2016 ISU CVM SSRP Mentor Abstract #10

Project Title: Novel mechanisms of host manipulation by parasitic nematodes Principle Investigator: Michael Kimber (BMS) Abstract: Lymphatic filariasis (LF) is a mosquito-borne Neglected Tropical Disease caused by filarial nematodes including Brugia malayi; over 120 million people worldwide are infected, with over 1.4 billion at risk in 70 endemic countries. Current control strategies employing mass drug administration have reduced prevalence in many areas, but LF remains a significant global health concern. There is a recognized need for new strategies to control LF and other diseases caused by parasitic nematodes. This project focuses on the host-parasite interface during parasite infection, development and persistence of LF and represents an exciting new direction for investigating this field. We have identified a novel mechanism by which B. malayi modulates the host immune system, through small, regulatory RNAs and proteins delivered via a specific type of extracellular vesicle called exosomes. Our preliminary data demonstrate that infective stage larvae of B. malayi secrete exosomes, that these exosomes contain a diverse miRNA and protein cargo and that distinct parasite miRNA potentially target host genes. Further, these exosomes are internalized by host macrophages and elicit a specific modulatory phenotype. The overall goal of this summer scholar project is to better understand the mechanistic basis for parasite exosome bioactivity in the host. We propose to examine uptake of parasite exosomes by host macrophages to reveal how these vesicles are internalized by host cells, then leverage the genetic capacity of the murine model by using genetic knock outs to generate mechanistic insight into the modulatory phenotype elicited in host macrophages by exosome internalization. The long-term impact of the project will be new knowledge of B. malayi pathobiology that may be exploited in novel LF control strategies. Further, the mechanisms we describe here may be conserved across animal, human and plant parasitic nematodes and could be utilized for broad-spectrum control applications.

2016 ISU CVM SSRP Mentor Abstract #11

Project Title: Structure and function of the regulatory endonuclease, CvfA, from Streptococcus pyogenes Principle Investigator: Brian Lee (BMS) Collaborating Investigator: Gabriela Pérez-Alvarado (VMPM) Abstract: Streptococcus pyogenes is associated with a superficial infections including scarlet fever, impetigo, and pharyngitis, which may develop into invasive diseases such as necrotizing fasciitis or streptococcal toxic shock syndrome. Sequelae that may develop after streptococcal infection include rheumatic fever or glomerulonephritis. Our research is focused on the regulatory endonuclease, conserved virulence factor A (CvfA), which contains a metal-dependent phosphohydrolase domain and regulates the expression of glycolytic enzymes and virulence factors in response to growth phase and nutrient conditions. The nutrient dependent regulation of protein expression allows for adaptation to various host tissues and represents a potential switching mechanism in the mode of infection from commensal to invasive. Our overall goal is to understand this mechanism of gene regulation by defining the catalytic activity and RNA binding specificity of CvfA. Our approach includes structural studies of CvfA using nuclear magnetic spectroscopy and X-ray crystallography, enzyme catalytic assays to define the mechanism of the phosphohydrolase activity, and in vitro selection to define the mRNA sequence specificity of CvfA. The impact of our results may lead to new therapeutic strategies against streptococcal infections. Students may participate in all stages of this project and will receive training in molecular biology, protein chemistry, bioinformatics and structural biology.

2016 ISU CVM SSRP Mentor Abstract #12 Project Title: Immune dysfunction in dogs with mucopolysaccharidosis: Can they cast neutrophil extracellular traps (NETs)? Principle Investigator: Dana LeVine (VCS) Collaborating Investigator(s): N. Matthew Ellinwood (AnSci) Patricia Dickson (Harbor UCLA Pediatrics) Abstract:

The mucopolysaccharidoses (MPS) are inborn errors of metabolism that involve simple inherited loss of lysosomal enzyme function and subsequent lysosomal accumulation of undegraded glycosaminoglycans. The disorder MPS I, the most common and severe of these disorders, results in profound neurological disease and is invariably fatal. MPS I occurs spontaneously in dogs and cats as well as people. Recurrent respiratory infections such as pneumonia, bronchitis, and otitis media are common in MPS patients. Although these infections may be due to airway obstruction secondary to MPS-induced anatomic abnormalities like pharyngeal narrowing, the impact of MPS on the immune system is poorly understood. Little is known about how the innate immune cells are functionally affected in MPS. Since normal function of neutrophils relies on their lysosome-like granules, we hypothesize that MPS results in neutrophil dysfunction. Neutrophil extracellular traps, or NETs, are webs of DNA and granular proteins that are released by stimulated neutrophils to trap bacteria, serving an important role in innate immunity. This summer we will evaluate NET production in MPS I affected and normal dogs. The results may allow us to better understand, and therefore treat, infections in MPS patients. Through this project you will learn to isolate canine neutrophils from blood samples and stimulate neutrophils to generate NETs. You will also have the opportunity to work with an amazing colony of affected and control dogs and learn about the husbandry of a research dog colony.

2016 ISU CVM SSRP Mentor Abstract #13 Project Title: Effect of in-feed antibiotic versus resistant starch prebiotic on enteric immune status in pigs. Principal Investigator: Crystal L. Loving, USDA-ARS-NADC Collaborating Investigator: Heather K. Allen, USDA-ARS-NADC Abstract: There is a relationship between the gut microbiota and immune system, though the mechanisms modulating this interaction have not been fully delineated. Antibiotics have been shown to have an impact on the gut microbiota, and in turn, alter the host immune system. In addition, some antibiotics (tetracyclines) have been shown to have a direct effect on the host immune system. Antibiotics are commonly administered to swine for the prevention of disease, though the effects of antibiotic ingestion versus injection on the pig immune system have not been thoroughly explored. Given the recent limitations antibiotic use in livestock species, efficacious alternatives to antibiotics are sought to maintain health in pigs. One such alternative is the inclusion of prebiotics in-feed to modulate the gut microbiome, and subsequently improve host immunity. The summer project will include evaluating the effects of parenteral oxytetracycline versus in-feed oxytetracycline on swine gut health. In addition, changes in the immune system associated with addition of the probiotic resistant starch will be assessed. Dietary resistant starch has been shown to modulate the intestinal microbiota and enhance gastrointestinal health, though the mechanism in swine has not been clearly defined. The assessment will comprise evaluating changes in gene expression in enteric samples, including levels of cytokines, anti-inflammatory mediators, and anti-microbial defensins. It will also include evaluating changes in lymphocyte populations in the gastrointestinal tract. The work for this project will provide the opportunity to perform ELISA, RNA extractions, real-time RT-PCR, and subsequent data analysis. It will also include microscopic evaluation of intestinal tissues, paired with immunohistochemistry (staining for different lymphocyte populations). The goal of the project is to identify immune changes associated with antibiotic versus probiotic administration for identification of alternatives to maintain a healthy and safe food supply.

2016 ISU CVM SSRP Mentor Abstract #14 Project Title: Correlation between porcine dermatitis and nephropathy syndrome (PDNS) renal lesions and porcine circovirus type 2 infection Principle Investigator:

Darin Madson (VDPAM) Collaborating Investigator(s):

Eric Burrough (VDPAM)

Paulo Arruda (VDPAM) Abstract:

Porcine dermatitis and nephropathy syndrome (PDNS) has long been an associated syndrome of porcine circovirus type 2 (PCV2) infection in swine. The disease process is thought to be a type III hypersensitive reaction, and prior to the development of PCV2 vaccine, antigen could reliably be demonstrated in associated kidney lesions. More recently, diagnostic cases have failed to show a consistent link between PCV2 infection and PDNS renal changes. In 2015 alone, PCV2 was only confirmed in 7 of 52 coded cases of PDNS at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL). The objective of the study is to compare PDNS cases prior to and after PCV2 vaccine development by 1) mining the ISU VDL diagnostic data for summary information and demographics for the last 10 years, 2) determine the presence of PCV2 antigen or DNA in these triaged PDNS cases, 3) clearly describe and contrast renal lesions of both outcomes and 4) determined if swine IgG antibodies can be demonstrated in renal blood vessels by immunohistochemistry to link this each process to a type III hypersensitivity reaction. The working hypothesis of our group is PDNS can be or is associated with multiple pathogens, not PCV2 alone/solely. If true, our generated information will provide needed answers for swine diagnosticians and practitioners to establish multiple pathogens can be linked to PDNS.

2016 ISU CVM SSRP Mentor Abstract #15

Principle Investigator:

Lisa Nolan (VMPM)

Abstract from Bacterial Pathogenesis Laboratory:

Our laboratory research focuses on bacterial diseases of production animals, including their

effects on animal health, public health and food safety. The long-term goal of one of our

projects is to establish the molecular basis of virulence and antimicrobial resistance of

Extraintestinal pathogenic Escherichia coli (ExPEC), caused diseases. ExPEC are the causative

agents of such diverse diseases as avian colibacillosis and human urinary tract infection,

neonatal meningitis, and sepsis. Understanding ExPEC virulence, resistance, and host specificity

should lead to more rational approaches for future disease control.

Our laboratory uses a wide range of scientific techniques including:

- Basic tools of microbiology: bacterial isolation, bacterial growth rates, genomic DNA

isolation, plasmid DNA isolation

- Bacterial epidemiology: using basic molecular biology techniques such as polymerase

chain reaction

- Molecular biology techniques: gene mutation and complementation, transformation,

and cloning. As well as gene expression monitoring using β-gal, and DNA sequencing

- Bioinformatics techniques: such as gene alignment, protein binding prediction and

primer design

- Protein techniques: protein expression, purification, and other assays

- Cellular biology: cell culture procedures using amoebae and mammalian/avian

eukaryotic cells as models

During your research you will learn some of these techniques and may assist in developing new

methods and procedures.

Through this research, you will gain greater understanding of microbial research techniques and

host-pathogen interaction and learn skills necessary for veterinary microbiology research.

If you wish to spend your summer participating in and learning microbial research, don’t

hesitate to contact us! We look forward to hearing from you.

2016 ISU CVM SSRP Mentor Abstract #16 Project Title: Diagnosed Neoplastic Conditions in a Captive Population of Budgerigars (Melopsittacus undulates) Principle Investigator: June Olds, DVM (VCS) Collaborating Investigator(s): Darin Madson, DVM, PhD, Dipl ACVP, and multiple other VDL pathologists (VDPAM) Abstract: Twenty-seven cases of neoplasia have been identified in a captive population of Budgerigars (Melopsittacus undulates) housed within a zoological public feeding aviary in a 5 year period. Affected budgerigars were acquired from the same breeder and are presumed to be interrelated. Cases of neoplasia were identified in birds ranging from 1.5 to 5 years of age. Renal cancer has been previously reported in budgerigars, and was identified in 4 cases at this institution. However, very little information is publicized regarding other types of neoplasia in this species. The objective of this investigation is to characterize the location, type, variety, clinical presentation and incidence of neoplasia in this captive psittacine population.

2016 ISU CVM SSRP Mentor Abstract #17 Project Title: Seasonal variation of serum 25-hydroxy (OH) vitamin D3 in captive black rhinoceros (Diceros bicornis michaeli) housed in a North American Zoo Principle Investigator: June Olds, DVM (VCS) Collaborating Investigator(s): Steve Ensley (VDPAM), Jesse Goff (BMS) Abstract: The black rhinoceros (Diceros bicornis) is a critically endangered species, with only 60 individual animals housed in captivity within AZA-accredited zoos within the United States, and less than 5000 individual animals surviving in the wild. Recent human research has highlighted the importance of sub-clinical vitamin D deficiency with links to increased risks for developing a variety of diseases such as cancer, autoimmune conditions, cardiovascular disease and infectious diseases such as tuberculosis1. Subclinical vitamin D deficiency is also likely to reduce breeding success through diminished fertility and a reduced ability to maintain calcium levels sufficient for normal fetal growth2. There is minimal data available identifying “normal” serum vitamin D3 levels in captive rhinoceros3. Serum samples were collected opportunistically from two captive Eastern black rhinoceros (Diceros bicornis michaeli) housed with seasonal access outdoors in a North American Zoo to test for 25-hydroxy-vitamin D3 (25OHD3) levels over a three-year period. The rhinoceros were also fed a commercially prepared pelleted diet which included dietary Vitamin D3 supplementation. This pilot study correlates environmental UVB, oral dietary supplementation, and seasonal serum 25OHD3 levels to compare to known 25OHD3 levels in free-ranging African black rhinoceros3. 1 Cianferotti L, Marococci C. Subclinical vitamin D deficiency. Best Pract Res Clin Endocrinol Metab 2012; 26:523-537 2 Ullrey DE, Beranard JB. Vitamin D: metabolism, sources, unique problems in zoo animals, meeting needs. In: Fowler ME, Miller RE (eds.). Zoo and wild animal medicine, Volume 4, Current therapy. Philadelphia (PA): W.B. Saunders Company; 1999. P. 63-78 3 Claus M, Jessup DA, Norkus EB, Chen TC, Holick MF, Streich WJ, Dierenfeld ES. Fat Soluble Vitamins in Blood and Tissues of free-ranging and captive rhinoceros. J Wildl Dis. (2002) 38(2); 402-413

2016 ISU CVM SSRP Mentor Abstract #18 Project Title: A broadly protective vaccine against PRRSV Principle Investigator(s): Fernando Osorio (UNL, Swine Virology) Hiep Vu (UNL, Swine Virology) Collaborating Investigator(s): Asit Pattnaik (UNL, Molecular Virology) F Ma (UNL, Bioinformatics) Abstract: Our research centers on pathogenesis of and immune response to viral infections. Due to the significance of the subject for U.S. animal agriculture, since the late 90’s we have focused on a major viral agent that affects swine: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV, an arterivirus, ssRNA+ genome). Our laboratories focus on three fundamental aspects of PRRSV research: immuno-pathogenesis, viral virulence and protective immunity. Immunizing in an effective way against the great diversity of variable strains of PRRSV is one the most significant challenges for swine producers at this time.Overall, our comprehensive PRRSV research goal is to develop an improved, rationally designed broadly protective vaccine against PRRSV. Along this line we have developed a revolutionary concept that may have profound implications in successfully vaccinating swine herds against PRRSV : using bioinformatics and molecular biology we developed a synthetic virus ( i.e. a PRRSV strain non-existing in nature), with the broadest possible coverage against all the strains circulating in the field. A brief visual idea of the concept we work in is given in the following video: https://www.youtube.com/watch?v=WuVVhuQsG2Y&feature=youtu.be