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PDF file - 843K, Unsupervised hierarchical clustering of cBCLs.

PDF file - 881K, Western blot comparing phospho-NF-κB (p65) levels in "ABC-like" and "GCB-like" canine samples.

XLSX file - 40K, Canine genes identified by Ingenuity Pathway Analysis as part of the NF-kB or B-cell receptor signaling pathways (Fig. 5).

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PDF file - 41K, Distribution of lymphoma samples into "Ongoing" or "Static" categories based on the proportion of IGHV subclones that are identical at the CDR3 region for each sample.

XLSX file - 116K, Canine genes that distinguish "ABC-like" and "GCB-like" subtypes (Fig. 5).

Coinfections by avian pathogenic Escherichia coli (APEC) and Enterococcus faecalis in poultry with colisepticemia have become increasingly recognized. Here, we report draft genome sequences of 18 APEC and 18 E. faecalis strains coisolated from lesions of diseased poultry.

Cranial internal hemipelvectomy can be successful for excision of ilial CSA with minimal complications. Iliectomy with adjuvant radiation therapy was well tolerated in a dog with grade II ilial CSA. The dog survived 1,271 days postoperatively and supposedly succumbed to a disease process unrelated to the CSA., Cranial internal hemipelvectomy can be successful for excision of ilial chondrosarcoma in dogs, with minimal complications. Iliectomy was well tolerated and resulted in good hindlimb function postoperatively.

Virulent strains of Salmonella enterica subsp. enterica serovar Enteritidis (SE) harbored by poultry can cause disease in poultry flocks and potentially result in human foodborne illness. Two broiler flocks grown a year apart on the same premises experienced mortality throughout the growing period due to septicemic disease caused by SE. Gross lesions predominantly consisted of polyserositis followed by yolk sacculitis, arthritis, osteomyelitis, and spondylitis. Tissues with lesions were cultured yielding 59 SE isolates. These were genotyped by Rep-PCR followed by whole-genome sequencing (WGS) of 15 isolates which were clonal. The strain, SE_TAU19, was further characterized for antimicrobial susceptibility and virulence in a broiler embryo lethality assay. SE_TAU19 was resistant to nalidixic acid and sulfadimethoxine and was virulent to embryos with 100% mortality of all challenged broiler embryos within 3.5 days. Screening the SE_TAU19 whole-genome sequence revealed seven antimicrobial resistance (AMR) genes, 120 virulence genes, and two IncF plasmid replicons corresponding to a single, serovar-specific pSEV virulence plasmid. The pef, spv, and rck virulence genes localized to the plasmid sequence assembly. We report phenotypic and genomic features of a virulent SE strain from persistently infected broiler flocks and present a workflow for SE characterization from isolate collection to genome assembly and sequence analysis. Further SE surveillance and investigation of SE virulence in broiler chickens is warranted.

This chapter includes a collection of miscellaneous organisms that have caused disease in poultry or are a public health concern. The less common pathogens implicated in poultry loss included are Staphylococcosis, Streptococcus and Enterococcus, Erysipelas, avian intestinal spirochetosis, tuberculosis. Disease syndromes included in the chapter include beak necrosis, venereal disease of geese, and liver granulomas, but are not identified to a specific organism responsible because of the multifactorial nature of the disease. Staphylococcosis is diagnosed by culturing suspected clinical material including exudate from joints, yolk material, and stab swabs of internal organs. Staphylococcosis can resemble infection with Escherichia coli, Pasteurella multocida, Salmonella gallinarum, Mycoplasma synoviae, reoviruses, or any other infection of bones or joints that is hatchery‐related, associated with mechanical trauma, or causes septicemia. Prevention and control of Enterococcus infections require reducing stress and preventing immunosuppressive diseases and conditions. Proper cleaning and disinfection can reduce environmental enterococcal resident flora to minimize external exposure.

The objective of this study was to characterize the metabolomic profile of equine allantoic fluid in the pregnant mare with and without experimentally induced ascending placentitis with the goal of identifying biomarkers of this disease. We compared the onset of metabolomic changes with common modalities for diagnosis of ascending placentitis, including measurement of the combined thickness of the uterus and placenta (CTUP), hormonal profiling, and measurement of serum acute phase proteins. Ten pregnant pony mares were randomly divided into two groups: five healthy control mares (CONT) and five mares induced to develop ascending placentitis (PLAC) via inoculation with Streptococcus equi subsp. zooepidemicus bacteria at Days 280–285 of gestation. Allantoic fluid, whole blood, and serum were collected from both groups at 270–275 days of gestation and at the following time points postinoculation: 4 hours, Days 2, 4, 6, and 10. Differences between groups in identified metabolites, progesterone, estradiol-17β, lactate, and serum amyloid A (SAA) were assessed using an analysis of variance with repeated measures. A total of 27 metabolites were identified in allantoic fluid. No differences were detected between groups at any time point (P > .05) for any identified metabolite, progesterone, estradiol-17β, or lactate concentrations. Significant elevations in CTUP (P = .003) and SAA (P = .0001) were detected by Days 4 and 6 postinoculation, respectively. The results of this study established a database of equine allantoic fluid metabolites and confirmed the utility of uteroplacental ultrasound for detection of placentitis before the onset of hematologic changes.

Surgical procedures that maintain continence with minimal complication following resection of trigono-urethral urothelial carcinoma (UC) are limited in canines; therefore, palliative options are often pursued. A feasible tumor resection option may improve disease control and survival. The study's objective was to evaluate a continent urine reservoir created from the urinary bladder body and vascularized solely by omentum. We hypothesized that a viable urine reservoir could be created, and staged omentalization would provide improved vascularity. Nine normal female Beagles were randomized to one of three groups. Group A urinary bladders were transected cranial to the ureteral papillae to create a closed bladder vesicle which was concomitantly omentalized. Group B underwent omentalization two weeks prior to vesicle creation. Based on Group A and B results, Group C underwent neoureterocystostomy and omentalization followed by neoreservoir formation and tube cystostomy 2 weeks later. Serial ultrasounds and histopathology confirmed adequate omental neovascularization in Groups B and C with continent Group C neoreservoirs maintained for 2 months. Some pylectasia and ureteral dilation was documented in all Group C dogs at variable timepoints. Progressive hydroureteronephrosis developed in 2/6 kidneys. Transient azotemia was noted in only 1 Group C dog, although all developed treatable urinary tract infections. The sample size is limited, and the efficacy of this technique in providing disease control for UC is unknown. However, this novel option could allow for primary UC resection while providing continence and limiting complications. Postoperative local or systemic adjuvant therapy, ultrasonographic neoreservoir monitoring, and BRAF analysis would be indicated.

BACKGROUND Glioblastoma patient survival statistics have remained unchanged for more than three decades. Despite tumor resection and chemoradiotherapy, recurrence is inevitable. Moreover, the invasive behavior of glioblastoma confounds treatment. To improve patient survival statistics, a targeted therapy that can home to distant tumor foci is desperately needed. Induced neural stem cells (iNSCs) armed with cytotoxic payloads have proven efficacious against human xenograft models of glioblastoma. To further propel iNSCs to human clinical trials, we investigated the safety, toxicity, and persistence of iNSCs in a canine model. METHODS Autologous iNSCs generated from the skin of four non-tumor-bearing, purpose-bred, male beagles were engineered to express TRAIL and thymidine kinase (TK). iNSCs were loaded with ferumoxytol to facilitate MRI-tracking. Canines were divided into two cohorts to denote iNSC administration route: scaffold encapsulation or intracerebroventricular (ICV). Two dose levels were investigated: 1′106 iNSCs/kg or 3′106 iNSCs/kg. The scaffold cohort received a single dose of iNSCs while the ICV cohort received three doses of iNSCs via a Rickham reservoir. To activate TK, canines were administered valganciclovir. Canine health was assessed via neurological exams, MRI, and serial blood, urine, and CSF analyses. RESULTS No acute injection reactions were observed. Three of four canines exhibited surgery-induced blindness. Urine and CSF analyses were unremarkable. Unexpectedly, blood analyses showed transient neutropenia. Hypodense signal was observed on all MRI sequences through endpoint. Post-mortem histopathology of the spleen, liver, and lung were unremarkable. As expected, brain tissues exhibited gliosis, fibrous thickening, and inflammation. Spinal cords exhibited acute hemorrhaging, attributed to perimortem CSF draws. Surprisingly, significant testicular degeneration was observed; this was confirmed to be caused by valganciclovir. In conclusion, iNSCs exhibit limited toxicity and warrant further exploration. FUTURE DIRECTIONS Prospective studies will investigate the efficacy of autologous iNSCs in a spontaneous canine glioma model in preparation for human clinical trials.

In this study, we take an important step toward clinical translation by generating the first canine‐induced neural stem cells (iNSCs). We explore key aspects of scale‐up, persistence, and safety of personalized iNSC therapy in autologous canine surgery models. iNSCs are a promising new approach to treat aggressive cancers of the brain, including the deadly glioblastoma. Created by direct transdifferentiation of fibroblasts, iNSCs are known to migrate through the brain, track down invasive cancer foci, and deliver anticancer payloads that significantly reduce tumor burden and extend survival of tumor‐bearing mice. Here, skin biopsies were collected from canines and converted into the first personalized canine iNSCs engineered to carry TNFα‐related apoptosis‐inducing ligand (TRAIL) and thymidine kinase (TK), as well as magnetic resonance imaging (MRI) contrast agents for in vivo tracking. Time‐lapse analysis showed canine iNSCs efficiently migrate to human tumor cells, and cell viability assays showed both TRAIL and TK monotherapy markedly reduced tumor growth. Using intraoperative navigation and two delivery methods to closely mimic human therapy, canines received autologous iNSCs either within postsurgical cavities in a biocompatible matrix or via a catheter placed in the lateral ventricle. Both strategies were well tolerated, and serial MRI showed hypointense regions at the implant sites that remained stable through 86 days postimplant. Serial fluid sample testing following iNSC delivery showed the bimodal personalized therapy was well tolerated, with no iNSC‐induced abnormal tissue pathology. Overall, this study lays an important foundation as this promising personalized cell therapy advances toward human patient testing.

Enterococcus spp. (ENT) are frequently co-isolated with avian pathogenic E. coli (APEC) from poultry with colibacillosis, a leading cause of flock mortality. Although largely overlooked, ENT may play an active role in these infections. To assess the frequency of ENT co-isolation in colibacillosis, cultures were collected from birds with gross lesions of omphalitis, polyserositis, and septicaemia over a 3-year period from three turkey flocks and three broiler flocks. In birds diagnosed with colibacillosis based on gross findings and isolation of E. coli, ENT were co-isolated with APEC in 35.7% (n = 41/115) of colibacillosis mortality and 3.7% of total mortality (n = 41/1122). Co-isolated APEC and ENT pairs (n = 41) were further characterized using antimicrobial resistance phenotyping and in vitro co-culture assays. E. faecalis (EF) was the most commonly co-isolated species (68% n = 28/41) and tetracycline resistance was the resistance phenotype most commonly found among APEC (51% n = 21/41) and ENT (93% n = 38/41). Under iron-restricted conditions, EF enhanced APEC growth in a proximity-dependent manner and APEC grown in mixed culture with EF exhibited a significant growth and survival advantage (P ≤ 0.01). In an embryo lethality assay, APEC co-infection with EF resulted in decreased survival of broiler embryos compared to mono-infections (P ≤ 0.05). These data demonstrate that EF augmented APEC survival and growth under iron limiting conditions, possibly translating to the increased virulence of APEC in broiler embryos. Thus, ENT co-infections may be a previously unrecognized contributor to colibacillosis-related mortality. Further investigations into the mechanism of this interaction are warranted. RESEARCH HIGHLIGHTSEnterococcus is frequently co-isolated with avian pathogenic E. coli (APEC).Enterococcus faecalis (EF) enhances survival of APEC in iron restricted conditions.EF co-infection increases APEC virulence in broiler embryos. Enterococcus is frequently co-isolated with avian pathogenic E. coli (APEC). Enterococcus faecalis (EF) enhances survival of APEC in iron restricted conditions. EF co-infection increases APEC virulence in broiler embryos.

Pathogenic strains of Enterococcus cecorum cause symmetrical paralysis in broilers due to infection of the free thoracic vertebra. The disease caused by pathogenic E. cecorum, known as enterococcal spondylitis or "kinky-back" continues to be responsible for significant losses to the broiler industry worldwide. In outbreaks of pathogenic E. cecorum, gut colonization and sepsis occur in the first three weeks-of-life. Since maternal antibodies are present during this period, we postulated that vaccination of breeders with a polyvalent killed vaccine would protect chicks from challenge. To test this hypothesis, representative isolates from seven genotype groups of pathogenic E. cecorum circulating in the US were chosen to produce adjuvanted killed vaccines (bacterins) and given to broiler-breeder hens. No single strain produced high titres of antibodies to all other strains; however, the combination of serologic reactivity of pathogenic isolates (designated SA3 and SA7) was sufficient to react with all genotypes. Vaccination of commercial broiler-breeder hens with a bacterin composed of SA3 and SA7 did not have any adverse effects. Vaccinated hens developed E. cecorum specific antibodies; however, no significant difference in survival was observed in infected embryos from hens in vaccine or adjuvant only groups. Chicks from vaccinated hens also failed to resist homologous or heterologous challenge during experimental infection. In a macrophage killing assay, pathogenic E. cecorum were found to evade opsinophagocytosis with elicited antibodies. These data suggest that pathogenic strains of E. cecorum possess virulence mechanisms that confound antibody-mediated opsinophagocytosis, complicating vaccine development for this pathogen of broilers.

Pathogenic strains of Enterococcus cecorum (EC) expressing multidrug resistance have emerged. In National Antimicrobial Resistance Monitoring System (NARMS) data, EC is rarely recovered from chickens. Two NARMS methodologies (FDA and USDA) were compared with standard culture (SC) techniques for recovery of EC. NARMS methods failed to detect EC in 58 caecal samples, 20 chicken breast or six whole broiler samples. EC was recovered from 1 of 38 (2·6%) and 2 of 38 (5·2%) preharvest spinal lesions (USDA and FDA method, respectively). In contrast, using the SC method, EC was recovered from 44 of 53 (83%) caecal samples, all 38 (100%) spinal lesions, 14 of 20 (70%) chicken breast samples, and all three spinal lesions identified in whole carcasses. Compared with other Enterococcus spp., EC isolates had a higher prevalence of resistance to macrolides. The NARMS methods significantly affected recovery of enterococcal species other than EC. When the postharvest FDA method was applied to preharvest caecal samples, isolates of Enterococcus faecium were preferentially recovered. All 11 E. faecium isolates were multidrug resistant, including resistance to penicillin, daptomycin and linezolid. These findings confirm that current methodologies may not accurately identify the amount and range of antimicrobial resistance of enterococci from chicken sources. Significance and Impact of the Study Enterococci are an important reservoir for antimicrobial resistance. This study demonstrates how current culture methods underreport resistance to macrolides in enterococci by selecting against strains of Enterococcus cecorum in pre- and postharvest chicken. Further, the application of postharvest surveillance methods to preharvest samples resulted in selective recovery of Enterococcus faecium over Enterococcus faecalis. Isolates of E. faecium recovered exhibited multidrug resistance including penicillin, daptomycin and linezolid resistance. These findings suggest that culture methodology significantly impacts the range and amount of antimicrobial resistance detected in enterococci isolated from chicken.

The genetic relatedness and antimicrobial susceptibility profiles of Salmonella isolated from poultry and their environment were determined. One broiler breeder flock (BBF1) and 2 broiler flocks (BF1 and BF2) were reared over a 1.75-year period on the same poultry research farm. Hatching eggs were obtained from BBF1 to produce BF1 chicks, while BF2 chicks were progeny of a separate, unsampled broiler breeder flock. BF1 and BF2 were reared in the same housing facilities but 6 mo apart. Salmonella isolates were collected via litter sock sampling (BF1), cecal excision (BF1 and BF2), or cloacal swabs (BBF1). Serotyping identified Salmonella enterica subsp. enterica serovar Altona (SA) in BBF1 and S. enterica subsp. enterica serovar Senftenberg (SS) in BF1 and BF2. Genotypic fingerprinting was achieved with Rep-PCR using the (GTG)5 primer and revealed sequence homology among Senftenberg isolates from BF1 and BF2. For each isolate, the minimum inhibitory concentration was determined for 27 antimicrobial agents using Sensititre plates with formularies specific to antimicrobials used in poultry production or those used to control gram negative pathogens. Isolates from the 3 flocks were resistant to clindamycin, erythromycin, novobiocin, penicillin, and tylosin tartrate and demonstrated intermediate resistance to azithromycin, florfenicol, and spectinomycin. These data demonstrated that serovar Altona and Senftenberg were harbored by poultry, the latter appeared to persist in broiler flocks, and both serotypes shared similar patterns of antimicrobial susceptibility in an integrated research operation. In the case of multiple Salmonella isolates, combining genotypic fingerprinting methods with serotyping of representative isolates would reduce the number of samples required for serotyping and more clearly identify relatedness of isolates. These methods facilitate effective surveillance in poultry production systems, thus allowing for implementation of precise Salmonella control measures.

Since William Coley utilized bacterial immunotherapy to treat sarcomas in the late nineteenth century, an association between infection and improved survival has been reported for human and canine osteosarcoma patients. One of the reasons for this improved survival is likely a reactivation of the host immune system towards an inflammatory anti-tumour response, and one of the key players is the macrophage. Yet, despite their importance, the response of macrophages to infectious agents in the context of osteosarcoma has not been thoroughly evaluated. The aim of this study was to evaluate how in vitro exposure to a bacterial agent (Staphylococcus aureus) influenced canine and human macrophage differentiation in the presence of osteosarcoma. Our hypothesis was that S. aureus would, in the presence of osteosarcoma, induce a macrophage phenotype with significantly increased inflammatory signatures. Consistent with our hypothesis, human macrophages co-cultured with osteosarcoma and S. aureus exhibited increased IFN-γ, TNF-α and IL-12p70 cytokine secretion, decreased TGF-β cytokine secretion, and increased mRNA expression of TNF-α when compared to macrophages co-cultured with osteosarcoma and to macrophages cultured alone. Canine macrophages similarly exhibited increased IFN-γ and TNF-α cytokine secretion, decreased TGF-β cytokine secretion, increased mRNA expression of TNF-α, and increased surface receptor expression of CD80 when co-cultured with osteosarcoma and S. aureus. Collectively, the findings of this study suggest that infection upregulates the inflammatory immune response to counteract osteosarcoma-induced immune suppression. This work informs a potential therapeutic strategy to optimize inflammatory stimuli for triggering an anti-osteosarcoma macrophage response.

Wooden breast myopathy (WBM), or "woody breast" or "wooden breast" affects modern, rapidly growing, high breast-yield broiler chickens. Decreased meat quality due to undesirable organoleptic properties and condemnation of affected breast meat cause economic losses. The pathogenesis of WBM remains unknown. In this study, WBM lesion development was determined in three modern broiler strains and Athens Canadian Random Bred (ACRB) broilers, a 1950s unselected broiler chicken. Correlations between WBM severity and incubation temperature profile, sex, strain, body weight, and lymphocytic phlebitis were also determined. At 2, 4, 6, and 8 weeks of age, samples of breast muscle from 10 male and 10 female birds from each strain, incubated under optimal or low-early, high-late temperatures, were scored histologically for severity of WBM and lymphocytic phlebitis. WBM lesions, identified as early as 2 weeks, became progressively more severe with age and growth in the three commercial broiler strains. WBM severity was significantly correlated with lymphocytic phlebitis and body weight. Lymphocytic phlebitis and minimal WBM were present in the ACRB broilers at all samplings, but did not progress in severity over time. There were no significant differences in severity of WBM among the commercial broiler strains, between sexes, or between incubation temperature profiles. The positive correlation between WBM severity and lymphocytic phlebitis indicates vascular injury is likely an important factor in the pathogenesis. Mild muscle lesions in ACRB birds without overt clinical signs indicate subclinical muscle disease may have been present in broilers prior to the description of WBM.

Angiosarcoma is a highly aggressive cancer of blood vessel-forming cells with high fatality and few effective treatment options. It is both rare and heterogenous, making large, well powered genomic studies nearly impossible. In dogs, angiosarcoma is common, with breeds like the golden retriever carrying heritable genetic factors that put them at very high risk. If the clinical similarity of canine and human angiosarcoma reflects shared genomic etiology, dogs could be a critically needed model for advancing angiosarcoma research. We assessed the genomic landscape of canine angiosarcoma via whole exome sequencing (47 golden retriever angiosarcomas) and RNA sequencing (74 angiosarcomas from multiple breeds). The predominant mutational signature was the age-associated deamination of cytosine to thymine, and somatic coding mutations occurred most frequently in the tumor suppressorTP53(59.6% of cases) as well as two genes in the PI3K pathway: the oncogenePIK3CA(29.8%) and its regulatory subunitPIK3R1(8.5%). We compared the canine data to human data recently released by The Angiosarcoma Project, and found the same genes and many of the same pathways significantly enriched for somatic mutations, most notably protein kinases, glycoproteins, fibronectin Type III domains, EGF-like domains, and cell adhesion proteins such as cadherins. As in human angiosarcoma,CDKN2A/Bwas recurrently deleted andVEGFA, KDR, and KITrecurrently gained. Canine angiosarcoma closely models human angiosarcoma on a genomic level, and is a powerful tool for investigating the pathogenesis of this devastating disease.

Background Metabolomics may represent an avenue for diagnosis of equine ascending placentitis. Objectives To characterise the plasma metabolomic profile in healthy mares and mares with induced ascending placentitis, with the goal of identifying metabolites with potential clinical value for early diagnosis of placentitis. Study design Controlled in vivo experiment. Methods Placentitis was induced in 10 late-term pregnant pony mares via Streptococcal equi subsp. zooepidemicus inoculation in five mares between days 285 and 290 of gestation, while five mares served as healthy controls. Repeated ultrasound examinations and jugular venipuncture were performed to obtain combined thickness of the uterus and placenta (CTUP) and plasma for NMR spectroscopy. Mares with increased CTUP were diagnosed with placentitis and treated in accordance with published therapeutic recommendations. NMR metabolomic analysis was performed to identify and quantify plasma metabolites at each time point. Concentrations were compared using ANOVA with repeated-measures and PLS-DA analysis. Results Four hours post-inoculation, a significant increase was detected in the metabolites alanine, phenylalanine, histidine, pyruvate, citrate, glucose, creatine, glycolate, lactate and 3-hydroxyisobutyrate that returned to baseline by 12 hours. On day 4, a significant reduction in the metabolites alanine, phenylalanine, histidine, tyrosine, pyruvate, citrate, glycolate, lactate and dimethylsulfone was seen in infected mares compared with controls. Main limitations There were small numbers of mares within groups. In addition, this work compares healthy animals with animals treated with multimodal therapeutics following diagnosis of placentitis without an untreated cohort. Conclusions Two phases of metabolite changes were noted after experimental infection: An immediate rise in metabolite concentration involved in energy, nitrogen, hydrogen and oxygen metabolism within 4 hours after inoculation that was followed by a decrease in metabolite concentrations involved in energy and nitrogen metabolism at 4 days, coinciding with ultrasonographic diagnosis of placentitis.

An 11-year-old, 443-kg Haflinger mare was presented to the North Carolina State University Veterinary Teaching Hospital with a 2-week history of lethargy and a 3-day duration of anorexia, pyrexia, tachycardia, and ventral edema. Severe pitting edema, peripheral lymphadenopathy, and a caudal abdominal mass were noted on physical examination. An extreme leukocytosis (154.3 × 103 /μL) and microscopic hematologic findings suggestive of myelomonocytic leukemia were observed. Serum protein electrophoresis revealed a monoclonal gammopathy and urine protein electrophoresis revealed a monoclonal light chain proteinuria. Necropsy and histopathology confirmed widespread neoplastic infiltration in many organs with a heterogenous population of cells; there was no apparent evidence of bone marrow involvement. Immunohistochemistry confirmed presence of a majority of B cells with a limited antigen expression, admixed with a lower number of T cells. Molecular clonality analysis of IgH2, IgH3, and kappa-deleting element (KDE, B cell) on whole blood and KDE on infiltrated tissues revealed clonal rearrangements, and the KDE intron clones that amplified in blood and in infiltrated tissue were identical. In contrast, the clonality analysis of T-cell receptor γ revealed no clonality on blood cells and infiltrated tissues. In conjunction with the histopathologic changes, the lesion was interpreted to be composed of neoplastic B cells with a reactive T-cell population. Polymerase chain reaction testing for equine herpes virus 5 was negative. The final diagnosis was diffuse large B-cell lymphoma with a marked hematogenous component.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) found in some human tumors such as sarcomas. Canine tumors are not characterized for ALT and the study aim was to identify if the ALT phenotype exists in canine sarcomas. Sixty-four canine sarcoma samples (20 snap-frozen, 44 FFPE) as well as six canine sarcoma cell lines were screened for ALT by C-circle assay. ALT was further evaluated by measuring telomere length via qPCR and telomere restriction-fragments including pulsed-field electrophoresis. ALT-associated proteins were validated by immunohistochemistry. Further, telomerase activity (TA) and gene expression were analyzed by TRAP and qPCR. DNA from 20 human neuroblastomas and 8 sarcoma cell lines served as comparative controls. ALT was detected in 9.4% (6/64) canine sarcomas including aggressive subtypes as hemangiosarcoma, osteosarcoma, and histiocytic sarcoma. C-circle levels were comparable with human ALT-positive controls. All ALT tumors demonstrated loss of ATRX expression and 5/6 showed strong p53 expression. TA was detected in 93% (14/15) snap-frozen samples including a sarcoma with ALT activity. This tumor showed long heterogeneous telomeres, and a high level of colocalization of DAXX with telomeres. One sarcoma was ALT and TA negative. All canine and human sarcoma cell lines were ALT negative. In this study, we demonstrated that canine sarcomas use ALT. As in humans, ALT was identified in aggressive sarcomas subtypes and coexisted with TA in one tumor. Overall, canine sarcomas seem to share many similarities with their human counterparts and appear an attractive model for comparative telomere research. © 2016 Wiley Periodicals, Inc.

Enterococcal spondylitis (ES) is a disease of commercial broiler chickens, with a worldwide distribution. Symmetrical hind limb paralysis typical of ES results from infection of the free thoracic vertebra (FTV) by pathogenic strains of Enterococcus cecorum . To determine the pathogenesis of ES, birds with natural and experimental ES were studied over time. In natural disease, case birds (n = 150) from an affected farm and control birds (n = 100) from an unaffected farm were evaluated at weeks 1–6. In control birds, intestinal colonization by E. cecorum began at week 3. In case birds, E. cecorum was detected in intestine and spleen at week 1, followed by infection of the FTV beginning at week 3. E. cecorum isolates recovered from intestine, spleen, and FTV of case birds had matching genotypes, confirming that intestinal colonization with pathogenic strains precedes bacteremia and infection of the FTV. Clinical intestinal disease was not required for E. cecorum bacteremia. In 1- to 3-week-old case birds, pathogenic E. cecorum was observed within osteochondrosis dissecans (OCD) lesions in the FTV. To determine whether OCD of the FTV was a risk factor for ES, 214 birds were orally infected with E. cecorum, and the FTV was evaluated histologically at weeks 1–7. Birds without cartilage clefts of OCD in the FTV did not develop ES; while birds with OCD scores ≥3 were susceptible to lesion development. These findings suggest that intestinal colonization, bacteremia, and OCD of the FTV in early life are crucial to the pathogenesis of ES.

Nucleic acid-containing debris released from dead and dying cells can be recognized as damage-associated molecular patterns (DAMPs) or pattern-associated molecular patterns (PAMPs) by the innate immune system. Inappropriate activation of the innate immune response can engender pathological inflammation and autoimmune disease. To combat such diseases, major efforts have been made to therapeutically target the pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) that recognize such DAMPs and PAMPs, or the downstream effector molecules they engender, to limit inflammation. Unfortunately, such strategies can limit the ability of the immune system to combat infection. Previously, we demonstrated that nucleic acid-binding polymers can act as molecular scavengers and limit the ability of artificial nucleic acid ligands to activate PRRs. Herein, we demonstrate that nucleic acid scavengers (NASs) can limit pathological inflammation and nucleic acid-associated autoimmunity in lupus-prone mice. Moreover, we observe that such NASs do not limit an animal's ability to combat viral infection, but rather their administration improves survival when animals are challenged with lethal doses of influenza. These results indicate that molecules that scavenge extracellular nucleic acid debris represent potentially safer agents to control pathological inflammation associated with a wide range of autoimmune and infectious diseases.

BACKGROUND Understanding the influence of dietary iron deficiency and dietary iron oversupplementation on intestinal health is important for both animal production and human health. OBJECTIVE The aim of this study was to determine whether dietary iron concentration influences intestinal physiology, morphology, and inflammation in the porcine duodenum. METHODS Twenty-four male pigs (21 d old) were fed diets containing either 20 mg Fe/kg [low dietary iron (L-Fe)], 120 mg Fe/kg [adequate dietary iron (A-Fe); control], or 520 mg Fe/kg [high dietary iron (H-Fe)] by FeSO4 supplement (dry matter basis). After 32-36 d, the duodenum was harvested from pigs and mounted in Ussing chambers for the measurement of transepithelial electrical resistance (TER), short-circuit current, and (3)H-mannitol permeability. Intestinal morphology and inflammation were assessed by histologic examination, and proinflammatory gene expression was assessed by real-time polymerase chain reaction. RESULTS Compared with A-Fe-fed pigs, pigs fed L-Fe diets exhibited reduced TER (by 30%; P 100%) and H-Fe-fed (∼4-fold) pigs; the L-Fe-fed and H-Fe-fed groups did not differ significantly from one another. Compared with the L-Fe-fed pigs, the A-Fe-fed and H-Fe-fed pigs had malondialdehyde concentrations 1.4- and 2.5-fold higher in the duodenum and 4.4- and 6.6-fold higher in the liver, respectively (P < 0.05). Neutrophil counts were higher in both the L-Fe-fed (by 3-fold) and H-Fe-fed (by 3.3-fold) groups than in the A-Fe-fed group; the L-Fe-fed and H-Fe-fed groups did not significantly differ from one another. Duodenal mucosal tumor necrosis factor α (TNFA), interleukin (IL) 1β, and IL6 relative gene expression was upregulated by 36%, 28%, and 45%, respectively, in H-Fe pigs (P < 0.05), but not in L-Fe pigs, compared with A-Fe pigs. CONCLUSION These data suggest that adequate but not oversupplementation of dietary iron in pigs is required to maintain intestinal barrier health and function.

Follicular lymphomas (FLs) typically exhibit a chromosome translocation that induces constitutive expression of the anti-apoptotic bcl2 protein and accumulation of additional molecular defects. This rearrangement offers a promising therapeutic target, but its nature as a fundamental driver of FL pathogenesis remains unclear as 15% of cases lack the translocation. We performed an integrated immunohistochemical and genomic investigation of 10 naturally occurring FL cases from domestic dogs, showing that, as with human tumours, they exhibit marked heterogeneity in the frequency and intensity of bcl2 protein expression. Genomic copy number aberrations were infrequent and broadly consistent with those of other canine B-cell lymphoma subtypes. None of the canine FL specimens exhibited a rearrangement consistent with the hallmark translocation of human FL, despite their remarkable histomorphologic similarity. Parallel exploration of canine and human cases may reveal alternative tumour-initiating mechanisms other than BCL2 disruption, yielding a more complete definition of the molecular pathogenesis of FL.

The utility of the domestic cat as a model system for biomedical studies was constrained for many years by the absence of a comprehensive feline reference genome sequence assembly. While such a resource now exists, the cat continues to lag behind the domestic dog in terms of integration into the &lsquo, One Health&rsquo, era of molecular medicine. Stimulated by the advances being made within the evolving field of comparative cancer genomics, we developed a microarray platform that allows rapid and sensitive detection of DNA copy number aberrations in feline tumors using comparative genomic hybridization analysis. The microarray comprises 110,456 unique oligonucleotide probes anchored at mean intervals of 22.6 kb throughout the feline reference genome sequence assembly, providing ~350-fold higher resolution than was previously possible using this technique. We demonstrate the utility of this resource through genomic profiling of a feline injection-site sarcoma case, revealing a highly disrupted profile of DNA copy number imbalance involving several key cancer-associated genes including KIT, TP53, PTEN, FAS and RB1. These findings were supported by targeted fluorescence in-situ hybridization analysis, which identified major alterations in chromosome structure, including complex intrachromosomal reorganization events typical of those seen in aggressive soft-tissue sarcomas of other species. We then characterized a second mass that was identified at a nearby site in the same patient almost 12 months later. This mass demonstrated a remarkably conserved genomic profile consistent with a recurrence of the original tumor, however the detection of subtle differences reflected evolution of the tumor over time. These findings exemplify the diverse potential of this microarray platform to incorporate domestic cat cancers into comparative and translational research efforts in molecular oncology.

BACKGROUND Esophagitis with eosinophilia, inflammation, and fibrosis represent a chronic condition in humans with food allergies. OBJECTIVE In this investigation, we asked whether esophagitis with an eosinophilic component is observed in young pigs rendered allergic to hen egg white protein (HEWP). METHODS Food allergy was induced in young pigs using two protocols. In one protocol, sensitized pigs were challenged by gavage with a single dose of HEWP. Clinical signs were monitored for 24 hours, and then, gastrointestinal (GI) tissues were collected for histological examination. The phenotype of circulating, ovalbumin (OVA)-specific T cells also was examined in HEWP challenged animals. In the second protocol, sensitized animals were fed HEWP for 28 days. Animals were then examined by endoscopy and gastrointestinal tissues collected for histological examination. RESULTS In pigs challenged by gavage with HEWP, clinical signs were noted in 5/6 pigs including diarrhoea, emesis, and skin rash. Clinical signs were not seen in any control group. Histological analysis revealed significant levels of oesophageal eosinophilic infiltration (P

Occurrence of mortality, wooden breast, and pulmonary disease in broiler chickens during the last 16 days of production in a teaching flock of 4000 commercial broilers was determined. A new syndrome was identified, in which broilers fell over for an unknown reason and were unable to right themselves (dorsal recumbency). Birds affected by dorsal recumbency were alert and responsive and showed no clinical signs except for occasional mild to moderate dyspnea. When turned over, they resumed normal behavior. Mortality (14 culls; 49 dead) during the last 16 days of production accounted for 1.6% of the flock and 36% of total mortality. Among these, 71% were heavy males, 70% had wooden breast, and 71% had pulmonary congestion and edema. Gross lesions of concurrent wooden breast and pulmonary disease occurred in 68% of the mortality, including 21 of 22 dead birds found on their backs. These findings indicate that wooden breast is associated with mortality prior to processing as a result of pulmonary disease in heavy male broilers. When birds with wooden breast fall onto their backs for unknown reason(s), they are unable to right themselves. If not found and turned over, they may not survive. Based on these findings, wooden breast is likely greater than just a problem with meat quality and should be considered an animal well-being issue.Reporte de caso- Presencia de “pechuga de madera” en pollos de engorde comerciales asociada con mortalidad, recumbencia dorsal y enfermedad pulmonar. Se determinó la presentación de mortalidad, “pechuga de madera” y enfermedad pulmonar en pollos de engorde durante los últimos 16 días de producción en una parvada de 4000 pollos de engorde comerciales. Se identificó un nuevo síndrome en el que los pollos se postraban por una razón desconocida y no podían enderezarse (recumbencia dorsal). Las aves afectadas por la recumbencia dorsal estaban alertas y respondían, y no mostraban signos clínicos, excepto casos de disnea ocasional de leve a moderada. Cuando las aves se colocaban en posición normal, retomaban su comportamiento normal. La mortalidad (14 aves eliminadas; 49 muertas) durante los últimos 16 días de producción representó el 1.6% de la parvada y 36% de la mortalidad total. Entre estos, el 71% eran machos pesados, 70% tenían “pechuga de madera” y 71% tenían congestión pulmonar y edema. Lesiones macroscópicas concurrentes de enfermedad pulmonar y de “pechuga de madera” ocurrieron en el 68% de la mortalidad, incluyendo 21 de las 22 aves muertas que fueron encontradas postradas sobre sus dorsos. Estos hallazgos indican que la “pechuga de madera” se asocia con la mortalidad antes del procesamiento como resultado de enfermedad pulmonar en pollos de engorde machos pesados. Cuando las aves con “pechuga de madera” se caen de espaldas por razones desconocidas, no pueden enderezarse. Si no se encuentran y se colocan en posición normal, pueden no sobrevivir. Según estos hallazgos, la “pechuga de madera” es probablemente más que solo un problema con la calidad de la carne y también debe considerarse un problema de bienestar animal.

Enterococcus cecorum was initially identified as a harmless commensal of the gastrointestinal tract of chickens. However, over the past 15 yr, pathogenic strains of E. cecorum have become a significant cause of morbidity and mortality in broiler breeders, and repeated outbreaks occur, but an environmental reservoir for pathogenic E. cecorum has yet to be identified. Genetic analyses of E. cecorum demonstrate that strains with increased pathogenicity are genetically related and share several putative virulence genes. Pathogenic E. cecorum carry increased antimicrobial resistance compared to commensal strains. These pathogenic strains can be recovered from retail meat and may serve as a reservoir for further spread of antimicrobial resistance among other Enterococcus spp. This review presents the current understanding of the pathogenesis of E. cecorum and briefly discusses antimicrobial resistance in E. cecorum due to the role of Enterococcus spp. in nosocomial infections in people.

Phosphorylated histone 3 (PH3) and cleaved caspase 3 (CCASP3) were used to detect proliferating and apoptotic cells, respectively, in the jejunums of female sibling poults, with and without enteritis and depressed growth, from hatch to day 35. Poults that developed enteritis and depressed growth (SIB flock) were raised on a commercial farm in eastern North Carolina, whereas poults with normal growth and no enteritis (TAU flock) were raised in the Teaching Animal Unit at North Carolina State University College of Veterinary Medicine. Beginning on day 5 through day 35 and at processing, TAU poults were significantly heavier than SIB poults. Jejunal weights, relative jejunal weights, and jejunal densities were greater in SIB poults from day 10 through 35. Jejunal efficiency (body weight /jejunal length) was higher in TAU poults at day 5 and days 10 through 35. Mucosal thickness was greater in SIB poults between days 7 and 21 but greater in TAU poults at days 28 and 35. From day 7 to 35, villus-to-crypt ratios were higher for TAU poults and lower for SIB poults because hyperplastic crypts formed a greater percentage of the mucosa in SIB poults. By day 7, PH3- and CCASP3-positive cells were increased in SIB poults, showing that mucosal changes resulted from combined crypt epithelial hyperplasia and increased apoptosis of villous enterocytes. Findings in this study confirm that enteritis, in the absence of clinical signs, and depressed growth in turkey poults begins by day 7, can be identified microscopically, persists for at least 35 days, is associated with lower processing weights, and has a profound negative effect on turkey growth.

OBJECTIVE: To evaluate the effects of nanoparticle hyperthermia therapy on monocyte function and tumor-derived factors associated with macrophage polarization in a murine osteosarcoma model. STUDY DESIGN: Experimental study. ANIMALS: Female C3H mice. METHODS: Peripheral blood monocyte cell surface phenotype, monocyte chemotaxis, tumor messenger RNA expression, and survival were compared among osteosarcoma (OS)-bearing mice treated with nanoparticle hyperthermia therapy, OS-bearing mice with osteomyelitis, OS-bearing mice, vehicle control mice, and normal control mice. RESULTS: OS-bearing mice with osteomyelitis had a higher proportion of “nonclassical” monocytes (Ly6C(lo)) compared with all other experimental groups. There were alterations in monocyte expression of multiple chemokine receptors among experimental groups including CXCR2, CCR2, and CXCR4. Monocytes from OS-bearing mice treated with hyperthermia therapy exhibited greater chemotaxis compared with monocytes from OS-bearing mice with osteomyelitis. CONCLUSION: OS likely induced alterations in monocyte phenotype and function. Nanoparticle hyperthermia therapy increased in vitro monocyte chemotaxis. CLINICAL IMPACT: Enhancing monocyte/macrophage function in dogs with OS may enhance antitumor immunity.

Mast cells are well established as divergent modulators of inflammation and immunosuppression, but their role in inflammatory bowel disease (IBD) remains to be fully defined. While previous studies have demonstrated a proinflammatory role for mast cells in acute models of chemical colitis, more recent investigations have shown that mast cell deficiency can exacerbate inflammation in spontaneous colitis models, thus suggesting a potential anti-inflammatory role of mast cells in IBD. Here, we tested the hypothesis that in chronic, spontaneous colitis, mast cells are protective. We compared colitis and intestinal barrier function in IL10−/−mice to mast cell deficient/IL10−/−(double knockout (DKO): KitWsh/Wsh × IL10−/−) mice. Compared with IL10−/−mice, DKO mice exhibited more severe colitis as assessed by increased colitis scores, mucosal hypertrophy, intestinal permeability, and colonic cytokine production. PCR array analyses demonstrated enhanced expression of numerous cytokine and chemokine genes and downregulation of anti-inflammatory genes (e.g.,Tgfb2,Bmp2,Bmp4,Bmp6, andBmp7) in the colonic mucosa of DKO mice. Systemic reconstitution of DKO mice with bone marrow-derived mast cells resulted in significant amelioration of IL10−/−-mediated colitis and intestinal barrier injury. Together, the results presented here demonstrate that mast cells exert anti-inflammatory properties in an established model of chronic, spontaneous IBD. Given the previously established proinflammatory role of mast cells in acute chemical colitis models, the present findings provide new insight into the divergent roles of mast cells in modulating inflammation during different stages of colitis. Further investigation of the mechanism of the anti-inflammatory role of the mast cells may elucidate novel therapies.

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis . Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene ( gelE ). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2 −/− ) mice were used to investigate the role of this receptor in E. faecalis -induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2 −/− mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2 −/− mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2 −/− mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.

An A10 aptamer (Apt)-functionalized, sub-100 nm doxorubicin-polylactide (Doxo-PLA) nanoconjugate (NC) with controlled release profile was developed as an intravenous therapeutic strategy to effectively target and cytoreduce canine hemangiosarcoma (cHSA), a naturally occurring solid tumor malignancy composed solely of tumor-associated endothelium. cHSA consists of a pure population of malignant endothelial cells expressing prostate-specific membrane antigen (PSMA) and is an ideal comparative tumor model system for evaluating the specificity and feasibility of tumor-associated endothelial cell targeting by A10 Apt-functionalized NC (A10 NC). In vitro, A10 NCs were selectively internalized across a panel of PSMA-expressing cancer cell lines, and when incorporating Doxo, A10 Doxo-PLA NCs exerted greater cytotoxic effects compared to nonfunctionalized Doxo-PLA NCs and free Doxo. Importantly, intravenously delivered A10 NCs selectively targeted PSMA-expressing tumor-associated endothelial cells at a cellular level in tumor-bearing mice and dramatically increased the uptake of NCs by endothelial cells within the local tumor microenvironment. By virtue of controlled drug release kinetics and selective tumor-associated endothelial cell targeting, A10 Doxo-PLA NCs possess a desirable safety profile in vivo, being well-tolerated following high-dose intravenous infusion in mice, as supported by the absence of any histologic organ toxicity. In cHSA-implanted mice, two consecutive intravenous infusions of A10 Doxo-PLA NCs exerted rapid and substantial cytoreductive activities within a period of 7 days, resulting in greater than 70% reduction in macroscopic tumor-associated endothelial cell burden as a consequence of enhanced cell death and necrosis.

Background Reports of histiocytic sarcoma (HS) involving the central nervous system (CNS) are sparse and consist mainly of case reports describing 1–3 animals. Objective The objective of this study was to report the signalments, clinical signs, clinicopathologic and diagnostic imaging findings, treatment, and outcome of a series of dogs with HS and CNS involvement. Animals Nineteen dogs with HS examined at veterinary referral hospitals. Methods Retrospective case series. Medical records were reviewed and cases with a histopathological diagnosis of CNS HS were included in the study. Diagnostic imaging studies of the CNS were evaluated and histopathologic samples were reviewed to confirm the diagnosis. Results Retrievers and Pembroke Welsh Corgis were overrepresented in this cohort of dogs. Tumors involved the brain in 14 dogs and the spinal cord in 5. In 4 dogs, HS was part of a disseminated, multiorgan process whereas it appeared confined to the CNS in 15 dogs. Diagnostic imaging had variable appearances although extraaxial masses predominated in the brain. There was meningeal enhancement in all dogs that was often profound and remote from the primary mass lesion. Pleocytosis was present in all dogs with CSF evaluation. Median survival was 3 days. Conclusions and Clinical Importance Breed predispositions appear to vary from reports of HS in other organ systems. Some unique imaging and clinicopathologic characteristics, particularly brain herniation, profound meningeal enhancement, and pleocytosis in combination with 1 or more mass lesions, might help to differentiate this neoplasm from others involving the CNS, although this requires further study.