University of Saskatchewan

Western College of Veterinary Medicine

2012 Graduate Student Poster Day

The 2012 WCVM Graduate Student Day takes place at the Western College of Veterinary Medicine (second floor) on March 13-14, 2012. The following is the list of graduate students participating in the event. Please click on graduate students' names to view their research project abstracts.


Title: Obesity is associated with adverse cardiovascular outcomes and insulin resistance in dogs

Team members:

Abstract:

Obesity and cardiovascular disease are strongly linked in humans, but this association is less clear in dogs. The purpose of this study was to evaluate the effects of obesity on cardiac structure and function as well as insulin resistance in dogs.

Cardiovascular variables were measured and oral glucose tolerance tests were performed before and after weight gain in 9 beagles. At baseline, dogs were fed a commercial diet in measured amounts to maintain an ideal body weight. Subsequently, for 12 weeks the dogs were allowed free access to the diet to allow weight gain. Echocardiography was used to analyze left ventricular function. Blood pressure was measured by high-definition oscillometry. Serum glucose and insulin were measured before and at specific time points after an oral glucose challenge to determine glucose tolerance and insulin sensitivity. Computed tomography (CT) was used to quantify total and visceral fat accumulation in the dogs in the obese state.

Body weight increased from 9.8 ± 0.6 kg at baseline to 12.1 ± 0.7 kg (123 ± 3% of ideal body weight) after ad libitum feeding. Systolic left ventricular free wall thickness, heart rate, area under the glucose response curve and peak glucose significantly increased in the obese state compared to the lean state (p<0.05). Systolic free wall thickness was positively correlated with total fat (r=0.7, p=0.02) and visceral fat (r=0.7, p=0.03). Area under the insulin response curve was associated with visceral fat (r=0.8, p=0.03). Increased systolic free wall thickness, coupled with elevated heart rate, in dogs likely reflects hyperdynamic cardiac function which may have negative long term cardiovascular consequences.

Thus, this study supports the hypothesis that visceral fat is linked to detrimental changes in cardiac function and insulin sensitivity in obesity. These results are particularly remarkable considering that the dogs were obese for only 12 weeks.

Funding sources: NSERC, Saskatchewan Pulse Growers, Horizon Pet Nutrition, Western College of Veterinary Medicine Vitamin Settlement Grant, Canadian Foundation for Innovation.


Title: Bovine adenovirus (BAdV)-3 pVIII, a core protein, inhibits capped cellular mRNA translation

Team members:

Abstract

Although several viruses inhibit capped cellular mRNA translation during infection to augment their life cycles, very little is known about the mechanisms involved in this process. Our preliminary data on bovine adenovirus (BAdV)-3 also showed a dramatic reduction in the translation of capped cellular mRNA during the course of virus infection.

Based on this data, we hypothesized that late proteins of BAdV-3 are responsible for the inhibition of translation of cellular capped mRNA. To assess the viral proteins and mechanisms involved in the inhibition of capped cellular mRNA translation, we determined the reduction of capped cellular mRNA translation in cells transfected with plasmid DNAs expressing late BAdV-3 proteins. The results indicated that pVIII (a core protein) reduces global cellular capped mRNA translation in a dose dependent manner. pVIII also inhibited the in-vitro translation of capped mRNA. 

In addition, cells co-transfected with a bicistronic luciferase plasmid DNA (IRES-Renilla Luciferase and capped-Firefly luciferase) and pVIII-expressing plasmid DNA suggested a significant reduction in the translation of  capped firefly luciferase indicating the involvement of pVIII in suppressing translation of cellular capped mRNA.

These results imply that pVIII is one of the late proteins involved in inhibiting capped cellular mRNA translation. Presently, we are working to determine the mechanisms pVIII uses to inhibit the translation of cellular capped mRNA.


Title: Expression of TLR10 in human lungs and neutrophils

Team members:

Abstract:

Toll-like receptors (TLRs) are conserved immune receptors that play critical roles in innate immunity. TLR10 dimerize with TLR1 or TLR2 on the plasma membrane but the identity of its ligand remains unclear. There also is a lack of information regarding expression of TLR10 in the lungs and neutrophils.

Because functional TLR10 gene is present in human and chicken but not in mice, we examined TLR10 protein expression in normal and inflamed human and chicken lungs by immunohistochemistry and human neutrophils stimulated with E. coli lipopolysaccharide (1μg/ml), which binds TLR4, was analysed for expression of TLR10 protein at 60, 90 and 120 minutes using western blotting. The surface expression of TLR10 was examined by flow cytometry and confocal microscopy used for the subcellular localization of TLR10 in LPS activated neutrophils.

Immunohistochemistry showed TLR10 in the vascular endothelium in the human and chicken lungs. Immunohistochemistry and Western blots detected an increase in TLR10 protein in lungs of chicken infected with E. coli or Fowl Adenovirus. LPS activated neutrophils showed increased expression of TLR10 protein at 60 and 120 minutes but a reduction at 90 minutes with Western blots. Flow cytometry mirrored the changes in TLR10 surface expression observed with Western blots. Confocal microscopy showed cytosolic and nuclear TLR10 in normal neutrophils. TLR10 in activated neutrophils co-localized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, to suggest its endocytosis.

We conclude that TLR10 is expressed in human and chicken lung vasculature, and that LPS alters expression of TLR10 and induce its endocytosis in human neutrophils.

Funding: NSERC


Title: A new approach to assess ovarian structures in live rabbits: surgical translocation and ultrasound biomicroscopy.

Team members:

Abstract:

This study was designed to develop an approach that permits serial evaluation of ovarian structures in vivo using ultrasound biomicroscopy (UBM). Female New Zealand rabbits (n=12) underwent surgical ovarian translocation. Each ovary was exteriorized through a lateral flank incision on each side, without compromising vascular supply. The mesovarium at both poles of the ovary was transfixed to the abdominal wall musculature to keep the ovary in subcutaneous position. Finally, the subcutaneous and skin layers were closed. The surgical site healed for 2 weeks, and the skin sutures were removed.

To evaluate ovarian structures, acoustic gel was applied to the skin previously shaved and translocated ovaries were scanned transcutaneously on 3 consecutive days using a 25 MHz UBM transducer. Both ovaries were clearly distinguished in all 12 rabbits. Antral follicles ranging from 0.6 to 3.1 mm were detected and number of ovarian follicles ≥0.6 mm ranged from 7 to 18. Data (mean ± SEM) were analyzed by ANOVA. The number (11.0 ± 0.82, 11.8 ± 0.85, 12.3 ± 0.75) and diameter (1.3 ± 0.07, 1.3 ± 0.07, 1.4 ± 0.07 mm) of follicles ≥0.6 mm per rabbit did not differ among 3 examinations. Follicles ≥2 mm in diameter were detected in 3/12 rabbits on the first two days, and 5/12 on the third day. One or more corpora lutea were detected in 2/12 rabbits during the examinations. Two months post-surgery, ovarian structures were clearly discernible by UBM in 11/24 translocated ovaries. Increased skin thickness and growth of subcutaneous fat and connective tissue around ovaries deteriorated image quality.

In summary, translocated ovaries continued to function in their new location and transcutaneous UBM permitted serial visualization of ovarian structures in live rabbits. Therefore, this new approach provides a non-terminal alternative for repetitive examination of rabbit ovaries.

Funding: Natural Sciences and Engineering Research Council of Canada.


Title: Improving molecular diagnostic strategies for detection and identification of Brachyspira in swine

Team members:

Abstract:

Brachyspira species can cause colitis and diarrhea in grow-finish pigs. It is a worldwide concern, as it results in great economic losses. Brachyspira hyodysenteriae is the agent associated with mucohaemorrhagic diarrhea (Swine Dysentery). Recently, a novel Brachyspira strain capable of causing a dysentery-like syndrome emerged in commercial farms across western Canada and the midwestern USA. Current diagnostic tools for Brachyspira spp. lack the specificity and sensitivity for both diagnostic and research needs. It is imperative, for clarification of pathogenesis and epidemiology, to improve diagnostic methods.

The current study involves the development of a quantitative real-time PCR for detection of the novel Brachyspira sp., comparing it to current available diagnostic methods, and determining optimal ante-mortem sampling techniques. A probe based assay was developed, employing a probe-linked DNA Minor Groove Binder, with the goal of maximizing detection limits and overcoming the challenge of targeting an organism with a low G+C content genome. Targeting the phosphogluconate mutase (pgm) gene, the assay was able to detect 10 target DNA copies/reaction. Specificity testing and comparison to culture and SYBR Green based qPCR assay are underway.

Investigating the appropriateness of rectal swabs and feces as sampling materials will elucidate their possible diagnostic value. The assay is expected to be suitable for diagnostic and research use, capable of detecting low levels of Brachyspira DNA in clinical samples.

Funding: Canadian Swine Health Board


Title: Effect of follicular aging on the nuclear maturation and distribution of lipid droplets in bovine oocytes

Team members:

Abstract:

The objective of this research was to study the effect of ovarian superstimulation protocols on nuclear maturation and lipid distribution in the ooplasm. We hypothesized that follicular aging after FSH starvation will result in maturation failure with accumulation of larger lipid droplets as compared to superstimulation with continued FSH support.

Hereford heifers (n=12) were given prostaglandin F (PGF) injection to induce ovulation. Five days later, a progesterone releasing device (CIDR) was placed into vagina and transvaginal ultrasound-guided follicular ablation was done to synchronize emergence of a new follicular wave. FSH treatments were started 24h after ablation (Day0). Standard FSH and FSH starvation groups were given 8 im injections of FSH at 12h intervals, while long FSH group was given 14 im injections (n=4 each group). All animals were given two PGF injections and CIDR’s were removed (on Day 3 for standard FSH and Day 6 for FSH starvation and long FSH) followed by LH injections 24h later.

Oocytes were collected 18-20 hours after LH injection by follicular aspiration and were fixed and stained for nucleus (Lamin/DAPI) and lipid droplets (Nile Red). Oocytes were imaged with Zeiss LSM 710 confocal microscope. Oocyte volume sets were deconvoluted using Autoquant X2 and lipid droplets segmented using Imaris Pro 7.4.The proportion of mature oocytes were compared using Mantel-Haenszel Chi square test and lipid droplet data were compared using ANOVA. Long FSH group had greater proportion (59/100, 59%, P<0.001) of mature oocytes compared to standard FSH and FSH starvation groups (5/23, 21.7% and 2/25, 8.0%, respectively).The FSH starvation and long FSH treatments did not cause significant changes in the total number (P=0.39), total volume (P=0.58) and distribution (P=0.66) of lipid droplets compared to standard FSH treatment. Average volume of lipid droplets in oocytes was higher in FSH starvation group (11.5±1.5µm3, P=0.03) compared to standard FSH and long FSH groups (7.2±0.6µm3and 8.0±0.8µm3). Average surface area of lipid droplets in oocytes was higher in FSH starvation group (20.3±1.5 µm2 P=0.04) compared to standard FSH group (14.6±1.1µm2) and was not different from long FSH group (17.1±1.1µm2). In conclusion, our hypothesis was supported in that FSH starvation caused maturation failure and accumulated larger lipid droplets in the ooplasm.

Funding: Natural Sciences and Engineering Research Council of Canada


Title: Development and characterization of a murine retinal explant tissue culture system as an in vitro model for ocular gene therapy

Team members:

Abstract:

Purpose. To develop and characterize a retinal explant culture system to facilitate investigation of novel methods of improving retinal gene therapy.

Methods. Retinal explants from adult mice of two different age groups were co-cultured with retinal pigment epithelium, choroid and sclera in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, propidium iodide uptake, activated caspase-3 expression and immunohistochemistry. Additionally, explants were exposed for 48 hours to variable concentrations of transducing units (TU) of a lentiviral vector construct with a green fluorescing protein (GFP) gene to model ocular gene therapy. Explants were compared with whole globes, with or without intravitreal gene transfer in vivo.

Results.  Explants remained viable for a minimum of 4 days. Compared with other existing techniques, our protocol detailed here is an easily manageable alternative with minimum disturbance of the tissue. Following gene transfer, findings in vivo and in vitro correlated in regards to targeted cell layers and influence of age of the animals used.

Conclusion.  Retinal explants prepared using the described techniques are viable for at least 4 days ex vivo and mimic in vivo reactivity to gene therapy. This system provides an efficient in vitro model for investigating methods of enhancing retinal gene therapy under controlled conditions and may aid to refine and reduce the number of animals required for in vivo use.

Funding: Heather L. Ryan and David Dube Research Fund


Title: Does the unfolded protein response trigger reactivation of latent equine herpesvirus type 1 in horse white peripheral blood cells?

Team members:

Abstract:

Equine herpesvirus type 1 (EHV-1) establishes latency in peripheral blood leukocytes (PBL) and nerve cells of infected horses. Re-activation of latent infections and subsequent virus shedding may play an important role in disease occurrence and transmission. The mechanisms leading to virus reactivation are poorly understood.

We hypothesize that activation of the “unfolded protein response (UPR)” can result in reactivation of EHV-1 from latently infected PBL. UPR is a cellular stress response that is triggered by a number of cell stressors. The role of the UPR in herpes simplex virus reactivation from sensory neurons is being studied and it has been propose that two UPR related proteins (Zhangfei and Luman) could play a role in latency establishment and viral reactivation.

In this preliminary experiment, we isolated PBL from a horse known to be latently infected with EHV-1. DNA was extracted from a proportion of cells and conventional (nested) PCR for EHV-1 detection of glycoproteinB genes and differentiation from EHV-4 was performed. PBL were treated for 4 hours with Thapsigargin, a drug capable of inducing the UPR. RNA was extracted and expression of down-stream UPR genes (X- binding protein XBP1; Glucose regulated protein 78 GRP78; C/EBP homologous protein CHOP; Homocysteine-induced ER protein HERP) detected and quantified using qRT-PCR optimized for equine samples. Activation of the UPR was detected as an increased expression of genes consistent with decreased ct values.

The next step will be to stimulate latently infected PBL with Thapsigargin and to test for EHV-1 reactivation through co-cultivation with equine fibroblasts (E. Derm). Cells stimulated with IL-2, which has been shown to reactivate EHV-1 from latency in PBL, will be used as a positive control. We propose that this research will serve to understand mechanisms of virus reactivation and may demonstrate potential new targets for drug therapies in latently infected horses. 

Funding:


Title: Interaction of bovine adenovirus-3 protein VIII with eukaryotic translation initiation factor

Team members: 

Abstract:

Viruses depend entirely on their host cell translation machinery for synthesis of their own proteins. To ensure that viral proteins are produced, viruses have developed various strategies to recruit host ribosomes to viral mRNAs. These strategies involve interaction of viral proteins with cellular proteins that regulate or are part of the host protein synthesis machinery. Identification and characterization of virus host interactions that regulate protein synthesis in virus infected cells will not only reveal key steps in viral life cycle but will also help to develop strategies to combat viral infection. Eukaryotic initiation factor-6 (eIF6) is an essential cellular protein that is necessary for 60S ribosome biogenesis and assembly. eIF6 possesses a unique anti association activity because of its ability to bind to 60S subunit, which prevents interaction of 40S ribosomal subunit with 60S subunits.

Results of our initial yeast two hybrid assay indicated that protein VIII (pVIII) of bovine adenovirus-3 (BAdV-3) interacts with cellular protein eIF6.  We have further confirmed the interaction of pVIII of BAdV-3 with cellular protein eIF6 by using bimolecular fluorescence complementation (BIFC), co-immunoprecipitation assay and GST-pulldown assay. Interaction of pVIII with eIF6 may be significant because at later times in infection adenovirus makes use of a ribosome shunting method to promote the translation of its structural protein at expense of cellular mRNA translation. Since eIF6 plays a major role in regulating ribosome assembly, it may be utilized by adenovirus to maximize the suitability of the infected cell for preferential translation of viral mRNA.


Title: Acute effects of β-naphthoflavone on swim performance, cardiorespiratory function, and energy stores in adult zebrafish (Danio rerio)

Team members: 

Abstract:

The class of chemicals known as polycyclic aromatic hydrocarbons (PAHs) are known agonists of the aryl hydrocarbon receptor (AHR). They are also contaminants of rivers, lakes, and marine shorelines, making fish a primary target species, but acute adult fish toxicity is thought to be minimal or absent.

In the present study adult zebrafish (Danio rerio) were aqueously exposed to solvent control (DMSO) or three increasing concentrations of the commonly used model PAH β-naphthoflavone (BNF; 0.1, 10, and 1000 µg/L) for a 48 hour period. Following exposure fish were subjected to swimming tests with concurrent oxygen consumption measurement (n=10 fish/treatment) or echocardiography to determine cardiac function (n=10 fish/treatment). Oxygen consumption (MO2) was increased at all exposure concentrations compared to control, reaching statistical significance at the second highest BNF exposure at a swim speed of 23.1 cm/s (p<0.01 in Fisher’s LSD test after two-way ANOVA). MO2 at zero water velocity was positively correlated with ventricular volume at diastole (r=0.957) and ejection fraction (r=0.859), but negatively correlated with acceleration of blood through the ventricle (r=-0.988) in resting fish. In contrast, BNF had no significant effect on Ucrit, tissue triglyceride, or glycogen concentrations. The effect of BNF on MO2 is likely to be physiologically important given that fish have a critical need for adequate oxygen to fuel aerobic activities such as swimming.

Future studies should be directed at examining the effects of more toxic and environmentally relevant PAHs on fish cardiorespiratory function and determining possible mechanisms of toxic effect.

Funding:


Title: Sensitivity of a real time PCR assay for bovine trichomonosis in pooled preputial samples

Team members:

Abstract: 

The objective of the present study was to evaluate the sensitivity of a commercially available RT-PCR assay for trich in pooled preputial samples at different ratios.

Steers (n=147) were sampled by means of the aspiration method and rinsed into the upper chamber of an InPouchTMTF. Pouches were transported to the lab within 4 h and incubated at 37°C for 7 days. In addition, weekly samples were collected and processed by the method described above from a naturally infected bull. Pouches were examined microscopically on days 1, 3, 5 and 7. On day 3 an aliquot was taken from all samples and tested individually by RT-PCR at a commercial diagnostic lab. Upon day 7 samples were frozen until later use. Pools were made by including known positive and negative samples in ratios of 1/2, 1/3, 1/5, 1/10, 1/15, 1/20, and 1/25 (n=31 per ratio). Positive samples used were quantified and evenly distributed so as to assure that all ratios contained high and low concentration samples. Statistical analysis was performed by Proc Genmod.

Sensitivity and 95% CI for each pool ratio were: 96.8% (83.8-99.4) for pool ratios 1/3 and 1/5; 93.5% (79.3-98.2) for pool ratios 1/2, 1/15, 1/20 and 1/25; and 90.3% (75.1-96.6). There were no differences (P=0.39) among any of the pool ratios evaluated. Sixty nine percent of the pools testing negative, contained the same positive sample.  The media in this sample had evidence of contamination.  

In conclusion, the use of pooled samples for the detection of Tritrichomonas foetus carrier bulls appears to be relatively sensitive in pools of up to 25 bulls.  Furthermore, this strategy provides the possibility of screening large number of groups at a more affordable cost. However, individual characteristics of each sample in a pool may, by the presence of inhibitors alter the result of the test.

Funding:


Title: Acute intermittent hypoxia improves ladder performance in spinal-injured animals

Team members:

Abstract:

Most spinal cord injuries (SCI) in humans and animals are incomplete and partial recovery arises as a result of plasticity within neural circuitry. After experimental SCI at the cervical level in rats, recovery of respiratory capacity can be improved through alternating exposure to brief periods of mild hypoxia (acute intermittent hypoxia (AIH)). The effect of AIH on forelimb functional recovery has not been explored.

Our hypothesis is that daily acute intermittent hypoxia (dAIH) induces spinal plasticity to improve forelimb function in rats with cervical spinal cord injury.

Rats were trained to cross a horizontal ladder and forelimb performance (measured as # of correct steps made/total steps made) was assessed prior to unilateral transection of the dorsolateral spinal funiculus at C2 (Pre-Sx), at 4 week after spinal surgery (Pre-IH), each day of the dAIH treatment, and I, 2 and 4 weeks after treatment. dAIH consisted of exposure to ten episodes of intermittent hypoxia (5 min 11% O2: 5 min  21% O2 n=5) or normoxia control (21% O2 n=3) for 7 days.

Sustained improvement in forelimb performance was observed in IH-treated rats in that they made fewer errors on ladder crossing compared to normoxia-treated control rats. Therefore intermittent hypoxia improves ladder performance in a rat model of cervical spinal injury.


Title: Cellular trafficking of helical rosette nanotubes in dendritic cells

Team members:

Abstract:

Biologically inspired, self-assembling helical rosette nanotubes (HRNs), composed of cytosine and guanine, have multiple desired therapeutic applications including drug delivery. HRN interactions with dendritic cells (DCs), sentinels of the immune system, are key in determining HRN safety and viability.

Splenic classical and plasmacytoid DCs were isolated from female C57BL/6 mice using magnetic cell sorting. HRNs were conjugated with the RGD (arginine, glycine, aspartic acid) peptide and the fluorophore FITC in a ratio of 1:10uM respectively and delivered to the isolated DCs for 15 minutes at a further 1:10uM dilution. Confocal microscopy was used to visualize the endocytic pathways, HRN intracellular localization and the real-time uptake process. 3-D imaging to better characterize HRN intracellular localization also was completed. Brefeldin A, a fungal metabolite, was added to the HRN-exposed DCs to disrupt endosomal-lysosomal intracellular transport.

It was found that HRNs used multiple pathways to take residence within clathrin and caveolin-coated vesicles, early endosomes, and lysosomes with brefeldin A not appearing to dramatically reduce HRN intracellular transport. Able to recognize RGD, integrin alpha-V-beta-3 is a potential receptor for receptor-mediated endocytosis. HRN exposure stimulated DC maturation as illustrated by dendrite formation and maturation marker (CD40, CD83) expression. Cell viability was confirmed through microscopy and caspase-3 and -9 expression.  These data demonstrate that HRN engage integrin alpha-V-beta-3 to enter DCs through multiple, presumably redundant, pathways.


Title: Induced regulatory T cells are distinctly superior to natural regulatory T cells of the same specificity in their abilities to induce tolerance

Team members:

Abstract:

We assessed the relative efficiency with which naturally-occurring CD25+Foxp3+ T cells (nT reg) and induced CD25+Foxp3+ cells (iT reg) from GFP-Foxp3/B6.CD45.1/OVA TCR-transgenic OT2 mice tolerize T effector (T eff) cells from asthmatic mice. The iT reg were induced either by culture of T eff cells with OVA-presenting IL-10-differentiated dendritic cells (DC10) or by injecting these T eff cells into DC10-treated OVA-asthmatic mice.  We purified nT reg from allergen-naïve mice and also cultured them with DC10 before use, or co-injected them into DC10-treated recipients. 

Both populations were then sorted and assessed for their therapeutic efficacy.  The iT reg were 64-72% more effective than analogous nT reg in reducing T eff cell proliferation and IL-4/IL-5 secretion in vitro.  Neutralization of IL-10, but not TGF-b, eliminated the suppressive activities of iT reg, which also expressed higher levels of PD-1, LAG3 & CTLA4.  Transfer of 5×105 nT reg had no impact on airway hyperresponsiveness (AHR) or IgE levels in asthmatic recipients, but reduced the airway eosinophil and IL-4/IL-5 responses by 4-26%, while the iT reg normalized AHR and reduced all airway responses to allergen challenge by 80-96%. 

These data suggest that iT reg and nT reg of the same antigen specificity employ distinct mechanisms to effect tolerance and indicate that iT reg are distinctly superior in induction of tolerance.


Title: Snatch-farrowed, porcine-colostrum-deprived (SF-pCD) pigs as a model for swine infectious disease research

Team members:

Abstract:

Snatch-farrowed, porcine colostrum deprived (SF-pCD) pig derivation is an alternative to conventional specific pathogen free (SPF), cesarean-derived colostrum-deprived (CDCD) and gnotobiotic pig derivation for infectious disease research. Previously reported SF-pCD protocols however resulted in 20 – 50% mortality. (Oliveira et al, 2003; Blanco et al, 2004) The current experiments evaluated the benefit of commercially available spray-dried bovine colostrum (The Saskatoon Colostrum Co. Ltd.) for use in raising SF-pCD pigs. 

In experiment 1, 12 SF-pCD pigs received a liquid diet made from mainly bovine colostrum from birth to day 10. Six remained on the same liquid diet (COL), and the other 6 were fed a diet mainly comprised milk replacer (RPL) until weaning. In experiment 2, 12 SF-pCD pigs were fed mainly bovine colostrum before weaning.  After weaning, 6 were fed a starter diet containing 20% (w/w) bovine colostrum powder (STARTER-COL) and 6 pigs were fed a starter diet without any bovine colostrum (STARTER-CTRL) until termination (day 42 or day 49). 

In experiment 1, COL pigs had significantly fewer fever-days than did RPL pigs.  In experiment 2, STARTER-COL pigs developed diarrhea, typhlocolitis and pancreatic degeneration after weaning.  In both experiments, all pigs fed mainly bovine colostrum before weaning survived until termination.  All pigs tested free of porcine reproductive and respiratory syndrome virus (PRRSV), H1N1 and H3N2 swine influenza virus (SIV) and porcine parvovirus (PPV).  In experiment 2, all pigs tested free of type 2 porcine circovirus (PCV2) but some in both groups tested positive for Torque Teno virus (TTV) genogroups 1 and 2. 

In conclusion, using snatch-farrowing and bovine colostrum, pigs can be raised in the absence of porcine maternal antibodies with 100% survival and freedom from most porcine pathogens of biological relevance. This model is potentially suitable for porcine infectious disease research.

Funding: Saskatchewan Agricultural Development Fund


Title: Definition of phenotypically distinct subtypes of Gardnerella vaginalis using cpn60 sequences and their distribution in women with and without bacterial vaginosis

Team members:

Abstract:

Gardnerella vaginalis has long been considered a hallmark organism of bacterial vaginosis (BV) and yet it is also found in clinically healthy women.  A large amount of genotypic and phenotypic diversity has been described in G. vaginalis but the clinical significance of these subtypes is unknown. Metagenomic profiles based on cpn60 sequences consistently reveal four subgroups within G. vaginalis (A-D).

The objective of the study was to determine if these cpn60 defined groups correspond to phenotypically and genomically distinct populations, and perhaps even different species. Analysis of G. vaginalis isolates and whole genome sequence data corresponding to groups A-D revealed that cpn60 sequences were good predictors of whole genome sequence similarity, and three groups (A, B and C) could be distinguished on the basis of sialidase gene presence and an amplified ribosomal DNA restriction analysis (ARDRA) genotyping scheme. No correspondence of cpn60 subtypes was observed with an established biotyping scheme. Distribution of frequency of G. vaginalis cpn60 subtype sequences in Kenyan women (n=44) grouped as BV, Intermediate and Normal based on Nugent score indicated that subtype B was significantly (z = -3.372, n =39, p = 0.001) more abundant in women with BV than in the normal group. This cpn60-metagenomic approach allows direct detection of G. vaginalis subtypes in vaginal samples.

In summary, G. vaginalis strains can be subdivided into four cpn60 subtypes and the study provides evidence for the proposal of these categories as different species of Gardnerella.  Establishment of a quantifiable relationship between G. vaginalis subtype distribution and clinical status in different clinical cohorts as above could have significant diagnostic implications.

Funding:


Title: CRISPR diversity in Enterococcus hirae isolates from pig feces

Team members:

Abstract:

Clustered regularly interspaced palindromic repeats (CRISPRs) are DNA arrays containing multiple unique spacers separated by short direct repeats. The structure generally contains 4–10 repeats ranging in size from 25 to 45 bp, separated by spacers of similar length containing specific genomic material that is not present elsewhere in the genome and has probably been imported from plasmids or viruses. Diversity in spacer sequences is an indication of exposure to mobile genetic elements, and can be used as a strain typing method.

A combination of PCR and sequence analysis was used study diversity of CRISPR arrays in commensal E. hirae isolates from pig feces obtained at three, nine and 15 weeks (N=164). CRISPR arrays with (N=64) and without CRISPR associated genes (CRISPR cas) were identified in our culture collection. CRISPR arrays without cas genes (N=164) was identified in all the isolates.

Preliminary sequence analysis of all CRISPR arrays shows that unique spacers exist in each animal at different time points. Maximum number of spacers was identified in isolates obtained from animals aged 15 weeks providing evidence that maximum exposure to foreign DNA occurs in adult animals that have complex diets. This study is the first description of CRISPR arrays in commensal Enterococcus spp. from pig feces.

Funding:


Title: Role of leukocyte specific protein 1 in neutrophil recruitment in lipopolysaccharide-induced lung inflammation

Team members:

Abstract:

Leukocyte specific protein 1 (LSP-1), an F-actin binding protein, has an important function in leukocyte chemotaxis into inflamed organs.While significant data are available on the role of LSP-1 in leukocytes, especially neutrophil recruitment in inflamed cremaster muscles and the peritonium, there has not been data on the effects of LSP-1 in infiltration of neutrophils into acute inflamed lungs. Acute lung inflammation accompanies many infectious and non-infectious lung diseases.

In this study, lipopolysaccharide (LPS) from E. coli was instilled intranasally to induce acute lung inflammation in wild type 129/SVJ and LSP-1 deficient mice. Data show that there was no significant difference in different leukocytes percentages in peripheral blood between wild type and LSP1 deficient mice after 9h saline treatment (7 wild type mice, 10 LSP-1 deficient mice). Significant neutrophilia was observed in both types of mice (10 wild type mice, 11 LSP-1 deficient mice) after 9h LPS administration.

In bronchoalveolar lavage fluid, the total leukocytes as well as neutrophil proportion in both phenotypes of mice treated with LPS increased dramatically compared to saline controls. However, the number of leukocytes of LPS-treated wild type mice was three - fold higher than that of LSP-1 deficient mice (P < 0.001). Lung sections stained with hematoxyline and eosin showed acute inflammation in LPS-treated mice but not in saline-treated animals. Compared to LSP1-/- mice, more neutrophils infiltration in lung sections from wild type mice were observed. Immunohistochemistry showed LSP1 expression on neutrophils, macrophages, endothelium and epithelium of wild type mouse lungs after LPS administration. Based on these data, LSP-1 plays a role in the recruitment of neutrophils in LPS-induced acute lung inflammation.

Funding: NSERC


Title: Demonstration of virulence of Canadian isolates of “variant” infectious bursal disease virus in specific pathogen free birds and commercial broilers.

Team members:

Abstract: 

“Variant” strains of Infectious bursal disease virus (IBDV) are highly immunosuppressive in young chickens which causes significant economic losses to the poultry industry. Protection against IBD in broiler chickens is achieved by hyperimmunization of their broiler breeder parents. Recent studies conducted in Canada have demonstrated that majority of IBD virus strains circulated in Canada are “variant” strains and are capable of immunosuppression in broilers.

The objective of this study was to investigate the virulence of “variant” IBDV strains isolated in Canada in commercial broilers and specific pathogen free (SPF) birds. Groups of SPF and commercial broilers were orally administered with three (i.e. NC171 “like”, Del-E “like” or 05SA8 “like”) “variant” strains of IBDV at day-6 post-hatch. The percentage of bursal weight to body weight (BW: BW), bursal histopathology and antibody titers were taken at day 20 and 35. Groups of SPF and commercial broilers that were not infected with IBDV had a percentage of BW: BW of 0.58± 0.16 and 0.19 ± 0.04 respectively. In contrast, SPF and commercial birds infected with IBDV had a percentage of BW: BW of 0.12±0.02 and 0.09 ± 0.08 respectively. A severe bursal atrophy was noted on microscopic examination of bursae in both SPF and commercial broilers at the age of day-20 post-hatch. The average antibody titer against IBDV was 8,768 in SPF birds at day-35 post-hatch. Similarly, the average antibody titer against IBDV was 4,795 in commercial broilers at day-35 post-hatch.

This study demonstrates that “variant” strains of IBDV circulated in Canada were capable of causing severe bursal atrophy and significantly low percentage of BW: BW ratio in both SPF and commercial broilers.


Title: Putative biomarkers of feline pancreatic neoplasia

Team members:

Abstract:

As in people, the early diagnosis of feline malignant exocrine pancreatic neoplasia is challenging, and delayed identification typically results in a paucity of treatment options, widespread metastasis, and a high mortality rate.

The objective of this study was to characterize the proteome in cats with malignant and inflammatory pancreatic disease in comparison to age-matched healthy adult cats, in order to identify differences in protein expression and putative biomarkers for pancreatic adenocarcinoma.

Plasma from 6 cats with pancreatic neoplasia, 6 with pancreatitis, and 6 healthy adult cats was subjected to standardized 2-dimensional SDS PAGE electrophoresis (iso-electric focusing with subsequent separation based on molecular weight). Resultant gels were stained with Coomassie Blue to visualize separated proteins and comparatively analyzed with specialized software (Image Master 2D Platinum 7.0, GE healthcare).  The relative volume of each protein spot was calculated as a percentage of the total volume of all the protein spots in each gel to minimize variations due to protein loading and staining. Proteins of interest were identified by liquid chromatography mass spectrometry.  Haptoglobin, apolipoprotein A-I, and α1-acid glycoprotein were identified as potential markers, with differential expression between study groups.

Results of our study indicate that there are significant differences in the proteomes of cats with malignant pancreatic neoplasia, pancreatitis, and healthy controls, some of which may act as putative biomarkers of disease.

Further testing to investigate their diagnostic utility in a larger group of cats is warranted, and improved diagnostic accuracy may be achieved by combining proteins into a panel.

Funding: WCVM Companion Animal Health Fund


Title: Use of environmental sites by mule deer as a measure of risk of CWD transmission in southern Saskatchewan

Team members:

Abstract

Chronic wasting disease (CWD) is a fatal and progressive transmissible spongiform encephalopathy (TSE) caused by prions. Routes of transmission include animal-animal and animal-environment-animal pathways. Prions deposited into the environment by infected animals are persistent and may continue to infect free-ranging cervids for years. Transmission dynamics of CWD are poorly understood  and this lack of understanding is hampering development of appropriate disease management responses.

Since 2006, we have captured, radio-collared, and established the CWD status of more than 350 free-ranging mule deer and 44 white-tailed deer from Southern Saskatchewan. We have been studying their individual behaviour and social interactions, along with their movement patterns and home ranges. The goal of the present poster is to present preliminary findings on the relative rates of use of environmental sites by mule deer as a measure of relative risk of CWD transmission.

Since July 2009, 22 trail cameras have been used to monitor 169 different locations of 7 different site types, and to monitor 20 mule deer carcasses that died of CWD. The cameras have taken more than 60 thousand photographs and over 12 thousand of these contain one or more deer. We are able to determine the most frequently visited site types, as well as the characteristics of the visitors: deer age class, gender and sometimes CWD status. With data collected from the mortality sites, we can identify mammalian and avian scavengers and visitors potentially exposed to CWD prions from mule deer carcasses, and can determine the length of time CWD positive mule deer carcasses persist in the environment.

Preliminary findings on how deer use anthropogenic and non-anthropogenic environmental sites and the implications for CWD transmission are discussed.

Funding:


Title: Superovulation and embryo collection in wood bison during the anovulatory season

Team members:

Abstract:

This study was designed to determine if embryo collection is feasible in wood bison during the anovulatory season (May-July), and to test if progesterone priming is required for superovulation.

A 2-by-2 design was used to determine the effectiveness of LH or hCG for induction of ovulation with or without progesterone releasing intravaginal device (PRID) in wood bison. Follicular wave emergence was synchronized among bison by follicular ablation. Synchronized bison were assigned to PRID+LH (n=12), PRID+hCG (n=4), no-PRID+LH (n=12) and no-PRID+hCG (n=4) groups. A single s.c. dose of 400 mg FSH in a slow-release formulation was given the day after follicular ablation (Day 0) and either 25 mg LH or 2000 IU hCG was given i.m. on Day 5. A PRID was inserted on the day of follicular ablation and removed on Day 4 in respective groups. Artificial insemination was done at 24, 36, and 48 h after LH or hCG treatment.

Embryos were collected non-surgically on Day 13. Ultrasonography was done on Days 0, 5, 6, 7, 8, and 13 to record follicular and ovulatory responses. Data (mean±SEM) were analyzed by two-way ANOVA and proportions by chi square. The number of ovulatory-sized follicles (≥10mm) did not differ among groups (P=0.33). Ovulation rate was greater (P<0.05) in PRID+hCG (78%) and no-PRID+hCG (71%) than PRID+LH (24%) and no-PRID+LH (38%) groups. Number of corpora lutea on Day 13 was greater (10.3±1.9) in the no-PRID+hCG group (P<0.05). No differences in number of ova/embryos and transferable embryos were found among groups (P=0.36, P=0.52, respectively).

In conclusion, progesterone priming (PRID) had no effect on ovarian superstimulation in wood bison during the anovulatory season. The ovulatory response was satisfactory only in bison treated with hCG. Embryo collection is feasible in wood bison, but the reasons for a low embryo collection rate in all groups remain unclear.

Funding: Advancing Canadian Agriculture and Agri-Food and Agri-Food Innovation.


Title: Comparison of direct and indirect blood pressure monitoring in cats

Team members:

Abstract:

Rationale: Accurate blood pressure measurement is important but especially difficult in cats.  It is measured by direct and indirect methods.  This study was performed to compare these methods to pressure in the aortic root, the gold standard.  Two newer oscillometric devices HDO (High Definition Oscillometry, Memodiagnostic S+B medvet, Babenhausen Germany), PC-VetGard+, (DVM Solutions, San Antonio, TX), and ultrasonic Doppler (Parks Medical) were utilized.

Methods: Six cats aged 1.5 to 13 years, median 1.5 years, were anesthetized, had the carotid artery surgically isolated and a transducer-tip catheter (Millar 3.5 Fr) inserted then advanced to approximately the level of the aortic root.  The dorsal pedal artery (DP) was surgically isolated, after application of EMLA cream, and an open-ended 24 gauge catheter inserted and attached to an external pressure transducer.  Blood pressure data were recorded continuously (Powerlab26T, ModelML856, LabChart 7.2 software ADInstruments).  Each catheter was calibrated using a mercury sphygmomanometer before placement. 

Comparable numbers of readings were obtained from forelimb and tail for both oscillometrics.  Doppler readings were obtained from the forelimb alone.  Blood pressure was adjusted using isoflurane at various concentrations, colloids and dobutamine to achieve hypotension (<80 mmHg), normotension (81-110 mmHg), and hypertension (> 110 mmHg).  Bias (DP minus aortic root for direct and aortic root minus indirect) and precision (Standard Deviation of difference) were calculated.

Results:  Aortic root was compared to the DP (551 paired readings), HDO (265) and VG (236) in hypo-, normo-, and hypertensive states for Systolic, Diastolic and MEAN pressures.  The results were tabulated.

The Doppler technique (66), when using Return to Sound as the measurement reading, was accurate for MEAN [bias (-0.5) (SD 11.0)] but not systolic pressure [bias (-25.3) [SD 16.5]. 

The Doppler technique (66), when using Return to Sound as the measurement reading, was accurate for MEAN [bias (-0.5) (SD 11.0)] but not systolic pressure [bias (-25.3) [SD 16.5]. 

Conclusion:  The HDO was the most accurate device while Doppler was accurate for of MEAN and DP was not accurate.

Funding: Companion Animal Health Fund (CAHF)


Title: Radiographic landmarks for measurement of cranial tibial subluxation in the canine cruciate ligament deficient stifle
 
Team members: 

Abstract:

Rationale – The primary objective of this ex vivo study was to determine and test the intra- and interobserver repeatability of different radiographic anatomic landmarks for assessing cranial tibial subluxation (CTS).  A secondary objective involved determining the effects of digital radiographic magnification on CTS accuracy. 

Methods – Twenty-three normal canine hind limbs were used to determine the magnitude of CTS before and after transection of the cranial cruciate ligament (CCL).  For each specimen, radiographs were taken before and after transection of the CCL in a normal mediolateral view, with and without fiduciary bone markers in place.  CTS was measured using various radiographically visible anatomic landmarks (two on the femur and four on the tibia).  Three different observers measured the CTS on all radiographs at two different magnifications. The total observed variabilities were assessed with reference to interobserver and intraobserver differences.  Paired t-tests were used to determine the effect of magnification and marker presence on CTS measures. 

Results –Measurement of CTS from the caudal limit of the intercondylar fossa on the femur to either the intercondylar eminence or caudal tibial plateau was most reliable based on intra- and interobserver variability. Poor correlation was observed between the anatomic landmarks and the fiduciary bone markers. We found no effect of magnification or presence or absence of bone markers on measurement of CTS. 

Conclusion – CTS can be detected most reliably between observers by measuring either from the proximo-caudal margin of the intercondylar fossa on the femur to the caudal tibial plateau or from the proximo-caudal margin of the intercondylar fossa on the femur to the intercondylar eminence.  Magnification of the digitized radiographic image has minimal effect on reliability of measurement of CTS.  This information can be used to assess various methods of stabilization for the canine cruciate ligament deficient stifle.

Funding:


Title: Epithelial cell signaling in innate immune responses to bacterial infection

Team members:

Abstract:  

Intestinal epithelial cells are the first host cells to interact with enteric pathogens and are the primary target for the invasion of Salmonella into the host.  These cells act as sentinels, sensing the presence of various pathogen-associated molecular patterns (PAMPs), and influencing cell signaling.  The influence of intestinal epithelial cells on the development and regulation of innate immunity to bacterial infection is still largely unknown.  Generally, after infection with enteroinvasive bacteria, intestinal epithelial cells quickly up-regulate the expression of an array of host genes, the products of which activate inflammation and immune responses, and alter epithelial cell functions. 

Our laboratory has developed a bovine intestinal epithelial cell line which we will use to study the response of intestinal epithelial cells to bacterial infection.  Using quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) we analyzed the expression of pro-inflammatory cytokines in cultured cells stimulated with select Toll-like receptor (TLR) agonists.  Stimulation of cells with lipopolysaccharide (LPS) and bacterial flagellin leads to increased expression of pro-inflammatory cytokines interleukin (IL)-6 and IL-8. 

Infection of cells with Salmonella bacteria also results in increased expression.  Pre-treatment of stimulated cells with a TLR4-specific inhibitor results in decreased expression of IL-6 and IL-8 in response to stimulation with LPS (a TLR4 agonist), while there is no decrease in expression in cells that are stimulated with bacterial flagellin (a TLR5 agonist).  Cytokine expression is decreased in Salmonella-infected cells, but not to the same degree that it is inhibited in LPS-stimulated cells.  This is likely due to the fact that Salmonella bacteria harbor multiple TLR agonists on their surface and are able to stimulate multiple receptors at one time.


Title: Effect of Zhangfei on estrogen receptor positive breast cancer cells

Team members:

Abstract:

Breast cancer is a common form of cancer among women and female dogs. Breast cancers with cells that have nuclear receptors for hormones such as estrogen (ER) are commonly treated with hormone antagonists. However, despite treatment with the ER-antagonist tamoxifen, ER positive tumours recur.  According to a recent study ER positive cells develop an alternative tamoxifen-independent endocrine pathway after prolonged chemotherapy and this gives them a better ability to survive, proliferate and invade. For ER, the co-receptor PELP1 plays an important role in neoplasia by phosphorylating extracellular receptor kinase (ERK1/2), a member of the mitogen activated protein kinase pathway. The nuclear receptors and co-receptors for the tamoxifen-independent pathways are not known.

Cancer cells also activate stress-response pathways that allow them to survive unfavourable micro-environmental stressors such as hypoxia, nutrient deprivation, etc. Zhangfei (ZF) is a basic-leucine zipper (b-zip) motif containing protein. It also possesses a nuclear receptor-binding domain and can influence the activity of a variety of nuclear receptors, such as the ER, likely by displacing co-regulators and targeting them for proteosomal degradation. ZF can also inhibit the activity of several b-zip transcription factors including those that induce important cellular stress responses such as the Unfolded Protein Response (UPR). The purpose of this study is to find if Zhangfei can inhibit the growth of ER positive breast cancer cells by displacing PEPL1 from the activated ER and render these cells more susceptible to the effects of chemotherapeutic agents, hypoxia and radiation by suppressing the stress responses such as the UPR.

We will test this hypothesis by examining the effect of ZF on estrogen dependent and independent growth, as well as the UPR, in human and dog breast cancer cells. We will also examine interaction of ZF with nuclear receptors and co-regulators and the components of the UPR.

This study can propose a novel way to counteract or suppress the resistance developed by estrogen receptor positive breast cancer cells undergoing prolonged hormone therapy and hence expect to achieve better results from hormone therapy.

Funding:


Title: Characterizing the use of differentiated medulloblastoma cells to examine Herpes Simplex Virus latency and reactivation.

Team members:

Abstract:

Herpes simplex virus (HSV) replicates via a lytic cycle in epithelial cells but is also able to establish and reactivate from a latent state within the context of sensory neurons. How HSV is able to navigate its latency-reactivation cycle has not as of yet been fully elucidated, due in part to the lack of a suitable model of human neurons in which to study the phenomenon. Medulloblastoma tumor cell lines may provide a useful tool for the study of HSV latency as they are of neuronal lineage and are able to be differentiated into non-proliferating neuron-like cells. In this study, a medulloblastoma cell line was differentiated and assayed for its ability to host latent HSV infection.

The results demonstrate that while this particular cell line is not able to host latent HSV infection (when using standard protocols for establishing HSV latency in cell culture), it may be a useful tool in studying the events permitting reactivation of the virus in a neuronal context.

Funding:


Title: Parasitic zoonoses and veterinary public health in northern Saskatchewan

Team members:

Abstract:

Rationale: Canadians living in northern or remote Indigenous areas might be at increased risk for exposure to zoonotic pathogens. We investigate parasite prevalence, and possible links between animal and human health.

Methods: In 2011 we collected blood from and administered surveys to 201 individuals residing in an Indigenous community in northwestern Saskatchewan.  Human serum was analysed for exposure to four parasites of zoonotic concern, Toxocara, Toxoplasma gondii, Echinococcus, and Trichinella. Surveys focused on identifying personal risk factors for parasite exposure, and barriers to accessing veterinary services.  We introduced a veterinary public health intervention whereby pets were treated for roundworms over two consecutive years, and measured the effect by collecting the feces of pets and free-roaming dogs in the region (2010, N=92; 2012, N=104).

Results: Serum analysis suggests that this northern population has exposure levels equal to or higher than levels previously reported in other Canadian Indigenous communities.  Administration of dewormer to dogs brought to a mobile veterinary clinic decreased the fecal prevalence of roundworm eggs by 20% between 2010 and 2011; however tapeworm egg prevalence in dogs increased.  Respondents were generally unaware of animals as a potential source of human disease, and 33% fed raw meat or fish to their dogs.  Vaccination and deworming rates were lower than urban rates, despite the availability of a free veterinary service clinic on an annual basis. 

Conclusions: Risk factors that contribute to parasite transmission gaps in public health messaging were noted.  Veterinary interventions, such as deworming and desexing pet populations, benefit animal and public health.    Dialogue among veterinarians, medical professionals and public health professionals is necessary to effectively improve the overall health of northern communities.

Funding:


Title: Pentraxin 3 expression in normal and inflamed lungs

Team members:

Abstract: 

Bacterial lung diseases cause significant mortality and morbidity, causing enormous economic damage to the animal industry and cost billions of dollars in human health care. The innate immune system plays a critical role in the initiation of the immune response in the lung.

Increasing our understanding of the mechanisms involved in the immune response will aid in the development of treatments for respiratory disease. Pattern recognition receptors (PRRs) are programmed to recognize microbial structures unique to pathogens and mount an immune response. Pentraxin 3 (PTX3) is a PRR that is produced at sites of inflammation by many cell types upon stimulation by proinflammatory cytokines, and agonists such as endotoxins. PTX3 recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells.

Because there are very few data on the expression of PTX3 in the lungs, we examined PTX3 expression in normal and inflamed lungs of the horse, pig, human, and cattle using light and electron microscopic immunochemistry. We show PTX3 staining in bronchial epithelial cells and vascular endothelium in normal and inflamed lungs. Alveolar macrophages and inflammatory cells recruited into the lungs of bacteria-infected animals stain intensely for PTX3. Immuno-gold electron microscopy on horse and calf lungs showed PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells and pulmonary intravascular macrophages. These data show PTX3 is expressed in normal lungs and its expression is increased in inflamed lungs.

Funding: Alberta Livestock and Meat Agency (ALMA)


Title: Mechanisms of adjuvant activity of polyphosphazenes in porcine monocytes

Team members:

Abstract:  

Adjuvants are essential components of vaccines. Current research suggests that combination adjuvants increase the duration of the protective immune response, reduce number of immunizations required, lower antigen dose requirements, overcome antigen competition in combination vaccines, and increase the breadth of immune response.

A novel adjuvant platform established at VIDO-Intervac is comprised of CpG-ODN or Poly I:C, innate defense regulator peptides, and Polyphosphazene: poly[di(carboxylatophenoxy)-phosphazene] (PCPP), or poly[di(sodiumcarboxylatoethylphenoxy)-phosphazene] (PCEP) both of which are synthetic, biodegradable polymers which are capable of being formulated into microparticles which can encapsulate multiple antigens.

However the mechanisms by which the polyphosphazenes stimulate the immune response are not currently understood. The Nod-like Receptors (NLRs) are pattern recognition receptors expressed in antigen presenting cells. Current theory suggests they are activated through a two signal mechanism requiring both TLR ligation and NLR stimulation to activate caspase-1 to cleave pro-IL-1b, pro-IL-18 and pro-IL-33 into their mature forms. RT-PCR, MACS, and ELISA were methods used in this study.

Additionally, Caspase-1 inhibitor is currently being assessed to determine whether the polyphosphazenes signal through the NLR pathway. We found that antigen presenting cells express increased levels of NLRs. Cells frequently in contact with foreign antigen, and tissues rich in these cells such as mucosa and its associated lymphoid tissue have been demonstrated to exhibit elevated levels of NLR genes.

We observed reduced gene expression of the NLRs in the Peyer’s Patches and Lamina Propria of 6 pigs. Increased titers of IL-1b were observed in monocytes which were stimulated in the presence of TLR ligands and PCEP (two-signal theory) as opposed to those which were stimulated with TLR or PCEP.

Funding:

 


 

Title: Effects of dietary selenomethionine exposure on repeat swim performance, metabolic rate and energy metabolism in adult zebrafish (Danio rerio)

Team members:

Abstract:

Selenomethionine (SeMet) is the dominant form of selenium (Se) present in food. In a previous study we reported impaired swimming and elevated stored energy (triglycerides and glycogen) in adult zebrafish after sublethal dietary SeMet exposure.

In the present study, we investigated effects of sublethal dietary SeMet exposure on repeat swimming performance, oxygen consumption (MO2), cost of transport (COT) and energy metabolism in adult zebrafish. Fish were fed varying concentrations of Se (1, 3, 10 and 30 μg Se/g, dry weight) in the form of SeMet for 90 days. At the end of the exposure period, fish from each treatment group were divided into three subgroups: a) no swim, b) swim, and c) repeat swim. Fish from the no swim group were euthanized immediately at 90 days and whole body triglycerides and glycogen were determined. Individual fish from the swim group were placed in a swim tunnel respirometer and swim performance was determined using the critical swimming speed (Ucrit) method.

After both Ucrit and MO2 analyses, fish were euthanized and whole body energy stores were determined. Similarly, individual fish from the repeat swim group were subjected to two Ucrit tests performed with a 60 min. recovery period between tests, followed by determination of energy stores. Impaired Ucrit was observed in fish fed greater than 3 μg Se/g in the form of SeMet. Both resting metabolic rate and COT were significantly greater in fish fed elevated dietary SeMet. Whole body triglycerides increased with increasing dietary SeMet exposure. Fish from all treatment groups were able to mobilize stored glycogen during repeat swimming.

Our ongoing studies are investigating mRNA expression of key enzymes involved in energy metabolism in zebrafish liver. Overall results from this study should provide a better understanding of mechanism(s) underlying altered energy homeostasis in fish exposed to SeMet.

Funding: NSERC
Title: Profiling the microbial consortium of anaerobic digesters processing agricultural waste

Team members:

Abstract:

Anaerobic digestion of agricultural waste involves a complex and dynamic microbial consortium which is currently poorly understood. Incorporating molecular tools such as deep sequencing and quantitative PCR can provide valuable information about the composition of these communities and how they correlate to digester performance. A deeper understanding of the dynamic shifts in these communities over the course of the digestion cycle could provide opportunities for intervention and optimization of this process.

Distillery waste products were combined with dairy manure and processed in a thermophilic anaerobic digestion system for methane production. Total genomic DNA from the end product digestates were used as templates for pyrosequencing analysis using the Chaperonin 60 universal gene target for organism identification. These results provided insight into the taxonomy, richness and diversity of the digestate communities. Pyrosequencing detected 1272 organisms present in the digestate samples. Several organisms of interest had a significant positive correlation to both methane production and volatile solids reduction.

These results were confirmed by quantifying the most abundant of these organisms, a Clostridium sp., using a species-specific quantitative PCR assay. The taxonomic breakdown of the organisms correlating to positive digester outcome dictated culture and media conditions to isolate potentially beneficial organisms from the digestate samples. Three such organisms have been isolated and cultured from these samples and are undergoing detailed phenotypic evaluation.

This molecular approach will enhance understanding of the microbial consortium involved in biogas production and will allow for the monitoring and manipulation of microbial communities during anaerobic digestion to optimize performance and yield.

 


 

Title: Bovine seminal plasma induces ovulation in llamas

Team members:

Abstract:

It has been reported that 2 mL of bovine seminal plasma given intramuscularly induced ovulations in llamas, but in a less proportion than llama seminal plasma. It has been also reported ovulation-inducing factor (OIF) from llama seminal plasma had a dose-dependent effect on ovulation rate and luteal function in llamas.

The present study was designed to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas in a comparable manner as llama seminal plasma when OIF dose is adjusted. Within species, seminal plasma was pooled from 1 to 4 ejaculates per male (n=160 bulls, n=4 llamas). The concentration of OIF in the pooled seminal plasma was measured, and treatment was adjusted to reach a total dose of 250 µg of OIF. Mature female llamas were assigned randomly to recieve single intramuscular dose of 10 ml of phosphate buffered saline (PBS, n=5), 50 µg GnRH (Fertiline, n=5), 3 ml of llama seminal plasma (n=6) and 12 ml of bull seminal plasma (n=6). Ovulation and CL development were monitored by transrectal ultrasonography. Ovulation rates were compared among groups by fisher’s exact test. Non-serial data were compared among groups by analyses of variance. The diameter of the preovulatory follicle at treatment did not differ among groups (P= 0.10). Ovulation rates were 0/5, 4/5, 3/6, 4/6 in PBS, GnRH, llama seminal plasma, and bovine seminal plasma respectively (P<0.05). Incidence of ovulation did not differ among llamas treated with GnRH, llama or bovine seminal plasma (P>0.05).

Among the treatments that elicited ovulation, neither the maximum CL diameter, nor the day of maximum CL diameter were different (P=0.30 and P=0.24, respectively). In addition, no difference was detected in the day of first detection of the CL (P=0.25). Seminal plasma from both llama and bull showed similar ovulation and luteotrophic effect after treatment.

Funding: Natural Sciences and Engineering Research Council of Canada.


Title: The effect of PTEN on HCV infection and its role in carcinogenesis

Team members:

Hepatitis C virus (HCV) infection causes serious global public health problems. The World Health Organization (WHO) has established that there are more than 170 million chronic HCV (CHC) patients worldwide. Phosphatase and tensin homologue (PTEN) gene, a tumor suppressor, is frequently mutated or deleted in several malignancies including human hepatocellular carcinoma (HCC). PTEN consists of a phosphatase domain and a C2 domain.

One mechanism of tumor suppression by PTEN is through inactivation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway.It has been shown that PI3K-Akt activation and PTEN inactivation are associated with HCV infection. Furthermore, it has been shown that HCV up-regulates the activity of sterol regulatory element binding protein (SREBP) and fatty acid synthase (FAS) through the PI3K-Akt pathway. This mechanism may play an important role in steatogenesis, cell proliferation and carcinogenesis. This study will make a significant contribution to using PTEN as a drug target for HCV therapy.

In this study, I will determine the effect of PTEN on HCV replication and establish the mechanism of how the inactivation of PTEN induced by HCV leads to steatosis, cell proliferation and thus HCC. Our prelimary results suggest that PTEN plays an important role in modulating HCV replication. Future experiments will determine the effect of PTEN on steatosis and cell proliferation associated with HCV infection.

 


Title: Effect of a prolonged aromatase inhibitor treatment on pre-ovulatory ovarian follicles in cattle

Team members:

Abstract: 

Letrozole, a non-steroidal aromatase inhibitor, prevents the body from producing its own estrogen. The potential use of letrozole for herd synchronization is supported by previous studies in which treatment increased mean plasma LH concentrations, prolonged the period of dominance of the extant dominant follicle, delayed emergence of the next follicular wave and exerted a luteotrophic effect.

The objective of the present study was to test the hypothesis that letrozole treatment during the preovulatory follicular wave will delay ovulation. Post-pubertal beef heifers where synchronized to follicular wave emergence by follicular ablation (Day -1=follicular ablation, Day 0=wave emergence) and a luteolytic dose of PGF was given 60 and 72 hr later. On Day 1, heifers were divided randomly into two groups (n=15/group) and given an intravaginal device containing 1 g of letrozole or a blank device (control). The devices were removed on Day 7, or at the time of ovulation, whichever occurred first. Transrectal ultrasonography and blood sample collection were performed daily from day of ablation to 12 days post-ovulation. Single point measurements were analyzed by t-test, and serial data were analyzed by ANOVA for repeated measures. In letrozole treated heifers the interval from device placement to ovulation was longer (6.1±0.25 vs 5.1±0.26 days, P<0.01), the day-to-day diameter profile of the ovulatory dominant follicle was larger (P<0.05) and the maximum diameter greater (14.6±0.51 vs 12.4±0.53 mm; P<0.01). Plasma progesterone concentrations were higher (P<0.01) in heifers treated with letrozole.

In summary, an intravaginal letrozole device delayed ovulation by 24 h and induced the formation of a CL that secreted higher levels of progesterone. A slow-release intravaginal letrozole device may become useful for the control ovulation for herd synchronization and to enhance fertility by increasing circulating progesterone concentrations during the first 7 days post-ovulation in cattle. Supported by NSERC and Bioniche Life Sciences Inc. 


Title: Molecular mechanism for the Zhangfei-mediated suppression of osteosarcoma cells

Team membrs:

Abstract:

Some spontaneous canine tumors, which have many similarities with human cancers, are excellent models for human cancer biology and cancer therapeutic strategies. Osteosarcomas (OS) are the most common malignant bone tumors in humans and dogs. Although there has been dramatic progress in treating OS in both species by surgery and chemotherapy, these therapies often fail leading to recurrence of the tumor and metastatic spread. Cancer cells exist in a harsh microenvironment that lacks adequate oxygen and nutrients. These micro-environmental stressors force cancer cells to activate stress responses that allow tumors to survive, develop, metastasize and escape therapy. The unfolded protein response (UPR) is one such tumor-sparing pathway.

Zhangfei is a basic region–leucine zipper transcription factor that can induce the differentiation and death of OS cells. However, the mechanism by which Zhangfei inhibits these cells is still unclear. The objective of this study is to determine the effect of Zhangfei on different canine and human OS cell lines and characterize the molecular mechanism responsible.  

Methods: We examined the cell viability and the UPR signaling pathways in different canine and human OS cells infected with adenovirus vectors expressing Zhangfei.

Results: Ectopic expression of Zhangfei in different unstressed canine OS cells suppressed their growth and eventually caused them to commit apoptosis. However, in human OS cell lines, Zhangfei either had no effect or its effect was not as pronounced. We also found that in canine OS cells, in which the ER was artificially stressed, Zhangfei inhibited the UPR.  

Conclusion: Our results suggest that while Zhangfei has the ability to selectively suppress the UPR and it may also suppress the growth of tumor cells by influencing other signaling pathways.

Funding: