BVDV COMPROMISES FETAL IMMUNE ORGAN DEVELOPMENT LEADING TO POST-NATAL PREDISPOSITION TO SECONDARY INFECTIONS.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1019423
• Grant No.: 2019-67015-29866
• Cumulative Award Amt.: $390,000.00
• Proposal No.: 2018-06840
• Project Start Date: Jul 1, 2019
• Project End Date: Jun 30, 2023
• Grant Year: 2019
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Biomedical Sciences

Non Technical Summary
BVDV outbreaks became a significant problem in the 1990s and in 2008, BVDV was announced as a research priority for USDA. Since then, an estimated 80% of cattle producers, including a majority of feedlot operators, vaccinate their cattle with inactivated or modified live viral (MLV) vaccines to limit losses from BVDV. Despite a US regional as well as a worldwide push to control the spread of BVDV, an estimated 59% of US cow-calf operators and 25% of dairy operators do not routinely vaccinate against BVDV, contributing to the continued prevalence of BVDV and its devastation on the US cattle industry. In addition to the lack of routine control measures and prevention, vaccine efficacy and safety is questionable due to the antigenic variation, mutagenicity and variable virulence of BVDV strains. US cattle producers who do vaccinate, rely and trust those vaccinations, assuming complete protection of their herds; however, the vaccines are not 100% efficacious. As evidence, a study correlating herd vaccination and calf BVDV viral titers found that in herds vaccinating with the inactive vaccine, 29.6% of them had at least one calf persistently infected (PI) with BVDV and the herds using MLV, 20% of them had at least one PI calf.PI animals have been identified as the primary vectors of BVDV, exposing and possibly infecting naïve animals within the PI's vicinity. An estimated 0.2-0.4% of cattle entering a feedlot are BVDV PI, equating to direct exposure of approximately 62% of feedlot animals to BVDV. PI animals themselves are low performing animals, weighing approximately 75 kg less than non-PI calves at weaning. Most importantly PI cattle spread the infection to cattle in proximity causing immunosuppression, secondary infections, resulting in more performance loss and exacerbating the seemingly low prevalence into a larger problem.Cattle TI with BVDV have stunted growth, increased susceptibility to other infections, and increased morbidity relative to uninfected calves. In a 2008 study, calves previously exposed to BVDV were 8.6-15.1 kg lighter at weaning than contemporary non-infected control calves. The insidious aspect of TI calves is that they are not easily distinguished from uninfected calves; however, they are 2.3 times more likely than uninfected calves to have morbid episodes, contributing to 56.5% of a herd's morbidity. From a population viewpoint, TI calves make up a higher proportion of calves in BVDV-infected herds. A study performed on dairy calves revealed that 10% of the calves were TI for BVDV compared to 0.5% PI calves. Therefore, TI animals represent the greatest number of cattle negatively impacted by BVDV fetal infections.Economic losses caused by BRD are associated with the costs of treatment and mortality, but also can occur through decreased performance in the feedlot and diminished carcass characteristics. Causes of BRD are complex, but often can be attributed to BVDV and Mannheimia haemolytica (M. haemolytica) (reviewed in [13]). Most bacteria, M. haemolytica, included, are not able to induce BRD alone without other predisposing factors such as concurrent or prior infection with BVDV. Infections secondary to immunosuppression by BVDV such as BRD contribute another $800-900 million annual loss in revenue. The economic impact of BVDV in beef and dairy industries dictates the need for further research to understand the development and consequences of PI and TI BVDV animals.The proposed experiments directly test the hypothesis that: Infection of fetuses with bovine viral diarrhea virus (BVDV) in utero permanently alters fetal gene expression through epigenetic mechanisms resulting in life-long impaired postnatal immune responses, thus resulting in predisposition of both TI and PI calves to post-natal bovine respiratory disease.Aim 1 identifies fetal genes regulating immune responses that are permanently altered-modified because of fetal infection with BVDV.Aim 2 identifies postnatal gene regulating immune responses that are permanently altered-modified because of fetal infection with BVDV.Aim 3 tests the hypothesis that impaired function of immune response genes contributes to more damaging effect of secondary infection with BRD. It not only examines immune response genes, but also examines feedlot performance and carcass characteristics in steers exposed to BVDV in utero.The research is directly related to understanding processes that support or disturb health (BVDV infection and immune responses) in cattle, including reducing harmful disruptions (epigenetic changes in gene expression) leading to disease. One positive outcome of these studies would be to improve disease prevention through educating producers about the long-term negative ramifications of PI and TI with BVDV. Also, future diagnoses of calves at risk for secondary infections may be based on the discovery of epigenetically modified genes in response to fetal PI and TI with BVDV. Likewise, development of therapeutic interventions may be possible through better understanding how immunotolerance is manifest to fetal PI and how epigenetic alteration of immune genes to fetal PI and TI impacts postnatal immune susceptibility to secondary infections. Finally, the research is directly relevant to USDA NIFA longer-term goals of enhancing sustainability of animal production agricultural systems.The information gained in these studies will aid in the design of improved diagnostic tests, control measures and therapeutic agents. All of which is ultimately directed towards educating stakeholders in 100% vaccination compliance and keeping BVDV infected animals out of production circulation.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1101	80%
304	3310	1090	20%

Knowledge Area
311 - Animal Diseases; 304 - Animal Genome;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1090 - Immunology; 1101 - Virology;

Keywords

bovine

epigenetics

fetus

immunology

pregnancy

virus

Goals / Objectives
Bovine viral diarrhea virus (BVDV), is a single stranded, positive sense RNA virus, in the Pestivirus genus of the Flavivirus family, first identified in 1946. Since then, BVDV has been shown to be the source of significant losses to the cattle industry and is estimated to cause an estimated 376 million to 1.5 billion dollars in annual losses. In healthy, immunocompetent adult animals, most BVDV infections cause subclinical disease with viral clearance in 14 days. However, acute BVDV infections can also cause profound immunosuppression in infected animals resulting in susceptibility to secondary infections such as bovine respiratory disease (BRD), which is associated with reduced feedlot performance and additional industry losses. BRD is the primary infectious disease of feedlot cattle, and costs the feedlot industry millions of dollars each year.If fetal infection occurs prior to gestational day 125, the fetus becomes persistently infected (PI) with BVDV. Postnatally, PI animals shed the virus continually throughout life and serve as the main reservoir of BVDV in cattle populations. The persistence of BVDV and the absence of BVDV-specific antibodies at birth suggest that the PI fetuses are unable to recognize viral antigens as foreign, and consequently do not mount a BVDV-specific immune response, referred to as a state of immunotolerance. In contrast, the immune system of fetuses infected with BVDV after day 160 of gestation is more mature, and therefore, able to mount an effective immune response to BVDV resulting in viral clearance. If these fetuses survive the transient fetal infection (TI), the calves will be born with evidence of BVDV-specific immunity, and may also be immunocompromised for life.BRD is associated with a variety of viruses and bacteria, and influenced by stress such as transportation and inclement weather. One of the viruses associated with BRD is bovine viral diarrhea virus (BVDV). The particular importance of BVDV in BRD is attributed to its ability to reduce the immune system's responses to other viruses and bacteria increasing the likelihood of pneumonia. One of the most common bacteria associated with pneumonia in BRD cases is Mannheimia haemolytica. M. haemolytica is a naturally occurring bacteria in the bovine nose and throat. The immune system of healthy cattle keeps M. haemolytica from causing pneumonia. However, when the immune system is impaired by BVDV, M. haemolytica may cause pneumonia.The purpose of this project is to determine the effect of bovine viral diarrhea virus (BVDV) infections on bovine fetal immune organ development during the last trimester of pregnancy and to determine whether the damage caused by BVDV to the developing fetal immune system increases the susceptibility of postnatal calves to BRD in a feedlot setting. To simulate BRD that occurs in feedlots, steers previously exposed to BVDV in utero, will be infected post-weaning with BVDV followed by an M. haemolytica challenge.Hypothesis and Specific Aims Hypothesis. Infection of fetuses with bovine viral diarrhea virus (BVDV) permanently alters immune gene expression through epigenetic mechanisms resulting in life-long impaired immune responses, thus resulting in predisposition of postnatal calves to bovine respiratory disease (BRD). Specific Aim 1. Test the hypothesis that fetal immune gene expression is altered following BVDV infection due to epigenetic methylation. Fetal thymic and splenic tissue from PI (infected day 75), TI (infected day 175) and control fetuses will be examined on day 190 of gestation using whole genome methylation approaches. Methylation of differentially expressed innate and adaptive immune response genes will be confirmed using targeted bisulfite sequencing analysis and RT-qPCR. Specific Aim 2. Test the hypothesis that epigenetically altered fetal immune gene expression continues to be impaired in post-natal PI and TI steers. Postnatal control and PI steer blood and control and TI postnatal steer spleen and liver will be analyzed using whole genome DNA methylation analysis, targeted bisulfite sequencing analysis and RT-qPCR. Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections such as bovine respiratory disease complex. Pregnant heifers will be inoculated with PBS or BVDV on day 175 of gestation to generate control or TI steer calves, respectively. After weaning, these calves will be challenged with BRD pathogen, M. Haemolytica. Consequences of the infections will be assessed by clinical assessment, immune cell gene expression by RT-qPCR and antibody approaches, clearance of the BRD infection, lung scores, feedlot growth performance, and complete carcass characteristics at slaughter.

Project Methods
Specific Aim 1. Test the hypothesis that fetal gene expression is negatively altered, through epigenetic methylation, following PI and TI in utero.Approach. Upregulated innate response genes in spleen and downregulated adaptive immune response genes in thymus will be discovered using whole genome methylation approaches and confirmed using targeted bisulfite sequencing analysis and directed RT-qPCR. We will use the custom Methyl-MiniSeq® (expanded Reduced Representation Bisulfite Sequencing, or RRBS) platform offered by Zymo Research Corporation which couples well-established bisulfite conversion technologies with the power of next-generation sequencing (NGS) and a fully developed bioinformatics analysis pipeline to provide the most comprehensive, unique, and genome-wide DNA methylation profiling analysis service available.This service includes all steps from library construction of genomic DNA to bioinformatics and statistical analysis of NGS data. The approach is unique in that it employs two restriction enzymes (RRBS protocols typically digest with only MspI) to target CpG motifs and extend coverage to include ~80% of all CpG islands and gene promoters. Gene bodies and other regulatory elements are also well represented in the sequencing libraries. Fragments enriched for CpG sites are then bisulfite-converted and prepared for NGS.Bioinformatics analysis will include read alignment, methylation calling, pairwise comparisons, browser tracks (read track & methylation track), histograms, box plots, scatterplots, and clustering heat maps. Methylated genes of critical immune function (see Fig. 11) will be followed up using a targeted bisulfite sequencing approach. Primers will be designed to target post-bisulfite treated DNA, and then simultaneously amplified across dozens of samples using a microfluidics device. The PCR products will be sequenced and then bioinformatically analyzed to confirm DNA methylation changes. It is proposed to examine global methylation of genome in day 190 control, TI and PI fetal spleen and thymus DNA.Specific Aim 2. Test the hypothesis that post-natal PI blood and TI blood immune response gene expression is epigenetically impaired.Approach. It was reasoned that whole genome methylation approaches will provide an inclusive view of each gene that is impacted epigenetically by fetal infection with BVDV. Peripheral blood mononuclear cells (PBMC) from age-matched PI and control feedlot steers will be processed to yield DNA that will be analyzed using whole genome DNA Methylation analysis. Postnatal control and TI PBMC DNA will be examined from Aim 3. The negative impact of fetal TI and PI on postnatal immune gene expression will be further examined using RT-qPCR.Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections such as bovine respiratory disease complex. Note that animal experiments will be completed in this aim, but most of the supporting analysis will not be completed because of budget reductions.Approach. Thirty BVDV-naive yearling beef heifers will be synchronized for estrus and artificially inseminated with sexed (Y- chromosome bearing) semen. Pregnancy will be diagnosed by ultrasound on day 32 of pregnancy. On day 175 of pregnancy, 12 heifers will be inoculated with a noncytopathic type 2 BVDV (4.4 log10 TCID50 in 2 ml of culture media) by intranasal route to produced transiently infected (TI) fetuses, and 12 heifers will receive 2 ml of culture media by intranasal route to produced control fetuses. Blood samples will be collected on days 175, 182, 190, 197 and 203 of pregnancy. The heifers will calve and delivery will be facilitated if necessary. The calves will have their naval cord dipped in 0.3% iodine solution, weighed and a nasal swab and blood sample collected prior to nursing. Blood samples and nasal swabs will collected 24 hours later and the serum assayed for adequate passive transfer of colostral antibodies. Blood will be collected at 1 week of age and 4 months of age. At 4 months of age, calves will be weighed and vaccinated with Clostridial toxoids. Calves will be weaned at 7 months, and vaccinated 2 weeks later with a live attenuated BRSV vaccine. Blood samples will be collected on day 0 (day of vaccination) and 3, 5, 7 & 14 days later.Two weeks after BRSV vaccination, 6 control and 6 BVDV TI calves (Challenge group) will be inoculated with a noncytopathic type 1b BVDV (5.0 log10 TCID50 in 4 ml of media) by intranasal route, and 6 control and 6 BVDV TI calves will receive 4 ml of media (no challenge group: controls) by intranasal route. Blood samples will be collected on the day of inoculation and 3 days later. On day 3 following type 1b BVDV inoculation, the challenged calves will receive 6 x 10^9 CFU of Mannheimia haemolytica in 10 ml of media by intratracheal instillation. Blood samples will be obtained on days 0, 1, 3, 5, 7, 14 and 28 days following M. haemolytica inoculation in the challenge calves and in control calves. Calves will be fed until they reach 1200 to 1350 lbs and then sent to slaughter to collect carcass data.Methylation studies will help validate or clarify which hypothesized genes function in innate and adaptive immune responses. Collectively, these approaches will provide a very strong and novel analysis of the mechanisms of impaired immune response to secondary infection in postnatal TI calves.Rate of gain in the feedlot is expected to be negatively impacted in TI calves challenged with BRD. Likewise, we suspect that carcass characteristics such as yield grade and quality grade will be inferior in the TI-BRD challenged, compared to the control calves. If the clinical symptoms are mild following BRD challenge, then we may consider a secondary BRD challenge with BHV1 followed by M. haemolytica challenge as mentioned previously. However, based on work by other groups, we expect a mild response following in controls following BRD challenge and a more pronounced fever, clinical score and presentation of bacterial infection and impaired immune response following BRD challenge in the TI calves.

Progress 07/01/19 to 06/30/23

Outputs
Target Audience:The target audiences were large animal veterinary medicine practitioners and academics, animal agriculture vaccine companies, outreach educators, beef and dairy producers and managers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Six undergraduate students were provided opportunities for professional development in context of learning how to manage, sample blood and weigh postnatal calves. Three graduate students assisted with these tasks, but also learned how to collect, store and analyze tissues at harvest such as spleen, thyroid, liver, lung, bone marrow, etc. One postodoctoral fellow also assisted with tissue collections. There were several abstracts-conference proceedings presented at national and regional-local venues. Opportunities for training in bioiformatics, as well as R statistics, Ingenuity Pathway Analysis , Kegg, GO and other software were provided. How have the results been disseminated to communities of interest?Results have been disseminated in the form of journal papers and abstract-conference presentations. What do you plan to do during the next reporting period to accomplish the goals?Final Report

Impacts
What was accomplished under these goals? Aim 1. As mentioned previously, because of budget reductions, this aim was deleted in the revised budget justification when accepting the award. However, we were able to demonstrate differential DNA methylation in PI compared to control fetal spleens in "Georges, et al., Viruses 2022, 14, 506. https://doi.org/10.3390/ v14030506". These findings support our hypothesis that fetal BVDV infection causes epigenetic changes and altered gene expression via differential DNA methylation. Aim 2. Peripheral blood mononuclear cell (PBMC) DNA from 12 control, 11 PI and 11 TI postnatal 4-month-old heifers was assayed for whole genome DNA methyl seq using bisulfite sequencing (global methylation). TI calves had massive epigenetic changes compared to controls: A total of 3745 CpG sites were differentially methylated with 2010 hypermethylated (≥ 25.0%) and 1735 hypomethylated (< 25.0%) regions in TI compared to control PBMC. Pathway analysis using IPA and GO-KEGG predicted life-long damage to the nervous, hepatic, metabolic, cardiovascular, pulmonary endocrine and immune systems. Tissues including thymus, spleen, bone marrow and liver were also collected from these heifers at slaughter. DNA methyl sequencing will be performed on these tissues in future studies. Aim 3. Pregnant heifers inoculated with BVDV2 had one fetal death (mummification), one weak calf, 12 control and 11 TI heifer calves. Serology was performed on day 0 serum to confirm BVDV fetal infections. All TI calves were seropositive for BVDV antibodies, and all control calves were seronegative for BVDV. IgG quantitation by RID on serum obtained 24 to 60 hours after birth confirmed colostrum transfer in all calves (>2,000 mg/dl). The percentage of T cells, T helper cells, CTLS, double positive, double negative and γδ T cells, B cells, granulocytes or monocytes were not found between TI calves and controls in PBMCs by flow cytometry did not differ. DNA methyl seq analysis of PBMC DNA demonstrated 2861 differentially methylated sites (DMS) (p≤0.05) comprised of 1739 hypomethylated and 1122 hypermethylated sites, and 217 differentially methylated regions (DMR) were found in TI calves compared to controls. Five differentially expressed genes (DEG) were found in TI PBMC RNA seq data at birth compared to controls including decreased expression of 4 IFN-related genes, and increased expression of a gene involved in FCGR activation. At 8 months of age, half of the control (n=6) and TI calves (n=5) were inoculated intranasally with a type 1b noncytopathic BVDV followed 3 days later by intratracheal inoculation with Mannheimia haemolytica. Clinical signs of respiratory disease, rectal temperatures, pulse, and respiration rates were recorded. Control (n=5) and TI calves (n=6) were sham inoculated with PBS; control (n=7) and TI calves (n=5) were inoculated with BVDV/M. haemolytica. There were no significant differences in the weights of calves in each group at the beginning of the BRD challenge and at 28 days after the start of the BRD challenge. There were no significant differences in clinical scores between TI and Control BRD challenged calves. Increased rectal temperatures and respiratory rates and effort were observed in both TI and control BRD challenged calves compared to the TI and control sham inoculated calves. During the feedlot portion of the experiment, the mean weight of TI calves was 45 kg less and the mean rate of gain 0.17 kg less per day than control calves (Table 1). A conservative estimate of the cost of the 50 kg reduction in weight at $2.00/kg live weight for heifers equals $100.00 per head. A reduction in ADG of 0.17 kg/day translates into 30 more days in the feedlot and, at $2.00/day, $60.00 added expense per TI calf. The appearance between TI and control calves born on the same day is illustrated in Fig. 1. The TI heifer was shorter, fine-boned and had a rough, brown-tinged hair coat compared to the larger, slick-coated control heifer. At harvest, the TI heifer weighed 125 kg less than that of the control heifer which at current market price of $1.77/lb ($3.89/kg) for live weight represents a difference in value of $486.75. Fig. 1. Representative TI (left) and control (right) heifer calf (right) born on the same day. Total tract dry matter digestibility (DMD) estimate: Titanium dioxide (TiO2) was added to the diet of 6, age-paired, individually fed, heifers (3 control and 3 TI heifer; approximately 1 year of age) as described [52, 53]. Heifers were limit fed at a rate of 2% of BW for 15 days. Rectal fecal collection began on day 16 and was conducted twice daily for 6 consecutive days with collections occurring 12 h apart. For every 24 h period, the time of collection was advanced 2 hours to minimize effects of diurnal variation. The fecal samples were then analyzed for TiO2 and used to estimate DMD. Control heifers had a 2.2% greater DMD when compared to TI heifers (74.9% vs. 72.7%±0.65 DMD). These data suggest that total tract digestibility was impaired in TI animals either due to morphophysiological changes in the digestive tract that interfere with nutrient absorption and/or due to an altered microbiome in the rumen. In total, genes affecting growth, nutrient metabolism and feed efficiency may also be affected by the fetal TI BVDV infection. At one year of age, jugular blood samples were obtained from all heifers. All samples were analyzed for biomarkers of inflammation (Table 2). Ceruloplasmin (p<0.03), an acute phase protein, and the oxidized form of glutathione (GSSG; p<0.01) were greater and the reduced form of glutathione (GSH) lesser (p<0.02) in TI compared to control heifers. These data suggest that TI cattle may be experiencing chronic inflammation affecting the production of ceruloplasmin and oxidative stress in the liver and suggests a role for inflammation in the decreased performance of TI cattle. The increased GSSG and decrease in GSH:GSSG ratio suggests that the thiol redox status in hepatocytes may be altered in turn activating oxidant response transcriptional elements such as NF-?β and activator protein most likely due to a rise in pro-oxidants such as H2O2. At harvest, TI calves that were BRD challenged were significantly lighter in weight (584 kg) compared to control calves that were BRD challenged (650 kg) (p<0.05) and hot carcass weights were lower in TI calves (mean = 338.2 kg) compared to control calves (mean = 376.1kg) (p<0.0065). Differences were not found between TI and control carcasses for ribeye area, preliminary yield grade, fat thickness, kidney pelvic heart fat, final yield grade or marbling score. All TI and control carcasses were scored as Choice (USDA Quality Score). Liver abscesses were the most common lesion observed at harvest followed by mild adhesions between lung and pleural surfaces. There were no differences in the number of calves and number of abscesses between groups. The percentage of TI calves with liver abscesses (41.7%) compared to controls (41.7%) is within the range of liver abscesses for feedlot cattle in the U.S. One TI calf that was challenged with the BRD agents (BVDV/M.haemolytica) had the most severe lesions at harvest. This calf had very large abscesses in the lung and liver extending into adjacent tissues and pericarditis, which suggests a systemic bacterial infection had occurred, whereas two TI age-matched sham-challenged heifer calves did not have any lesions. An effect of TI was not found in the BRD challenge study. In the future, the effect of TI on BRD outcomes could be investigated in naturally occurring outbreaks of BRD in feedlots with larger numbers of cattle. This type of investigation would depend on having a test to identify TI postnatal calve. Future studies will be focused on Identifying potential blood biomarkers of fetal TI with BVDV.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Van Campen H, Burke B, Eder JM, McDonald EM, Deines DM, Wright B, Bishop JV, Henao-Tamayo M, Brink Z, Engle TE, Gonzalez-Berrios CL, Georges HM, Hansen TR. Intrauterine growth restriction, epigenomic and transcriptomic changes following late-term bovine viral diarrhea virus fetal infections. Viruses 2023; To be submitted, October 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Kincade JN, Vancampen H, Engle TE, Bishop JV, Georges HM, Gonzalez-Berrios CL, Hansen TR. Global epigenomic changes observed postnatally elucidate lifelong defects following fetal BVDV infection. In: Conference of Research Workers in Animal Disease, vol. Session 41; 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Engle TE, Van Campen H, Kincade JN, Hansen TR. Epigenetic changes in transient fetal BVDV infection impacts postnatal growth, immune system, inflammation and feed effiiciencey. In: Annual USDA-NIFA Project Directors Meeting, vol. 104th Conference. Chicago, IL: Conference for Workers in Animal Disease; 2023.
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Van Campen H, Hansen TR, Guimaraes O, Loh HY, Eder JM, Engle TE. Effect of late term maternal infection with bovine viral diarrhea virus on feedlot calf performance and carcass characteristics. J. Anim. Sci. 2023; (to be submitted in October, 2023)
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Kincade JN, Bishop JV, Eder JM, Deines DM, Van Campen H, Hansen TR. Persistence of epigenomic alterations into the postnatal period in calves persistently infected with bovine viral diarrhea virus. Viruses 2023; To be submitted by December, 2023 to a Special Pestivirus Issue in Viruses.

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:The target audiences are veterinary professionals, veterinary technicians, large animal research faculty and students, beef and dairy producers, large animal extension and outreach professionals and animal pharmaceutical companies interested in bovine health and disease. Changes/Problems: We planned to assess and compared B cell functions in control and TI calves following vaccination with an intranasal modified live BRSV vaccine (BRSV Vac). This vaccine was no longer available and an intranasal modified live IBR (BHV1), BRSV and PI3 vaccine (Inforce 3, Zoetis) was substituted. Calves were vaccinated with this product at 7.5 months of age, however, BRSV serum neutralization assays performed on serum samples collected on the day of vaccination indicated that the calves had already been infected with BRSV. We speculate that BRSV circulating in other cattle on the same premises was transmitted to the calves. One control heifer calf was accidentally vaccinated with a modified live viral vaccine (Bovishield Gold 5, Zoetis) containing BVDV, IBR, BRSV and PI3 prior to BRD challenge. Her samples are excluded from the BRD challenge analysis. What opportunities for training and professional development has the project provided? Several undergraduate and two veterinary medicine students assisted with calving these heifers. Students rotated on 3 h shifts to watch for calving. All calves were weighed, and blood samples were collected prior to nursing, 1 to 2 and 7 days of age. Several undergraduate and graduate students in Animal Sciences and Biomedical Sciences participated in weighing, sampling, feeding, observing calves for health or clinical signs of respiratory disease, taking rectal temperatures, pulse, and respiratory rates, collecting blood, nasal swabs, and fecal samples from the calves from 4 months of age through the day of harvest at 16 months of age. Meat Science students have trained in harvest techniques when these calves go to slaughter. Animal Sciences graduate students and a Biomedical Science graduate student assisted with collection, weighing and photography of tissues after harvest. How have the results been disseminated to communities of interest?The results have been disseminated to target audiences in a peer-reviewed publication and abstracts at local and national meetings. What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1. Test the hypothesis that fetal immune gene expression is altered following BVDV infection due to epigenetic methylation. DNA methyl seq data obtained from PBMCs for 4-month-old control, PI and TI heifer calves has demonstrated differential DNA methylation in immune response pathways in PI and TIs compared to controls. This data is the subject of a manuscript in progress. Specific Aim 2. Test the hypothesis that epigenetically altered fetal immune gene expression continues to be impaired in post-natal PI and TI steers. DNA methyl seq data from PBMCs for 4-month-old control and TI heifer calves has already been analyzed (see specific aim 1). We plan to complete control and TI tissue analysis using whole genome DNA methylation analysis, targeted bisulfite sequencing analysis from the same animals at harvest in 2023 pending new funding. DNA methyl seq data will be analyzed and compared to the data from the same animals obtained at 4 months of age. Manuscripts analyzing flow cytometry and RNA seq data for control and TI calves at birth, and flow cytometry, DNA methyl seq and RNA seq data on post-natal (4-month-old) control, PI and TI calves are currently in progress and scheduled for submission during the next reporting period. Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections such as bovine respiratory disease complex. Half of the TI and control calves were challenged with BRD pathogens, type 1b ncp BVDV and M. Haemolytica or PBS-inoculated (BRD controls). Clinical scores, rectal temperatures, pulse, and respiration rates were recorded. The results will be compared with lung scores to evaluate clearance of the BRD infection. Complete carcass characteristics at slaughter will be completed in 2023. In addition, CBCs, PBMC DNA methyl seq and immune cell gene expression by RT-qPCR and serology will be compared between calves in the 4 treatment groups: 1) control/BRD control, 2) control/BRD challenged, 3) TI/BRD control and TI/BRD challenged, to assess the susceptibility of TI calves to BRD. In addition to documenting lung and liver lesions, samples will be collected for histopathology, DNA methyl seq and RNA seq. Processing, data collection and analysis are planned for the next reporting period. In collaboration with others for multiplexed flow cytometry, RNA-Seq and single-cell RNA-Seq will be analyzed over all time points from birth (day0) through harvest pending new funding. Feedlot performance and carcass characteristics will continue to be collected and analyzed.

Impacts
What was accomplished under these goals? Specific Aim 1. Test the hypothesis that fetal immune gene expression is altered following BVDV infection due to epigenetic modification. Aim 1 Accomplishments: As mentioned in our 2019 report, because of budget reductions from our original proposed experiments, this aim was deleted in our revised budget justification when accepting the award. However, we were able to demonstrate differential DNA methylation in PI compared to control fetal spleens in the publication entitled, "Georges, H.M., Van Campen, H., Bielefeldt-Ohmann, H. and Hansen, T.R. Epigenomic and proteomic changes in fetal spleens persistently infected with bovine viral diarrhea virus: repercussions for the developing immune system, bone, brain, and heart. Viruses 2022, 14, 506. https://doi.org/10.3390/ v14030506." These findings support our hypothesis that fetal BVDV infection causes epigenetic changes and altered gene expression via differential DNA methylation. Specific Aim 2. Test the hypothesis that epigenetically altered fetal immune gene expression continues to be impaired in post-natal PI and TI steers. Postnatal control, TI and PI heifer blood and control and TI heifer postnatal spleen and liver will be analyzed using whole genome DNA methylation analysis, targeted bisulfite sequencing analysis and RT-qPCR. Aim 2 Accomplishments: Peripheral blood mononuclear cell (PBMC) DNA from 12 control, 11 PI and 11 TI postnatal 4-month-old heifers was assayed for whole genome DNA methyl seq using bisulfite sequencing (global methylation). Differential DNA methyl seq results were found in PBMC DNA between control, PI, and TI calves at 4 months of age. Four-month-old TI calves have massive epigenetic changes compared to controls: A total of 3745 CpG sites were differentially methylated with 2010 hypermethylated (≥ 25.0%) and 1735 hypomethylated (< 25.0%) regions in TI compared to control PBMC. Pathway analysis using IPA and GO-KEGG predicts life-long damage to the nervous, hepatic, metabolic, cardiovascular, pulmonary endocrine and immune systems. The results have been presented as posters by Jessica Kincaide et al. I 2022 (see above Abstracts). Tissues including thymus, spleen, bone marrow and liver were collected from control (n=6) and TI heifers (n=3) at slaughter in August 2022. Tissues from the remaining control (n=4) and TI heifers (n=6) will be harvested in November 2022. DNA methyl sequencing will be performed on these tissues, differential methylation and pathway analysis will compared between control and TI heifer calves. Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections such as bovine respiratory disease complex. Aim 3 Accomplishments: Pregnancies were achieved using sexed X-sperm and artificial insemination. Heifers inoculated with BVDV2 had one fetal death (mumification), one weak calf, 12 control and 11 TI heifer calves and one bull calf. Serology was done at day 0 to confirm BVDV fetal infections. All TI calves were seropositive for BVDV antibodies, and all control calves were seronegative for BVDV. IgG quantitation by RID on serum obtained 24 to 60 hours after birth confirmed colostrum transfer in all 24 calves (>2,000 mg/dl). At 8 months of age, half of the control (n=6) and TI calves (n=5) were inoculated with a type 1b noncytopathic BVDV followed 3 days later by intratracheal inoculation with Mannheimia haemolytica. Clinical signs of respiratory disease, rectal temperatures, pulse, and respiration rates were recorded. These data will be analyzed and compared with lesions found at harvest which will be completed in 2023. To date, 1 TI calf that was challenged with the BRD agents (BVDV/M.haemolytica) has been examined at harvest. This calf had large abscesses in the lung and liver extending into adjacent tissues and pericarditis, evidence for an increased susceptibility to bacterial infections, whereas the 2 TI age-matched BRD-control heifer calves did not have lesions at harvest. Flow cytometry analysis of PBMCs indicated an increase in T helper cells (CD4+ CD8-) (p ≤ 0.01) and a decrease in double negative (DN) T cells (CD4-CD8-) (p ≤ 0.02) in TI calves at birth compared to controls; CD3+ T cells were decreased in TI calves compared to controls in 4-month-old TI calves. A significant decrease in DN T cells could have several significant outcomes: peripheral T cells with a DN phenotype have been shown to be involved in immune regulation and tolerance as well as in host defense and inflammation. This suggests that fetal BVDV infection impacts postnatal T cell development. Notably, a decrease in DN inflammatory T cells could severely affect protection against intracellular pathogens, negatively impacting calf development. Ceruloplasmin (p<0.03), an acute phase protein, and the oxidized form of glutathione (GSSG; p<0.01) were greater and the reduced form of glutathione (GSH) was lesser (p<0.02) in TI compared to control heifers. Blood cytokines IFN-γ TNF-α, IL-6, and the acute phase protein haptoglobin were similar between control and TI heifers. These data suggest that TI calves may be experiencing chronic inflammation affecting production of ceruloplasmin and oxidative stress in the liver. The elevated levels of ceruloplasmin and GSSG with a decrease in GSH in the blood of TIs suggests a role for inflammation in the decreased performance of TI cattle (see below). Additional accomplishments Day 0 heifer calf weights were lower (P<0.05) in TI compared to controls. TI calves' weights continued to be less than control calves at 4, 5.5 and 7 months of age (p<0.05). During the feedlot portion of the experiment to date, TI calves weighed 45 kg less and gained 0.10 kg less per day than control calves. A conservative estimate of the cost of the 45 kg reduction in weight at $2.00/ kg live weight for heifers equals $90 per head. A reduction in ADG of 0.10 kg/day translates into 30 days of additional time in the feedlot and, at $2.00/day, $60.00 added expense per TI calf. The differences in weight and rate of gain between control and TI heifer calves continue to widen with time. Currently, TI heifer calves weigh 60.95 kg less (p=0.006) and have an ADG of 0.1288 (p=0.005) less than control heifer calves during the feedlot portion of the project. During the feedlot study, the inflammatory marker, ceruloplasmin, was elevated in TI calves' plasma compared to controls suggesting that the reduced feedlot performance in TI calves might be due to chronic inflammation. To pursue this hypothesis we plan to assay markers of inflammation in serum and liver collected at harvest from control and TI calves. Previous DNA methyl seq data analysis (Georges et al., 2022) and from the 4-month-old calves' PBMCs (Kincaide et al., 2022) indicated a potential negative effect of fetal BVDV infection on cardiac pathways. To examine possible effects of TI on cardiac function, pulmonary arterial pressures were measured on 10 control and 8 TI calves at 16 months of age. TI calves had higher diastolic pressure in the right atrium (p<0.05) and lower systolic pressure in the right ventricle (p=0.05) compared to control calves. These findings support a role for fetal BVDV infection in predisposing TI calves to congestive right heart failure, high-altitude disease (HAD) or "brisket disease". HAD affects 3 to 5% of cattle raised high elevations in the U.S. or approximately 75,000 head. Economic losses due to HAD including mortality in the feedlot and carcass condemnation are significant. In the next reporting period, we plan to continue measuring PAP on the remaining calves, measure heart weights, collect heart samples for histopathology, DNA methyl seq, RNA seq and proteomics at harvest.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kincaide, J.N., Van Campen, H., Engle, T.E., Bishop, J.V., Georges, H.M., Gonzalez-Berrios, C., Eder, J., McDonald, E., Bally, A., and T.R. Hansen. 2022. Maternal BVDV infections and fetal phenotypes. Annual CVMBS Research Day, Ft. Collins, CO.1st place advanced stage Basic Research (platform).
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kincaide, J.N., Van Campen, H., Engle, T.E., Bishop, J.V., Georges, H.M., Gonzalez-Berrios, C., Eder, J., McDonald, E., Bally, A., and T.R. Hansen. 2022. Fetal BVDV infections and postnatal outcomes. Annual meeting of the Rocky Mountain Reproductive Society, Ft. Collins, CO. (poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kincaide, J.N., Van Campen, H., Engle, T.E., Bishop, J.V., Georges, H.M., Gonzalez-Berrios, C., Eder, J., McDonald, E., Bally, A., and T.R. Hansen. 2022. Fetal BVDV Infections and unique fetal phenotypes. Annual meeting for the Society for Reproductive Investigation, Denver, CO. (poster).
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Georges HM, Van Campen H, Hansen TR. BVDV fetal immunotolerance; a unique story of epigenetics and osteoimmunology. In: Rocky Mountain Reproductive Sciences Symposium, vol. 14th Annual Meeting. Fort Collins, CO; 2021: 10; Platform talk. Invited by the American Association of Veterinary Immunologists as a featured talk at the 2022 Conference for Research Workers in Animal Disease meeting, Chicago, IL
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Epigenetic changes in transient fetal BVDV infection impacts postnatal growth, immune system, inflammation and feed efficiency. Engle, TE, Van Campen, H, Kincade, JN and Hansen TR. USDA-NIFA Annual PI Meeting, Conference for Researcher Workers in Animal Disease, Chicago, IL
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Georges, H.M., Van Campen, H., Bielefeldt-Ohmann, H. and Hansen, T.R. Epigenomic and proteomic changes in fetal spleens persistently infected with bovine viral diarrhea virus: repercussions for the developing immune system, bone, brain, and heart. Viruses 2022, 14, 506. https://doi.org/10.3390/ v14030506

Progress 07/01/20 to 06/30/21

Outputs
Target Audience:Target audience is veterinary professionals, veterinary technicians, large animal research faculty and students, beef and dairy producers, and animal pharma companies interested in bovine health and disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several undergraduate and two veterinary medicine students assisted with calving these heifers last Spring. Students rotated on 3 h shifts to watch for calving and to assist with collecting data. All calves were weighed and blood samples were collected prior to nursing. Students will continue to assist with sample collections during this Fall Semester. How have the results been disseminated to communities of interest?We presented two abstracts at scientific meetings. It is still quite early in context of collecting and analyzing samples, particularly for Aim 3. What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 2. Test the hypothesis that epigenetically altered fetal immune gene expression continues to be impaired in post-natal PI and TI steers. We plan to complete preliminary 4 month old postnatal control, TI and PI tissue analysis using whole genome DNA methylation analysis, targeted bisulfite sequencing analysis and RT-qPCR. Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections, such as bovine respiratory disease complex. After weaning, these calves will be challenged with BRD pathogens, type 1b ncp BVDV and M. Haemolytica. Consequences of the infections will be assessed by clinical assessment, blood immune cell gene expression by RT-qPCR and antibody approaches, clearance of the BRD infection, lung scores, feedlot growth performance, and complete carcass characteristics at slaughter. More specifically, blood samples will be obtained at day 0, 7 and 4 months of age for clinical pathology, and in collaboration with others for multiplexed flow cytometry, RNA-Seq and single-cell RNA-Seq. Blood will also be collected from weaned calves after vaccination with BRSV at day 0, 3, 5, 7 and 14. Two weeks after BRSV, calves will be challenged with type 1b BVDV and blood will be collected on 0 and 3 days. This will be followed by a challenge with m. Haemolytica with blood collected on days 0, 1, 3, 5, 7, 14 and 28. In collaboration with others, we will profile immune cell responses using multiplexed flow cytometry and single-cell RNA-Seq. Feedlot performance and carcass characteristics will be collected and analyzed.

Impacts
What was accomplished under these goals? Specific Aim 1. Test the hypothesis that fetal immune gene expression is altered following BVDV infection due to epigenetic methylation. Aim 1 Accomplishments. As mentioned in our 2019 report, because of budget reductions from our original proposed experiments, this aim was deleted in our revised budget justification when accepting the award. We are however focused in Aim 2 on postnatal gene expression and methylation studies and of course the postnatal challenge with BRD studies in Aim 3. Specific Aim 2. Test the hypothesis that epigenetically altered fetal immune gene expression continues to be impaired in post-natal PI and TI steers. Postnatal control, TI and PI heifer blood and control and TI heifer postnatal spleen and liver will be analyzed using whole genome DNA methylation analysis, targeted bisulfite sequencing analysis and RT-qPCR. Aim 2 Accomplishments. We have now collected peripheral blood mononuclear cell (PBMC) DNA from 12 control, 11 PI and 11 TI postnatal 4 month old heifers. PBMCl DNA will be examined for modification of genes using bisulfite sequencing (global methylation) approaches: TI Tissues (i.e., spleen and liver) for methylation studies will be collected from heifers at slaughter in year 3 of the proposal. Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections such as bovine respiratory disease complex. Aim 3 Accomplishments. Pregnancies were achieved using sexed X-sperm and artificial insemination. Heifers inoculated with BVDV2 had one fetal death (mumification), one weak calf, 10 heifer calves and one bull calf. Serology was done at day 0 to confirm BVDV fetal infections. All TI calves were seropositive for BVDV. All control calves were seronegative for BVDV. IgG quantitation RID d 1-2 confirmed colostrum transfer in all calves (>2,000 mg/dl). Day 0 calf weights were lower (P<0.05) in TI compared to controls. Calf weights at 4 months of age tended (P=0.11) to be lower in TI compared to controls.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Hansen TR, Van Campen H, Engle TE, Bishop JV, Brink Z, Georges HM, Gonzalez-Berrios CL. BVDV compromises fetal immune organ development leading to post-natal predisposition to secondary infections. 2020 USDA NIFA Awardee Administrative Meeting, Special Session in the Conference for Workers in Animal Diseases Virtual Conferences 2020; Platform Talk.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Georges HM, Van Campen H, Hansen TR. BVDV Infection Epigenetically Alters T-Cell Transcription Factors In Persistently Infected Fetal Spleens. Society for the Study of Reproduction Virtual Abstracts 2020; 53rd Annual Meeting, July 8-12, 2020:Page 81 and 82. Abstract # 2183, USDA NIFA Merit Award.

Progress 07/01/19 to 06/30/20

Outputs
Target Audience:Target audience is veterinary professionals, veterinary technicians, large animal research faculty and students, beef and dairy producers, and animal pharma companies interested in bovine health and disease. Changes/Problems:Our original budget for this project was reduced from $500,000 to $390,000. We essentially eliminated aim 1 and reduced aim 2 in scope as well as Aim 3 (see budget justification for final award). Regardless, the orignal intent of the proposal is maintained in context of study of impact of transient fetal BVDV infection during pregnancy on postnatal response to secondary infections. A primary interest is discovery of immune genes that may be impaired because of epigenetic modification. What opportunities for training and professional development has the project provided?Two PhD students have been helping and participating with estrous synchronization, AI, pregnancy and sex of fetus ultrasound exams. They also will assist with infecting pregnant heifers, on day 175 of pregnancy to generate TI fetuses, assistant with calving and with sampling not only at birth but also at weaning and following secondary bovine respiratory disease challenge. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1. Test the hypothesis that fetal immune gene expression is altered following BVDV infection due to epigenetic methylation. This aim, centered on fetal gene methylation studies was deleted in the revised budget justification for this grant with focus placed on methylation studies in postnatal tissues from heifers at slaughter. Specific Aim 2. Test the hypothesis that epigenetically altered fetal immune gene expression continues to be impaired in post-natal PI and TI steers. We plan to complete preliminary postnatal control and PI steer tissue analysis using whole genome DNA methylation analysis or RNA-Seq approaches. Primary methylation studies are planned on tissues collected from Aim 3 at slaughter during year 3 of the project. Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections, such as bovine respiratory disease complex. Pregnancies are being generated in naïve heifers. These pregnant heifers will be inoculated with PBS or BVDV on day 175 of gestation to generate control or TI heifer sex-selected calves, respectively. After weaning, these calves will be challenged with BRD pathogen, M. Haemolytica. Consequences of the infections will be assessed by clinical assessment, blood immune cell gene expression by RT-qPCR and antibody approaches, clearance of the BRD infection, lung scores, feedlot growth performance, and complete carcass characteristics at slaughter.

Impacts
What was accomplished under these goals? Specific Aim 1. Test the hypothesis that fetal immune gene expression is altered following BVDV infection due to epigenetic methylation. Fetal thymic and splenic tissue from PI (infected day 75), TI (infected day 175) and control fetuses will be examined on day 190 of gestation using whole genome methylation approaches. Methylation of differentially expressed innate and adaptive immune response genes will be confirmed using targeted bisulfite sequencing analysis and RT-qPCR. Accomplishments. Because of budget reductions from our original proposed ~$499,975 to the actual $390,000 award, this aim was deleted in our revised budget justification. Although this change in focus on methylation of genes in post-natal samples was very clear in our revised budget justification, this change was not reflected in our original CRIS submission for this project. Specific Aim 2. Test the hypothesis that epigenetically altered fetal immune gene expression continues to be impaired in post-natal PI and TI steers. Postnatal control and PI steer blood and control and TI postnatal steer spleen and liver will be analyzed using whole genome DNA methylation analysis, targeted bisulfite sequencing analysis and RT-qPCR. Accomplishments. Whole blood (EDTA) samples were obtained from 9 yearling Corriente steers and heifers including: 3 persistently infected (PI), 3 transiently infected (TI) and uninfected control animals. PI status was determined by BVDV ACE positive test on an ear notch sample followed by a BVDV RT PCR positive test on whole blood sample taken >4 weeks later. TI animals were characterized by BVDV ACE positive test on an ear notch sample followed by a BVDV RT PCR negative test on whole blood taken > 4 weeks later. Control animals had ear notch samples which were BVDV ACE negative and whole blood samples which were BVDV RT PCR negative. DNA was extracted from PBMCs and RNA was extracted from PBMCs or whole blood using Trizol (Ambion) and DNeasy (Qiagen) methods. Tissues for methylation studies will be collected from heifers at slaughter in year 3 of the proposal. Specific Aim 3. Test the hypothesis that fetal TI with BVDV results in post-natal epigenetic silencing of genes associated with impaired immune responses to secondary infections such as bovine respiratory disease complex. Accomplishments. Unvaccinated, seronegative, BVDV antigen negative (i.e., naive) weaned Hereford heifers were purchased in November 2019. They were transported to CSU in April 2020 and their seronegative status confirmed by serum neutralization (SN) assay. Heifers were vaccinated and then boostered 4 weeks later with a 7 way Clostridial toxoid and with Clostridium perfringens C&D with tetanus toxoid (CDT). Their weights were monitored until they reached an average weight of 766 lbs. Heifers were started on an estrous synchronization breeding regime. They were artificially inseminated (AI) with X-selected semen and are being checked for pregnancy at day 32 and for pregnancy and sex of fetus on day 60 post-breeding by ultrasound.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Hansen, TR., Georges HM, Van Campen H, Engle TE. BVDV infection during late pregnancy may produce offspring prone to infection after birth. Conference of Research Workers in Animal Disease 2019; 100th Annual Conference,Chicago IL, Abstract P104:214.