College of Agriculture & Natural Resources
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item COMPARATIVE STUDY OF LIPOPROTEIN METABOLISM IN MAREK'S DISEASE SUSCEPTIBLE AND RESISTANT LINES(2010) Yuan, Ping; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Marek's disease virus (MDV) infection causes atherosclerosis, and prior vaccination prevented the development of this disease. Two main strategies to resist Marek's disease (MD) have been demonstrated: vaccination and genetic resistance. However, little is known about the role of genetic resistance in the progression of MDV induced atherosclerosis. Atherosclerosis is primarily associated with lipoprotein metabolism. The purpose of this study was to investigate whether lipoprotein metabolisms are different in distinct MD susceptible and resistant chicken lines. Here, we studied different backgrounds of lipoprotein metabolism in the two lines and the changes of lipoprotein levels in response to MDV infection. The results showed that during chicken growth, the increase in total cholesterol was mostly due to the increasing (LDL+VLDL) in MD susceptible line, whereas it was mainly due to the elevating HDL in MD resistant line. These results suggested that different lipoprotein metabolisms exist in MD susceptible and resistant lines.Item Genetics of Avian Paramyxovirus serotype 2(2010) Subbiah, Madhuri; Samal, Siba K.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian Paramyxovirus (APMV) serotype 2 is one of the nine serotypes of APMV that infect a variety of bird species around the world. In chickens and turkeys, APMV-2 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-2, the complete genome sequences of prototype strain Yucaipa and strains Bangor, England and Kenya were determined. The genome lengths of APMV-2 strains Yucaipa, Bangor, England and Kenya are 14904, 15024, 14904, 14916 nucleotides (nt), respectively. Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Sequence comparison of APMV-2 strains England and Kenya with prototype strain Yucaipa show 94-98% nt and 90-100% aggregate amino acid (aa) identities. However, strain Bangor shares low level of nt and predicted aa sequence identities with the other three strains. The phylogenetic and serological analyses of all four strains indicated the existence of two subgroups: strains Yucaipa, England and Kenya represented one subgroup and strain Bangor represented the other subgroup. All four strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenicity of APMV-2, a reverse genetics system for strain Yucaipa was established in which infectious recombinant APMV-2 was recovered from a cloned APMV-2 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to wild type virus. Recombinant APMV-2 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of mutant viruses by replacing the fusion protein (F) cleavage site of APMV-2 with those of APMV serotypes 1 to 9 demonstrated that the amino acid composition at F protein cleavage site does not affect the pathogenicity of APMV-2. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-2 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-2 would be of considerable utility for introducing defined mutations into the genome of this virus and develop vaccine vector for animal and human pathogens.Item MOLECULAR BASIS OF VIRULENCE IN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) USING A REVERSE GENETICS APPROACH(2009) Ammayappan, Arun; Vakharia, Vikram N; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Infectious hematopoietic necrosis virus (IHNV) is a pathogen of major economic importance to the aquaculture industry. The long-term goal of our work is to develop a safe and effective recombinant IHNV vaccine and possibly use IHNV as a virus vector to express foreign genes. To achieve this goal, the complete genome of IHNV 220-90 virulent strain was sequenced and characterized. Subsequently, a full-length cDNA clone of IHNV was generated by constructing the full length cDNA clone, between the cytomegalovirus (CMV) promoter and the autocatalytic hammerhead and hepatitis delta virus ribozymes. Transfection of a full-length plasmid, along with the supporting plasmids resulted in the recovery of infectious rIHNV-220-90. Characterization of the rIHNV-220-90 showed that its growth characteristics in tissue culture were comparable to those of the parental virus. The possible role of IHNV proteins in virulence was explored to some extent. For this, the entire genome of attenuated virus (IHNV-61) was sequenced and compared with its virulent strain. The comparative sequencing analysis studies revealed that majority of differences were located in the glycoprotein gene. The M and G genes, and the trailer region between virulent and attenuated viruses were exchanged; recombinant chimeric viruses were recovered and studied for their pathogenicity in rainbow trout. The results obtained from in vivo studies indicate that the glycoprotein plays a major role in IHNV virulence in fish, whereas the M gene and trailer region play a negligible role in virulence of IHNV. The potential of rIHNV to serve as a viral vector was explored by expressing the VP2 protein of IPNV and hemagglutinin-estrase (HE) protein of ISAV. The recovered rIHNV-VP2 and rIHNV-HE viruses stably expressed the VP2 and HE proteins respectively for at least five serial passages and showed characteristics comparable to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively to examine the molecular determinants of virulence, pathogenesis, and new approaches for vaccine development.Item Proteomic Profiling and Label-Free Quantification of Bovine Milk Proteins during Experimentally Induced Escherichia coli Mastitis(2009) Boehmer, Jamie Layne; Peters, Robert R; Bannerman, Douglas D; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Coliform mastitis has been a primary focus of dairy cattle disease research due to staggering affiliated losses, severe systemic complications arising from host inflammatory response to lipopolysaccharide, and the poor response of coliform pathogens to antimicrobials. Reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis, and to aid in the approval of new veterinary drugs. The aims of the current analyses were to utilize proteomic methodologies to evaluate protein expression in whey from cows with experimentally induced coliform mastitis, and to employ label-free quantification strategies to estimate changes in relative abundance of proteins identified in milk over the course of clinical infection. Two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry (MS) resulted in the identification of complement factors, antimicrobial proteins, and acute phase proteins in mastitic milk. Analysis using liquid chromatography (LC) inline with electrospray ionization - quadrupole TOF tandem mass spectrometry (MS/MS) resulted primarily in the identification of abundant whey and casein proteins, and the transient detection of proteins related to host response. Nano-LC- nanospray-MS/MS using a linear ion trap, however, led to the robust discovery of over fifty inflammatory proteins in whey from mastitic milk, including the novel markers kininogen-2 and inter-alpha trypsin inhibitor heavy chain-4. Normalized spectral counts were compared to enzyme-linked immunosorbant assay (ELISA) for select proteins to assess the accuracy of the spectral count data. Similar expression patterns were detected using spectral counts and ELISA. Results indicate that proteomic methodologies can detect biomarkers of coliform mastitis in bovine milk during clinical infections, and that spectral counts are a viable means of evaluating relative changes in protein biomarkers of mastitis, including those for which no antibody currently exists.Item FUNCTIONAL CHARACTERIZATION OF THE INTERACTION OF HEPATITIS E VIRUS ORF3 PRODUCT WITH THE CYTOSKELETON(2008) Kannan, Harilakshmi; Zhang, Yan-Jin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hepatitis E virus (HEV) causes several outbreaks of hepatitis in humans. Many aspects of HEV pathogenesis are not well understood. The HEV ORF3 product (henceforth known as vp13) is a multifunctional protein essential for infection of animals. To better understand the vp13 functions, this study was performed. We observed that vp13 protein was associated with the microtubules (MT) in transfected cells. Mutational studies revealed that both hydrophobic domains at the N-terminal region of vp13 are required for the vp13-MT interaction. Our studies also showed that HEV vp13 protein increased the stability of the MT, activated the apoptotic pathway, and, increased the levels of tumor suppressor gene p53 and its downstream effector p21Cip/WAF1 in the transfected cells. However, no noticeable effect on cell survival was observed. These results indicated that HEV vp13 protein may act as a viral regulatory protein.Item MILK AND BLOOD CONCENTRATIONS OF LIPOPOLYSACCHARIDE-BINDING PROTEIN IN COWS WITH NATURALLY-OCCURRING SUBCLINICAL AND CLINICAL MASTITIS(2008-08-22) ZENG, RONGYU; Bequette, Brian J; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objective of this thesis was to evaluate the blood and milk concentrations of lipopolysaccharide-binding protein (LBP) from cows with naturally-occurring mastitis as biomarkers of this disease. Milk and blood samples were collected from 101 clinically healthy dairy cows and 17 dairy cows with clinical mastitis. The concentrations of LBP, haptoglobin and serum amyloid A (SAA) were determined in these samples by enzyme-linked immunosorbent assay. Both LBP and haptoglobin concentrations were higher in the milk and blood of quarters and cows with clinical mastitis respectively than in those that were healthy. Whereas haptoglobin concentrations differed between uninfected and subclinically-infected quarters, LBP concentrations only differed between them when milk somatic cell counts were low. Unlike haptoglobin and SAA, blood concentrations of LBP in cows with a subclinical intramammary infection were not significant from those of cows with all healthy quarters. Thus, haptoglobin may be a preferred biomarker of subclinical intramammary infection.Item The role of Newcastle disease virus internal proteins in pathogenesis(2007-09-24) Rout, Subrat N; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The internal proteins, nucleocaspid protein (NP), phosphoprotein (P) and large polymerase protein (L) of Newcastle disease virus (NDV), play an important role in transcription and replication of the viral genome. However, their role in NDV pathogenesis has not been explored. In this study, the importance of internal proteins in NDV virulence was evaluated through a chimeric approach using an established reverse genetics technique. The L gene between an avirulent NDV strain LaSota and a moderately virulent NDV strain Beaudette C (BC) was exchanged, recombinant chimeric viruses were recovered and studied for their pathogenicity in the natural host, chicken. The results obtained from in vivo studies indicated that the L gene of NDV modulate role in NDV virulence in chickens. The NP and P genes of NDV were exchanged between BC and LaSota individually as well as in combination; chimeric viruses were recovered, indicating that heterologous NP and P genes were functional. In vitro replication of chimeric NP and P recombinant viruses in DF-1 cells indicated that the exchange of NP or P gene in NDV did not affect the replication of the chimeric viruses. The in vivo studies in chickens showed that the change in pathogenicity of these chimeric viruses was minimal and homotypic interaction between NP and P proteins is necessary for optimum pathogenicity of the virus.Item Molecular Epidemiology and Surveillance of Avian Influenza in Wild and Domestic Birds(2006-05-09) Pascua, Annabelle Morano; Tablante, Nathaniel L; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Surveillance of the existence of avian influenza virus in birds is essential in understanding its epidemiology and potential zoonosis. Point surveillance was made on December 2004 and May to August 2005 in wild birds, domestic poultry and environment. Seven out of 67 samples were positive for avian influenza infection resulting to a 10.4 % isolation rate during the winter. Partial sequencing revealed that all isolates were of H11N3 subtype. In the summer, a total of 584 tracheal, cloacal and environmental swabs were tested in the laboratory through virus isolation, real- time PCR and RT-PCR. All samples were negative. To understand the evolution and ecology of the isolated virus, further sequencing was done for all eight genes of H11N3 and each gene sequence was phylogenetically analyzed with available sequences in the Influenza Sequence Database. Replication and transmission of H11N3 were also investigated through experimental infection of chicken and quail.Item REVERSE GENETICS OF AVIAN METAPNEUMOVIRUS(2005-12-06) Dhanasekaran, Govindarajan; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses to the US turkey industry. With a long-term goal of developing a better vaccine for controlling AMPV in the US, a reverse genetics system to produce infectious AMPV entirely form cloned cDNA was established. To achieve this, the unpublished sequences of the G gene, the L gene, the leader and trailer region were first determined to complete the entire genome sequence of AMPV subgroup C strain Colorado (AMPV/CO). Our results showed that the full-length AMPV/CO genome was 14,150 nucleotides (nt) in length, denoting that AMPV/CO possessed the longest genome among known metapneumoviruses. Subsequently, a cDNA clone encoding the entire 14,150 nt genome was generated by assembling 5 cDNA fragments, representing the entire genome, between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a low-copy number transcription plasmid pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1 and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. The recovered virus was observed to contain the genetic markers that were artificially introduced during cloning. Characterization of the recombinant AMPV/CO showed that its growth characteristics in tissue culture were similar to those of the parental virus. These results demonstrate that infectious AMPV can be generated entirely from cloned DNA using reverse genetics techniques. The potential of AMPV/CO to serve as a viral-vector was examined using green fluorescent protein (GFP) as a reporter. The recovered rAMPV/CO-GFP virus stably expressed GFP for at least five serial passages and showed characteristics similar to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively for various studies involving AMPV molecular biology, pathogenesis and vaccine development.Item EPIDEMIOLOGIC ANALYSIS OF RISK FACTORS FOR LOCAL DISAPPEARANCES OF NATIVE RANID FROGS IN ARIZONA(2005-08-11) Witte, Carmel Lee; Kane, Andrew S.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This study used epidemiologic case-control methodology to examine habitat and environmental factors contributing to amphibian declines in Arizona. Risk factors were compared between sites where frogs disappeared (cases) and persisted (controls) using univariate and multivariable logistic regression analyses. Thirty-six percent (117/324) of all sites became cases during the study period. Elevation, non-native predators, hydrologic characteristics, aspect, and effects of nearby sites were significantly associated with frog persistence or disappearance. In the final multivariable model, risk for disappearance increased with increasing elevation (OR=2.7 for every 500 meters, P<0.01). Sites where disappearances occurred were 4.3 times more likely to have other nearby sites that also experienced disappearances (P<0.01), while having an extant population nearby decreased risk of disappearance by 85% (OR=0.15, P<0.01). Sites experiencing disappearances were 2.6 times more likely to have crayfish than control sites (P=0.04). Identification of risk factors associated with frog disappearances will guide future research and conservation efforts.