Evidence of viral survival in representative volumes of feed and feed ingredients during long-distance commercial transport across the continental United States.

The hypothesis that feed ingredients could serve as vehicles for the transport and transmission of viral pathogens was first validated under laboratory conditions. To bridge the gap from the laboratory to the field, this current project tested whether three significant viruses of swine could survive in feed ingredients during long-distance commercial transport across the continental US. One-metric tonne totes of soybean meal (organic and conventional) and complete feed were spiked with a 10 ml mixture of PRRSV 174, PEDV and SVA and transported for 23 days in a commercial semi-trailer truck, crossing 29 states, and 10,183 km. Samples were tested for the presence of viral RNA by PCR, and for viable virus in soy-based samples by swine bioassay and in complete feed samples by natural feeding. Viable PRRSV, PEDV and SVA were detected in both soy products and viable PEDV and SVA in complete feed. These results provide the first evidence that viral pathogens of pigs can survive in representative volumes of feed and feed ingredients during long-distance commercial transport across the continental United States.

An evaluation of additives for mitigating the risk of virus-contaminated feed using an ice-block challenge model.

The role of animal feed as a vehicle for the transport and transmission of viral diseases was first identified during the porcine epidemic diarrhoea virus (PEDV) epidemic in North America. Since that time, various feed additives have been evaluated at the laboratory level to measure their effect on viral viability and infectivity in contaminated feed using bioassay piglet models. While a valid first step, the conditions of these studies were not representative of commercial swine production. Therefore, the purpose of this study was to evaluate the ability of feed additives to mitigate the risk of virus-contaminated feed using a model based on real-world conditions. This new model used an 'ice-block' challenge, containing equal concentrations of porcine reproductive and respiratory syndrome virus (PRRSV), Senecavirus A (SVA) and PEDV, larger populations of pigs, representative commercial facilities and environments, along with realistic volumes of complete feed supplemented with selected additives. Following supplementation, the ice block was manually dropped into designated feed bins and pigs consumed feed by natural feeding behaviour. After challenge, samples were collected at the pen level (feed troughs, oral fluids) and at the animal level (clinical signs, viral infection, growth rate, and mortality) across five independent experiments involving 15 additives. In 14 of the additives tested, pigs on supplemented diets had significantly greater average daily gain (ADG), significantly lower clinical signs and infection levels, and numerically lower mortality rates compared to non-supplemented controls. In conclusion, the majority of the additives evaluated mitigated the effects of PRRSV 174, PEDV and SVA in contaminated feed, resulting in improved health and performance.

Comparison of the sensitivity of laryngeal swabs and deep tracheal catheters for detection of Mycoplasma hyopneumoniae in experimentally and naturally infected pigs early and late after infection.

Detection of Mycoplasma hyopneumoniae infection in live pigs is a critical component to measure the success of disease control or elimination strategies. However, in vivo diagnosis of M. hyopneumoniae is difficult and the imperfect sensitivity of diagnostic tools has been deemed as one of the main challenges. Here, the sensitivity of laryngeal swabs and deep tracheal catheters for detection of M. hyopneumoniae early and late after infection was determined using inoculation status as a gold standard in experimentally infected pigs and a Bayesian approach in naturally infected pigs. Three-hundred and twenty 8-week old seeder pigs were intra-tracheally inoculated with M. hyopneumoniae strain 232 and immediately placed with 1920 contact pigs to achieve a 1:6 seeder-to-contact ratio. A subset of seeders and contacts were longitudinally sampled at 7, 28, 97, and 113 days post-inoculation (dpi) and at 28, 56, 84, and 113 days post-exposure (dpe), respectively, using laryngeal swabs and deep tracheal catheters. Samples were tested for M. hyopneumoniae by a species-specific real-time PCR. The sensitivity of deep tracheal catheters was higher than the one obtained in laryngeal swabs at all samplings (seeders: 36% higher than laryngeal swabs at 7 dpi, 29% higher at 97 dpi, and 44% higher at 113 dpi; contacts: 51% higher at 56 dpe, 42% higher at 84 dpe, and 32% higher at 113 dpe). Our study indicates that deep tracheal catheters were a more sensitive sample than laryngeal swabs. The sensitivity of both sample types varied over time and by exposure method, and these factors should be considered when designing diagnostic strategies.

A randomized controlled trial to evaluate performance of pigs raised in antibiotic-free or conventional production systems following challenge with porcine reproductive and respiratory syndrome virus.

The trial objective was to compare the performance and animal health parameters of pigs raised according to one of 3 antibiotic (AB) protocols: standard AB medication consisting of mass treatment on days 4 and 21 and judicious AB therapy given therapeutically thereafter as group medication in water and feed or by individual injection (group T1, N = 702); modified AB medication identical to group T1 but with mass treatment only on day 4 and without subsequent therapeutic feed medication (group T2, N = 675); or an antibiotic-free (ABF) regimen (group T3, N = 702). All pigs were vaccinated with a modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine 3 days after weaning. Using a seeder pig model to mimic endemic field infection dynamics, pigs were contact-challenged with virulent PRRSV lineage 1 strain 174 four weeks after vaccination. At finishing, average daily gain (ADG) and mean feed conversion ratio (FCR) were significantly better (p ≤ 0.05) for the T1 and T2 groups compared to the T3 group. There were no significant differences in post-weaning ADG and FCR between the T1 and T2 groups. Mortality and removals significantly favored (p ≤0.05) the T1 and T2 groups (20.94% and 24.89%, respectively) versus the T3 group (57.98%). Net revenue per pig was $105.43, $98.79, and $33.81 for the T1, T2 and T3 groups, respectively. Under the conditions of this study, these results indicate that in a PRRSV-endemic setting involving bacterial co-infections, an ABF production strategy may leave pigs at considerable risk of exposure to severe clinical disease and that judicious use of antibiotics can significantly improve animal health.

An XRCC4 splice mutation associated with severe short stature, gonadal failure, and early-onset metabolic syndrome.

CONTEXT: Severe short stature can be caused by defects in numerous biological processes including defects in IGF-1 signaling, centromere function, cell cycle control, and DNA damage repair. Many syndromic causes of short stature are associated with medical comorbidities including hypogonadism and microcephaly. OBJECTIVE: To identify an underlying genetic etiology in two siblings with severe short stature and gonadal failure. DESIGN: Clinical phenotyping, genetic analysis, complemented by in vitro functional studies of the candidate gene. SETTING: An academic pediatric endocrinology clinic. PATIENTS OR OTHER PARTICIPANTS: Two adult siblings (male patient [P1] and female patient 2 [P2]) presented with a history of severe postnatal growth failure (adult heights: P1, -6.8 SD score; P2, -4 SD score), microcephaly, primary gonadal failure, and early-onset metabolic syndrome in late adolescence. In addition, P2 developed a malignant gastrointestinal stromal tumor at age 28. INTERVENTION(S): Single nucleotide polymorphism microarray and exome sequencing. RESULTS: Combined microarray analysis and whole exome sequencing of the two affected siblings and one unaffected sister identified a homozygous variant in XRCC4 as the probable candidate variant. Sanger sequencing and mRNA studies revealed a splice variant resulting in an in-frame deletion of 23 amino acids. Primary fibroblasts (P1) showed a DNA damage repair defect. CONCLUSIONS: In this study we have identified a novel pathogenic variant in XRCC4, a gene that plays a critical role in non-homologous end-joining DNA repair. This finding expands the spectrum of DNA damage repair syndromes to include XRCC4 deficiency causing severe postnatal growth failure, microcephaly, gonadal failure, metabolic syndrome, and possibly tumor predisposition.

Identifying biological pathways that underlie primordial short stature using network analysis.

Mutations in CUL7, OBSL1 and CCDC8, leading to disordered ubiquitination, cause one of the commonest primordial growth disorders, 3-M syndrome. This condition is associated with i) abnormal p53 function, ii) GH and/or IGF1 resistance, which may relate to failure to recycle signalling molecules, and iii) cellular IGF2 deficiency. However the exact molecular mechanisms that may link these abnormalities generating growth restriction remain undefined. In this study, we have used immunoprecipitation/mass spectrometry and transcriptomic studies to generate a 3-M 'interactome', to define key cellular pathways and biological functions associated with growth failure seen in 3-M. We identified 189 proteins which interacted with CUL7, OBSL1 and CCDC8, from which a network including 176 of these proteins was generated. To strengthen the association to 3-M syndrome, these proteins were compared with an inferred network generated from the genes that were differentially expressed in 3-M fibroblasts compared with controls. This resulted in a final 3-M network of 131 proteins, with the most significant biological pathway within the network being mRNA splicing/processing. We have shown using an exogenous insulin receptor (INSR) minigene system that alternative splicing of exon 11 is significantly changed in HEK293 cells with altered expression of CUL7, OBSL1 and CCDC8 and in 3-M fibroblasts. The net result is a reduction in the expression of the mitogenic INSR isoform in 3-M syndrome. From these preliminary data, we hypothesise that disordered ubiquitination could result in aberrant mRNA splicing in 3-M; however, further investigation is required to determine whether this contributes to growth failure.

Exploring the spectrum of 3-M syndrome, a primordial short stature disorder of disrupted ubiquitination.

3-M syndrome is an autosomal recessive primordial growth disorder characterized by small birth size and post-natal growth restriction associated with a spectrum of minor anomalies (including a triangular-shaped face, flat cheeks, full lips, short chest and prominent fleshy heels). Unlike many other primordial short stature syndromes, intelligence is normal and there is no other major system involvement, indicating that 3-M is predominantly a growth-related condition. From an endocrine perspective, serum GH levels are usually normal and IGF-I normal or low, while growth response to rhGH therapy is variable but typically poor. All these features suggest a degree of resistance in the GH-IGF axis. To date, mutations in three genes CUL7, OBSL1 and CCDC8 have been shown to cause 3-M. CUL7 acts an ubiquitin ligase and is known to interact with p53, cyclin D-1 and the growth factor signalling molecule IRS-1, the link with the latter may contribute to the GH-IGF resistance. OBSL1 is a putative cytoskeletal adaptor that interacts with and stabilizes CUL7. CCDC8 is the newest member of the pathway and interacts with OBSL1 and, like CUL7, associates with p53, acting as a co-factor in p53-medicated apoptosis. 3-M patients without a mutation have also been identified, indicating the involvement of additional genes in the pathway. Potentially damaging sequence variants in CUL7 and OBSL1 have been identified in idiopathic short stature (ISS), including those born small with failure of catch-up growth, signifying that the 3-M pathway could play a wider role in disordered growth.

The genetics of 3-M syndrome: unravelling a potential new regulatory growth pathway.

3-M syndrome is an autosomal recessive primordial growth disorder characterised by severe postnatal growth restriction caused by mutations in CUL7, OBSL1 or CCDC8. Clinical characteristics include dysmorphic facial features and fleshy prominent heels with a variable degree of radiological abnormalities. CUL7 is a structural protein central to the formation of an ubiquitin E3 ligase that is known to target insulin receptor substrate 1 for degradation. CUL7 also binds to p53 and may be involved in the control of p53-dependent apoptosis. OBSL1 is a cytoskeletal adaptor protein that was thought to play a central role in myocyte remodelling, and CCDC8 has no defined function as yet. However, the physical interaction of OBSL1 with both CUL7 and CCDC8 and its potential role in the regulation of CUL7 expression suggest all three proteins are members of the same growth-regulatory pathway. Future work should be directed to investigating the function of the 3-M syndrome pathway and in particular the role in the insulin like growth factor I signalling pathway with a view of potentially revealing new therapeutic targets and identifying key regulators of cellular growth.

Exome sequencing identifies CCDC8 mutations in 3-M syndrome, suggesting that CCDC8 contributes in a pathway with CUL7 and OBSL1 to control human growth.

3-M syndrome, a primordial growth disorder, is associated with mutations in CUL7 and OBSL1. Exome sequencing now identifies mutations in CCDC8 as a cause of 3-M syndrome. CCDC8 is a widely expressed gene that is transcriptionally associated to CUL7 and OBSL1, and coimmunoprecipitation indicates a physical interaction between CCDC8 and OBSL1 but not CUL7. We propose that CUL7, OBSL1, and CCDC8 are members of a pathway controlling mammalian growth.

The primordial growth disorder 3-M syndrome connects ubiquitination to the cytoskeletal adaptor OBSL1.

3-M syndrome is an autosomal-recessive primordial growth disorder characterized by significant intrauterine and postnatal growth restriction. Mutations in the CUL7 gene are known to cause 3-M syndrome. In 3-M syndrome patients that do not carry CUL7 mutations, we performed high-density genome-wide SNP mapping to identify a second locus at 2q35-q36.1. Further haplotype analysis revealed a 1.29 Mb interval in which the underlying gene is located and we subsequently discovered seven distinct null mutations from 10 families within the gene OBSL1. OBSL1 is a putative cytoskeletal adaptor protein that localizes to the nuclear envelope. We were also able to demonstrate that loss of OBSL1 leads to downregulation of CUL7, implying a role for OBSL1 in the maintenance of CUL7 protein levels and suggesting that both proteins are involved within the same molecular pathway.

Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation.

FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.

Tumor necrosis factor-alpha stimulates focal adhesion kinase activity required for mitogen-activated kinase-associated interleukin 6 expression.

Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-alpha (TNFalpha)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK(-/-)) and FAK(+/+) mouse embryo fibroblasts. FAK promoted TNFalpha-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFalpha-mediated nuclear factor-kappaB or c-Jun N-terminal kinase activation. TNFalpha-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK(-/-) fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFalpha-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK(-/-) and A549 FAK short hairpin RNA-expressing cells rescued TNFalpha-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFalpha-stimulated IL-6 expression. Moreover, analyses of Src(-/-), Yes(-/-), and Fyn(-/-) fibroblasts showed that Src expression was inhibitory to TNFalpha-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression.