Kansas State University College Research Articles

Serum amyloid A increases following routine vaccination of healthy adult horses.

To measure the effect of routine vaccination on serum amyloid A (SAA) concentration in apparently healthy horses. We hypothesized that routine vaccination would increase SAA in healthy horses. 21 apparently healthy client-owned horses and 15 Kansas State University College of Veterinary Medicine-owned horses. In experiment 1 (n = 8 horses), a blinded, randomized, prospective, crossover study was performed. Horses were either vaccinated (rabies, tetanus, West Nile, Eastern and Western equine encephalomyelitis, equine herpesvirus-1/-4, influenza) or administered saline, and SAA was measured at 6, 12, and 24 hours and daily until day 10 with a commercial lateral-flow immunoassay. In experiment 2 (n = 28 horses), a prospective, observational study measured SAA after vaccination at 12 and 24 hours and daily until day 10. A linear mixed-effect model with repeated measures over time blocked by horse tested the effect of treatment on SAA. A repeated-measures correlation tested the correlation between SAA and temperature. Over time, vaccinated horses had increased model-adjusted SAA compared to unvaccinated horses without clinical evidence of adverse reaction (P < .01). In experiment 1, the model-adjusted SAA after vaccination peaked on day 2 (median, 1,872 µg/mL; IQR, 1,220.8 to 2,402.5 µg/mL) and returned to normal (< 20 µg/mL) by day 9 (median, 6 µg/mL; IQR, 0.8 to 23.5 µg/mL) after vaccination. In experiment 2, vaccinated horses had increased SAA over time; temperature and SAA were not correlated (P = .78). Results of this study indicated that routine vaccination results in increased SAA concentration and provided evidence for a period of convalescence following vaccination. Measuring SAA for 10 days following vaccination cannot be used as an indicator of illness.

Effects of aging on vasoconstrictor responses of diaphragm arterioles

Introduction: In health, the diaphragm is the most important inspiratory muscle and the only skeletal muscle that is continuously recruited throughout life. Because of the diaphragm’s importance to generating the exercise hyperpnea and wealth of evidence that diaphragm fatigue and exhaustion can impair exercise tolerance, we were interested in whether the exercise intolerance that attends older populations might be associated with altered diaphragm vasomotor control. While it is known that aging compromises peripheral skeletal muscle vasomotor control, it is not known if similar impairments are present in the respiratory muscle vasculature, namely the diaphragm. Therefore, we tested the hypothesis that advanced age decreases diaphragm arteriole contractile responses to alpha-adrenergic agonists. Methods: 4–5 month old Fischer rats and 22–24 month old Fischer rats were divided by age into two groups: young (Y) and old (O). In Y and O, diaphragm first order (1A) arterioles (~200μm diameter, ~2μm in length) were isolated, cannulated, and pressurized to develop spontaneous tone. Following this, contractile responses to cumulative doses of norepinephrine (NE) and phenylephrine (PE) (10−9 to 10−4M) were determined. Results and Conclusion: The physical characteristics of the two groups differed as O were heavier than Y. However, with respect to the diaphragm 1A arterioles, there were no differences in spontaneous tone development (%) or maximal diameter (μm) between Y (38 ± 4%; 132 ± 19μm) and O (37 ± 4%; 157 ± 14 μm; both P &gt;0.05). In diaphragm 1A arterioles, the maximal vasoconstrictor response to NE was also not different between O and Y animals (O: 37 ± 8% vs. Y: 45 ± 1%; p &gt; 0.05). However, the maximal vasoconstrictor response to PE was greater in Y compared to O (O: 29 ± 3% vs. Y: 39 ± 5%; p ≤ 0.05). These data support that aging may reduce a1 adrenergic receptor mediated vascular control, altering the vasoconstrictive responsiveness of diaphragm resistance vessels. We are increasing our group sizes to test the robustness of this conclusion. This work was supported by the Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants awarded to T.I.M and D.C.P. by Kansas State University College of Veterinary Medicine and the National Institute on Aging 1R15AG078060 awarded to B.J.B and D.C.P. A.G.H and K.M.S. were financially supported by Ruth L. Kirschstein National Research Service Awards 1F31HL167618-01 and 1F31HL170643-01, respectively. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

106 Serial Evaluation ofA. Marginale Positivity Rate Within a Single Herd of Mature Beef Cows

Abstract Anaplasmosis is an economically devastating disease in cattle that is caused by the rickettsial pathogen Anaplasma marginale. Infected cattle can serve as biological reservoirs for the organism, thus enabling further transmission of the disease to naive individuals via ticks, biting flies and contaminated needles. It is estimated that this disease causes over $300 million in expenses for the U.S. cattle industry annually. As a result, a better understanding of this disease and its prevalence is needed. As part of an ongoing collaborative effort between the University of Arkansas and the Kansas State University (KSU) College of Veterinary Medicine, general surveillance testing of beef cattle herds in Washington county, Arkansas previously indicated a possible 30% positivity rate for A. marginale in 2019. The objective of this study was to utilize both quantitative polymerase chain reaction (qPCR) and competitive enzyme-linked immunosorbent assay (cELISA) tests to determine the whole-herd prevalence of A. marginale within the University of Arkansas Washington county research herd. In the fall of 2020, the entire fall-calving herd (168, apparently healthy, mature beef cows) was sampled and tested. Both whole blood and serum samples were collected from each animal. qPCR testing was completed on each whole blood sample submitted at the KSU College of Veterinary Medicine, while cELISA testing was completed on each serum sample by the University of Arkansas. Of the68 cows tested, 20 cows tested seropositive, with a % Inhibition of 30 or above (mean = 45%). Confirmatory qPCR testing on all seropositive samples, however, indicated that all cows were negative for active infection with A. marginale. Subsequently, in the spring of 2022, all available cows that had previously tested seropositive for A. marginale were retested using the same protocol as previously described, except that all qPCR testing was completed by University of Arkansas Veterinary Entomology Lab. Of the 17 mature cows still in inventory, that had previously tested seropositive for A. marginale, only 7 were still seropositive with a % inhibition of 30 or above (mean = 39%). However, confirmatory qPCR testing on all 7 seropositive samples indicated that all cows were negative for active infections with A. marginale. Because of its lower cost, compared with qPCR testing, ELISA testing is commonly recommended to and utilized by producers and veterinarians as a first-line diagnostic for individual and herd health evaluation and management. Information gathered by this study indicates that cELISA testing alone may not provide an accurate depiction of actual infection rates within a herd, and that further investigation into possible causes of false positivity are required. As part of this study, additional assays investigating the specificity of the commercial Anaplasmosis cELISA and possible cross-reaction with other endemic rickettsial pathogens will be performed.

Authentic Conversations about Self-Care with Fourth-Year Veterinary Medical Students.

Expanding literature on well-being within veterinary medicine has been instrumental in recognizing the prevalence of psychological distress among students and practitioners and promoting awareness and advocacy for well-being within teaching institutions, professional associations, and the workplace. However, greater focus on distress has also been critiqued for overemphasizing illness and reactive interventions, and a call has been made for more balanced conversations about veterinary well-being, with focus on strengths and proactive interventions. This Best Practices article highlights a proactive, strengths-focused intervention, aimed at increasing fourth-year students' awareness of self-care while in training and in their transition to the profession. Authentic conversations about self-care is a required part of clinical training at Kansas State University College of Veterinary Medicine. All students attend a private meeting with a behavioral scientist, engaging in an authentic conversation about their experience of stress and coping strategies. Current practices in providing stress management interventions are restricted to pre-clinical training. Authentic conversations about self-care are presented here as an alternative to current practices, which the authors argue are not adequately addressing students' needs during clinical training and the transition to the profession. Potential implications of providing self-care interventions during the clinical year of training include greater student engagement, increasing awareness, and self-efficacy as students make efforts to maintain well-being both in training and in the professional realm.

Abstract 2588: Oral administration of water extract from Euglena gracilis prevents lung carcinoma growth in mice by alteration of intestinal microbiota

Abstract Euglena gracilis, a single-celled alga, is rich in nutrients and thus, used as a nutritional dietary supplement. This alga can be found in both fresh and saltwater and possesses characteristics of both plants and animals. Euglena gracilis extracts have a wide range of medicinal properties including stimulation of anticancer immunity against multiple types of cancers; however, the anticancer mechanism has not yet been fully elucidated. Therefore, a water extract from Euglena gracilis devoid of water-insoluble mature paramylons was evaluated as an anticancer agent against lung carcinoma. Two different extracts were prepared using dried powder from whole Euglena gracilis. First, partially purified water extract (EWE) was prepared by suspending dry powder in PBS and centrifugation at 10,000g for 20 min followed by a filtration using a 0.22µm pore size membrane. Second, boiled EWE (bEWE) was prepared by immersing unfiltered EWE in boiling water for 12 min followed by centrifugation at 10,000g for 10 min and filtration using 0.22µm pore size membrane. Both EWE and bEWE treatments inhibited the growth of lung carcinoma cells in vitro in a dose- and time-dependent manner. Furthermore, both extracts significantly inhibited the three-dimensional growth of Lewis Lung Carcinoma (LLC) cells. Flow cytometry analysis showed that the EWE treatment attenuates granulocytic myeloid-derived suppressor cells (MDSCs) in bone marrow cell cultures. The in vivo study was conducted using a mouse LLC orthotopic allograft model. Oral administration of EWE and bEWE (100-200 mg/kg/day) three weeks prior to LLC cell inoculation attenuated the tumor growth in the lungs of immunocompetent mice while decreasing the peripheral granulocytes. However, this attenuation was not seen for the extract treatment initiated after LLC cell inoculation. The tumor growth attenuation was more efficient with the bEWE treatment than with EWE treatment. The fecal microbiomes of the mice were analyzed by 16s rRNA gene amplicon sequencing which revealed that alpha diversity in three groups (EWE, bEWE, and PBS control) was similar, however, the microbial compositions of the EWE- and bEWE-treated mouse groups were more diversified than in the PBS group. Specifically, an increase in the ratio of Bacteroidetes to Firmicutes was observed, and a significant increase in Akkermansia and Muribaculum was detected in EWE- and bEWE- treated mice compared to PBS treated mice. These studies suggest that oral administration of partially purified water extracts from Euglena gracilis altered the intestinal microbiome and the alteration may attenuate host MDSCs, thereby preventing lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Deepa Upreti, Susumu Ishiguro, Mayme Loyd, Nicole Robben, Paige Cote, Morgan Phillips, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Oral administration of water extract from Euglena gracilis prevents lung carcinoma growth in mice by alteration of intestinal microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2588.

Extension Contribution to Anaplasmosis Surveillance in Arkansas: A Story of Collaboration

Abstract The emergence of resistant bacteria forced the medical and animal agriculture communities to rethink how antibiotics are used. In Arkansas, medicated feed is mostly used to treat or control Anaplasmosis in beef cattle herds. Bovine Anaplasmosis is a tick-borne disease caused by the rickettsial bacteria, Anaplasma marginale. This disease causes over $300 million in losses annually for the U.S. cattle industry. With beef cattle being the fifth largest agricultural commodity in Arkansas, it is important to know the prevalence of Anaplasmosis infection in the state. The project described is a collaborative effort between the University of Arkansas-Fayetteville campus, the University of Arkansas Extension, and the Kansas State University (KSU) College of Veterinary Medicine. Extension agents from 33 Arkansas counties were trained to recruit producers for the study and to coordinate sample collection. On the day of blood collection, Extension agents discussed the purpose of the project and appropriate disclosures with the producers. A total of 578 mature beef cattle were randomly selected from six geographical regions for sampling between the months of November 2019 and February 2020. Both whole blood and serum samples were collected from each animal. PCR testing was completed at the KSU College of Veterinary Medicine andcELISA testing was completed at the University of Arkansas Veterinary Diagnostic Laboratory. Blood samples from 335 cattle (58.7%) were positive for Anaplasmosis on at least one test with the majority of animals testing positive (229; 68.4%) on both the cELISA and PCR tests. Rates of regional prevalence ranged from 36.7% to 93.8%. The overall results were discussed with Extension agents via Zoom prior to discussing results with individual producers. Data from this study were added to previous surveillance data collected by Kansas State University and will direct Extension education efforts pertaining to Anaplasmosis management in regional beef cattle herds.

Abstract 3459: Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocytes

Abstract Phytochemicals have been developed into treatments against many diseases, including various cancers. Euglena is a genus of single-cell flagellate eukaryotes having features of both animals and plants. These organisms, which are found in both fresh and salt water, have been used as a dietary supplement for their rich nutrient content. Although recent studies suggest that Euglena extracts have anti-tumor properties, the mechanism is yet to be clarified. Accordingly, the anti-tumor and immunomodulatory effects of partially purified water extract from Euglena gracilis (EWE) was evaluated in cell culture and a mouse orthotopic lung carcinoma allograft model. The EWE was prepared by a PBS extraction at 37°C for 30 min, centrifugation at 2,000 g for 20 min, and filtration through a membrane filter (0.22 μm pore size). Primary components of this extract are water soluble materials and exclude mature paramylons, intact chloroplasts, and oil droplets. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose (1-100 μg/ml) and time (24-72 h)-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs) were dose (1-100 μg/ml) and time (24-72 h)-dependently stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. Flow cytometry revealed that the EWE treatment attenuated granulocytic myeloid-derived suppressor cells in BMCs. In a mouse LLC orthotopic allograft model, pretreatment with EWE (100-200 mg/kg/day, via drinking water) three weeks prior to LLC cell inoculation significantly attenuated the growth of LLC tumors in the lungs of immunocompetent syngeneic mice. However, this attenuation was not seen for EWE treatment initiated after LLC cell inoculation. The tumor growth attenuation by EWE pretreatment coincided with a significant decrease of myeloid-derived cells, primarily neutrophils. EWE pretreatment appears to inhibit lung carcinoma growth mainly by stimulating host anti-tumor immunity via an attenuation of the host myeloid-derived immune suppressor cells. Hence, the present study suggests that the partially purified extract derived from Euglena gracilis contains bioactive materials that prevent lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), a Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Susumu Ishiguro, Deepa Upreti, Nicole Robben, Page Cote, Riley Burghart, Damilola Ogun, Tran Le, Jennifer Delzeit, Jayson Carswell, Arashi Nakashima, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3459.

Perceptions Of Caffeine Use Among Kansas High Intensity Functional Training Participants

Caffeine is a psychoactive drug that decreases perceived effort and improves exercise tolerance. Yet, high-doses of caffeine can result in negative symptoms, which may affect exercise performance. High caffeine users likely perceive positive effects with caffeine-use. While high intensity functional training (HIFT) has expanded rapidly, the perceived effects of caffeine-use during HIFT is unknown. PURPOSE: To compare perceived and actual responses to caffeine during HIFT between low and high caffeine users. METHODS: Seventeen HIFT-experienced men were recruited (age = 26.9 ± 6.5 years, weight=84.7±10.1kg). Participants were randomized in a double-blind, crossover design to consume 5 mg/kg body mass of caffeine pills or placebo 60-minutes prior to a HIFT workout,. Perceptions of caffeine’s effect, successful blinding, and actual positive- and negative-symptoms were determined with surveys. Three chi-square tests were conducted to determine differences between caffeine-users (low- vs. high-users) for perceptions of caffeine’s effects (positive vs. negative), blinding to the treatment (successful vs. unsuccessful), and symptoms after caffeine supplementation (positive and negative). RESULTS: Nine participants were low-users (< 200 mg caffeine/day), and 8 high-users (> 200 mg caffeine/day). Chi-square tests were non-significant (p > 0.05). Descriptively, 2 low-users and 0 high-users perceived negative-effects, while 6 low-users and 7 high-users perceived positive-effects of caffeine use at baseline. One low-user perceived both positive- and negative-effects. Four participants from each group correctly identified the caffeine condition. After study caffeine consumption, 1 participant from each group reported negative-symptoms, while 2 low-users and 4 high-users reported positive-symptoms. CONCLUSION: No significant differences in perceived and actual responses to caffeine supplementation were found between low and high caffeine-users. However, we found that caffeine supplementation may result in negative-symptoms, so individual effects should be considered. Future studies should investigate perceived and actual responses for popular caffeinated pre-workout supplements on; specifically for negative symptoms. Funding: Kansas State University College of Human Ecology

Growing Our Mindset: A Diversity Summit Assessment

In 2003, the Kansas State University College of Agriculture established a diversity programs office to assist the college with recruitment, retention, and graduation of underrepresented students. The Kansas State University Tilford Group has established standards for multicultural competency development that are founded in three primary discussion areas: knowledge, personal attributes, and skills. After several campus incidents, a university-wide event, KSUnite, was created to educate students and improve campus climate. Because diversity is an invaluable element of education, the Kansas State University College of Agriculture Diversity Programs Office seeks to educate students, faculty, and staff in the areas of cultural competency. To attain this objective, the Kansas State University College of Agriculture Diversity Programs Office co-hosted a diversity summit entitled “Growing Our Mindset” in the fall of 2017. At the conclusion of the summit, participants completed a Likert scale questionnaire that measured their development of cultural competencies as a result of forum participation. Questionnaire results and demographic information were analyzed using descriptive statistics. Overall, attendees responded favorably to the forum and indicated that diversity discussions should continue via an ongoing series. To continue this effort, the diversity programs office will utilize the obtained information to further develop cultural competencies within the College of Agriculture.

Evaluation of Fluorescence Microsphere Immunoassay for Antibody Detection to Rift Valley Fever Nucleocapsid Protein and Glycoproteins

Rift Valley Fever Virus (RVFV) is a zoonotic disease that infects ruminants including cattle, sheep, goats, camels and buffalo. There is a need to create a safe, rapid, and accurate assay that can simultaneously detect antibodies directed against multiple RVFV antigens. We describe the improvement and validation of a fluorescence microsphere immunoassay (FMIA) for the detection of IgG antibodies against the RVFV glycoproteins and the immunodominant nucleocapsid (N) protein. The purpose of this assay is to simultaneously screen and confirm sera from animals for positive or negative disease status. Well‐characterized vaccinated and experimentally infected sheep and calf sera were used for the evaluation of the assay. Recombinant viral proteins were produced then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. An unrelated viral antigen coupled control bead set was added to the reaction in order to account for non‐specific binding of antibodies to the antigen of the antigen‐bead complex. Median Fluorescence Intensity (MFI) results were converted to Sample/Positive ratios in order to standardize test results. Our results revealed highest MFI values for the detection of IgG antibodies against the RVFV N protein, indicating that this RVFV antigen could serve as a good candidate for a screening assay. The glycoprotein Gn target has been shown to differentiate vaccinated from non‐vaccinated animals in a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The FMIA demonstrated a high correlation (R2) of 0.91 when compared side by side with plaque reduction neutralization tests. The addition of further RVFV targets such as the non‐structural proteins NSs and NSm is currently under investigation. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay will provide the basis for the development of a multiplex sero‐diagnostic assay system that can simultaneously screen for several ruminant diseases including transboundary animal diseases.Support or Funding InformationThis material is based upon work supported by the Kansas State NBAF Transition Funds, Kansas State University College of Veterinary Medicine, University of Pretoria Department of Veterinary Tropical Diseases, the USDA Agricultural Research project number 5430‐32000‐005‐00D and the U.S. Department of Homeland Security under Grant Award Number DHS‐2010‐ST‐061‐AG0001. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security.

Howard H. Erickson: contributions to equine exercise physiology and veterinary medicine

For over four and a half decades Howard Erickson has been at the forefront of scientific discovery. As a veterinary cardiovascular specialist with the United States Air Force he made fundamental progress to developing a working artificial heart. Subsequently as a retired US Air Force Colonel and Professor at Kansas State University College of Veterinary Medicine Erickson detected the immensely high pulmonary vascular pressures in the horse during exercise. These observations were essential to resolving the mechanistic bases for exercise-induced arterial hypoxemia and pulmonary haemorrhage (EIPH) that afflict all racehorses. Subsequently, Erickson pioneered the scientific proof-of-concept of the equine Nasal Strip™ which reduces lung damage and epistaxis, and constitutes the only effective non-pharmaceutical treatment for EIPH available today. We owe much of our understanding of equine cardiorespiratory physiology to this remarkable scientist.

Public health campaign to promote hand hygiene before meals in a college of veterinary medicine.

Veterinary students can be exposed to environmental infectious agents in school that may include zoonotic pathogens. Encouraging effective hand hygiene can minimize the spread of zoonoses and promote public health and the One Health concept among veterinary students. The purpose of this study was to determine if a campaign could improve hand hygiene among veterinary students at extracurricular meetings serving meals. Nine Kansas State University College of Veterinary Medicine (KSU-CVM) extracurricular organizations participated in the study, sanitizer was provided at each meeting, and baseline hand-hygiene data were observed. A hand-hygiene opportunity was defined as any student observed to approach the buffet food line. Sanitizer use (yes/no) and gender (male/female) were recorded. Campaign interventions included a 3.5-minute educational video and a novel motivational poster. The video was presented to all first-year, second-year, and third-year veterinary students. Posters encouraging hand sanitization were displayed on doors and tables alongside sanitizers at each meeting. Observational hand-hygiene data were collected immediately after introduction of interventions and again 3 months later. Environmental sampling for presence of bacteria in and around meeting locations was also performed. Observed hand hygiene was lowest during baseline (11.0% ± 1.7), improved significantly post-intervention (48.8% ± 3.2), and remained improved at 3-month follow-up (33.5% ± 4.0). Females had higher probability of hand sanitizing (35.9% ± 2.2) than males (21.4% ± 2.4) (p<.01). Clostridium perfringens was isolated from 2/42 samples, and Salmonella spp. were isolated from 4/42 samples. A short-term public health campaign targeting veterinary students successfully improved hand hygiene before meals.

Abstract 220: Human umbilical cord matrix-derived stem cells control tumor growth by tumor suppressor gene expression.

Abstract Umbilical cord matrix derived stem cells (UCMSC) have the potential to treat various diseases including cancer. We have shown that naïve human and rat UCMSC significantly attenuate proliferation of multiple cancer cells (Ganta et al., Cancer Res., 2009. Ayuzawa et al., Cancer Lett., 2009). However, the growth attenuation ability of rat UCMSC is stronger than that of human UCMSC. To clarify their different tumoricidal abilities, differential gene expression profiles of these two cells were studied by micro-array analysis using Illumina HumanRef-8 V2 for human and RatRef-12 BeadChip for rat UCMSC. The gene expression profiles were compared between UCMSC untreated and co-cultured with either human (MDA-231) or rat breast carcinoma cells (Mat B III). Selection criteria used for the screening of candidate genes associated with UCMSC-dependent tumoricidal ability are as follows; 1) gene expression difference should be at least a 1.5 fold between naive UCMSC and those co-cultured with mammary tumor cells; 2) they must encode secretory proteins; and 3) they must encode cell growth regulation-related proteins. These analyses screened 16 common genes from both human and rat UCMSC. The comparison between two sets of gene expression profiles identified that two genes, adipose-differentiation related protein (ADRP) and follistatin (FST), were specifically up-regulated in rat UCMSC, whereas they were down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast cancer cells. The suppression of either protein by adding specific neutralizing antibody in culture of rat UCMSC significantly abrogated their ability to attenuate cell growth. Over-expression of both genes by adenovirus vector in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. FST over-expression in MDA 231 cells also decreased their growth significantly. In the experimental lung metastasis model with MDA 231 cells in immunodeficient mice, the treatment with FST-over-expressing human UCMSC was administered three times, at one week intervals, via tail vein injection, starting 6 days after tumor cell inoculation. Examination 4 weeks after tumor inoculation showed a significant decrease in the number of tumor nodules in the lungs. Both ADRP and FST are considered to be tumor suppressor genes. These results suggest that both ADRP and FST may play important roles in exhibiting a stronger tumoricidal ability in rat UCMSC than human UCMSC. These results imply that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH RR017686, RR15563, CA135599, Kansas Bioscience Authority research grant. Citation Format: Naomi Ohta, Susumu Ishiguro, Atsushi Kawabata, Deepthi Uppalapati, Marla Pyle, Deryl Troyer, Supriyo De, Yongqing Zhang, Kevin G. Becker, Masaaki Tamura. Human umbilical cord matrix-derived stem cells control tumor growth by tumor suppressor gene expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 220. doi:10.1158/1538-7445.AM2013-220

Factors affecting student performance in an undergraduate genetics course1

The objective of this study was to determine some of the factors that affect student success in a genetics course. Genetics for the Kansas State University College of Agriculture is taught in the Department of Animal Sciences and Industry and covers Mendelian inheritance, molecular genetics, and quantitative/population genetics. Data collected from 1,516 students over 7 yr included year and semester of the course; age; gender; state of residence; population of hometown; Kansas City metro resident or not; instructor of course; American College Testing Program (ACT) scores; number of transfer credits; major; college; preveterinary student or not; freshman, sophomore, junior, and senior grade point average (GPA); semester credits when taking genetics; class standing when enrolled in genetics; cumulative GPA before and after taking genetics; semester GPA in semester taking genetics, number of semesters between the biology prerequisite and genetics; grade in biology; location of biology course; and final percentage in genetics. Final percentage in genetics did not differ due to instructor, gender, state of residence, major, or college (P > 0.16). Transfer students tended to perform better than nontransfer students (P = 0.09), and students from the Kansas City metro outscored students from other areas (P = 0.03). Preveterinary option students scored higher in genetics than non-preveterinary students (P < 0.01). Seniors scored higher than juniors and sophomores, who scored higher than freshmen (P < 0.02). We observed a tendency for students with higher grades in biology to perform better in genetics (P = 0.06). Students who took biology at Kansas State University performed better in genetics than students who transferred the credit (P < 0.01). There was a negative regression of hometown population on score in genetics (P < 0.01), and positive regressions of ACT score, all measures of GPA, course load, and cumulative credits on final percentage in the course (P < 0.02). To maximize chances for success in genetics, students should take biology from Kansas State, perform well in biology, and wait until at least sophomore standing to enroll in genetics.

Abstract 5653: A novel cationic peptide nanoparticle-based gene therapy with angiotensin II type 2 receptor gene significantly attenuates growth of lung adenocarcinoma xenografts in mouse model

Abstract A successful gene therapy requires targeted gene delivery, high gene transfection efficiency at the target tissue, and low toxicity of the vectors. We have demonstrated that the HIV-1 TAT peptide is suggested to be an effective gene delivery vector in various cultured cells (Baoum, et al., Pharm Res, 2009; Baoum, et al., Int J Pharm, 2011). The objectives of the present study were to examine the gene transfection efficacy of modified TAT peptide nanoparticle (two TAT peptides connected in tandem i.e. dTAT NP) as a vector, and to examine the efficiency of the tumor-targeted gene delivery after intratracheal administration. Expression efficiency of the dTAT NP encapsulating luciferase or angiotensin II type 2 receptor (AT2R) plasmid DNA (pDNA) was evaluated using Lewis Lung carcinoma (LLC) cells in culture and orthotopic autografts in C57BL/6 mice lungs. Cell culture studies clarified that the dTAT NP caused effective gene transfection with negligible cytotoxicity until approximately 4 mg dTAT/mL (IC50= 4.075 mg/mL), whereas polyethyleneimine (PEI) showed much stronger cytotoxicity (IC50= 0.028 mg/mL). The in vitro study also revealed that the addition of calcium and glucose to the dTAT/pDNA NP caused effective DNA transfection. The in vitro transfection of pAT2R by dTAT NP caused marked attenuation of the growth of LLC cells. Immunohistochemical analysis of the in vitro transfection and in vivo mouse gene expression studies with dTAT/luciferase pDNA NP revealed that dTAT NP vector successfully delivered the dTAT/pDNA complex to the tumor tissues and caused gene expression primarily in the tumor cells and bronchial epithelium. Gene expression in the tumor tissues lasted for at least 14 days post-administration. Bolus intratracheal administration of dTAT/pAT2R as well as dTAT/pTRAIL markedly attenuated tumor growth of LLC autografts in the mouse lungs. Immunohistochemical analysis of dissected tumors revealed that the AT2R expression was predominantly located in the tumor cells of the LLC tumor bearing mouse lung. Apoptotic index was significantly higher in the treated tumors than PBS or luciferase treated control tumors, suggesting that AT2R pDNA was successfully delivered to the tumor tissues by dTAT NP vector and caused apoptosis of tumor cells. Taken together, the current study suggests that a gene delivery system using intratracheal administration of dTAT/pAT2R NP offers an effective strategy for lung cancer gene therapy. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH P20 RR017686, p20 RR1556, R21CA135599 and Kansas Bioscience Authority Collaborative Cancer Research grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5653. doi:1538-7445.AM2012-5653

Abstract B46: Umbilical cord matrix-derived stem cells control tumor growth by their tumor suppressor and promoter gene expression

Abstract Umbilical cord matrix-derived stem cells (UCMSC) have the potential to treat various diseases including cancer. We have shown that naïve human and rat UCMSC significantly attenuate proliferation of multiple cancer cells. However, our previous study showed that the growth attenuation ability of rat UCMSC is stronger than that of human UCMSC. To clarify their different tumoricidal abilities, differential gene expression profiles were studied by microarray analysis using IIIumina HumanRef-8 V2 BeadChip for human and RatRef-12 BeadChip for rat UCMSC. The differential gene expression profile between untreated human UCMSC and those co-cultured with MDA-MB 231 human breast carcinoma cells was compared with that between untreated rat UCMSC and those co-cultured with Mat B III rat mammary gland carcinoma cells. Strict screening criteria used to identify putative genes associated with UCMSC-dependent tumoricidal activity were as follows: gene expression should (1) be over than 1.5 fold different, (2) encode secretory proteins, and (3) be associated with cell growth regulation. Seventeen genes were identified as being associated with either human or rat UCMSC-dependent tumor growth regulation. Among these genes, eight were up-regulated in both human and rat UCMSC (two being known tumor suppressor and six being putative tumor promoter genes). Seven out of seventeen genes were up-regulated in human UCMSC but not in rat UCMSC (three were identified to be tumor suppressor and four were tumor promoter genes). Two out of the seventeen genes, adipose-differentiation related protein (ADRP) and follistatin (FST), which are known tumor suppressor genes, were specifically up-regulated in rat UCMSC, whereas they were down-regulated in human UCMSC when they were co-cultured with carcinoma cells. These results strongly suggest that the balance of the up-regulation of tumor suppressor genes and down-regulation of tumor promoter genes in UCMSC appear to control tumor growth. Since both ADRP and FST are considered to be tumor suppressor genes and were specifically up-regulated in only rat UCMSC, these two genes expression may play central role in strong tumoricidal activity by rat UCMSC. In support of this hypothesis, suppression of ADRP and FST protein by adding a neutralizing antibody (4 µg/ml) in culture medium of rat UCMSC significantly abrogated their ability to attenuate DNA synthesis. Over-expression of ADRP and FST genes by adenoviral vector (100 MOI) in human UCMSC promoted their ability to suppress the DNA synthesis of MDA-MB 231 cells in [3H]-thymidine uptake assay. Interestingly, ADRP expression in human UCMSC stimulated differentiation to adipose type cell morphology (cell enlargement and oil droplet accumulations in cytoplasm). This result suggests that ADRP may stimulate adiponectin production in vivo thereby attenuating tumor growth. Taken together, these results suggest that both ADRP and FST may be key genes that exhibit stronger tumoricidal ability in rat UCMSC than human UCMSC. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH RR017686, RR15563, CA135599, Kansas Bioscience Authority Collaborative Cancer Research grant and by the Intramural Research Program of the NIH, National Institute on Aging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B46.

Abstract 3355: Identification of unique tumoricidal genes in rat umbilical cord matrix stem cells

Abstract Umbilical cord matrix stem cells (UCMSC) represent a promising source of therapeutics for various diseases including cancer. We have shown that naïve rat UCMSC significantly attenuate Fisher 344 rat-derived mammary adenocarcinoma (Mat B III) cell proliferation both in vivo and in vitro. To study the underlying mechanisms and genes involved in the Mat B III growth attenuation, we investigated gene expression profiles of naive rat UCMSC alone co-cultured with Mat BIII cells by cDNA microarray analysis. Total RNA was extracted from the naïve rat UCMSC alone and those co-cultured with Mat B III in transwell culture dishes. The comparison of gene expression profiles between untreated and co-cultured rat UCMSC identified five up-regulated (Follistatin (FST), Sulfatase1 (SULF-1), Glucose phosphate isomerase (GPI), HtrA serine peptidase (HTRA1), and Adipocyte differentiation-related protein (ADRP)) and two down-regulated (Transforming growth factor, beta-induced (TGFBI), Podoplanin (PDPN)) candidate genes based upon the following screening criteria: 1) candidate gene expression should show at least a 1.5 fold change in rat UCMSC co-cultured with Mat B III cells; 2) candidate genes encode secretory proteins; and 3) candidate genes encode cell growth-related proteins. Following confirmation of gene expression by real time-PCR, ADRP, SULF-1 and GPI were selected for further analysis. Addition of specific neutralizing antibodies against these three gene products individually or all together in the co-culture of Mat B III cells and 1/20 cell number of rat UCMSC significantly increased [3H] thymidine uptake in the Mat B III cells implying that these gene products are functionally produced under the co-cultured condition and attenuate cell growth. Immunoprecipitation followed by Western Blot analysis demonstrated that these proteins are indeed secreted into the culture medium. We are investigating the siRNA-dependent down regulation of these genes in rat UCMSC to confirm their tumor suppressor function. These results suggest that ADRP, SULF-1 and GPI act as tumor suppressor genes and these genes might be involved in rat UCMSC-dependent growth attenuation of rat mammary tumor. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH P20 RR017686, p20RR1556, R21CA135599, Kansas Bioscience Authority research fund and by the Intramural Research Program of the NIH, National Institute on Aging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3355. doi:10.1158/1538-7445.AM2011-3355

Abstract 3328: Cytotherapy with naïve rat umbilical cord matrix stem cells significantly attenuates growth of murine pancreatic cancer cells and increases mouse survival in syngeneic mice

Abstract Pancreatic cancer is one of the most aggressive human malignancies, with a very poor prognosis. It is the fourth leading cause of cancer-related morbidity and mortality in the United States. MSC derived from the human umbilical cord matrix (UCMSC) have a great potential for therapeutic use in cancer. UCMSC-based targeted gene/therapeutic-delivery and successful tumor attenuation has been demonstrated. However, the targeted stem cell therapy would be significantly safer and more applicable to human patients if unmodified postnatal stem cells alone were shown to cause tumor cell death and regress tumors, without any foreign gene transfection. Accordingly, the aim of this study was to determine the anti-cancer effect of un-engineered naïve rat UCMSC on the growth of murine pancreatic ductal carcinoma in vitro and in vivo. A mouse peritoneal model was used to test the ability of un-engineered rat UCMSC to control growth of pancreatic cancer. In addition, MTT, direct cell count, [3H] thymidine uptake, and soft agar colony assays were carried out as in vitro studies. Co-culture of rat UCMSC with PAN02 murine pancreatic carcinoma cells (UCMSC:PAN02, 1:6 and 1:3) caused G0/G1 arrest and significantly attenuated the proliferation of PAN02 carcinoma cells as monitored by MTT assay, direct cell counts, and [3H] thymidine uptake assay. Rat UCMSCs also significantly reduced PAN02 colony size and number as measured by soft agar colony assay. The in vivo mouse studies showed that rat UCMSC treatment significantly decreased the peritoneal PAN02 tumor burden 3 weeks after tumor transplantation and increased mouse survival time. Histological study revealed that intraperitoneally administered rat UCMSC survived for at least 3 weeks and the majority were found near or inside the tumor. These results indicate that naïve rat UCMSC alone remarkably attenuate the growth of pancreatic carcinoma cells in vitro and in a mouse peritoneal model. Thus, these studies imply that UCMSC could be a potential tool for targeted cytotherapy for pancreatic cancer. This work was supported by Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's fund, Kansas State Legislative Appropriation, NIH P20RR017686, P20RR015563 and R21 CA135599. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3328.

Swine CAFOs &amp; Novel H1N1 Flu: Separating Facts from Fears

With cases documented in more than 170 countries, the global swine flu pandemic that erupted in spring 2009 remains a serious public health problem. Caused by a strain of H1N1 influenza virus, which is normally found in pigs, the flu now known as novel H1N1 has so far been less severe than regular seasonal flu in terms of deaths and hospitalizations. Yet given its remarkable capacity for human-to-human transmission and a widespread lack of immunity among potentially exposed people, it’s likely the number of cases will rise during the flu season later this fall and winter, according to many public health experts. Given that possibility, enormous resources are being mobilized to address novel H1N1, with an emphasis on vaccine development, education, and efforts to its limit its movements among human communities. Yet one potential source of the original outbreak—factory swine farming in concentrated animal feeding operations (CAFOs)—has received comparatively little attention by public health officials. CAFOs house animals by the thousands in crowded indoor facilities. But the same economy-of-scale efficiencies that allow CAFOs to produce affordable meat for so many consumers also facilitate the mutation of viral pathogens into novel strains that can be passed on to farm workers and veterinarians, according to Gregory Gray, director of the Center for Emerging Infectious Diseases at the University of Iowa College of Public Health. “When respiratory viruses get into these confinement facilities, they have continual opportunity to replicate, mutate, reassort, and recombine into novel strains,” Gray explains. “The best surrogates we can find in the human population are prisons, military bases, ships, or schools. But respiratory viruses can run quickly through these [human] populations and then burn out, whereas in CAFOs—which often have continual introductions of [unexposed] animals—there’s a much greater potential for the viruses to spread and become endemic.” Gray says workers exposed routinely to livestock can pass these zoonotic infections—which transmit readily among humans and animals—on to the wider public. However, public health agencies that monitor risks from zoonotic infections routinely overlook CAFO workers, according to Ellen Silbergeld, a professor at the Johns Hopkins Bloomberg School of Public Health. And animal disease sampling data collected by the food animal industry typically are not shared publicly, according to Gray, although such data could reveal how novel pathogens evolve in CAFOs and how they might move among animals, workers, and the broader community. Experts believe that without these data, society has a diminished capacity to detect and respond to new zoonotic threats before they become more widespread.

Congenital Diseases and Abnormalities of Pigs

Several congenital developmental anomalies are not listed in this review because they have no proposed cause. It is the author's hope that this article will serve practicing veterinarians and the swine industry by being an informative guide for stimulating independent thought toward diagnosing congenital diseases. Remember that the cause may have exerted its effects weeks or months before being observed. A detailed history of the dam as well as the rest of the herd is important. If possible, a history of neighboring herds should also be examined for similar problems. Either conduct a thorough necropsy with appropriate tissues submitted for diagnostic analysis, or send representative pigs to a diagnostic center for a complete evaluation. If chromosome evaluation is desired, contact the evaluating laboratory for submission procedures. Breeding records should be evaluated for intragenerational frequencies and intergenerational patterns typical of genetic transmission. When evaluating pedigrees, the frequent occurrence of an individual is not proof of its involvement. The frequency of occurrence in the diseased population must be significantly greater than it is in a random population of pigs. Dr. H.W. Leipold, in the pathology department at the Kansas State University College of Veterinary Medicine, maintains an extensive database of congenital anomalies in domestic animals. I recommend that he be contacted in all cases of undiagnosed or unusual cases of congenital disease. He possibly can help with a definitive diagnosis and your contact will help expand this important central database of congenital diseases.