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Introduction: Targeted single nucleotide polymorphisms (SNPs) have been used in genomic prediction methodologies to enhance the accuracy of associated genetic transmitting abilities in Holstein cows. The objective of this study was to identify and validate SNPs associated with fertility traits impacting early embryo mortality. Methods: The mRNA sequencing data from day 16 normal (n = 9) and embryo mortality (n = 6) conceptuses from lactating multiparous Holstein cows were used to detect SNPs. The selection of specific genes with SNPs as preliminary candidates was based on associations with reproductive and fertility traits. Validation of candidate SNPs and genotype-to-phenotype analyses were conducted in a separate cohort of lactating primiparous Holstein cows (n = 500). After genotyping, candidate SNPs were filtered using a quality control pipeline via PLINK software. Continuous numeric and binary models from reproductive traits were evaluated using the mixed procedure for a generalized linear model-one way ANOVA or logistic regression, respectively. Results: Sixty-nine candidate SNPs were initially identified, but only 23 passed quality control procedures. Ultimately, the study incorporated 466 observations for statistical analysis after excluding animals with missing genotypes or phenotypes. Significant (p <0.05) associations with fertility traits were identified in seven of the 23 SNPs: DSC2 (cows with the A allele were older at first calving); SREBF1 and UBD (cows with the T or G alleles took longer to conceive); DECR1 and FASN (cows with the C allele were less likely to become pregnant at first artificial insemination); SREBF1 and BOLA-DMB (cows with the T allele were less likely to be pregnant at 150 days in milk). It was also determined that two candidate SNPs within the DSC2 gene were tag SNPs. Only DSC2 SNPs had an important allele substitution effect in cows with the G allele, which had a decreased age at first calving by 10 days. Discussion: Candidate SNPs found in this study could be used to develop genetic selection tools to improve fertility traits in dairy production systems. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhea virus (BVDV) infections cause USD 1.5–2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle. [ABSTRACT FROM AUTHOR]

Fetal infection of bovine viral diarrhea virus (BVDV) before d 125 to 150 of gestation results in the birth of immunotolerant, persistently infected (PI) calves. Infection of BVDV during late gestation results in transient fetal infections (TI). Unvaccinated, yearling Hereford heifers (n = 25), seronegative for antibodies to BVDV1 and BVDV2, were bred by artificial insemination with X chromosome-bearing sperm from an Angus sire to examine the impact of TI on postnatal growth, estimated dry matter digestibility, blood glucose concentrations, and carcass characteristics. On d 175 of pregnancy, heifers were intranasally inoculated with either DMEM + 2% horse serum (sham control) to generate control calves or 4.0 log TCID50 noncytopathic type2 BVDV to generate TI calves. All sham-inoculated control dams remained seronegative, and all BVDV-inoculated dams seroconverted by d 14 post-inoculation. Sham-inoculated control dams (n = 12) and BVDV-inoculated dams (n = 12) gave birth to live calves. All control offspring were seronegative, and all TI offspring were seropositive for antibodies to type 2 BVDV at birth. All offspring were raised on pasture until weaning. At weaning, all calves were transported to our feedlot research facility, housed in one group feedlot pen, and transitioned to a high-energy concentrate-based diet until reaching an approximate BW of 600 kg. Upon arrival at the feedlot, all animals received a standard heifer growth implant, a modified live viral vaccine containing IBR-BRSV-PI3 and were dewormed. Heifer BW and jugular blood samples were collected every 28 d. On d 84 of the feeding period, titanium dioxide was added to the diet of 12, age-paired, individually fed, heifers (3 control and 3 TI heifers; approximately 1 yr of age) for 28 d and used to estimate dry matter digestibility. After approximately 280 d on feed heifers were transported to a USDAinspected abattoir and harvested. The TI heifers hadlighter birth weights (P < 0.03) and final BW (P < 0.04) when compared with control heifers. Average daily gain was greater (P < 0.01) in control compared with TI heifers. Blood glucose concentrations were similar between control and TI heifers at all sampling time points. Dry matter intake of individually fed heifers was similar across treatments. TI heifers had a 2.2% lesser (P < 0.05) dry matter digestibility and lighter (P < 0.01) hot carcass weights compared with controls. These data suggest that TI fetal BVDV infection negatively impacts growth throughout the feeding period, possibly by impacting gastrointestinal tract function. This research was supported by USDA-NIFA Grant # 2019-67015-29866. [ABSTRACT FROM AUTHOR]

The objective of this experiment was to examine the impact of dams infected with bovine viral diarrhea virus (BVDV) during late gestation on inflammatory blood biomarkers in their offspring. Fetal infection with BVDV before d 125 to 150 of gestation results in the birth of immunotolerant, persistently infected (PI) calves. Infection of BVDV during late gestation results in transient fetal infections (TI). It was hypothesized that offspring of dams transiently infected with BVDV on d 175 of gestation would have greater inflammatory blood biomarkers than control offspring. Unvaccinated, yearling Hereford heifers (n = 24), seronegative for antibodies to BVDV1 and BVDV2 were bred by artificial insemination with X chromosomebearing sperm from an Angus sire. On d 175 of pregnancy, heifers were intranasally inoculated with either Dulbecco’s Modified Eagle Medium + 2% horse serum (sham control) or 4.0 log TCID50 noncytopathic type 2 BVDV to generate control and transient infected calves, respectively. All BVDV-inoculated dams seroconverted by d 14 post-inoculation and all sham-inoculated control dams remained seronegative. Twelve, seronegative control heifer calves were born to the control dams, and 11 seropositive (to type 2 BVDV) TI heifer calves were born to BVDV-inoculated dams. All offspring were raised on pasture until weaning. At weaning, all calves were transported to our feedlot research facility, housed in one group feedlot pen, and transitioned to a high-energy concentrate-based diet until reaching an approximate body weight (BW) of 600 kg. Upon arrival, all animals received a standard heifer growth implant containing 200 mg of testosterone propionate and 20 mg of estradiol benzoate, a modified live viral vaccine containing IBR-BRSV-PI3, and were dewormed. Jugular blood samples were obtained every 28 d until harvest (280 d on feed) and analyzed for biomarkers of inflammation. Data were analyzed as a randomized block design with the MIXED procedures of SAS. There was a treatment x time interaction (P < 0.05) for plasma ceruloplasmin and the oxidized form of glutathione. On d 0 and 28 of the feeding period, all biomarkers were similar across treatments. By d 56 of the feeding period, ceruloplasmin (P < 0.03) and the oxidized form of glutathione (P < 0.01), indicators of chronic inflammation, were increased in plasma samples from TI heifers compared with controls. These parameters remained greater in TI heifers compared with controls throughout the feeding period. There were no treatment or treatment x time interactions for plasma interferon (INF)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and haptoglobin. These data suggest that TI heifers may be experiencing a greater level of oxidative stress later in the finishing period. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhea virus (BVDV) infection during early gestation results in persistently infected (PI) immunotolerant calves that are the primary reservoirs of the virus. Pathologies observed in PI cattle include congenital defects of the brain, heart, and bone as well as marked functional defects in their immune system. It was hypothesized that fetal BVDV infection alters T cell activation and signaling genes by epigenetic mechanisms. To test this, PI and control fetal splenic tissues were collected on day 245 of gestation, 170 days post maternal infection. DNA was isolated for reduced representation bisulfite sequencing, protein was isolated for proteomics, both were analyzed with appropriate bioinformatic methods. Within set parameters, 1951 hypermethylated and 691 hypomethylated DNA regions were identified in PI compared to control fetuses. Pathways associated with immune system, neural, cardiac, and bone development were associated with heavily methylated DNA. The proteomic analysis revealed 12 differentially expressed proteins in PI vs. control animals. Upregulated proteins were associated with protein processing, whereas downregulated proteins were associated with lymphocyte migration and development in PI compared to control fetal spleens. The epigenetic changes in DNA may explain the immune dysfunctions, abnormal bone formation, and brain and heart defects observed in PI animals. [ABSTRACT FROM AUTHOR]

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: BOLADMB (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 1st artificial 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. [ABSTRACT FROM AUTHOR]

Infection of pregnant cows with noncytopathic (ncp) bovine viral diarrhea virus (BVDV) induces rapid innate and adaptive immune responses, resulting in clearance of the virus in less than 3 weeks. Seven to 14 days after inoculation of the cow, ncpBVDV crosses the placenta and induces a fetal viremia. Establishment of persistent infection with ncpBVDV in the fetus has been attributed to the inability to mount an immune response before 90–150 days of gestational age. The result is ‘immune tolerance’, persistent viral replication and shedding of ncpBVDV. In contrast, we describe the chronic upregulation of fetal Type I interferon (IFN) pathway genes and the induction of IFN-γ pathways in fetuses of cows infected on day 75 of gestation. Persistently infected (PI) fetal IFN-γ concentrations also increased at day 97 at the peak of fetal viremia and IFN-γ mRNA was significantly elevated in fetal thymus, liver and spleen 14–22 days post maternal inoculation. PI fetuses respond to ncpBVDV infection through induction of Type I IFN and IFN-γ activated genes leading to a reduction in ncpBVDV titer. We hypothesize that fetal infection with BVDV persists because of impaired induction of IFN-γ in the face of activated Type I IFN responses. Clarification of the mechanisms involved in the IFN-associated pathways during BVDV fetal infection may lead to better detection methods, antiviral compounds and selection of genetically resistant breeding animals. [ABSTRACT FROM PUBLISHER]

An epizootic of hemorrhagic disease associated with Epizootic hemorrhagic disease virus serotype 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. [ABSTRACT FROM PUBLISHER]

Summary The central nervous system (CNS) is a major target of several important human and animal viral pathogens causing congenital infections. However, despite the importance of neuropathological outcomes, for humans in particular, the pathogenesis, including mode of neuro-invasion, remains unresolved for most congenital virus infections. Using a natural model of congenital infection with an RNA virus, bovine viral diarrhoea virus in pregnant cattle, we sought to delineate the timing and mode of virus neuro-invasion of and spread within the brain of foetuses following experimental respiratory tract infection of the dams at day 75 of pregnancy, a time of maximal risk of tissue pathology without foetal death. Virus antigen was first detected in the foetal brains 14 days postinfection of dams and was initially restricted to amoeboid microglial cells in the periventricular germinal layer. The appearance of these cells was preceded by or concurrent with vasculopathy in the same region. While the affected microvessels were negative for virus antigen, they expressed high levels of the type I interferon-stimulated protein ISG15 and eventually disappeared in parallel with the appearance of microcavitary lesions. Subsequently, the virus spread to neurons and other glial cells. Our findings suggest that the virus enters the CNS via infected microglial precursors, the amoeboid microglial cells, in a 'Trojan horse' mode of invasion and that the microcavitary lesions are associated with loss of periventricular microvasculature, perhaps as a consequence of high, unrestricted induction of interferon-regulated proteins. [ABSTRACT FROM AUTHOR]

Citation Hansen TR, Smirnova NP, Van Campen H, Shoemaker ML, Ptitsyn AA, Bielefeldt-Ohmann H. Maternal and fetal response to fetal persistent infection with bovine viral diarrhea virus. Am J Reprod Immunol 2010 Problem Infection of naïve pregnant cows with non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) results in transplacental infection of the fetus. Infection of the pregnant cow with ncp BVDV late in gestation (after day 150) results in transient infection (TI), as both the dam and fetus can mount an immune response to the virus. In contrast, if the fetus is infected with ncp BVDV early in gestation (before day 150), the fetal immune system is undeveloped and unable to recognize the virus as foreign. This results in induction of immune tolerance to the infecting BVDV strain and persistent infection (PI). Methods Infection of naïve pregnant heifers with ncp BVDV2 on day 75 was hypothesized to induce differential gene expression in white blood cells of the dams and their fetuses, adversely affecting development and antiviral immune responses in PI fetuses. Results Gene expression differed in maternal blood cells in the presence of PI versus uninfected fetuses. PI adversely affected fetal development and antiviral responses, despite protective immune responses in the dam. Conclusion Fetal PI with BVDV alters maternal immune function, compromises fetal growth and immune responses, and results in expression of maternal blood biomarkers that can be used to identify cows carrying PI fetuses. [ABSTRACT FROM AUTHOR]

The article reports on the Hepatopancreatic rickettsial infection of the penaeid shrimp. Frozen Penaeus (P.) marginatus tissues were screened for pathogens by shrimp bioassay. Preserved shrimp were processed for routine histopathology and stained with haematoxyiin and eosin. The rickettsial agent from P. marginatus was found to be highly infectious for juvenile P. stylirostris. Based on the data presented, P. stylirostris bioassay would appear to be of use as a method to determine the presence of this rickettsia in wild-caught P. marginatus, and juvenile P. stylirostris could be used as a suitable host for experimental culture of this rickettsia under laboratory conditions.

Two adult female bison, housed in an outdoor research facility and observed daily, died suddenly three days apart. Minimal coordination and behavioral changes were observed in one animal the evening before being found in a moribund state.Malignant catarrhal fever was suspected in both bison due to a recent confirmed MCF case with similar course. The cause of death was not apparent from necropsy, but brains of both animals were strongly positive for rabies virus antigen by fluorescent antibody and/or immunohistochemical tests. Minimal to mild encephalitis with Negri bodies was observed on histopathology. The bison were located in an area that had not been endemic for skunk rabies; however, a case of rabies in a skunk had been discovered 1.6 km north of the bison paddock two months prior to the bison cases. [ABSTRACT FROM AUTHOR]

Maternal influenza A viral infections in humans are associated with low birth weight, increased risk of pre-term birth, stillbirth and congenital defects. To examine the effect of maternal influenza virus infection on placental and fetal growth, pregnant C57BL/6 mice were inoculated intranasally with influenza A virus A/CA/07/2009 pandemic H1N1 or phosphate-buffered saline (PBS) at E3.5, E7.5 or E12.5, and the placentae and fetuses collected and weighed at E18.5. Fetal thymuses were pooled from each litter. Placentae were examined histologically, stained by immunohistochemistry (IHC) for CD34 (hematopoietic progenitor cell antigen) and vascular channels quantified. RNA from E7.5 and E12.5 placentae and E7.5 fetal thymuses was subjected to RNA sequencing and pathway analysis. Placental weights were decreased in litters inoculated with influenza at E3.5 and E7.5. Placentae from E7.5 and E12.5 inoculated litters exhibited decreased labyrinth development and the transmembrane protein 150A gene was upregulated in E7.5 placentae. Fetal weights were decreased in litters inoculated at E7.5 and E12.5 compared to controls. RNA sequencing of E7.5 thymuses indicated that 957 genes were downregulated ≥2-fold including Mal, which is associated with Toll-like receptor signaling and T cell differentiation. There were 28 upregulated genes. It is concluded that maternal influenza A virus infection impairs fetal thymic gene expression as well as restricting placental and fetal growth. [ABSTRACT FROM AUTHOR]

Bovine Viral Diarrhea Virus (BVDV) fetal infections occur in two forms; persistent infection (PI) or transient infection (TI), depending on what stage of gestation the fetus is infected. Examination of lymphoid organs from both PI and TI fetuses reveals drastically different fetal responses, dependent upon the developmental stage of the fetal immune system. Total RNA was extracted from the thymuses and spleens of uninfected control, PI, and TI fetuses collected on day 190 of gestation to test the hypothesis that BVDV infection impairs the innate and adaptive immune response in the fetal thymus and spleen of both infection types. Transcripts of genes representing the innate immune response and adaptive immune response genes were assayed by Reverse Transcription quatitative PCR (RT-qPCR) (2−ΔΔCq; fold change). Genes of the innate immune response, interferon (IFN) inducible genes, antigen presentation to lymphocytes, and activation of B cells were downregulated in day 190 fetal PI thymuses compared to controls. In contrast, innate immune response genes were upregulated in TI fetal thymuses compared to controls and tended to be upregulated in TI fetal spleens. Genes associated with the innate immune system were not different in PI fetal spleens; however, adaptive immune system genes were downregulated, indicating that PI fetal BVDV infection has profound inhibitory effects on the expression of genes involved in the innate and adaptive immune response. The downregulation of these genes in lymphocytes and antigen-presenting cells in the developing thymus and spleen may explain the incomplete clearance of BVDV and the persistence of the virus in PI animals while the upregulation of the TI innate immune response indicates a more mature immune system, able to clear the virus. [ABSTRACT FROM AUTHOR]

This article reports the occurrence of B. penaei in an additional host species, Penaeus marginatus Randall, and geographical area, Hawaii. Penaeus marginatus or Opai Lolo is the only member of the genus Penaeus indigenous to the Hawaiian Islands. Additional juvenile (01 to 4-0 g) P. marginatus were collected from the Kuliouou tidal flats from January to March 1984. These shrimp were held in aquaria for 7-10 days prior to examination. Shrimp were sacrificed by injection and immersion in Davidson's fixative, and after 24 h transferred to 50% ethanol for storage. Preserved hepatopancreata were processed for routine histopathology and stained with haematoxylin and eosin.