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Effective Bovine Viral Diarrhea Virus (BVDV) control on dairy farms is multifaceted and includes methods to accurately detect virus, remove BVDV persistently infected (PI) cattle, prevent virus introduction using comprehensive biosecurity plans, and optimize herd immunity through continuous vaccination against BVDV. The work in this thesis takes into consideration the above means to achieve effective BVDV control more specifically by attempting to: 1) determine the herd infection status by screening newborn calves for precolostral BVDV serum antibodies; 2) eradicate BVDV from a large commercial dairy herd through a combination of test and removal procedures and biosecurity measures; 3) characterize BVDV in PI calves from the Upper Midwestern United States by nucleic acid sequencing in order to more fully understand the changes that are occurring in the BVDV genome that may affect detection and elimination protocols, and; 4) implement a quantitative real-time RT-PCR (qRT-PCR) for quantification of BVDV RNA in a variety of clinical samples obtained from PI cattle. Although many tests have been developed to detect BVDV PI cattle, there are few strategies to detect endemic BVDV infections at the herd level, especially in those herds that routinely administer BVDV vaccines. Many BVDV infections result from direct exposure to BVDV PI cattle. The detection and removal of BVDV PI cattle are essential steps towards reducing virus exposure within the herd and are critical components of national BVDV eradication efforts, such as those in Scandinavia. Veterinary diagnosticians and researchers have developed a variety of accurate tests to detect BVDV PI cattle in dairy herds. For example, screening bulk milk from dairy herds for BVDV by RT-PCR is popular due to the ease of sample collection and the large number of animals that can be screened with one sample. A disadvantage of screening bulk milk is that it will not detect PI cattle in the non-lactating herd nor in youngstock. Alternatively, non-vaccinated sentinel calves can be used to detect BVDV PI exposure in youngstock; however, the sensitivity of sentinel calves in large herds with multiple groups of cattle is not known. Chapter 2 of this thesis investigated a novel screening approach to detect BVDV by screening newborn calves for BVDV serum antibodies prior to colostrum feeding. Newborn calves that are seropositive for BVDV antibody prior to colostrum feeding indicate fetal infection during the last two trimesters of gestation. The number of newborn calves seropositive for BVDV serum antibodies at birth is estimated to be greater than the number of PI calves. Because the number of BVDV seropositive calves is greater than the number of BVDV PI calves fewer calves need to be tested by precolostral serum antibody screening to detect BVDV fetal infections and the probability of one or more PI cattle in the pregnant herd is likely. In addition to requiring fewer test animals, precolostral screening detects infections in lactating, non-lactating, and pregnant youngstock populations and is not confounded by vaccination. Rapid consolidation of the United States dairy industry has resulted in fewer and larger dairy herds. Chapter 3 of this thesis describes the elimination of BVDV PI animals in a large commercial dairy herd with a RT-PCR test. Previous testing in the study herd indicated that approximately 5% of the calves were born with BVDV precolostral serum antibodies. The birth of BVDV seropositive calves also roughly coincided with an increase in post-partum diseases that failed to respond to proven therapies. The herd owners elected to test all animals for BVDV PI with a serum BVDV RT-PCR test. Accurate detection of BVDV PI cattle is important in all herds, but less than perfect sensitivity and the potential of a false negative result are amplified in large herds with PI cattle. False negative test results would lead to the retention of one or more PI cattle and ultimately the continued persistence of BVDV within the herd. Serum samples from all cattle on the premises, and heifer calves born during the following 9 months, were tested for BVDV by RT-PCR and those determined to be BVDV PI on confirmatory tests were removed from the herd. Whether or not BVDV persisted in, or was eliminated from, this herd was determined by monitoring newborn calf precolostral serum antibodies for BVDV one year after the test and removal of all PI cattle. The chapter describes the detection of BVDV PI cattle, genetic characterization of BVDV isolated from the PI cattle, the detection of BVDV acute infections, and the precolostral monitoring results before and after the removal of PI cattle. Bovine viral diarrhea virus is a single-stranded RNA virus that lacks a proof-reading mechanism resulting in mutations and recombination of the viral genome. Point mutations and recombination of viral RNA can result in novel, unique viruses. Few animal disease laboratories perform nucleic acid sequencing for BVDV, thus changes in the BVDV genome are not well described. The objective of chapter 4 was to successfully sequence a portion of the viral RNA and compare the viral genome sequences of forty PI cattle detected on dairy farms in the Upper Midwestern United States. The 5' untranslated region (5'UTR) region of the BVDV genome contains conserved regions and is commonly used for PCR detection tests. This project described the use of primers targeting 5'UTR that produce a PCR product for nucleic acid sequence comparisons between vaccine and field strains and allow for differentiation between subgenotypes BVDV 1a, BVDV 2a, and BVDV 1b. Testing many animals for BVDV PI requires appreciable amount of supplies and labor. The ear notch (skin) sample is a convenient tissue for testing and detecting BVDV PI animals because it is an easy sample to collect and requires minimal amounts of supplies and equipment. Ear notch skin samples offer some flexibility because they can be tested for BVDV by immunohistochemistry (IHC), antigen-capture ELISA (ACE), or RT-PCR. Pooling ear notch phosphate buffered saline (PBS) supernatant for RT-PCR is a popular method to screen large numbers of animals at a reduced cost. This method involves soaking the ear notch in a small amount (~2 ml) of PBS and then pooling the supernatant. The pooled supernatant is then tested for BVDV by RT-PCR. If the pooled supernatant is positive, the originally submitted samples can be tested individually to determine the PI animal. While ear notches have become the sample of choice for PI testing, there is little information available on the quantity of viral RNA in ear notches and the PBS supernatant that contains the soaking ear notch. The objective outlined in Chapter 6 was to implement a quantitative real-time RT-PCR (qRT-PCR) for quantification of BVDV RNA in a variety of clinical samples obtained from PI cattle. Serum, whole blood, nasal swabs and skin samples were collected from PI cattle and analyzed by qRT-PCR. The data derived from qRT -PCR allowed for an estimation of RNA copies in the variety of samples obtained from PI calves. This thesis will give bovine veterinarians, diagnosticians, and researchers additional information on the dynamics and manifestations of BVDV in dairy herds. The precolostral screening method of newborn calves appears feasible and has potential application in commercial dairy herds. The performance and utility of a highly sensitive and specific RT-PCR test was assessed and appeared successful in a large commercial dairy herd. Additionally, nucleic acid sequence analysis of BVDV obtained from PI dairy cattle were compared to PI cattle from other farms and well-described viruses strains listed in GenBank. Quantification of BVDV RNA in clinical samples provided essential information needed to estimate the size of pools and potential variations in detectable RNA from diagnostics samples.

Methanotrophs are a group of microorganisms found in diverse environments that utilize methane for energy and biomass. These microorganisms have long been studied for their practical applications as well as for their significant contributions to the global carbon and nitrogen cycles. The scope of this study was to closely examine the physiological and genetic properties of different groups of methanotrophs to the utility of these cells for: (1) pollutant degradation by these cells and (2) removal of methane from the atmosphere. Finally, a new assay was created to screen methanotrophs for production of methanobactin, a unique copper chelating biomolecule that has significant potential industrial applications engineering practices or development of biological engineering devices and processes de novo utilizing these bacteria. Biodegradation of TCE, trans-DCE, and VC by methanotrophs was closely examined in this study. Significance of selective expression of sMMO and pMMO in biodegradation activity of Methylosinus trichosporium OB3b was examined at a realistic in situ groundwater temperature condition to support the argument that pMMO is responsible for methane-augmented bioremediation. Also Methylocystis daltona SB2 was examined for the expression of pMMO and degradation of chlorinated ethene compounds under growth on acetate in absence of methane using quantitative RT–PCR technique. Considering the significant contribution of methane emission to the greenhouse effect, the ability of methanotrophs to use methane as the electron donor has great significance in environmental perspective. In this study, a methanotrophic biotrickling filtration system was designed to actively utilize methanotrophic activity to remove atmospheric methane from local methane “hotspots”. Mathematical modeling was performed to assess the technological and economical feasibility of this biofiltration system. Methanobactin, a copper chelating biomolecule, has been hypothesized to have significant roles in methanotrophy, as well as interesting potential applications. To enable the genetic studies on methanobactin, we have adopted CAS assay for screening methanotrophs for production of methanobactin. The assay was successful in screening the methanotrophic strains for differential capability to abstract copper. Altogether, we expect the findings in this study to serve as basis for further research that will allow better understanding and more effective applications of methanotrophs.