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Our objective was to test the hypothesis that sorting Holstein dairy cows with high direct genomic value for daughter pregnancy rate (DGV-DPR) and low services per conception records improves interferon tau production and endometrial signaling for maternal recognition of pregnancy.

Investigating the influences of single nucleotide polymorphisms (SNP) associated with conceptus death (CD) on day 16 of pregnancy could distinguish cows who have inferior value for fertility traits. We hypothesized that CD is associated with SNP genotypes that disrupt maternal recognition and lead to early pregnancy loss. RNA sequences from Holstein cow pregnancies (n = 15) with normal conceptuses (Norm) or CD were subjected to SNP discovery via Qiagen CLC Genomics Workbench. Selected SNP were based on previous fertility studies, differentially expressed genes within pregnancy development analyses and proximity to SNP associated with fertility traits in the Cattle QTL Database. A second cohort of Holstein cows (n = 500) was used to conduct a validation and genotype to phenotype analysis of candidate SNP (cSNP) via DNA from blood samples and farm records. PLINK software was used to remove cows (n = 34) missing more than >20% cSNP and remove cSNP with monomorphic alleles, minor allele frequency (MAF) < 10% and not in Hardy-Weinberg Equilibrium (>1e-15). The GLM-one way ANOVA was used for all statistical models in SAS. Statistically significant models (P <0.05) were further analyzed for LSMEANS, Bonferroni adjustment of P-values, additive allele effect and predicted effect of amino acid (aa) change on protein function via SIFT analyses tool in the Ensembl variant table (scoring 0-1.0; <0.05 being significant). Previously, we identified 69 cSNP on the RNA sequences of Norm or CD pregnancies from the initial group of Holstein cows and in proximity to >1 SNP associated with fertility traits from the Cattle QTL database. Quality control measures in PLINK reduced the number of Holstein cows in the second cohort to 466 and identified 4 cSNP associated with non-binary reproductive traits. Herein, we report an additional 4 cSNP discovered and validated with binary reproductive traits. The cSNP within genes were associated with the following roles: BOLA-DMB (antigen loading in the immune system), DECR1 (lipid metabolism), FASN (reconstitution of body reserves during pregnancy) and SREBF1 (synthesis of fatty acids). Cows with T allele, in BOLA-DMB and SREBF1, were less likely (P <0.05) to become pregnant <150 days in milk (DIM). For DECR1 and FASN, cows were less likely (P <0.05) to become pregnant at 1startificial insemination with C or A allele, respectively. Only the cSNP in SREBF1 had both an additive allele effect and a predicted effect of aa change on protein function. When cows had C allele, in the SREBF1 cSNP, there was 6% greater (P <0.01) probability of becoming pregnant <150 DIM. While SIFT revealed the cSNP in SREBF1 have a predicted significant effect with a score of 0.03 for the aa change of proline/leucine. The reported cSNP, specifically those influenced by CD, add valuable information for the development of improved genetic tools for the dairy industry. USDA-NIFA#2019-07133.

Bovine viral diarrhea virus continues to cost the cattle industry millions of dollars each year despite control measures. The primary reservoirs for bovine viral diarrhea virus are persistently infected animals, which are infected in utero and shed the virus throughout their lifetime. The difficulty in controlling the virus stems from a limited understanding of transplacental transmission and fetal development of immunotolerance. In this study, pregnant bovine viral diarrhea virus naïve heifers were inoculated with bovine viral diarrhea virus on day 75 of gestation and fetal spleens were collected on gestational days 82, 97, 190, and 245. Microarray analysis on splenic RNA from days 82 and 97 revealed an increase in signaling for the innate immune system and antigen presentation to T cells in day 97 persistently infected fetuses compared to controls. Reverse transcription quantitative polymerase chain reaction on select targets validated the microarray revealing a downregulation of type I interferons and lymphocyte markers in day 190 persistently infected fetuses compared to controls. Protein was visualized using western blot and tissue sections were analyzed with hematoxylin and eosin staining and immunohistochemistry. Data collected indicate that fetal immunotolerance to bovine viral diarrhea virus developed between days 97 and 190, with mass attenuation of the immune system on day 190 of gestation. Furthermore, lymphocyte transcripts were initially unchanged then downregulated, suggesting that immunotolerance to the virus stems from a blockage in lymphocyte activation and hence an inability to clear the virus. The identification of lymphocyte derived immunotolerance will aid in the development of preventative and viral control measures to implement before or during pregnancy.

An epizootic of hemorrhagic disease associated with Epizootic hemorrhagic disease virusserotype 2 (EHDV-2) infections in yaks from 5 herds occurred in Colorado between August 21 and October 3, 2012. Affected yaks presented with fever, lethargy, anorexia, dyspnea, and swollen conjunctivae. Ulcerated dental pads, mucoid sanguineous nasal discharge, petechial hemorrhages in multiple organs, pulmonary edema, and serosanguinous fluid in the thorax, abdomen, and pericardial sac were observed at necropsy. Blood and tissue samples from 8 yaks with similar clinical signs and necropsy findings were positive for EHDV-2 by reverse transcription polymerase chain reaction and 5 yaks were seropositive for EHDV. Tests for malignant catarrhal fever (Ovine herpesvirus 2), Bovine viral diarrhea virus, Bovine herpesvirus 1, Foot-and-mouth disease virus, and Vesicular stomatitis virus were negative. The findings indicate that yaks are susceptible to infection with EHDV-2 and exhibit the clinical signs, and gross and histologic lesions of hemorrhagic disease observed in other ruminant species.

Bovine viral diarrhea virus(BVDV) is a significant viral pathogen of domestic cattle. Worldwide, there is evidence of BVDV exposure and infection in wild ungulates; however, the frequency and significance of such events are unknown. To determine the prevalence and distribution of Colorado deer, elk, and moose persistently infected (PI) with BVDV, a cross-sectional study was conducted using full-thickness ear tissue samples collected from animals presented to the Colorado Division of Wildlife for chronic wasting disease surveillance in the 2005–2006 hunting season. Tissue from 5,597 harvested animals (2,934 mule deer, 2,516 elk, 141 white-tailed deer, and 6 moose) was paraffin-embedded and stained for BVDV using immunohistochemistry. A single adult male mule deer had BVDV antigen in the skin; staining distribution was consistent with that seen in PI cattle. Skin and lymph node were also positive for viral RNA by polymerase chain reaction, and the virus was determined to be a type 1. The prevalence of BVDV PI cervids in Colorado is very low. However, the identification of a naturally infected adult PI animal in the wild suggests that the virus infects free-ranging populations. The source of the BVDV is unknown and is assumed to be spillover from cattle or maintenance within wildlife populations. Consideration of a potential wild animal reservoir is important in the design and implementation of BVDV management practices in cattle.

To reduce the cost of whole herd screening for bovine viral diarrhea virus persistently infected animals, the sensitivity and specificity of an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a microtiter virus isolation ELISA using saline from ear notch samples or pooled serum was determined. Pooled saline from ear notch samples, assayed by AC-ELISA, gave a sensitivity and specificity of 98% and 94%, respectively, for pools containing 2 samples and 72% and 100%, respectively, for pools of 5. The sensitivity of pooled ear notch or serum samples for bovine viral diarrhea virus detection by microtiter virus isolation (sensitivity < 5%) or serum samples for detection by AC-ELISA (sensitivity < 15%) is too low to be used for whole herd screening. Pooling saline from ear notch samples from 2 animals tested by AC-ELISA, however, could provide a less expensive, reliable method for whole herd screening for bovine viral diarrhea virus.

A virus that could not be identified as a previously known equine virus was isolated from the mononuclear cells of a horse. Electron microscopy revealed enveloped virions with nucleocapsid structures characteristic of viruses in the Paramyxoviridae family. The virus failed to hemadsorb chicken or guinea pig red blood cells and lacked neuraminidase activity. Two viral genes were isolated from a cDNA expression library. Multiple sequence alignments of one gene indicated an average identity of 45% as compared to Morbillivirus N protein sequences. A weaker relationship was found with Tupaia paramyxovirus (TPMV) and Hendra virus (HeV) N proteins. In the second gene, multiple open reading frames (ORFs) were identified, corresponding to the arrangement of the P, V, and C ORFs in the Morbillivirus and Respirovirus viruses. Short stretches in the C-terminal regions of the P and C proteins showed limited homologies to viruses in the Morbillivirus genus but no obvious relationship to viruses in other genera. The V ORF translation product contained a highly conserved, cysteine-rich domain that is common to most viruses in the Paramyxovirinae subfamily. Sequencing of P gene cDNA clones confirmed the use of a cotranscriptional editing mechanism for the regulation of P/V expression. Based on the location of its origin it has been named Salem virus (SalV).

An in vitro fertilization assay employing zona-free hamster eggs was used to analyze human spermatozoal fertilizing ability. Human spermatozoa were preincubated for 18 to 20 hours in Biggers, Whitten, and Whittingham’s medium (1971) at a concentration of 1 x 107sperm/ml prior to the addition of zona-free superovulated hamster eggs. Eggs were examined microscopically 2 hours later for evidence of swelling or decondensing sperm heads in the cytoplasm. A total of 6266 eggs were examined in assays for both suspected fertile and infertile donors; 50 eggs/sample were examined. The percentage fertilization was found to range from 14% to 100% in the suspected fertile group with an average of 56.3%. The sperm concentration in this fertile group ranged from 22 to 303 million/ml with an average of 114. The suspected infertile samples yielded fertilization rates of 10% or less and an average count of 50.6 million/ml. These data suggest that human spermatozoa fuse with the vitelline membrane of zona-free hamster eggs and decondense with varying efficiencies. The percentage of fertilization in this cross-species system did not show a significant correlation with sperm concentration or motility. However, suspected infertile samples always yielded 10% or less fertilization in this assay. This method may have potential value as a diagnostic tool in evaluating human spermatozoal fertilizing capacity which avoids the ethical and logistical problems associated with fertilization of human eggs in vitro.

The isolation of endothelial cells from the aortae of chickens and maintenance in culture is described. The uptake of acetylated low-density lipoprotein labeled with 1,1′-dioctadecyl-1,3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate was used to purify the endothelial cells by fluorescence-activated cell sorting (FACS). The morphology of the purified cells is similar to that of endothelial cells from other species; however, these characteristics change rapidly with continued culture and passage.

The role of tumor necrosis factor α (TNFα) in the pathogenesis of influenza A viral pneumonia was examined. CD-1 male mice were challenged intranasally with influenza A virus A/PR/8/34 (H1N1) and administered rabbit anti-mouse TNFα-specific-neutralizing antibodies intraperitoneally. The effect of treatment on virus titer, TNFα levels, morbidity, mortality, and on pathologic lung lesions were compared with sham-treated controls. The severity of gross and histologic lung lesions positively correlated with the peak bronchoalveolar TNFα levels and was ameliorated with anti-TNFα treatment. Survivorship was prolonged in mice given a lethal dose of virus by treatment with TNF-α neutralizing antibodies. Reduction of TNFα levels by treatment with TNFα-antibodies did not affect virus titers in the lung. These results suggest that TNFα is a mediator of pulmonary inflammation during influenza A viral pneumonia, but may not play a significant anti-viral role in influenza pneumonia.

The mechanisms of persistent infection are incompletely understood, and bovine viral diarrhea virus (BVDV) provides a useful disease model for studying the complex host-virus interactions involved during the prenatal infection period. The noncytopathic BVDV biotype (ncpBVDV) has the ability to establish infection early in fetal development (<150 d.), creating a persistently infected (PI) calf that is immunotolerant to the virus and unable to resolve the infection. A viremia persists in the animal after birth, and continuous viral shedding in body effluents ensures the continuation of the infection cycle. In contrast, infection after the development of a competent immune system (fetal >150 d. or postnatal) results in a transient infection (TI) that is associated with clearance of the virus and seroconversion. It has been suggested that the establishment of persistent infection with BVDV may involve viral evasion of innate and subsequent adaptive immune responses. Screening of maternal blood cell mRNA on day 190 of pregnancy by microarray analysis revealed an intense upregulation of interferon stimulated gene 15 K (ISG15) after BVDV infection. ISG15 is considered a prominent marker in the cascade of events that occur during the innate immune response. BVDV naï ve heifers (n=6 per treatment group) were infected with ncpBVDV type 2 virus at d. 75 or d. 175 of pregnancy creating PI and TI fetuses, respectively. A control group was maintained free of BVDV. Maternal blood was collected at multiple time points during the experiment, and cesarean sections were performed on d. 190. Infection was confirmed by ELISA and qtRT-PCR. A ponderal index of fetal growth was calculated (body weight/crown rump length3), indicating intrauterine growth restriction of PI fetuses (p<0.05). Decreased megakaryocyte numbers were noted in PI spleen tissues, in addition to a prominent occurrence of BVDV antigen in the spleen, liver, and brain of PI fetuses. No viral antigen was detected in TI or control fetuses. qtRT-PCR analysis of fetal spleen mRNA revealed a dramatic upregulation of ISG15 expression in TI fetuses (p<0.01) in contrast to the low expression of ISG15 in PI and control fetuses. These findings were further confirmed by Western Blot analysis of spleen samples using anti-ISG15 antibody. The strong induction of ISG15 in TI fetuses is characteristic of acute viral infection and the consequent innate immune response, however the mild ISG15 response of PI fetuses, despite their viremic state, indicates a deficient IFN pathway activation in the host tissues. This lack of a robust response by the PI host during prenatal development may contribute to the establishment of the persistent infection, and the associated inhibition of normal fetal growth. NcpBVDV infection during gestation clearly has numerous adverse effects on the developing fetus, in addition to a complex interaction with the innate immune response. Further understanding of the relationship between BVDV and its infected host is crucial to identifying the impact of viral infection on fetal growth and immune development, and the subsequent control of this and other viral pathogens. National Research Initiative Competitive Grant 2006-03907 from the USDA Cooperative State Research, Education, and Extension Service (T.R.H & A.L.V.). (platform)