut immunity. RNAseq and gene expression analysis revealed significant differential gene expression in AFB1-treated animals compared to control-fed domestic and wild birds and in within-treatment comparisons between bird types. Significantly upregulated expression of the primary hepatic AFB1-activating P450 (CYP1A5) as well as transcriptional changes in tight junction proteins were observed in AFB1-treated birds. Numerous pro-inflammatory cytokines, TGF-ß and EGF were significantly down regulated by AFB1 treatment in DT birds and pathway analysis suggested suppression of enteroendocrine cells. Conversely, AFB1 treatment modified significantly fewer unique genes in EW birds; among these were genes involved in lipid synthesis and metabolism and immune response. This is the first investigation of the effects of AFB1 on the turkey gastro-intestinal tract. Results suggest that in addition to the hepatic transcriptome, animal resistance to this mycotoxin occurs in organ systems outside the liver, specifically as a refractory gastrointestinal tract.

Thermal challenge alters the transcriptional profile of the breast muscle in turkey poults.

Extremes in temperature represent environmental stressors that impact the well-being and economic value of poultry. As homeotherms, young poultry with immature thermoregulatory systems are especially susceptible to thermal extremes. Genetic variation and differences in gene expression resulting from selection for production traits, likely contribute to thermal stress response. This study was designed to investigate in vivo transcriptional changes in the breast muscle of young turkey poults from an unselected randombred line and one selected for 16 wk body weight under hot and cold thermal challenge. Newly hatched turkey poults were brooded for 3 d at one of 3 temperatures: control (35°C), cold (31°C), or hot (39°C). Samples of the pectoralis major were harvested and subjected to deep RNA sequencing. Significant differential gene expression was observed in both growth-selected and randombred birds at both temperature extremes when compared to control-brooded poults. Growth-selected birds responded to thermal stress through changes in genes predicted to have downstream transcriptional effects and that would result in reduced muscle growth. Slower growing randombred birds responded to thermal stress through modulation of lipid-related genes, suggesting reduction in lipid storage, transport, and synthesis, consistent with changes in energy metabolism required to maintain body temperature.

Comparative Response of the Hepatic Transcriptomes of Domesticated and Wild Turkey to Aflatoxin B1.

The food-borne mycotoxin aflatoxin B1 (AFB1) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys (Meleagris gallopavo) are especially sensitive, whereas wild turkeys (M. g. silvestris) are more resistant. AFB1 toxicity entails bioactivation by hepatic cytochrome P450s to the electrophilic exo-AFB1-8,9-epoxide (AFBO). Domesticated turkeys lack functional hepatic GST-mediated detoxification of AFBO, and this is largely responsible for the differences in resistance between turkey types. This study was designed to characterize transcriptional changes induced in turkey livers by AFB1, and to contrast the response of domesticated (susceptible) and wild (more resistant) birds. Gene expression responses to AFB1 were examined using RNA-sequencing. Statistically significant differences in gene expression were observed among treatment groups and between turkey types. Expression analysis identified 4621 genes with significant differential expression (DE) in AFB1-treated birds compared to controls. Characterization of DE transcripts revealed genes dis-regulated in response to toxic insult with significant association of Phase I and Phase II genes and others important in cellular regulation, modulation of apoptosis, and inflammatory responses. Constitutive expression of GSTA3 was significantly higher in wild birds and was significantly higher in AFB1-treated birds when compared to controls for both genetic groups. This pattern was also observed by qRT-PCR in other wild and domesticated turkey strains. Results of this study emphasize the differential response of these genetically distinct birds, and identify genes and pathways that are differentially altered in aflatoxicosis.

Response of Turkey Muscle Satellite Cells to Thermal Challenge. II. Transcriptome Effects in Differentiating Cells.

Background: Exposure of poultry to extreme temperatures during the critical period of post-hatch growth can seriously affect muscle development and thus compromise subsequent meat quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells by thermal challenge during differentiation. Our goal is to better define how thermal stress alters breast muscle ultrastructure and subsequent development. Results: Skeletal muscle satellite cells previously isolated from the Pectoralis major muscle of 7-wk-old male turkeys (Meleagris gallopavo) from two breeding lines: the F-line (16 wk body weight-selected) and RBC2 (randombred control line) were used in this study. Cultured cells were induced to differentiate at 38°C (control) or thermal challenge temperatures of 33 or 43°C. After 48 h of differentiation, cells were harvested and total RNA was isolated for RNAseq analysis. Analysis of 39.9 Gb of sequence found 89% mapped to the turkey genome (UMD5.0, annotation 101) with average expression of 18,917 genes per library. In the cultured satellite cells, slow/cardiac muscle isoforms are generally present in greater abundance than fast skeletal isoforms. Statistically significant differences in gene expression were observed among treatments and between turkey lines, with a greater number of genes affected in the F-line cells following cold treatment whereas more differentially expressed (DE) genes were observed in the RBC2 cells following heat treatment. Many of the most significant pathways involved signaling, consistent with ongoing cellular differentiation. Regulation of Ca2+ homeostasis appears to be significantly affected by temperature treatment, particularly cold treatment. Conclusions: Satellite cell differentiation is directly influenced by temperature at the level of gene transcription with greater effects attributed to selection for fast growth. At lower temperature, muscle-associated genes in the satellite cells were among the genes with the greatest down regulation consistent with slower differentiation and smaller myotubes. Fewer expression differences were observed in the differentiating cells than previously observed for proliferating cells. This suggests the impact of temperature on satellite cells occurs primarily at early points in satellite cell activation.

Response of turkey muscle satellite cells to thermal challenge. I. transcriptome effects in proliferating cells.

BACKGROUND: Climate change poses a multi-dimensional threat to food and agricultural systems as a result of increased risk to animal growth, development, health, and food product quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells cultured under cold or hot thermal challenge to better define molecular mechanisms by which thermal stress alters breast muscle ultrastructure. RESULTS: Satellite cells isolated from the pectoralis major muscle of 7-weeks-old male turkeys from two breeding lines (16 weeks body weight-selected and it's randombred control) were proliferated in culture at 33 °C, 38 °C or 43 °C for 72 h. Total RNA was isolated and 12 libraries subjected to RNAseq analysis. Statistically significant differences in gene expression were observed among treatments and between turkey lines with a greater number of genes altered by cold treatment than by hot and fewer differences observed between lines than between temperatures. Pathway analysis found that cold treatment resulted in an overrepresentation of genes involved in cell signaling/signal transduction and cell communication/cell signaling as compared to control (38 °C). Heat-treated muscle satellite cells showed greater tendency towards expression of genes related to muscle system development and differentiation. CONCLUSIONS: This study demonstrates significant transcriptome effects on turkey skeletal muscle satellite cells exposed to thermal challenge. Additional effects on gene expression could be attributed to genetic selection for 16 weeks body weight (muscle mass). New targets are identified for further research on the differential control of satellite cell proliferation in poultry.

Targeted capture enrichment and sequencing identifies extensive nucleotide variation in the turkey MHC-B.

Variation in the major histocompatibility complex (MHC) is increasingly associated with disease susceptibility and resistance in avian species of agricultural importance. This variation includes sequence polymorphisms but also structural differences (gene rearrangement) and copy number variation (CNV). The MHC has now been described for multiple galliform species including the best defined assemblies of the chicken (Gallus gallus) and domestic turkey (Meleagris gallopavo). Using this sequence resource, this study applied high-throughput sequencing to investigate MHC variation in turkeys of North America (NA turkeys). An MHC-specific SureSelect (Agilent) capture array was developed, and libraries were created for 14 turkeys representing domestic (commercial bred), heritage breed, and wild turkeys. In addition, a representative of the Ocellated turkey (M. ocellata) and chicken (G. gallus) was included to test cross-species applicability of the capture array allowing for identification of new species-specific polymorphisms. Libraries were hybridized to ∼12 K cRNA baits and the resulting pools were sequenced. On average, 98% of processed reads mapped to the turkey whole genome sequence and 53% to the MHC target. In addition to the MHC, capture hybridization recovered sequences corresponding to other MHC regions. Sequence alignment and de novo assembly indicated the presence of several additional BG genes in the turkey with evidence for CNV. Variant detection identified an average of 2245 polymorphisms per individual for the NA turkeys, 3012 for the Ocellated turkey, and 462 variants in the chicken (RJF-256). This study provides an extensive sequence resource for examining MHC variation and its relation to health of this agriculturally important group of birds.

Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics.

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9 Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6 % of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.

Conserved MHC gene orthologs genetically map to the turkey MHC- B.

The avian MHC-associated gene set includes orthologs to genes found throughout the human major histocompatibility complex (MHC), including some loci of the evolutionarily conserved class III region. In the turkey and other Galliformes, genes linked to the MHC have been identified because they are closely associated with class I or class II genes. This study was designed to evaluate additional class III genes for linkage to the avian MHC to further determine conservation of these loci in birds. BLAST searches were used to locate sequences in the turkey genome with similarity to genes shared between the MHC of Xenopus and humans. Primers were designed to target 25 genes, and putative orthologs were amplified by PCR and sequenced. Sequence polymorphisms were identified for 15 genes in turkey reference mapping families, and 8 genes showed significant genetic linkage to the turkey MHC-B locus. These new genetic markers and linkage relationships broaden our understanding of the composition of the avian MHC and expand the gene content for the turkey MHC-B.

Response of the hepatic transcriptome to aflatoxin B1 in domestic turkey (Meleagris gallopavo).

Dietary exposure to aflatoxin B1 (AFB1) is detrimental to avian health and leads to major economic losses for the poultry industry. AFB1 is especially hepatotoxic in domestic turkeys (Meleagris gallopavo), since these birds are unable to detoxify AFB1 by glutathione-conjugation. The impacts of AFB1 on the turkey hepatic transcriptome and the potential protection from pretreatment with a Lactobacillus-based probiotic mixture were investigated through RNA-sequencing. Animals were divided into four treatment groups and RNA was subsequently recovered from liver samples. Four pooled RNA-seq libraries were sequenced to produce over 322 M reads totaling 13.8 Gb of sequence. Approximately 170,000 predicted transcripts were de novo assembled, of which 803 had significant differential expression in at least one pair-wise comparison between treatment groups. Functional analysis linked many of the transcripts significantly affected by AFB1 exposure to cancer, apoptosis, the cell cycle or lipid regulation. Most notable were transcripts from the genes encoding E3 ubiquitin-protein ligase Mdm2, osteopontin, S-adenosylmethionine synthase isoform type-2, and lipoprotein lipase. Expression was modulated by the probiotics, but treatment did not completely mitigate the effects of AFB1. Genes identified through transcriptome analysis provide candidates for further study of AFB1 toxicity and targets for efforts to improve the health of domestic turkeys exposed to AFB1.

Aflatoxicosis chemoprevention by probiotic Lactobacillius and lack of effect on the major histocompatibility complex.

Turkeys are extremely sensitive to aflatoxin B1 (AFB1) which causes decreased growth, immunosuppression and liver necrosis. The purpose of this study was to determine whether probiotic Lactobacillus, shown to be protective in animal and clinical studies, would likewise confer protection in turkeys, which were treated for 11 days with either AFB1 (AFB; 1 ppm in diet), probiotic (PB; 1 × 10(11) CFU/ml; oral, daily), probiotic + AFB1 (PBAFB), or PBS control (CNTL). The AFB1 induced drop in body and liver weights were restored to normal in CNTL and PBAFB groups. Hepatotoxicity markers were not significantly reduced by probiotic treatment. Major histocompatibility complex (MHC) genes BG1 and BG4, which are differentially expressed in liver and spleens, were not significantly affected by treatments. These data indicate modest protection, but the relatively high dietary AFB1 treatment, and the extreme sensitivity of this species may reveal limits of probiotic-based protection strategies.

Evaluation of CHD7 as a candidate gene for choanal atresia in alpacas (Vicugna pacos).

Choanal atresia (CA) is a craniofacial malformation characterized by obstruction of the posterior nasal aperture, resulting in laborious respiratory inspiration and exhalation. Alpaca crias with CA typically develop fatal pneumonia, frequently as the result of milk aspiration during nursing, and euthanasia is usually inevitable. Nonsense or missense mutations in the CHD7 gene cause a comparable condition (CHARGE syndrome) in humans. In this study, the coding region of CHD7 was sequenced in six CA-affected alpacas. Forty-nine sequence variants were identified, of which 10 would result in amino acid changes (non-synonymous), some with potentially deleterious effects. However, none of the observed variants would result in the obvious deleterious effects caused by nonsense or missense mutations. Although a role for CHD7 mutations in CA cannot be definitively dismissed, these do not appear to be the primary cause of CA in alpacas.

Heterologous expression and functional characterization of avian mu-class glutathione S-transferases.

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB1-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB1 detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.