Brief Summary of Minutes of Annual Meeting

Stations reporting but not attending: Louisiana, Nebraska. Adopted agenda: SEPTEMBER 20: 8:00-8:30, registration; 8:30, welcome (Dr. Chowdury); welcome to Minnesota (Dr. Maheswaran). 8:45-10:30: Address and discussion with Dr. Bert Stromberg, Associate Dean for Research, University of Minnesota College of Veterinary Medicine. 10:30-10:50: Break. 10:50-12:00: Station reports. 12:00-1:00 PM: Lunch. 1:00-4:00: Station reports. 4:00-5:30: Address and discussion with Dr. Gary Sherman, USDA CSREES. 5:30: Adjourn SEPTEMBER 21: 8:00-8:20: station reports. 8:20-9:00: Business Meeting. 9:00-10:30: Open forum with AABP attendees regarding needs in bovine respiratory research. 10:30-10:50: Further discussion of plans for national summit on future of BRD research. 10:50: Adjourn. Key discussions: Meeting with Dr. Stromberg, Associate Dean for Research, College of Veterinary Medicine, University of Minnesota: Dr. Stromberg presented an overview of research activities at the University of Minnesota College of Veterinary Medicine. As he is an Administrative Advisor for another CSREES Regional Project, he presented a short summary of the current status of the Regional Project Program: funding has been cut, but NC-107 was recognized to be a historically strong project and administrators were glad the project was renewed. Project members presented Dr. Stromberg with concerns regarding the loss of funds for the project, other research historically funded through Hatch money, and lack of funding for BRD research specifically and animal health research in general. Dr. Stromberg agreed with these concerns; noted that CSREES held a listening meeting in Kansas City in 2005, and there was some support from industry for BRD research. Dr. Stromberg pointed out that government research funding will always be directed at what producers and their interest groups consider most important. If we want more funding for BRD research, we need to get the cattle industry behind us. After Dr. Stromberg left, the group continued to discuss the problem of lack of funding for BRD research and apparent decreased interest in participation in the regional project. Attendance has been relatively poor for the past two years, and it has become rare for voting members to bring guests (other researchers, graduate students) with them. The consensus of those attending this year was that the group needed to find ways to get more and more consistent funding for BRD research, which could then (we hope) improve the attendance at this meeting. If funding and attendance do not improve, it does not appear that the project will survive much longer. Discussion led to the idea that NC-1027 should host a national summit on the future of BRD research. We would invite representatives of state and national producer organizations (NCBA, national dairy heifer rearers association, others) as well as reps from USDA, and other relevant officials. The point of the summit would be to ask the industry whether they think BRD warrants ongoing research, and, if they do, to ask that they make coordinated efforts to get Congress, USDA, and others to improve research funding. If producer organizations are not willing to make this effort, the group agreed that this would be a signal that there was no real support by the cattle industry for BRD research, and the small amount of money available through the project would not warrant the effort required to keep NC-1027 going. Other reasons for the national summit: solicit input from the cattle industry on what should be the focus of BRD research in coming years; discuss the problem of relative lack of funding for animal research as a whole (point was made that USDA research budget is reportedly about $1 billion, while NSF research budget is about $12 billion, and NIH is even morewhy the disparity when agriculture is a major portion of the U.S. economy?), and the problem of lack of young scientists going into animal health research. Point was raised that most people being hired into basic science departments at vet schools are not people with a primary research focus on animal disease, but rather people with research focus that enables them to compete for NIH R01 grants. Thus, as members of NC-1027 retire, it is expected that they will not be replaced by new people with a focus on BRD. Thus a serious threat to animal health research is attrition of scientists. The meeting with Dr. Sherman on Wednesday afternoon also focused much on the problem of diminished funding available to members of NC-1027 for bovine respiratory disease research. Many project members get no support, or only travel support, for their involvement in NC-1027. It was noted that at some schools, Formula Funds are used to support start-up funds for faculty writing NIH grantspeople doing animal health research may get little or no support from Formula Funds; this depends on the discretion of their administrators. Dr. Sherman was attentive and sympathetic to these problems. He noted that in a climate in Washington D.C. where many budgets are being cut, animal health research is actually getting small increasesbut he agreed with the point that the problems NC-1027 members are experiencing are symptomatic of the larger problem of the relatively serious lack of funds for animal health research in general. He reminded us that he is in charge of Formula Funds, does not have influence at NRIbut he realized that lack of funds through both funding arms of USDA is a problem. He asked that NC-1027 draft a short letter outlining current problems with the funding for the regional research project, and he will try his best to use our input to improve the way funds are made available. Open forum for AABP attendees was generally considered a success, although miscommunication with AABP organizers led to forum not being advertised widely. Only a small number of people attended (no more than 10 people outside the technical committee), and only 3 were producers, but those who attended contributed much useful input. Major points made by the producers who attended were that they perceived currently available vaccines and antibiotics to be relatively effective, but BRD is still a widespread problem, at least in part due to younger calves being brought into feedyards. Cheap corn allows feedlots to bring in more higher risk cattle than in past. Also had good discussion among rep from FDA, rep from biologicals industry, NC-1027 members, and producers on issues of concern to allrequirements for vaccine and antibiotic labelling, particularly. Research needs identified by producers who attended: more research on ways to prevent disease at the level of the cow-calf producer (before animals leave for feedlot); more development of cow-side tests to identify BRD etiologies and biological markers that predict prognosis; and more research on causes and control of respiratory disease in nursing beef calves. Little discussion of BRD in dairy animals, probably only because no dairy producers attended. Detailed notes of discussion were taken by Dr. Woolums; contact her if you would like to review them. Business meeting: Meeting opened by Chairman Dr. Chowdhury at 8:20 AM on Thursday September 21. Minutes of 2005 meeting approved. Discussion of who will serve as Chairman next year. Dr. Briggs from NADC was supposed to serve as Secretary this year but no one has heard from him. Group agreed that he should be called to see if he will be able to serve as Chair next year. If he cannot, Dr. Chris Chase has agreed to serve as Chair next year; if Dr. Briggs can serve as Chair, Dr. Chase will serve as Secretary. Discussion of plans for national summit (see above) began, but had to be cut short due to time (due to open forum scheduled at 9 AM). Business meeting was concluded at 9 AM, but further discussion of national summit occurred from 10:30-10:50 AM. It was agreed that the national summit should be held in 2007. Dr. Grooms and Chowdhury agreed to write a conference proposal to NRI for the November 2006 deadline to try to get funds for the conference. Pros and cons of having conference in conjunction with AABP (in Vancouver, Canada in 2007) were discussedconcluded that the national summit should be held at a time separate from AABP or other meetings to ensure that discussion is focused, and attendance not diluted by other distractions. Consensus: national summit will be held in Washington D.C. Representatives from national and state cattle associations, USDA, AAVMC, and other relevant and influential organizations will be invited; travel will be supported for individuals we wish to have speak (e.g., President of NCBA Gary Webber, who spoke in support of needs for BRD research at 2005 CSREES Listening Meeting). Drs. Chase and Woolums will plan agenda with input from committee and plan local arrangements. Tentative date: first week of October, 2007. 2007 NC-1027 Technical Committee meeting will be held in conjunction with the national summit. Consensus that meetings after 2007, if they occur, should be held in conjunction with AABP, but members would most likely not have time and money to travel both to the summit and also to a Technical Committee meeting in conjunction with AABP in one year. Meeting of Technical Committee adjourned Sept. 21, 10:50 AM. Assigned responsibilities/deadlines/target dates: Dr. Woolums will record and distribute minutes of 2006 meeting. Dr. Chowdhury will prepare annual report (due 90 days from the date of our meeting). Dr. Woolums will draft a short letter to Dr. Gary Sherman, outlining current problems for the regional project, and will send to group for comment. Dr. Grooms and Dr. Chowdhury will write USDA NRI conference grant to obtain support for national summit on future of BRD research; due November 29, 2006. Dr. Woolums and Dr. Chase will prepare draft agenda and list of invitees for national summit and distribute to membership within the next 1-2 months. Dr. Woolums will identify and reserve an appropriate location for the summit in the Washington D.C. area within the next month. Next meeting information: To be held in conjunction with an NC-1027 sponsored national summit on the future of BRD research in Washington D.C., first week of October 2007. More details to follow. INTERSTATION COLLABORATION AND INTERACTIONS (2001-2006) SD, NE, OK and AL collaborated on an issue of Veterinary Clinics of North America focused on bovine viral diarrhea virus SD and MI collaborated with CA on a book titled 5-Minute Veterinarian MI worked with SD, KS, OK, AL, OH and NADC in identifying cattle persistently infected with BVDV for natural fetal challenge study. SD, GA, CA, and NE collaborated on BRSV challenge models SD, AL, MI, and NADC shared various BVDV strains and collaborated on developing new challenge models. SD and GA collaborated on bovine immunology studies. SD shared BHV-1 strains with KS, U of Pennsylvania and Princeton. NE, GA and CA collaborated on a vaccinology course for veterinarians and veterinary students. OH worked with SD, KSU, CA, IA, WCVS and Pfizer in developing and teaching a graduate level course in vaccinology. GA collaborated with University of Montreal and NIH on new vaccine vehicles and delivery systems. GA collaborated with Merial in measuring vaccine efficacy across a multi assay platform. KS and NE shared reagents related to BHV-1 and BHV-5 research. CA shared BRSV isolates with SD and U of Tennessee. NADC and OK in constructing and evaluating new mutant P. multocida vaccine. NADC, OK and New Mexico State on evaluating mucosal immunity to oral M. haem/P. multi vaccine NADC and MN collaborated on generating leukotoxin knock out mutants of M. haemolytica. NADC, NE, OK, WI and MN share monoclonal antibodies directed against M. haemolytica leukotoxin. OK and NADC collaborated on sequencing of multiple BVDV isolates.

OBJECTIVE 1: IDENTIFY EMERGING AND RE-EMERGING AGENTS AND DEVELOP DIAGNOSTIC METHODS FOR BOVINE RESPIRATORY DISEASE (BRD). CALIFORNIA: Infection with Hemophilus somnus on day 6 of BRSV infection leads to a longer febrile period, persistent cough, and a more severe lesion at necropsy. GEORGIA: 1. In collaboration with IA, PCR and IFA assays are being developed to identify pathogenic bovine mycoplasmas. 2. The prevalence of Mycoplasma bovis in nasal swabs of cattle arriving at backgrounding/stocker operations in Georgia was evaluated. The herd level prevalence of M. bovis shedding was high (6 of 9 operations positive), but the animal-level prevalence was low, with 2% - 6% of animals shedding. At the individual level, agreement between culture of nasal swabs and direct PCR of nasal swabs was only moderate, while agreement at the farm level for identification of any animal shedding M. bovis was excellent. Calves shedding M. bovis were significantly more likely to have a fever (p < 0.01, odds ratio = 10.6) than calves not shedding M. bovis. IOWA: 1. Nasal sampling for Mycoplasma bovis was predictive of the biotypes of the mycoplasma in tracheal wash samplings from the same calf. Phenotyping of strains by antibiotic sensitivity was more discriminating than genomic fingerprinting. 2. VspA antigen of M. bovis was used as specific target for ELISA serology and immunohistochemistry (IHC). Data suggested that ELISA serology based on VspA may not be a sensitive indicator of exposure to M. bovis. 3. A whole cell ELISA was more sensitive than a peptide ELISA. Detection of M. bovis in the noses of calves was most sensitive when culture was followed by immunoblot. A generic mycoplasma PCR demonstrated that mycoplasmas other than M. bovis are common in most calves. KANSAS STATE: The relationship between BVDV biotype and genotype and characteristics of the resulting disease were examined for 117 isolates. Specific disease syndromes were not confined to specific virus biotypes or genotypes. LOUISIANA: Over 300 bovine coronavirus strains have been isolated from animals that experienced acute respiratory disease during two shipping fever epizootics. Comparing the sequences of the enteric and lung genomes has revealed numerous single nucleotide substitutions. MICHIGAN: 1. The usefulness if IHC for identifying neonatal calves persistently infected with BVDV was evaluated and determined to be very accurate. 2. A rapid detection of BVDV in cell culture media using a biosensor was demonstrated. Detection of cattle persistently infected with BVDV using ear notches was shown to be most consistent when compared to serum and nasal swabs. 3. Mycoplasma bovis is an emerging pathogen in feedlot calves and is believed to be responsible for an increasing incidence of nonresponsive chronic pneumonia, polyarthritis and mortality. MINNESOTA: The infectious agents associated with BRD were monitored by bacterial culture, virus isolation, viral serum neutralization tests, viral serum hemagglutination inhibition (HI) test,direct fluorescent antibody techniques (DFA), immunohistochemistry (IH), and PCR tests. MISSISSIPPI: 1. Persistent BVD virus infection was more prevalent in light weight placement cattle than in heavier weight placement cattle. Most positive cattle were identified as sick and pulled within the first 10 days on feed. 2. By comparing transcriptional regulator gene sequences from the P. multocida genome with other DNA sequences at GenBank, two genes (Pm0762 and Pm1231) were identified that are uniquely present in this species. NADC: 1. Characterization of goat adenovirus isolates from Minnesota and Canada indicate that there may be across-species antigenic relationships among the ruminant Mastadenovirus, although they differ at the genetic level. 2. Several widespread outbreaks of severe acute BVD could be traced to a single strain of BVDV. Unlike most BVDV strains, this strain spread quickly from herd to herd and caused significant death losses. To date, the only way to determine if a virus caused severe disease or caused mild disease was to infect animals. A research model was developed that uses cultured cells rather that animals to differentiate viral virulence. This model will reduce the need to use live animals and will significantly cut the cost and difficulty of studying BVDV virulence. OKLAHOMA: 1. Serum concentrations of four acute phase proteins were evaluated as either prognostic or diagnostic tools regarding the occurrence of bovine respiratory disease in marketing and shipping stressed feedlot cattle. 2. In collaboration with NADC, bovine viral diarrhea virus (BVDV) subtype 1b was shown to be transmitted by persistently infected calves to both vaccinated and non-vaccinated calves, including BVDV1b seropositive calves. 3. An antigen capture ELISA (AC ELISA) test kit and a RT-PCR test were used to detect bovine coronavirus (BCV) in nasal swabs collected from four groups of cattle. While the AC ELISA is a useful screening test because of ease of use, the RT-PCR test identified more BCV positive cattle. 4. We continue to monitor for BVDV persistently infected (PI) cattle in feedlots by testing all cattle dying via immunohistochemistry (IHC) of formalin fixed ear. Ear notches stored in 10% neutral buffered formalin remained BVDV IHC positive after extended storage: up to one year (one calf positive after 4 years). Since 2003 we have tested samples from five feedyards in Oklahoma, Colorado, and Kansas. There were 3463 samples tested with 116 positive (3.3%). The range of IHC positives in the five feedyards was 2.6% to 5.4%. The owners of the cattle are being contacted to determine vaccination histories. The pen data for morbidity, mortality, treatment costs, number of treatments, and cost of gain are being collected for analysis. 5. The prevalence of BVDV subgenotypes and biotypes was determined from 131 BVDV positive samples from a diagnostic laboratory. There were 131 BVDV samples represented by 117 noncytopathic (NCP), 11 cytopathic (CP) and 3 cases with mixed NCP and CP biotypes. The NCP isolates were more common (P<0.05) than the CP and NCP/CP combination. There were more BVDV1b subgenotypes 60/131 (45.8%) than BVDV1a, 37/131 (28.2%) or BVDV2a 34/131 (26.0%). Only one of the 131 viruses was genetically similar to the strains present in the U.S. vaccines. 6. Tests were compared to identify BVDV PI calves entering feedlots. The antigen capture ELISA (ACE) test on initial test was followed by IHC, viral isolation, and PCR of the serum of ACE test positive calves. Two of 88 ACE test positive calves were negative with the other tests, yielding 2 false positives. The PI rate was 0.4% (86/21,743). SOUTH DAKOTA: 1. Principal respiratory pathogens identified were similar to preceding years. Pasteurella multicida, Mannheimia haemolytica, and noncytopathic BVDV were most frequently isolated. An interesting finding was the increase in IBR abortions, apparently related to the use of MLV IBR-BVDV vaccines in pregnant animals. 2. Immunoperoxidase BVDV outgrowth test for the detection of BVDV PI animals was used on 7409 animals and there were 17 positives. Ear notch BVDV tests were conducted on 21,048 samples and there were 150 positives. PCR tests were done for BVDV and 316 tests (1547 samples) were tested and 39 positives were detected. WISCONSIN: The Wisconsin Veterinary Diagnostic Laboratory provided data regarding testing for, and diagnosis of, bovine viral pathogens during the year 2004. As in past years, the vast majority of tests requested are for detection of BVDV. The highest percentage of positive tests is for BRSV. OBJECTIVE 2: CHARACTERIZE MECHANISMS AND INTERVENTION TARGETS IN PATHOGENESIS OF BRD AT THE MOLECULAR, CELLULAR, AND HOST LEVEL. CALIFORNIA: We have shown that BRSV facilitates development of IgE antibodies to inhaled allergen. BRSV infected allergen (ovalbumin, OA) aerosol exposed calves had detectable OA specific IgE by day 10 and initial analysis of results show a correlation between IgE responsiveness and IL-4 producing T cells in lymph.. GEORGIA: 1. Messenger RNA for IL-4 and IFN-gamma was measured using RT-cPCR of RNA from lung and bronchoalveolar lavage fluid cells from calves challenged with BRSV. The results show that BRSV infection induces a T helper type 1 response. 2. The progression of immune activities following vaccination with modified live virus (MLV) or killed bovine viral diarrhea (BVD) virus was evaluated. Comparison of different tests of immune response showed poor agreement among tests commonly used to measure cellular or humoral immunity. 3. A survey of the relationship between management practices and risk of acute interstitial pneumonia (AIP) at U.S. feedlots was undertaken. The total number of cattle placed by respondents was 2,495,439; representing approximately 10% of the cattle placed in feedlots in 2000. Feedlots in northern states were less likely to report AIP as a cause of morbidity/mortality, while larger feedlots and feedlots placing higher proportions of yearlings more often recognized AIP. Although heifers were recognized to account for 62% of AIP deaths, feedlots placing a large proportion of heifers were not more likely to recognize AIP as a cause of morbidity/mortality than feedlots placing a small proportion of heifers. Feedlots that vaccinated over 95% of cattle for Mannheimia/Pasteurella were less likely to recognize AIP as a cause of morbidity/mortality. 4. Studies undertaken with Iowa confirmed that field isolates of Mycoplasma bovis have more pronounced effect to impair bovine neutrophil function than high pass laboratory isolates, indicating that M. bovis likely causes disease in part through impairing neutrophils function. 5. The impact of whole, live maternal cells, freeze lysed maternal cells, and cell free colostrum on the phenotypic and functional development of innate and adaptive immune function in calves from birth to 6 weeks of age was studied. Colostrum containing whole live cells improved innate and adaptive immune function in calves. IOWA: 1. A lymphotoxic activity is described for Mycoplasma bovis, and this virulence factor was not observed in other species of bovine mycoplasmas. 2. Primary cell cultures of bovine lung alveolar type 2 cells were developed. These will be very valuable for the study of bovine lung pathogens. 3. Aortic endothelial cell cultures were infected with an abscessing or a non-abscessing strain of Mycoplasma bovis. Cells were examined for ICAM-1 up-regulation, nitric oxide synthase activation, and VCAM-1 up-regulation. Only VCAM-1 expression was upregulated. 4. A lymphocyte-suppressive activity of Mycoplasma bovis was found to reside in a peptide cleaved off a surface-exposed membrane protein of the organism. The native, or recombinant form of this peptide had suppressive activity. The immunosuppressive peptide was expressed on nearly all colonies from most strains of M. bovis. KANSAS: 1. The role of envelope proteins gE and US9 in the anterograde transport of BHV-1 following reactivation in TG neurons of calves was investigated. The data suggest that in the absence of Us9 or gE, the latent virus can reactivate in the TG, however the viral anterograde axonal transport in nasal and ocular epithelium is defective. During primary infection, calves infected with BHV-1 wild type, gE deleted, or US9 deleted recombinant viruses shed similar amounts of virus from the eye and nasal cavity. When reactivation form latency was initiated with dexamethasone, virus shedding was observed only in calves infected with wild type virus, but not in gE and US9-deleted BHV-1 viruses. We suggest that the gE and US9 play a role in anterograde transport from TG neurons back to the nasal epithelium, cornea and conjunctiva. 2. The genome of BHV-1 has been cloned as a bacterial artificial chromosome (BAC). Using a two step Red recombinase system a BHV-1 BAC mutant has been constructed in which the cytoplasmic tail of the gE gene has been truncated. The data show that gE cytoplasmic domain is required for posttranslational modification of the gE. 3. Bovine alveolar macrophage neurokinin-1 (NK-1) and the in vitro response of alveolar macrophages to Substance P (SP) exposure were investigated. The results suggest that bovine alveolar macrophages respond to SP at least in part by enhancing phagocytosis and TNF production. LOUISIANA: The spike glycoproteins encoded by two prototypic respiratory and enteric bovine coronaviruses were codon optimized. This codon optimization resulted in high gene expression in transient assays unlike the wild type genes that failed to significantly express in transient expression systems. MICHIGAN: Research determined that administration of a vaccine for leptospirosis in cattle (Spirovac") had no effect on the response of cattle in the CFT or INF³ tests for bovine tuberculosis. MINNESOTA: 1. Studies examined the role of the major ligand-binding domain in LFA-1 in both binding to M. haemolytica LktA and mediating LktA-induced effects. The results demonstrated that the LktA binding domain is within the CD18 molecule, and that binding site lies within the EGF-3 domain of CD18 which encompasses amino acids 541 to 581. However, cellular activation upon LktA binding requires the interaction of CD18 and CD11a. Recently, this group has successfully cloned and sequenced the bovine CD11a coding sequence which has 81.6% nucleotide identity to human CD11a. 2. We mapped the site within the bovine CD18 that is required for LktA binding and its biological effects. To accomplish this, we created several bovine (B) x human (H) chimeric CD18 constructs by domain swapping replacing the nucleotide sequences in the cDNA encoding the amino acids in the extracellular portion of human CD18 with corresponding sequences from the bovine CD18. binding site 3. Expression of the reputed M. haemolytica leukotoxin receptor, bovine LFA-1, in the human erythroleukemic K562 cell line was successfully accomplished. The expressed product was also found to be functionally active. MISSISSIPPI: 1. DNA adenine methylation in P. multocida and M. haemolytica have been demonstrated; data indicated that appropriate expression of Dam is required for virulence of P. multocida. 2. Prophylactic use of feed additive antibiotics in cattle significantly reduces respiratory morbidity/mortality, but the basis for benefit is not known. We compared the proteome of respiratory pathogen M. haemolytica from cultures grown in the presence of ¼ MIC of both antibiotic preparations to that obtained without the antibiotic treatment. Expression of 85 proteins was significantly altered by at least one of the antibiotic treatments. The most notable sub-MIC effect was a significant decrease in the expression of leukotoxin A. 3. We identified mannose receptor (MR) as important in endocytosis in bovine monocytes. We have also demonstrated that antigen uptake through MR-mediated endocytosis, an important APC function, is affected in monocytes in the early stage of BVDV infection, with both BVDV biotypes. We identified the following proteins related to antigen uptake: the galactose binding lectin, MyD-1, clathrin light chain A and MR precursor. We also identified 8 proteins related to cytoskeleton such as actin, beta actin and gamma actin. MISSOURI: Studies determined the expression of gelatinases in healthy calf lungs by examining MMP activity in bronchoalveolar lavage fluid from calves reared in the clinic. All calves possessed gelatinase activity as MMP 2 and MMP 9. NADC: 1. An immunoglobulin-binding protein was demonstrated in whole cell sonicate and culture supernatant preparations from an M. haemolytica serotype 1. 2. Serial analysis of gene expression (SAGE) was used to identify differential gene expression in cultured epithelial and lymphatic cells following infection with BVDV. Findings indicated increased protein translational potential, increased nascent peptide transport and enhanced intracellular membrane targeting and transport. 3. A method for isolation of respiratory tract dendritic cells from lung tissue of neonatal lambs as well as adult sheep was developed. This is the first documented isolation of dendritic cells from neonates, and shows that they are similar to adults phenotypically and functionally. This is in contrast to the current dogma. NEBRASKA: 1. Subpopulations of lymphocytes were more markedly altered in peripheral blood and lymphoid tissues from co infected calves compared to calves infected with either BRSV or BVDV alone. Extensive apoptosis of dendritic cells and lymphocytes occurred in the cortex of mesenteric lymph nodes of calves infected with BVDV. 2. We demonstrated that heat shock protein gp96 peptide complexes isolated from a murine cell line transduced with glycoprotein D (gD) of BHV 1 induce CTLs specific for BHV 1 in mice. Calves infected with the LR mutant exhibited mild clinical symptoms, but they sero converted. 3. Using a vaccinia virus T7-promoter expression system, expression of the single and combination N-glycosylation deletion F proteins of BRSV was characterized in COS-7 cells. 4. Translation efficiencies of the 5' untranslated region from BVDV type 2 isolates was evaluated using bicistronic reporter assays. Low virulence isolates translated with greater efficiency than high virulence isolates in rabbit reticulocyte lysates, We have now determined that in BT and BL-3 cells, the Npro coding region either had no influence or caused a significant decrease in IRES-mediated translational efficiency, with the exception of BVDV 890, where Npro increased IRES-mediated translational efficiency in BL-3 cells. Mutagenesis of IRES base 219 and/or 278 ameliorated suppression of the translational efficiency modulated by the Npro coding region. Results suggest interactions between the Npro coding region and nucleotides within and external to the IRES element influence translational efficiency. IRES element mutagenesis or addition of the Npro coding region to the 5 UTR enhanced the characteristic low translational efficiency of the high virulent isolate 890, while decreasing the translational efficiency of low virulent isolates, such as 7937. These results underscore the complex interplay of cellular factors influencing IRES-mediated translation. 5. Protection against systemic replication of BVDV type 1 in calves vaccinated with with a modified-live noncytopathic BVDV1 vaccine was evaluated. Post-challenge with NY-1, vaccinated calves were protected against systemic replication of challenge virus since they did not develop reduced leukocyte counts and were protected against viremia and infection of target lymphoid cells. 6. Several studies were undertaken to determine the mechanism by which BHV-1 establishes latency. a. A novel BHV-1 gene that is expressed in trigeminal ganglia of latently infected calves (ORF-E) was discovered. The BHV-1 LR gene encodes multiple functions that together regulate latency. The dominant function is performed by a protein encoded by ORF-2 that is absolutely required for the latency-reactivation cycle in cattle. The LR gene interferes with apoptosis, which promotes neuronal survival during the transition from acute infection to latency. The LR gene also appears to encode a small non-protein coding RNA that inhibits cell cycle progression. Finally, LR-RNA sequences or a small open reading frame appears to inhibit bICP0 expression (the gene that is anti-sense to the LR gene), and consequently can inhibit viral gene expression. b. bICP0 is considered to be most important transcriptional regulatory gene of BHV-1 and is antisense to the LR gene. In addition to regulating transcription, bICP0 is toxic to cells, but does not appear to directly induce apoptosis. bICP0 contains a C3HC4 zinc ring finger at its amino terminus. Since bICP0 does not specifically bind DNA, we have hypothesized that bICP0 interacts with transcription factors. Earlier studies demonstrated that bICP0 interacts with histone deacetylase 1 (HDAC1). HDAC1 represses transcription because it removes acetyl groups from histones, thus making chromatin transcriptionally inactive. We have also determined that bICP0 binds p300, a histone acetyltransferase that regulates transcription. We believe that the ability of bICP0 to interact with HDAC1 and p300 promotes productive infection. OKLAHOMA: 1. Three outer membrane proteins (OMPs) including a 95 kDa OMP from a bovine P. multocida grown in iron-depleted conditions were described. 2. Isolated bovine neutrophils were used to study the relationship between the duration and magnitude of the M. haemolytica leukotoxin-induced increase in intracellular calcium concentration and leukotriene B4 synthesis. High and sustained intracellular calcium concentration was necessary to stimulate production of leukotriene B4, which may play an important role in the pathogenesis of M. haemolytica infection. The gene for M. haemolytica outer membrane protein PlpE was cloned and expressed. Vaccination of cattle with rPlpE enhanced resistance against experimental challenge. 3. Calves given modified live virus (MLV) BVDV1a and 2a vaccine were protected against challenge using PI calves. Also antigen detection by the ACE test in fresh notches and IHC in formalin notches were negative in acutely infected calves. SOUTH DAKOTA: 1. BVDV infected macrophages down regulated MHC Class I and II and CD 14 surface markers. This down regulation correlated with the in vivo pathogenicity of the isolates with persistent infection strains down regulating MHC Class I and the acute disease strains down regulating MHC Class II and CD 14. IFN-alpha inhibited BVDV replication and RNA synthesis and its effect was dose, time and biotype dependent. CP/BVDV induced apoptosis as early as 24 hours post infection; p53 and caspase pathways were involved in the BVDV apoptosis. 2. BVDV strains that cause acute disease produce a soluble factor that down regulates un-infected macrophage functions and surface marker expression and nitric oxide (NO) production. 3. Depolymerization of the cytoskeleton (microtubules or actin) had a major effect on BHV-1 replication. We investigated the effect of wild-type (WT) or mutant strains on the integrity of cytoskeleton components, actin filaments, microtubules and intermediate filaments during the course of viral infection. 4. An RT-PCR test for the detection of Toll-like receptors 2 and 4 was developed. Monocyte-derived macrophages (MDM) were infected with BVDV strains of different virulence. The effect of BVDV strains on the mRNA expression level of MDMs of TLR-2 and -4 was evaluated. A BVDV strain from persistent infection significantly decreased the amount of mRNA of MDMs TLR-2 as compared to the mock and other strains. However, this virus has only minimal effect on TLR-4 mRNA expression. Thus, BVDV may modify the innate immune recognition by influencing the expression of toll-like receptors. 5. Forty-four calves were identified as BVDV positive and the strain was BVDV type 2a. Thirty-nine calves were transported to a university facility in September 2004; 36 were PI. These animals were analyzed over a 10-month period. The 5 UTR and E2 region of isolates were analyzed. The results indicate that the 5UTR was similar among all calves but one group of 7 calves had conserved changes in the E2 region. Twenty-four of the calves succumbed to mucosal disease during the experiment and CP-BVDV virus was isolated from 18 calves. The CP-BVDV is currently being sequenced to determine mutation differences. These results indicate that within a population of PI infected animals that the amount of genetic variation in the 5UTR and E2 that occurs over time is well conserved and that small variations in E2 region can occur within the same cohort but these changes occur very early in the infection. 6. The infectivity of BVDV white deer isolates in white tailed deer was evaluated. Eight white tailed deer fawns 4-6 weeks in age were placed in 2 groups: group 1 was inoculated with non-cytopathic (NCP) BVDV type 1b and group 2 was inoculated with NCP-BVDV type 2. A control group of two animals was also used. The results showed that white tail deer are susceptible to BVDV infection from white tail deer isolates. It remains to be determined if deer can transmit the virus to cattle or if deer can become persistently infected as calves. 7. Measurements of serum levels for 4 cytokines (TNF-alpha, IL-10, IL-1B and gamma interferon) were done in BVDV PI and control animals. Basal cytokine levels were similar between controls and PI animals. However TNF-alpha levels were elevated 10X in a calf 3 days prior to death. These same animals were analyzed over a 3 month period for the immunological changes. The most interesting finding was a 50% increase in B cells. TEXAS: There was no association of the specific polymorphisms in the AMPK gene family of the bovine and the incidence of BRD. WISCONSIN: 1. We continued investigations of the effects of M. haemolytica leukotoxin, cytokines, and BHV-1 on bovine leukocytes. Interaction of the leukotoxin (LKT) with LFA-1 was shown to be necessary for the previously described ability of cytokines to enhance LKT cytotoxicity. In collaboration with Minnesota, it was shown that preincubation of bovine PMNs with LKT or LPS enhanced cytotoxic activity of LKT. 2. M. haemolytica leukotoxin (LKT) was internalized by BL-3 cells and transported to the mitochondria, where it caused an increase in mitochondrial permeability. This process was dependent on LKT transport by lipid rafts and dynamin-2. 3. Studies of the molecular pathogenesis of Haemophilus somnus continued: a. H. somnus LOS caused activation of bovine platelets and upregulation of surface molecules that may contribute to the development of vasculitis and thrombosis seen in H. somnus infection. H. somnus and its LOS, as well as H. somnus activated platelets, can up-regulate expression of tissue factor and ICAM-1 (CD54), and down regulate thrombomodulin on bovine pulmonary artery and brain microvascular endothelial cells. We are currently evaluating whether these data reflect the release of PAF by bovine platelets in response to LOS, or suggest a role of the PAF receptor in binding to H. somnus LOS (as has been reported for the LOS of the human pathogen Haemophilus influenzae). b. Using primary cell culture and H. somnus, we have developed an in vitro model to study the sequence of events and mediators involved in the dysfunction of the blood brain barrier in bacterial infection. We have investigated the ability of H. somnus to attach to and invade primary cerebral microvascular endothelial cells (CMVECs). Attachment occurs in a dose dependent manner, but invasion is a rare event. 4. Studies of the VP22 of bovine herepesvirus-1 (BHV-1), a tegument protein that accumulates in the nucleus, indicate a possible regulatory function of VP22 within the nucleus, but how VP22 enters the nucleus is unknown. Despite the abundance of basic residues within this protein, no classic nuclear localization signal (NLS) motif has been identified. A mitochondrial targeting sequence in the C terminal 49 amino acids was identified, which overlaps with the sequence required for nuclear targeting. 5. Studies of intracellular pathways associated with leukocyte death following exposure to M. haemolytica LKT showed that LKT activates caspase-9 more than caspase-8, suggesting involvement of the mitochondrial pathway of apoptosis. The anti-apoptotic protein Bcl-2 decreased, while the pro-apoptotic proteins Bax and Bad increased. Phosphorylation of Akt-1, a signaling protein downstream of LFA-1 that usually leads to cell survival, was decreased following LKT exposure. OBJECTIVE 3: DEVELOP INTERVENTION STRATEGIES FOR CRITICAL CONTROL POINTS TO REDUCE THE IMPACT OF BRD. ALABAMA: Identified 6 potential antiviral compounds. Demonstrated that cattle persistently infected with BVDV negatively impact feedlot cattle CALIFORNIA: Research showed that a cDNA vaccine containing the BRSV N protein provided some degree of protection, and appeared to induce the greatest proliferation of peripheral blood lymphocytes after vaccination and during infection. GEORGIA: 1. Cytokine expression in calves vaccinated intranasally with modified live BRSV prior to virulent BRSV challenge was studied. Results showed evidence of both T helper type 1 (TH1) and T helper type 2 (TH2) cytokine production in vaccinated calves, in contrast to results in nonvaccinated calves, where TH1 responses predominated. 2. Acidic pH below 7.0 altered significantly the capacity of neutrophils to produce oxygen radicals and increased the level of neutrophil apoptosis in cattle. IOWA: 1. Calves infected intratracheally with Mycoplasma bovis responded with a Th2-like response characterized by IL-4 production by PBMCs and antigen-specific IgG1 serum antibody response; this may enhance disease due to slow clearance of the organism. In vitro activation of bovine gamma-delta-T cells was observed to occur when stimulated with M. bovis antigen, indicating activation of innate immunity. 2. Antimicrobial susceptibility of 223 recently recovered isolates of M. bovis was completed. There were no significant differences in susceptibility patterns that could correlate to geographical origin or type of disease presentation. Enrofloxacin, florfenicol, and spectinomycin were active. Oxytetracycline and chlortetracycline were active against half of the isolates, and very few were susceptible to tilmicosin. None were susceptible to erythromycin, ampicillin, or ceftiofur. KANSAS: 1. Near infrared spectroscopy (NIRS) was evaluated in a feedlot study as a means of noninvasively determining oxygen saturation StO2 of hemoglobin to identify animals as having undifferentiated bovine respiratory disease (UBRD). 2. Needle-free and conventional needle-andsyringe injection techniques were evaluated in two different studies of dairy calves and feedlot steers. Blood samples were collected from all animals at the time of vaccination and 21 days later, and the serum analyzed for antibody titers. On day 21 the serological response of heifers to the IBR fraction of the 5-way viral vaccine, MH bacterin and LP fraction of the 5-way Leptospira bacterin were not significantly different between routes of administration. On day 21 the serological response of steers to the IBR fraction of the 5-way viral vaccine and MH bacterin was significantly higher for the needle-free route of administration, while the serological response to the LP fraction was not significantly different between routes of administration. LOUISIANA: 1. Transfecting the upper respiratory tract of cattle with a gene coding for cecropin B does not result in a change in the indigenous and transient nasal flora. Results indicate that transfecting the upper respiratory tract with 100 ¼g of DNA per nostril inhibits colonization of a virulent strain of M. haemolytica 1:A. 2. The spike glycoproteins encoded by two prototypic respiratory and enteric bovine coronaviruses were codon optimized. MICHIGAN: In collaboration with AL, a study was undertaken to determine the effect that the continuous presence of PI cattle has on the overall performance of non-PI feedlot cattle, which shoed that BVDV PI cattle impact the performance of cohort feedlot cattle by increasing morbidity and decreasing average daily gain. NADC: 1. In collaboration with OK, a field trial was conducted on fall-shipped beef calves to evaluate a single-dose combination M. haemolytica/P. multocida edible modified-live vaccine formulation. Vaccination with a modified-live M. haemolytica/P. multocida product single-dose product top-dressed on feed improved weight-gain of calves over a 35 day feeding trial by 6.1 kg compared to unvaccinated ones. 2. New leukotoxin mutants of M. haemolytica serotypes 1 and 6 were constructed which express and transport the C-terminal approximately 1/3 of the leukotoxin structural gene. Mutants may be useful for vaccination of ruminant species. 3. Adenovirus appears to play a role the pathogenesis of outbreaks of respiratory disease in domestic and wild ruminants just as do the better known respiratory viruses. Virus infection may influence the function of pulmonary dendritic cells and thereby cause immunosuppression. OKLAHOMA: 1. Herds with a low morbidity rate had higher levels of BVDV1a antibodies than herds with a high morbidity rate. Calves with low levels of antibody to BVDV1a, P. multocida, and BVDV2 and BVDV2 had increased total treatment costs and decreased net value to owner. 2. The passive immunity transferred to calves from their dams was investigated in a beef herd. Health status at entry to a feedlot was evaluated in reference to performance and carcass value. There were increased numbers of treatments for the individual animals in calves with low antibody levels of M. haemolytica, BVDV1, BVDV2 and PI-3V. 3. During 2003 and 2004 there were reports of bovine herpesvirus-1 (BHV-1) disease in cattle with prior vaccination (often multiple doses) with modified live virus (MLV) vaccines. The disease was not associated with use of any one commercial vaccine. These 2003-2004 episodes had greater morbidity/mortality than expected. Serums were tested for BHV-1 antibodies in a 24-well plaque reduction assay using different BHV-1 strains. It appears that most of the vaccines induced serum antibodies that poorly neutralized the BHV-1 Cooper challenge strain and the field strain. High passages in cell culture may result in changes in virus neutralization properties of the BHV-1. The implications for differences in efficacy of the vaccines against field strains are unclear. Additional testing is in progress to detect genomic differences among the BHV-1 strains. SOUTH DAKOTA: 1. Isoflavones fed to cattle had a small effect on bovine herpesvirus 1 and no effect on NCP-BVDV type 1. 2. A study was done to compare the effect of two dairy cow vaccination programs with either an inactivated vaccine in the dry period vs. MLV in the fresh period. There was no difference in production parameters with the exception of somatic cell count that was higher in MLV animals. 3. Commercial modified live BHV-1 vaccine protected calves from challenge when administered 48, 72, and 96 hours (hr) prior to challenge. TEXAS: 1. Dual prophylaxis reduces the incidence and severity of BRD as compared to metaphylaxis alone. 2. Serum total antioxidant capacity (TACA) and serum malondialdehydes (MDA) were measured in transported cattle. Transportation stress decreased serum TACA and increased serum MDA. WISCONSIN: Research evaluated relationships between air quality, environmental risk factors, and calf respiratory health in 13 naturally ventilated calf barns. Factors that were significantly associated with a reduced prevalence of respiratory disease were reduced pen log10 cfu/m3 on BAP, presence of a solid barrier between each calf were significantly different pen, and increasing nesting score.

PEER REVIEWED PUBLICATIONS (2001) Brock KV, Cortese VS. 2001. Experimental fetal challenge using type II bovine diarrhea virus in cattle vaccinated with a modified-live virus vaccine. Vet therapeutics. 2:354-360. Butler JA, Pinnow CC, Thomson JU, Levisohn S, Rosenbusch RF. 2001. Use of arbitrarily primed polymerase chain reaction to investigate Mycoplasma bovis outbreaks. Vet. Microbiol. 78: 175-181. Chouljenko VN, Lin XQ, Storz J, Kousoulas KG. 2001. Elucidation of the genomic nucleotide sequence of bovine coronavirus and analysis of cryptic leader mRNA fusion sites. Adv. Exp. Med. Biol. 2002. 494: 49-55. Chouljenko VN, Lin XQ, Storz J, Kousoulas KG, Gorbalenya AE. 2001. Comparsion of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia. J. Gen. Virol. 82: 2927-2933. Confer AW, Suckow MA, Montelongo M, Dabo SM, Miloscio LJ, Gillespie AJ, Meredith GL. 2001. Intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membranes expressing iron-regulated proteins. Amer. J. Vet. Res. 62: 697-703. Confer AW, Montelongo M, Brown MJ, Fergen BJ, Clement JC. 2001. Onset of Serum Antibodies to Pasteurella (Mannheimia) haemolytica following vaccination with five commercial vaccines. Bov. Practitioner. 35: 141-148. DeBey BM, Lehmkuhl HD, Chard Bergstrom C, Hobbs LA. 2001. Ovine adenovirus serotype 7 associated mortality in lambs in the US. Vet. Pathol. 38: 644 648. Frazier M, Pence M, Mauel MJ, Ligget A, Hines ME, Sangster L, Lehmkuhl HD, Miller D, Styer E, West J, Baldwin CA. 2001. Endometritis in postparturient cattle associated with bovine herpesvirus 4 infection: 15 cases. J. Diagnos. Invest. 13: 502 508. Givens MD, Galik PK, Riddell KP, Stringfellow DA, Brock KV, Bishop MD, Eilertsen KJ, Loskutoff NM. 2001. Validation of a reverse transcription nested polymerase chain reaction (RTnPCR) to detect bovine virus diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells. Theriogenology. 56: 787-799. Grooms DL, Kaiser L, Walz PH, Baker JC. 2001. Study of cattle persistently infected with bovine viral diarrhea virus that lack detectable virus in their serum. J. Am. Vet. Med. Assoc. 219: 629-631. Hart ML, Mosier DA, Chapes SK. 2001. The response of Tlr-4-receptor positive cells to infection. 35th Annu. Soc. for Leuk. Biol., Honolulu, HI, J. Leukocyte Biol. supp. 67. Inman M, Zhange Y, Geiser V, Jones C. 2001. The zinc ring finger of bovine herpes virus 1 encoded bICP0 is necessary for transcriptional regulation and infection. J. Gen. Virol. 82: 483-492. Inman M, Lovato L, Doster A, Jones C. 2001. A mutation in the latency related gene of bovine herpesvirus 1 leads to impaired ocular shedding in acutely infected calves. J. Virol. 75: 8507-8515. Jeyaseelan, S, Kannan MS, Briggs RE, Thumbikat P, Maheswaran SK. 2001. Mannheimia haemolytica leukotoxin activates a non-receptor tyrosine kinase-signaling cascade in bovine leukocytes which induces biological activity. Infect. Immun. 69: 6131-6139. Jeyaseelan S, Kannan MS, Hsuan SL, Singh AK, Walseth TF, Maheswaran SK. 2001. Pasterella haemolytica leukotoxin  induced cytolysis of bovine leukocytes: Role of arachidonic acid and its regulation. Microb. Pathog. 30: 59-69. Kalfa VC, Jia HP, Kunkle RA, McCray Jr. PB, Tack BF, Brogden KA. 2001. Congeners of SMAP29 kill ovine pathogens and induce ultrastructural damage in bacterial cells. Antimicrob. Agents Chemother. 45: 3256-3261. Lafleur RL, Malazdrewich C, Jeyaseelan S, Bleifield E, Abrahamsen MS, Maheswaran SK. 2001. Lipopolysaccharide enhances cytotolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella (Mannheimia) haemolytica leukotoxin. Microb. Pathog. 30: 347-357. Leite F, Sylte M, OBrien S, Schultz R, Peek S, Van Reeth K, Czuprynski C. 2001. Effect of experimental infection of cattle with bovine herpesvirus-1 on the ex vivo interaction of bovine leukocytes with Mannheimia haemolytica leukotoxin. Vet. Immunol. Immunopathol. 84: 97-110. Malazdrewich C, Ames TR, Abrahamsen MS, Maheswaran SK. 2001. Pulmonary expression of tumor necrosis factor  alpha, interleukin  beta, and interleukin  8 in the acute phase of bovine pneumonic pasteurellosis. Vet. Pathol. 38: 297-310. Ramirez-Romero R, Brogden KA, Gallup JM, Sonea IM, Ackermann, MR. 2001. Mast cell density and substance P-like immunoreactivity during the initiation and progression of lung lesions in ovine Mannheimia (Pasteurella) haemolytica pneumonia. Microb. Pathogen. 30: 325-335. Walz PH, Bell TH, Wells J, Grooms DL, Kaiser L, Maes RK, Baker JC. 2001. Relationship between level of viremia and disease manifestations in calves experimentally infected with bovine viral diarrhea virus. Am. J. Vet. Res. 62: 1095-1103. Walz PH, Bell TH, Wells J, Grooms DL, Kaiser L, Maes RK, Baker JC. 2001. Platelet aggregation responses and virus isolation from platelets in calves experimentally infected with type I or type II bovine viral diarrhea virus. Can. J. Vet. Res. 65:241-247. Wang P, Hurley DJ, Braun LJ, Chase CCL. 2001. Detection of bovine herpesvirus 1 in peripheral blood mononuclear cells 8 months post infection. J. Vet. Diag. Invest. 13: 424-427. Wittum TE, Groteueschen DM, Brock KV, Kvasnicka WG, Floyd JG, Kelling CL, Odde KG. 2001. Persistent bovine viral diarrhea virus infection in U.S. beef herds. Vet. Prev. Med. 49: 83-94. Zhang Y, Jones C. 2001. The bovine herpes virus 1 immediate early protein (bICP0) is associated with histone deaetylase 1 to activate transcription. J. Virology. 75: 9571-9578. PEER REVIEWED PUBLICATIONS (2002) Akula S, Hurley DJ, Wixon RL, Wang C, Chase CCL. 2002. Effect of genistein on replication of bovine herpesvirus type 1. Am. J. Vet. Res. 63:1124-1128. Ambagala AP, Feng Z, Barletta RG, Srikumaran S. 2002. Molecular cloning, sequencing, and characterization of bovine transporter associated with antigen processing 2 (BoTAP2). Immunogenetics. 54:30-38. Carter JN, Meredith GL, Montelongo M, Gill DR, Krehbiel CR, Payton ME, Confer AW. 2002. Relationship of vitamin E supplementation and antimicrobial treatment with acute-phase protein responses in cattle affected by naturally acquired respiratory tract disease. Am. J. Vet. Res. 63: 1111-1117. Champlin FR, Shryock TR, Patterson CE, Austin FW, Ryals PE. 2002. Prevalence of a novel capsule-associated lipoprotein among Pasteurellaceae pathogenic in animals. Curr. Microbiol. 44: 297-301. Chowdhury SI, Onderci M, Bhattacharjee PS, Al-Mubarak A, Weiss ML, Zhou Y. 2002. Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis. J. Virol. 76: 3839-3851. Deshpande M, Ambagala TC, Ambagala APN, Srikumaran S. 2002. Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella) haemolytica leukotoxin induced cytolysis. Infection and Immunity. 70: 5058 5064. Evermann JF, Ridpath JF. 2002. Clinical and epidemiologic observations of bovine viral diarrhea virus in the northwestern United States. Vet Microbiol. 89(2-3): 129-39. Fales-Williams AJ, Gallup JM, Ramirez-Romero R, Brogden K, Ackermann MR. 2002. Increased anionic peptide distribution and intensity during progression and resolution of bacterial pneumonia. Clin. Diagn. Lab. Immunol. 1:28-32. Fent GM, Fulton RW, Saliki JT, Caseltine SL, Lehmkuhl HD, Confer AW, Purdy CW, Briggs RE, Loan RW, Duff GC. 2002. Bovine adenovirus-7 infections in postweaning calves. Am. J. Vet. Res. 63: 976-978. Flores EF, Risatti GR, et al. 2002. Expression of the mouse Fc receptor B2 in bovine cells rescues the infectivity of conditionally neutralized bovine viral diarrhea virus. Vet. Microbiol. 85: 99-109. Frank GH, Briggs RE, Duff GC, Loan RW, Purdy CW. 2002. Effects of pretransit vaccination and arrival medication with florfenicol on the health of transported calves and the presence of Mannheimia haemolytica organisms in nasal secretions. Am. J. Vet. Res. 63: 251 256. Fulton RW, Cook BJ, Step DL, Confer AW, Saliki JT, Burge LJ, Welsh RD, Blood KS, Payton MD. 2002. Evaluation of animal health status of calves and their impact on feedot performance: Assessment of a retained ownership program of postweaning calves. Can. J. Vet. Res. 66:173-180. Fulton RW, Ridpath JF, Saliki JT, Briggs RE, Confer AW, Burge LT, Purdy CW, Loan RW, Duff GC, Payton ME. 2002. Bovine viral diarrhea virus (BVDV)1b: Predominant subtype in calves with respiratory disease. Can. J. Vet. Res. 66: 181-190. Gatto NT, Dabo SM, Hancock RE, Confer AW. 2002. Characterization of, and immune responses of mice to, the purified OmpA-equivalent outer membrane protein of Pasteurella multocida serotype A:3 (Omp28). Vet Microbiol. 87: 221-235. Geiser G, Inman M, Zhang Y, Jones C. 2002. The latency related (LR) gene of bovine herpes virus 1 (BHV 1) can inhibit the ability of bICP0 to activate productive infection. J. of General Virology. 83: 2965 2971. Gopinath RS, Ambagala APN, Hinkley S, Srikumaran S. 2002. Effects of virion host shut off activity of bovine herpesvirus 1 on MHC class I expression. Viral Immunology. 15: 595 608. Grooms DL, Coe PH. Antibody response following vaccination for respiratory viruses in preconditioned calves. Vet. Therap. 3: 119-127 Grooms DL, Keilan E. 2002. Screening of neonatal calves for bovine viral diarrhea virus by immunohistochemistry on skin samples. Clin. Diagn. Lab. Immun. 9: 898-900. Guey-Chuen P, Maguen B, Jing L, Mott KR, Osorio N, Slanina SM, Yukht A, Ghiasi H, Nesburn AB, Inman M, Henderson G, Jones C, Wechsler SL. 2002. A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 LAT gene and restore wild type reactivation levels. J. Virol. 76: 1224-1235. Harding MJ, Cao X, Shams H, Johnson AF, Vassilev VB, Wheeler DW, Haines D, Sibert GJ, Nelson LD, Campos M, Donis RO. 2002. Role of bovine viral diarrhea virus biotype in the establishment of fetal infections. Am. J. Vet. Res. 63(10): 1455 63. Inman M, Lovato L, Doster A, Jones C. 2002. A mutation in the latency related gene of bovine herpesvirus 1 disrupts the latency reactivation cycle in calves. J. of Virology. 76: 6771 6779. Jeyaseelan, S, Srinand S, Maheswaran SK. 2002. Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis. Animal Health Research Reviews. 3: 69-82. Kelling CL, Steffen DJ, Topliff CL, Eskridge KM, Donis RO, Higuchi DS. 2002. Comparative virulence of bovine viral diarrhea virus type II in experimentally inoculated six to nine month old calves. Am. J. of Vet. Res. 63: 1379 1384. Kelling CL, Steffen DJ, Cooper VL, Higuchi DS, Eskridge KM. 2002. Effect of acute bovine viral diarrhea virus alone, bovine rotavirus alone, or concurrent infection with both on enteric disease in gnotobiotic neonatal calves. Am. J. of Vet. Res. 63: 1179 1186. Leite F, OBrien S, Sylte MJ, Page T, Atapattu D, Czuprynski CJ. 2002. Inflammatory cytokines enhance the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood neutrophils in vitro. Infect. Immun. 70:4336-4343. Maheswaran SK, Thumbikat P, Dileepan T. 2002. Current knowledge on pathogenesis of lung injury caused by Mannheimia haemolytica and Pasteurella multocida in the bovine. In: Recent Developments and Perspectives in Bovine Medicine. p. 160-167. Pillars RA, Grooms DL. 2002. Serological evaluation of five unvaccinated heifers for detecting herds persistently infected with bovine viral diarrhea virus. Am. J. Vet. Res. 63: 499-5. Purdy CW, Straus DC, Chirase N, Parker DB, Ayers JR, Hoover MD. 2002. Effects of aerosolized endotoxin in feedyard dust on weanling goats. Small Rum. Res. 446: 133. Vanden Bush TJ, Rosenbusch RF. 2002. Mycoplasma bovis induces apoptosis of bovine lymphocytes. FEMS Immunol. Med. Microbiol. 32: 97-103. Ward ACS, Weiser GC, Delong WJ, Frank GH. 2002. Characterization of Pasteurella spp isolated from healthy domestic pack goats and evaluation of the effects of a commercial Pasteurella vaccine. Am. J. Vet. Res. 63: 119 123. Winkler MTC, A Doster, J-H Sur, C Jones. 2002. Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1. Vet. Microbiol. 86: 139-155. ABSTRACTS (2002) Blackwood ER, Ayalew S, Confer AW. 2002. Molecular and Immunological analysis of outer membrane protein PlpE from Mannheimia haemolytica serotypes 1, 2, and 6. In 83th Annual Meeting of CRWAD. November 11-12. St. Louis, MO. Abstract nr 93. Brady RP, Topliff CL, Kelling CL. 2002. Analyses of plasmid DNA encoding the attachment glycoprotein of bovine respiratory syncytial virus. In: Conference of Research Workers in Animal Diseases Proceedings. Braun LJ, Braunschmidt M, Arias N, Wixon R, Wang CY, Chase CCL. 2002. Effect of genistein on bovine herpesvirus 1 and bovine viral diarrhea virus infections in vivo. In: 35th annual Conference of American Association of Bovine Practitioners. September 26-28. Rapid City, SD. p. 206. Dunn J, Kaneene JB, Grooms D, Bolin S, Bolin C, Bruning-Fann C. 2002. The effect of infection with Mycobacterium paratuberculosis on the reliability of the caudal fold tuberculin (CFT) test and the gamma-interferon test for bovine tuberculosis in cattle. In: Proceedings of the 83th Annual Meeting of The Conference of Research Workers in Animal Diseases. November 11. St. Louis, MO. Abstract nr 47. Elmowalid G, Ransom BL, Braun LJ, Young A, Ridpath J, Chase CCL. Bovine viral diarrhea virus interferes with macrophage surface marker expression in vitro. In: 83rd annual meeting of Conference of Research Workers in Animal Disease. November 11-12. St. Louis, MO. Abstract nr 185. Fulton RW, Step DL, Ridpath JF, Saliki JT, Confer AW, Johnson BJ, Welsh RD, Burge LJ, Hawley RV, Payton ME. 2002. Bovine Viral Diarrhea Virus (BVDV) Persistently Infected Calves: Response to Vaccinations and Diagnoses at Necropsy. In: 45th Annual Meeting of AAVLD, October 18-22. St. Louis, MO. Fulton RW, Step DL, Ridpath JF, Saliki JT, Confer AW, Johnson BJ, Welsh RD, Burge LJ, Hawley RV, Payton ME. 2002. Bovine Viral Diarrhea Virus (BVDV) Infections of Calves after Exposure to Persistently Infected Calves with BVDV Subtype 1b. In: 45th Annual Meeting of AAVLD. October 18-22. St. Louis, MO. Grooms DL, Brock KV, Norby B. 2002. Performance Of Feedlot Cattle Exposed To Animals Persistently Infected With Bovine Viral Diarrhea Virus. In: Proceedings of the 83th Annual Meeting of The Conference of Research Workers in Animal Diseases. November 11. St. Louis, MO. Abstract nr 186. Hassan, EAD, Braun LJ, Chase CCL. 2002. The effect of bovine herpesvirus 1 (BHV-1) on the organization of actin filaments and microtubules. In: 83rd annual meeting of Conference of Research Workers in Animal Disease. November 11-12. St. Louis, MO. Abstract 116-P. Hunsaker BD, Topliff CL, Achenbach JA, Kelling CL. 2002. Back passage of a commercial modified live vaccine (MLV) strain of type 1 noncytopathic bovine viral diarrhea virus did not result in reversion to virulence. In: Conference of Research Workers in Animal Diseases Proceedings. Lasarsky E, Fulton RW, Confer AW, Saliki JT, Briggs RE, Huchzermeier R. 2002. Bovine Coronavirus Infections in Cattle: Detection in Nasal Swabs by Antigen Capture ELISA and RT-PCR. In: CRWAD. November 10-12. St. Louis, MO. Lu X., Rosenbusch RF. 2002. 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Measurement of cytokine gene expression by RT-competitive PCR in BRSV vaccinated and infected calves. In: Proceedings of the Conference for Research Workers in Animal Disease. St. Louis, MO. Prado ME, Dabo SM, Confer AW. 2002. Cloning and characterization of HasR gene homologue from Pasteurella multocida A:3. In: Proceedings of HAP2002. Banff, Alberta, Canada. Topliff CL, Chon SK, Donis RO, Eskridge KM, Kelling CL. 2002. Translational Efficiencies of genotype 2 bovine viral diarrrhea virus field isolates using a T7 vaccinia virus expression system. In: Conference of Research Workers in Animal Diseases Proceedings. THESIS (2002) Elmowalid G. 2002. Unmasking the effect of bovine viral diarrhea virus on macrophage inflammatory functions. PhD Dissertation. South Dakota State University, 2002. PEER REVIEWED PUBLICATIONS (2003) Boudreaux CM, Corstvet RE. 2003. A novel strategy of controlling shipping fever. 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Identification of a novel transcript containing a small open reading frame that is expressed during latency, and is antisense to the latency related gene of bovine herpes virus 1 (BHV-1). J. of Virology. 5438-5447. Jiang Y, Inman M, Zhang Y, Posadas NA, Jones C. 2004. A mutation in the latency related gene of bovine herpesvirus 1 (BHV-1) inhibits expression of proteins encoded by ORF2 and Reading Frame C during productive infection. J. of Virology. 78: 3184-3189. Kalina WV, Woolums AR, Berghaus RD, Gershwin LJ. 2004. Formalin-inactivated bovine RSV vaccine enhances a Th2 mediated immune response in infected cattle. Vaccine. 22 (11-12): 1465-74. 2004 Leite F, Atapattu D, Kuckleburg C, Schultz R, Czuprynski CJ. 2004. Incubation of bovine PMNs with conditioned medium from BHV-1 infected peripheral blood mononuclear cells increases their susceptibility to Mannheimia haemolytica leukotoxin. Vet. Immunol. Immunopathol. 103: 187-193. Liu D, Lawrence ML, Austin FW. 2004. Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes. J. Microbiol. Methods. 58(2): 263-267. Lu X, Rosenbusch RF. 2004. Endothelial cells from bovine pulmonary microvasculature respond to Mycoplasma bovis preferentially with signals for mononuclear cell transmigration. Molecular Pathogenesis. 37: 253-261. Malazdrewich CP, Thumbikat MS, Abrahamsen MS, Maheswaran SK. 2004. Pharmacological inhibition of Mannheimia haemolytica lipopolysaccharide and leukotocin-induced inflammatory cytokine expression in bovine alveolar macrophages. Microbial Pathogenesis. 36: 159-169. Malazdrewich CP, Thumbikat MS, Maheswaran SK. 2004. Protective effects of dexamathasone in experimental bovine pneumonic pasteurellosis. Microbial Pathogenesis. 36: 227-237. Meyerholz DK, Grubor B, Gallup JM, Lehmkuhl HD, Anderson RD, Lazic T, Ackermann MR. 2004. Adenovirus-mediated gene therapy enhances parainfluenza virus 3 infection in neonatal lambs. J. Clin. 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Woolums AR, Brown C, Brown Jr JC, Cole D, Scott M, Williams S, Miao C. 2004. Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on resistance to subsequent viral challenge in calves. Am. J. Vet. Res. 65: 363-372. ABSTRACTS (2004): Chase CCL, Miskimins DM, Graham T, Braun L, Steen P, Ridpath J. 2004. Evidence of bovine viral diarrhea virus persistent infection in two white-tail deer in southeastern South Dakota. In: Proceedings of the 37th annual conference of American Association of Bovine Practitioners. September 23-25. Fort Worth, TX. p. 169. Confer AW, Fulton RW, Step DL, Johnson BJ, Ridpath JF. 2004. Viral Antigen Distribution in the Respiratory Tract of Cattle Persistently Infected with Bovine Viral Diarrhea Virus Subtype 2a. At: 47th Annual Meeting of AAVLD. October 22-24. Greensboro, NC. Donovan DC, Barton MH, Norton N, Ely LO, Hurley DJ. 2004. Modulation of innate immunity by in vitro treatment of keukocytes with CD14, lactoferrin and IgG. In: Proceedings of the 84th CRWAD meeting. November 14-16. Chicago, IL. p. 39. Donovan DC, Reber AJ, Parks RJ, Collier C, Ely LO, Hurley DJ. 2004. Extracellular pH alters the innate immune response by enhancing phagocytosis and decreases reactive oxygen species production. In: American Dairy Science Annual Meeting. St. Louis, MO. J. Dairy Sci. 87 (Suppl. 1): 179. Donovan DC, Collier C, Reber AJ, Parks RJ, Hurley DJ. 2004. Extracellular pH alters immunity by decreasing the production of reactive oxygen and nitrogen species, but enhancing phagocytosis in bovine neutrophils and monocytes. In: International Conference of Production Diseases in Farm Animals. July 18-22. Michigan State University. Abstract nr A-14. Donovan DC, Reber AJ, Parks RJ, Hurley DJ. 2004. Acidic extracellular pH enhances phagocytosis and decreases reactive oxygen species production in polymorphonuclear neutrophils and monocytes. In: 7th International Veterinary Immunology Symposium. July 25-29. Quebec City, Qc, Canada. Abstract nr WK 13.6.10 p. 415. Fogarty-Fairbanks K, Rinehart CL, Ohnesorge WC, Loughin MM, Chase CCL. 2004. Fetal protection against BVDV type 1 or 2 heterologous challenge. In: Proceedings of the 37th annual conference of American Association of Bovine Practitioners. September 23-25. Fort Worth, TX. p.171. Fogarty-Fairbanks K, Campbell J, Chase CCL. 2004. Subcutaneous dose of infectious bovine rhinotracheitis virus provides early protection against an intranasal challenge. In: Proceedings of the 37th annual conference of American Association of Bovine Practitioners. September 23-25. Fort Worth, TX. p. 289. Fulton RW, Burge LJ, dOffay JM, Funk R, Weaver GD, Van Campen H, Johnson BJ. 2004. Immune Response Differences in Serums from Bovine Herpesvirus-1 Vaccinated Cattle: Depenndence of Viral Strarin. At: 47th Annual Meeting of AAVLD. October 22-24. Greensboro, NC. Fulton RW, Ridpath JF, Ores S, Saliki JT, Burge LJ, Confer AW. 2004. Bovine Viral Diarrhea Virus (BVDV) Subtypes in Diagnostic Laboratory Accessions from Clinical and Necropsy Cases: Distribution of BVDV1a, BVDV1b, and BVDV2a Subtypes. At: 47th Annual Meeting of AAVLD. October 22-24. Greensboro, NC. Gershwin LJ, Corbeil LB, Berghaus LJ, Arnold KF, Anderson ML. 2004. IgE and cytokine profiles in calves concurrently infected with BRSV and Histophilus somni. Presented at 7th International Veterinary Immunology Symposium. July. Quebec City, Canada. Hunsaker BD, Steffen DJ, Topliff CL, Kelling CL. 2004. Characterization of protection in calves from systemic infection or disease by vaccination with modified-live noncytopathic bovine viral diarrhea virus type 1. At: Conference of Research Workers in Animal Disease. Topliff, C.L., S.K.Chon, R.O. Donis, K.M. Eskridge, C.L Kelling. 2004. Translational efficiencies of the 5' untranslated region from eight genotype 2 bovine viral diarrhea virus field isolates varying in virulence. Conference of Research Workers in Animal Disease. Johnson TK, Triche PC, OReilly KL. 2004. Immune response of newborn calves to intrabronchial infection with bovine coronavirus. In: Meeting of the Louisiana Biomedical Research Network. July 28. Baton Rouge, LA. Klink HA, Brady RP, Topliff CL, Eskridge KM, Srikumaran S, Kelling CL. 2004. Influence of bovine respiratory syncytial virus F glycoprotein N-linked glycans on in vitro expression and on antibody responses in BALB/c mice. At: Conference of Research Workers in Animal Disease. Nanduri B, Lawrence ML, Burgess SC. 2004. Determination of sub-MIC effects of antibiotics on Pasteurella multocida protein expression using cleavable isotope coded affinity tag reagents. In: 104th General Meeting of the American Society for Microbiology. New Orleans, LA. Okinaga T, Hurley DJ, Woolums AR, Miao C, Zarlinga DS. 2004. Comparison of bovine cytokine gene experession as measured by competitive and real-time PCR. In: Proceedings of the 84th CRWAD meeting. November 14-16. Chicago, IL. p. 41. Okinaga T, Hines II ME, Hurley DJ. 2004. Real-time PCR measurements of cytokine mRNA from cattle and goats. In: Proceedings of the 84th CRWAD meeting. November 14-16. Chicago, IL. p. 40 Wiggins M, Woolums A, Hurley DJ, Cole D, Sanchez S. 2004. The prevalence of Mycoplasma bovis in a subpopulation of Georgia cattle. In: Proceedings of the 84th CRWAD meeting. November 14-16. Chicago, IL. OReilly KL, Godeny GK, Kousoulas KG, Olcott BM, Paulsen DB, Harden TT, Smith S, Triche PC. 2004. Intrabronchial infection of newborn calves with respiratory bovine coronavirus. In: Animal Disease Research Workers of Southern States. February. Biloxi, MS. Reber AJ, Okinaga T, Tanner M, Woolums AR, Hurley DJ. 2004. Development of immunity after vaccination with MLV or killed BVD vaccines. In: Proceedings of the 84th CRWAD meeting. November 14-16. Chicago, IL. p. 40. Reber AJ, Okinaga T, Tanner M, Woolums A, Leard T, Milward F, Hurley DJ. 2004. Evaluation of multiple immune parameters during development of immunity after vaccination with MLV or killed BVD. In: 7th International Veterinary Immunology Symposium. Program and Book of Abstracts. July 25  30. Quebec City, Canada. Abstract nr WK.1.6.7. p. 150 DISSERTATIONS (2004): Fogarty-Fairbanks K. 2004. New clinical applications for three USDA licensed vaccines in the prevention of infectious bovine rhinotracheitis or bovine viral diarrhea virus. Master of Science Thesis. South Dakota State University. Hassan E. 2004. Investigation of the role of viral glycoproteins gE AND gI in bovine herpesvirus type 1 (BHV-1) pathogenesis: study of cytoskeleton and phosphorylation. PhD Dissertation. South Dakota State University. Kalina WV. 2004. The influence of Alternaria alternate Aerosol on the Development of Immune Responses in Calves During a Primary and Secondary Bovine Respiratory Syncytial Virus Infection. PhD Dissertation. University of California, Davis. PEER REVIEWED PUBLICATIONS (2005) Atapattu D, Czuprynski CJ. 2005. Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9 dependent mitochondrial pathway. Infect. Immun. 73: 5504-5513. Boudreaux CM, Corstvet RE, Cooper RK, Enright FM. 2005. Effects of cecropin B transgene expression on Mannheimia haemolytica serotype 1 colonization of the nasal mucosa of calves. Am. J. Vet. Res. 66(11): 1922-1930. Carter JN, Gill DR, Lalman DL, Krehbiel CR, Confer AW, Smith RA, Claypool PL, McDowell LR. 2005. Vitamin E supplementation in the diet of newly arrived feedlot cattle. J. Anim. Sci. 83: 1924-1932. Confer AW, Fulton RW, Step DL, Johnson BJ, Ridpath JF. 2005. Viral Antigen Distribution in the Respiratory Tract of Cattle Persistently Infected with Bovine Viral Diarrhea Virus Subtype 2a. Vet. Path. 42: 192-199. Dabo SM, Confer AW. 2005. Adherence of Pasteurella multocida to fibronectin. Vet. Microbiol. 110: 265-275. Dileepan T, Thumbikat P, Walcheck B, Kannan MS, Maheswaran SK. 2005. Recombinant expression of bovine LFA-1 and characterization of its role as the receptor for Mannheimia haemolytica leukotoxin. Microbial Pathogenesis. 38: 249-257. Dileepan T, Kannan MS, Walcheck B, Thumbikat P, Maheswaran SK. 2005. Mapping of the binding site for Mannheimia haemolytica leukotoxin within the bovine CD18. Infection and Immunity. 73: 5233-5237. Doyle CK, Labruna MB, Breitschwerdt EB, Tang Y, Corstvet RE, Hergarty BC, Bloch KC, Li P, Walker DH, McBride JW. 2005. Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene. J. Mol. Diagn. 7(4): 1-7. 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