Browsing by Author "Clark-Deener, Sherrie"
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- Advanced breeding techniques of small ruminants and camelidsClark-Deener, Sherrie (2015-05-14)After a producer has gained knowledge and experience with the breeding management of their herd or flock, they may decide to attempt a variety of assisted reproductive techniques. What exactly is included in the category of assisted reproductive techniques? The author has included timed artificial insemination – both transcervical and laparoscopic, embryo transfer, and semen collection and freezing as the techniques to be discussed in this manuscript. In technical terms, the use of various pharmaceuticals for manipulation of the estrous cycle is assisted reproductive technology, but most authors will include this material when discussing the breeding management of groups as was done in this series of proceedings articles.
- Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigsClark-Deener, Sherrie; Kang, Jung-Taek; Cho, Bumrae; Ryu, Junghyun; Ray, Caitlin; Lee, Eun-Jin; Ahn, SunMi; Lee, JinSeok; Ji, Dal-Young; Jue, Nathaniel; Lee, Kiho; Park, Kwang-Wook (2016-11-03)Background: Pigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype. Methods: First, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer. Results: Three live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen. Conclusions: Here, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research.
- Breeding management of small ruminants and camelidsClark-Deener, Sherrie (2015-05-14)Understanding the basic physiological characteristics of sheep and goats are essential for a successful breeding program. This knowledge will insure that the males and females are in the correct stage of their reproductive cycle during the proper season for the appropriate timing of an insemination. This proceedings article will outline the major characteristics affecting the reproduction of small ruminants and how to manage their breeding program.
- Cattle temperament: Effects on health and reproduction.Clark-Deener, Sherrie (2016-01-20)
- Characterization of the expression of angiogenic factors in the feline placenta during development and in feline cutaneous squamous cell carcinomaGudenschwager Basso, Erwin Kristobal Felipe (Virginia Tech, 2018-11-13)Throughout gestation, the blood vessel network of the placenta is formed sequentially by processes known as vasculogenesis and angiogenesis, which together meet the needs of the growing fetus. Normal placental angiogenesis is critical to support adequate fetal growth and assure the health of the offspring. Proper angiogenesis requires precise regulation of expression of agents that modulate this process; otherwise, pathologies of pregnancy such as preeclampsia may occur. The placenta is composed of different layers of tissue, including the lamellar (LZ), junctional, and glandular zones, each with a vascular morphology attuned to its function. We hypothesized that higher expression of pro-angiogenic factors is associated with increased morphological metrics in the LZ, the major vascularized zone. Thus, we aimed to characterize the major changes in morphology and vascular development in the placenta throughout pregnancy in cats, alongside a compressive analysis of the expression of major angiogenic factors and their receptors in the placenta, with an emphasis on the identification and interaction of different isoforms of the VEGF family. Microscopic analysis of tissue specimens from different stages of pregnancy revealed increased thickness of the LZ, especially during early to mid-gestation, at which time the tissue is composed of abundant materno-fetal interdigitations that appears rich in capillaries. VEGF proteins were detected in placental tissue in both fetal and maternal cells of the placenta, suggesting stimulatory interactions between different cell types to promote growth and angiogenesis. Gene expression analysis of placenta revealed upregulation of the pro-angiogenic factor VEGF-A in mid-pregnancy, followed by a steady decline toward term, consistent with morphologic changes in the LZ. In contrast, another pro-angiogenic factor, PlGF, showed a marked increase toward term; Flt-1, which acts as a receptor or reservoir for PLGF and VEGF A, was also upregulated at late pregnancy. Increased ratios of PLGF:VEGF-A may contribute to LZ proliferation in the last trimester. These findings are consistent with the creation of a proangiogenic placental state during gestation. Overall, we expect that this research will help elucidate mechanisms of placental vascularization, which can be applied to the design of improved strategies to treat vascular complications of pregnancy. Lastly, we applied the tools developed for placental studies to investigate pathologic angiogenesis in cutaneous squamous cell carcinoma (CSCC), a common skin cancer with major economic and medical impacts in humans and veterinary species. The creation of a new blood supply is essential for growth and metastasis of many tumor types. The goal of this study was to measure expression of variants of proteins that stimulate angiogenesis or transmit an angiogenic stimulus in feline CSCC. The results were mixed, with differences detected in expression of some regulatory agents and, for others, unexpectedly lower expression in CSSC compared to controls. Interestingly, the expression of VEGF-A relative to the protein that transmits its signal (KDR) was elevated in CSCC, suggestive of an altered signaling relationship. This finding supports our hypothesis and is consistent with human SCC studies. Our results encourage further studies on angiogenic factor variants in feline CSCC.
- Comparison of Luteolysis and Timed Artificial Insemination Pregnancy Rates after Administration of PGF2a in the Muscle or the Ischiorectal Fossa in CattleHolland, Sarah C. (Virginia Tech, 2015-09-09)Prostaglandin F2α (PGF2α) is commonly given to female cattle intramuscularly (IM) for the synchronization of estrus. A novel site for administration of PGF2α that improves beef quality assurance is the ischiorectal fossa (IRF). The objective of this study was to determine whether administration of PGF2α in the IRF results in a similar physiologic response to administration of PGF2α given IM. Yearling angus-cross heifers (n=112) were blocked by weight and randomly assigned within blocks to be injected with 5 mL PGF2α either IM in the neck or in the IRF. Blood samples were taken at 0, 8, 16, 24, 36, and 48 h post-injection. Serum samples were analyzed for progesterone concentration using a radioimmunoassay. Progesterone concentration curves for each heifer were plotted to determine luteolysis. The median times to luteolysis for neck and IRF injections were 18.1 hrs and 20.0 hrs, respectively (p=0.06). Angus cross commercial beef cows (n=1471) at least 30 days post-partum were blocked by age and randomly assigned to be injected with 5 mL PGF2α either IM in the neck muscle or in IRF as part of a 7-Day CO-Synch + CIDR ovulation protocol. Pregnancy diagnosis was performed via ultrasound at 60 days post insemination. Results were analyzed with Proc Glimmix (SAS). Pregnancy rates for neck and IRF injections were 52.6% and 57.2%, respectively (p=0.06). In summary, injection of PGF2α in the IRF for estrus synchronization and lysis of the corpus luteum did not differ from injection in the neck muscle. Utilizing the ischiorectal fossa as an injection site for PGF2α may be considered as an alternative that more closely aligns with beef quality assurance objectives.
- The Effects of Low Stress Cattle Handling and Weaning Training on Post-Weaning Weight Gain and Calf ActivityLigon, Jennifer Marie (Virginia Tech, 2015-02-04)The objective of this study was to assess the effect of low stress (LS) handling of beef calves on weight gain and calf activity associated with the weaning process. Cattle were of Angus and Angus cross breeding from two separate herds in Virginia. Handlers for the LS groups went through a short training session. Handlers for the Control (C) groups did not have any special training and handled their group as they would have with no adjustments. Handling and calf activity were monitored each time (6 times) the cows were worked from calving through one month post-weaning. Weights were taken from birth to one month post-weaning. During the week post-weaning the C calves averaged a gain of 4.38 lbs. and the LS calves averaged a gain of 16.94 lbs. One month post-weaning the C calves averaged a gain of 49.01 lbs., while the LS calves averaged a gain of 68.6 lbs. This showed a difference (p < 0.0001) between handling method for weight gain in calves for one week and one month post-weaning. Pedometers were used to assess calf activity post-weaning. Steps per hour (SPH) for the week post-weaning was numerically higher for those calves handled conventionally and not trained for weaning. The C calves averaged 1048 to 1629 SPH for the first three days, where the LS calves averaged 443 to 644 SPH for the first three days. Additionally, the artificial insemination conception rates (AICR) were calculated in each herd and treatment groups compared, however results were equivocal. This study demonstrated that handling cattle using low stress techniques can make significant improvements with regard to weaning weights and has potential to increase other areas of production in beef cattle.
- Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporationHendricks-Wenger, Alissa; Aycock, Kenneth N.; Nagai-Singer, Margaret A.; Coutermarsh-Ott, Sheryl; Lorenzo, Melvin F.; Gannon, Jessica; Uh, Kyungjun; Farrell, Kayla; Beitel-White, Natalie; Brock, Rebecca M.; Simon, Alexander; Morrison, Holly A.; Tuohy, Joanne L.; Clark-Deener, Sherrie; Vlaisavljevich, Eli; Davalos, Rafael V.; Lee, Kiho; Allen, Irving C. (Nature Research, 2021-04-07)New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.
- Evaluation of Different Concentrations of Egg Yolk in Canine Frozen Semen ExtenderTrout, Stephanie Williams (Virginia Tech, 2013-01-09)This study tested different concentrations of egg yolk in canine freezing extender void of glycerol, a commonly used cryoprotectant, by examining the motility and morphology throughout the freezing process: initial (baseline after extender added), post-cool (after three hours at 5"C) and post-thaw (after freezing.) Initial values of pH, osmolarity, motility and morphology were obtained for comparison of the samples. Spermatozoa from six normal dogs as determined by progressive linear motility > 70% and normal morphology > 60% was used. Semen was collected and pooled for five freezing trials. The concentrations of egg yolk used in the extender were: 0%, 10%, 20%, 30% and 40%. Assessment of each sample was blinded to the treatments until all results were obtained and statistics had been analyzed. Based on this study a 20% egg yolk concentration is slightly superior to a 30% egg yolk concentration when assessing post-thaw motility, morphology and longevity and significantly superior to a 0%, 10% or 40% egg yolk concentration. The study also showed motility and normal post-cool and post-thaw sperm morphology did not always correlate. Utilization of 0% and 10% concentrations of egg yolk has negative effects on semen quality as measured by the motility and/or morphology. Results confirm freezing does not affect secondary sperm abnormalities, abnormalities of the tail and distal section of the middle piece, during cooling or freezing. Primary abnormalities, abnormalities of the head and midpiece, increased in the 0% extender during cooling and all extenders during freezing. The pH of the extenders before the addition of sperm was significantly different. Once sperm was added to the extenders, there was no longer a significant difference in pH. There was a positive correlation for both motility and normal morphology percentages post-cool and post-thaw for the extenders with similar osmolarity to the semen.
- Ex Vivo Deformations of the Uterosacral LigamentsDonaldson, Kandace E. (Virginia Tech, 2023-02-24)The uterosacral ligaments (USLs) are important anatomical structures that support the uterus and apical vagina within the pelvis. As these structures are over-stretched, become weak, and exhibit laxity, pelvic floor disorders such as pelvic organ prolapse occur. Although several surgical procedures to treat pelvic floor disorders are directed toward the USLs, there is still a lot that is unknown about their function. These surgeries often result in poor outcomes, demonstrating the need for new surgical approaches and biomaterials. The first chapter of this dissertation presents a review of the current knowledge on the mechanical properties of the USLs. The anatomy, microstructure, and clinical significance of the USLs are first reviewed. Then, the results of published experimental studies on the {emph{in vivo}} and {emph{ex vivo}}, uniaxial and biaxial tensile tests are compiled. Based on the existing findings, research gaps are identified and future research directions are discussed. The second chapter proposes the use of planar biaxial testing, digital image correlation (DIC), and optical coherence tomography (OCT) to quantify the deformations of the USLs, both in-plane and out-of-plane. Using virgin swine as an animal model, the USLs were found to deform significantly less in their main direction (MD) of {emph{in vivo}} loading than in the direction perpendicular to it (PD) at increasing equibiaxial stresses. Under constant equibiaxial loading, the USLs deformed over time equally, at comparable rates in both the MD and PD. The thickness of the USLs decreased as the equibiaxial loading increased but, under constant equibiaxial loading, the thickness increased in some specimens and decreased in others. The third chapter presents new experimental methods for testing the {emph{ex vivo}} tensile properties of the uterosacral ligaments (USLs) in rats. USL specimens were carefully dissected to preserve their anatomical attachments, and they were loaded along their main {emph{in vivo}} loading direction (MD) using a custom-built uniaxial tensile testing device. This chapter reports the first mechanical data on the rat USLs in isolation from surrounding organs. It is also the first experimental study to provide measurements of the inhomogeneous deformations of the USLs during loading along their main textit{in vivo} loading direction, revealing that the USLs may behave as auxetic structures. The fourth and final chapter presents preliminary findings on novel imaging applications to characterize the evolving structure of the USLs before, during, and after tensile pulling along the ligaments' main textit{in vivo} axis of loading. Rat USLs were excised using the proposed novel dissection method and pulled uniaxially as was performed in the previous chapter. Before and after mechanical testing, second harmonic generation (SHG) was used to image collagen and muscle within the three anatomical regions of the USLs. During mechanical testing, OCT was used to collect out-of-plane images of the cervical/intermediate regions of the USL specimens, resulting in 3D volume scans of the regions. SHG images showed the USLs to have complex microstructures with significant wavy collagen bundles interwoven with muscle bundles. Preliminary observation of the microstructure during testing revealed interwoven sections of tissue with collagenous fibers that reoriented in all directions illustrating how the USLs may expand laterally during uniaxial loading, causing the auxetic properties documented in the previous chapter. Though more quantitative work remains to be done, the findings presented in this dissertation improve our understanding of how the USLs deform with increasing load, such as what occurs during pregnancy. Together, these studies serve as a springboard for future investigations on the supportive function of the USLs in animal models by offering guidelines on testing methods that capture their complex mechanical behavior.
- Feline Obesity: Food Toys and Owner Perceived Quality of Life During a Prescribed WeightDodd, Lauren (Virginia Tech, 2019-09-12)The prevalence of overweight and obesity in the feline population is estimated to be 25.7% and 33.8%, respectively. Feline obesity is associated with comorbidities such as insulin resistance and hepatic lipidosis. Several risk factors are associated with obesity including middle age, neuter status, decreased activity, and diet. Obesity management is multifaceted and includes client education, diet modification, and consistent monitoring. Successful obesity management may be dependent on owner perception of their cat's quality of life during a prescribed weight loss plan. Poor quality of life perception may result in failure to complete the weight loss process. Food toys may be used to enhance environmental enrichment, allow cats to express their natural predatory behavior and overall improve owner-perceived quality of life. Therefore, we set out to investigate the role of food toys in owner-perceived quality of life of obese cats during a prescribed weight loss plan. Fifty-five cats with a BCS > 7 were enrolled in a double-blinded weight loss study and randomized into one of two groups: food toy (n=26) or food bowl (n=29). Each cat was provided a prescribed weight loss diet and instructions. Body weight and body condition score were evaluated monthly. Additionally, owners completed a monthly questionnaire to assess their cat's quality of life. Of the 44 cats in the final analysis, 66% (n=29) successfully completed the study and lost > 2 BCS points and/or achieved an ideal BCS of 5/9. Low-calorie vegetables were offered to the majority of cats (n= 32) due to owner reports of disruptive food seeking behavior. Of the cats offered vegetables, 87.5% (n=28) cats required a commercial palatant to consume the vegetables. All enrolled cats had a higher (p<0.0000) owner-perceive quality of life at the final visit/recheck/end of study (median QOL=110.0), as compared to the initial weight loss appointment (median QOL=126.0). The increase in quality of life was primarily driven by improvement in moving from one place to another, grooming and scratching, engaging in social activities, and playing and hunting. There was no effect (p=0.27) of food toy on owner-perceived quality of life. Prescribed weight loss improves owner-perceived quality of life of obese cats. A single food toy (ball-style) was included in this study and did not appear to influence owner-perceived QOL. However, the role of food toys needs further investigation as there are several food toy styles that have not yet been investigated during a prescribed weight loss plan. We suspect that most/all of the 32 cats fed vegetables would have withdrawn from the study. Therefore, including vegetables in the prescribed weight loss plan appears to improve success of weight loss in obese cats.
- Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryosCarey, Kayla; Ryu, Junghyun; Uh, Kyungjun; Lengi, Andrea J.; Clark-Deener, Sherrie; Corl, Benjamin A.; Lee, Kiho (2019-05-06)Background The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. Results First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events. Conclusions The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.
- Hepatitis E virus infects brain microvascular endothelial cells, crosses the blood–brain barrier, and invades the central nervous systemTian, Debin; Li, Wen; Heffron, C. Lynn; Wang, Bo; Mahsoub, Hassan M.; Sooryanarain, Harini; Hassebroek, Anna M.; Clark-Deener, Sherrie; LeRoith, Tanya; Meng, Xiang-Jin (Proceedings of the National Academy of Sciences, 2022-06-14)Hepatitis E virus (HEV) is an important but understudied zoonotic virus causing both acute and chronic viral hepatitis. A proportion of HEV-infected individuals also developed neurological diseases such as Guillain–Barre syndrome, neuralgic amyotrophy, encephalitis, and myelitis, although the mechanism remains unknown. In this study, by using an in vitro blood–brain barrier (BBB) model, we first investigated whether HEV can cross the BBB and whether the quasi-enveloped HEV virions are more permissible to the BBB than the nonenveloped virions. We found that both quasi-enveloped and nonenveloped HEVs can similarly cross the BBB and that addition of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has no significant effect on the ability of HEV to cross the BBB in vitro. To explore the possible mechanism of HEV entry across the BBB, we tested the susceptibility of human brain microvascular endothelial cells lining the BBB to HEV infection and showed that brain microvascular endothelial cells support productive HEV infection. To further confirm the in vitro observation, we conducted an experimental HEV infection study in pigs and showed that both quasi-enveloped and nonenveloped HEVs invade the central nervous system (CNS) in pigs, as HEV RNA was detected in the brain and spinal cord of infected pigs. The HEV-infected pigs with detectable viral RNA in CNS tissues had histological lesions in brain and spinal cord and significantly higher levels of proinflammatory cytokines TNF-α and interleukin 18 than the HEV-infected pigs without detectable viral RNA in CNS tissues. The findings suggest a potential mechanism of HEV-associated neuroinvasion.
- Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A ReviewHendricks-Wenger, Alissa; Arnold, Lauren; Gannon, Jessica; Simon, Alex; Singh, Neha; Sheppard, Hannah; Nagai-Singer, Margaret A.; Imran, Khan Mohammed; Lee, Kiho; Clark-Deener, Sherrie; Byron, Christopher R.; Edwards, Michael R.; Larson, Martha M.; Rossmeisl, John H.; Coutermarsh-Ott, Sheryl; Eden, Kristin; Dervisis, Nikolaos G.; Klahn, Shawna L.; Tuohy, Joanne L.; Allen, Irving C.; Vlaisavljevich, Eli (IEEE, 2021-09-03)New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.
- Immunocontraceptive vaccines against brucellosis and population growth in feral swineSmith, Garrett Paul (Virginia Tech, 2016-10-03)Feral swine are a nuisance species across the United States that costs around $1.5 billion each year in agricultural, environmental, and personal property damages. In the last ten years the population of feral swine is estimated to have quadrupled and novel population control methods are needed. Furthermore, feral swine are known carriers of zoonotic diseases such as brucellosis, which threatens both livestock biosecurity and public health. Recombinant multimeric gonadotropin-releasing hormone (mGnRH) has been previously used as a subunit vaccine to induce immunocontraception in feral pigs. However, potent adjuvants and large amounts of purified antigen are needed to elicit a robust anti-GnRH immune response and current delivery methods are limited. Brucella suis strain VTRS2 can be used as a novel platform to deliver mGnRH without the use of antibiotic resistant markers. Strain VTRS2 was created by deletion of the LPS biosynthesis gene wboA as well as the leuB gene required for leucine biosynthesis inside the nutrient-depleted intracellular environment occupied by Brucella. Mutations in wboA are known to attenuate Brucella strains such as the vaccine strain B. abortus RB51, however strain RB51 is rifampin resistant and has poor efficacy in swine. Strain VTRS2 confers significant protection against B. suis challenge in mice and additionally shows evidence of protection in feral swine. Furthermore, the mGnRH antigen can be delivered using the pNS4 plasmid (which expresses leuB under its native promoter) thus maintaining the plasmid in strain VTRS2 under leucine-deficient conditions while expressing recombinant antigen in the host. The murine model was used to determine the clearance kinetics of strain VTRS2-mGnRH and to measure vaccine efficacy against challenge by virulent B. suis 1330. Subsequently the effects of the VTRS2-mGnRH vaccine on fertility were assessed in breeding trials in mice. Strains VTRS2 and VTRS2-mGnRH were found to be protective against virulent Brucella suis challenge. Strain VTRS2-mGnRH elicited an anti-mGnRH antibody response in vaccinated mice, though an effect on fertility was not observed. An improved vaccine against brucellosis in swine, which also confers immunocontraception without the introduction of antibiotic resistance, could become an important tool in the management of this nuisance invasive species.
- Immunological castration of boars temporarily reduces testosterone concentration, testis size and function, without long term effects on libido and sperm qualityLugar, Drew William (Virginia Tech, 2015-01-06)The objective was to determine the effects of immunocastration (Improvest) on reproductive characteristics in boars. Seventy-two boars were used in a randomized design with three treatments: single injection (SI) or double injection (DI) of Improvest, and intact controls (no Improvest; CNT) (n = 24/group). At wk 10, 15, 20, 25, and 40, blood was collected and serum harvested to evaluate testosterone concentrations via RIA. At wk 25, 18 pigs (n = 6/ group) were sacrificed and testes were removed, weighed, and measured. Libido was assessed at 32, 36, 47, 60, and 63+ wk of age (1 to 5; 1 = no libido; 5 = high libido) and semen collected beyond 60 wk of age. Testosterone concentrations were less for DI boars compared to CNT boars and SI boars at 20 and 25 wk of age (P < 0.001), but not different at 40 wk of age. All testicular measurements and weight were less for DI boars compared to SI and CNT boars (P < 0.001). There was no treatment effect on libido between 32 to 63+ wk of age. Semen volume, gel weight and total number of sperm cells were not different among treatments. Sperm concentration was greater for DI than SI (P = 0.011), and tended to be greater for DI compared to CNT (P = 0.102). Sperm motility tended to be greater for DI boars compared to CNT boars (P = 0.066). The results show that there are no permanent effects of immunocastration on reproductive characteristics in boars.
- Immunological castration temporarily reduces testis size and function without long-term effects on libido and sperm quality in boars.Clark-Deener, Sherrie; Lugar, Drew William; Rhoads, Michelle; Callahan, Stuart Russell; Prusa, Ken J.; Estienne, Mark J. (2016-10-27)The objective was to determine the effects of immunization against gonadotropin releasing hormone on reproductive characteristics in boars. Seventy-two boars were used in a randomized design with three treatments: single immunization (SI) or double immunization (DI) with Improvest® (Zoetis Animal Health, Florham Park, NJ, USA) and intact controls (no Improvest®; CNT) (n = 24/group). At 10, 15, 20, 25, and 40 wk of age, blood was collected and serum harvested to evaluate testosterone concentrations. Testosterone concentrations were less for DI boars compared to CNT boars and SI boars at 20 and 25 wk (P < 0.001), but not 40 wk of age. At wk 25, 18 pigs (n = 6/ group) were sacrificed and testes were removed, weighed, and measured and seminiferous tubules were examined and scored using histological slides of testes parenchyma. A sample of backfat was assessed for boar taint aroma. All testicular measurements and weights and seminiferous tubule scores were less for DI boars compared to SI and CNT boars (P < 0.001). More (P < 0.05) SI and CNT boars had detectable boar taint aroma than DI boars. Libido was assessed at 32, 36, 47, 60, and 63 wk of age and semen collected at 60 wk of age was analyzed for indicators of quality. There was no treatment effect (P = 0.41) on libido. Semen volume, gel weight and total number of sperm cells were not different among treatments. Sperm concentration was greater for DI than SI (P = 0.01), and tended to be greater for DI compared to CNT (P = 0.10). Sperm motility tended to be greater for DI boars compared to CNT boars (P = 0.066). The results show that there are no permanent effects of immunocastration on reproductive characteristics in boars.
- Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot StudyImran, Khan Mohammad; Tintera, Benjamin; Morrison, Holly A.; Tupik, Juselyn D.; Nagai-Singer, Margaret A.; Ivester, Hannah; Council-Troche, McAlister; Edwards, Michael; Coutermarsh-Ott, Sheryl; Byron, Christopher; Clark-Deener, Sherrie; Uh, Kyungjun; Lee, Kiho; Boulos, Paul; Rowe, Cliff; Coviello, Christian; Allen, Irving C. (MDPI, 2023-05-24)Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.
- Improving the development of bovine in vitro produced embryos cultured individuallyGibson, Bethany Gale (Virginia Tech, 2014-07-30)Previous research in bovine embryology has found that embryos cultured individually have limited ability to develop compared to their counterparts cultured in a group of other embryos. This investigation aimed to find if any of three different interventions over two experiments would increase development of individually cultured embryos to that of group cultured embryos. In the first experiment both the addition of serum/serum replacer and a co-culture with bovine granulosa cells were applied to individually cultured embryos in a 3x2 design. None of the interventions was found to be significantly different from the others, and all resulted in significantly lower development than embryos cultured as a group (avg. 4.7 +/- 1.93% individual vs. 21.7 +/- 3.76% group). However, a significant difference was found in the hatching rate between blastocysts cultured in media including cells (71.4 +/- 17.07%) and those cultured without cells (18.1 +/- 11.63%). In the second experiment, embryos were either cultured in standard droplets or microwells made at the bottom of culture droplets either in groups or individually for a 2x2 design. This experiment experienced poor development in all treatments including the group control, and none of the treatments were found to be significantly different from each other. However, the hatching rate of blastocysts cultured in multiple microwells was significantly higher than those cultured individually in droplets. To summarize, none of the treatments increased the development rate, but embryos cultured with granulosa cell co-cultures and in group microwells showed improvements in hatching rates.
- In Vitro Models of Cellular Dedifferentiation for Regenerative MedicineWilliams, Kaylyn Renee (Virginia Tech, 2018-06-22)Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts, or can be made by dedifferentiation of adult cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient's own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create in vitro cell lines with which to study regenerative medicine. First, we used siRNA targeted against myogenin to induce the dedifferentiation of murine C2C12 myotubes into myoblasts. Timelapse photography, immunofluorescence, and western blot analysis support successful dedifferentiation into myoblasts. However, the inability to separate the myotubes and myoblasts prior to siRNA treatment confounded the results. This system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing, but a better method for separating out the myoblasts needs to be developed before this will be achievable. Second, we used a doxycycline-inducible lentiviral vector encoding the transcription factors Oct4, Sox2, cMyc, and Klf4 to create a line of naive-like porcine induced pluripotent stem cells (iPSCs). This reprogramming vector was verified first in murine cells, the system in which it was developed. Successful production of both murine and porcine iPSC lines was achieved. Both showed alkaline phosphatase activity, immunofluorescence for pluripotency marker (Oct4, Sox2, and Nanog) expression, PCR for upregulation of endogenous pluripotency factors (Oct4, Sox2, cMyc, Klf4, and Nanog), and the ability to form embryoid bodies that expressed markers of all three germ layers. Additionally, we were able to create secondary porcine iPSC lines by exposing cellular outgrowths from embryoid bodies to doxycycline to initiate more efficient production of porcine iPSCs. The secondary porcine iPSCs were similar to the primary porcine iPSCs in their morphology, behavior, alkaline phosphatase expression, and Nanog expression with immunofluorescence. The porcine iPSCs were dependent on doxycycline to maintain pluripotency, indicating that they are not fully reprogrammed. Despite this dependence on doxycyline, this system can be used in the future to study the process of reprogramming, to develop directed differentiation protocols, and to model diseases.