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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gonzalez Berrios, Bowden, Saad, Bishop, Van Campen, Pinedo, Hansen and Thomas.)

Conceptus-derived interferon-tau (IFNT) initiates maternal recognition of pregnancy in ewes by paracrine actions on the endometrium and endocrine action on the corpus luteum (CL). To examine the effect of IFNT on the CL without inducing IFN stimulated genes (ISGs) in the endometrium, recombinant ovine IFNT (roIFNT) or bovine serum albumin (BSA) was delivered directly into CLs via osmotic pumps at a rate of 10, 50 or 100 ng/h from days 9 to 12 of the estrous cycle. Endometrial and CL samples were collected on day 12. Fifty ng/h of roIFNT induced ISG15 in the CL on day 12 without affecting endometrial ISG15 concentrations. In a second experiment, roIFNT (50 ng/h) was infused into the CL from days 10 to 17 of the estrous cycle and serum samples were collected daily. Serum progesterone concentrations were significantly higher on days 15 to 17 in roIFNT-infused ewes compared to controls. Levels of LHCGR, STAR, CYP11A1, HSL, OPA1 and PKA mRNA and proteins were higher in the roIFNT-infused CLs compared to the controls. Levels of ISG15 and MX1 mRNA increased in the CLs of roIFNT-infused ewes but not in the endometrium. Endometrial ESR1 mRNA and protein concentrations were higher in the controls compared to roIFNT-infused ewes. In conclusion, intra-luteal delivery of roIFNT induced ISGs, stabilized steroidogenesis in the CL and delayed luteolysis without inducing endometrial ISGs. Inhibition of ESR1 in the endometrium of roIFNT-infused ewes was observed suggesting that direct delivery of IFNT to the CL has an additional anti-luteolytic effect on the endometrium., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

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.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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.

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.

Development of transplacental infection depends on the ability of the virus to cross the placenta and replicate within the fetus while counteracting maternal and fetal immune responses. Unfortunately, little is known about this complex process. Non-cytopathic (ncp) strains of bovine viral diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, cause persistent infection in early gestational fetuses (<150 days; persistently infected, PI), but are cleared by immunocompetent animals and late gestational fetuses (>150 days; transiently infected, TI). Evasion of innate immune response and development of immunotolerance to ncp BVDV have been suggested as possible mechanisms for the establishment of the persistent infection. Previously we have observed a robust temporal induction of interferon (IFN) type I (innate immune response) and upregulation of IFN stimulated genes (ISGs) in BVDV TI fetuses. Modest chronic upregulation of ISGs in PI fetuses and calves reflects a stimulated innate immune response during persistent BVDV infection. We hypothesized that establishing persistent fetal BVDV infection is also accompanied by the induction of IFN-gamma (IFN-γ). The aims of the present study were to determine IFN-γ concentration in blood and amniotic fluid from control, TI and PI fetuses during BVDV infection and analyze induction of the IFN-γ downstream pathways in fetal lymphoid tissues. Two experiments with in vivo BVDV infections were completed. In Experiment 1, pregnant heifers were infected with ncp BVDV type 2 on day 75 or 175 of gestation or kept naïve to generate PI, TI and control fetuses, respectively. Fetuses were collected by Cesarean section on day 190. In Experiment 2, fetuses were collected on days 82, 89, 97, 192 and 245 following infection of pregnant heifers on day 75 of gestation. The results were consistent with the hypothesis that ncp BVDV infection induces IFN-γ secretion during acute infection in both TI and PI fetuses and that lymphoid tissues such as spleen, liver and thymus, serve both as possible sources of IFN-γ and target organs for its effects. Notably, induction of IFN-γ coincides with a decrease in BVDV RNA concentrations in PI fetal blood and tissues. This is the first report indicating the possible presence of an adaptive immune response in persistent BVDV infections, which may be contributing to the observed reduction of viremia in PI fetuses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

Transplacental viral infections are dependent upon complex interactions between feto-placental and maternal immune responses and the stage of fetal development at which the infection occurs. Bovine viral diarrhea virus (BVDV) has the ability to cross the placenta and infect the fetus. Infection early in gestation with non-cytopathic (ncp) BVDV leads to persistent infection. Establishment of fetal persistent infection results in life-long viremia, virus-specific immunotolerance, and may have detrimental developmental consequences. We have previously shown that heifers infected experimentally with ncp BVDV type 2 on d. 75 of gestation had transient robust up-regulation of the type I interferon (IFN) stimulated genes (ISGs) 3-15 days after viral inoculation. Blood from persistently infected (PI) fetuses, collected 115 days post maternal infection, demonstrated moderate chronic up-regulation of ISGs. This infection model was used to delineate timing of the development of innate immune responses in the fetus and placenta during establishment of persistent infection. It was hypothesized that: (i) chronic stimulation of innate immune responses occurs following infection of the fetus and (ii) placental production of the type I IFN contributes to up-regulation of ISGs in PI fetuses. PI fetuses, generated by intranasal inoculation of pregnant heifers with ncp BVDV, and control fetuses from uninfected heifers, were collected via Cesarean sections on d. 82, 89, 97, 192, and 245 of gestation. Fetal viremia was confirmed starting on d. 89. Significant up-regulation of mRNA encoding cytosolic dsRNA sensors -RIG-I and MDA5 - was detected on d. 82-192. Detection of viral dsRNA by cytosolic sensors leads to the stimulation of ISGs, which was reflected in significant up-regulation of ISG15 mRNA in fetal blood on d. 89, 97, and 192. No difference in IFN-α and IFN-β mRNA concentration was found in fetal blood or caruncular tissue, while a significant increase in both IFN-α and IFN-β mRNA was seen in cotyledons from PI fetuses on d. 192. It is concluded that fetuses respond to early gestational ncp BVDV infection by induction of the type I IFN pathway, resulting in chronic up-regulation of ISGs. Cotyledonary tissue contributes to up-regulation of ISGs by increased production of IFNs. The innate immune response might partially curtail viral replication in PI fetuses, but is not able to eliminate the virus in the absence of a virus-specific adaptive immune response., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one-step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine-thiocyanate-based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column-based purification coupled with one-step RT-PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri-phasic extraction method (Tri-reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT-PCR was as low as 0.505 to 2.84 TCID(50) for VSV-IND and VSV-NJ, respectively. The multiplex RT-PCR consistently detected VSV-IND and NJ RNA in as little as 0.1-1.0  fg of total RNA from spiked BHK-21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT-PCR assay was capable of detecting both types of VSV in a one-step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID(50) (IND), 0.0946 TCID(50) (NJ), and 0.057  fg (IND and NJ) per 2  µl PCR sample, which is significantly more sensitive than reported previously (0.28-2.8 TCID50/1  µl). So the present study improved the sensitivity of previously reported multiplex RT-PCR for the detection and differentiation of VSV-IND and VSV-NJ in a single assay., (© 2011 Wiley-Liss, Inc.)

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.

The apparent prevalence of bovine viral diarrhea virus (BVDV) persistently infected cattle has been found to be low in U.S. dairies, beef herds and feedlots. Current management practices within U.S. cattle industries that impact the epidemiology of BVDV infections include purchasing untested cattle, lack of biosecurity procedures, large herd sizes, mixing cattle from multiple sources, high cattle densities in dairy and feedlot operations, synchronous breeding of beef herds, communal grazing and widespread vaccination. Evidence for BVDV infection has been found in farmed and free-ranging wildlife in North America; however the risk of BVDV transmission from wildlife to cattle is not known. The perception of a low prevalence of BVDV herd infections, the unrestricted sale of PI cattle, lack of economic data, intensive marketing of vaccines, reluctance to accept federal regulations, and a "gambler's" attitude among producers are impediments to implementation of a national systematic BVD control program. Since 2004, voluntary BVDV control programs have been organized in nine states reflecting the recognition of BVD as an important and preventable problem in the U.S., (Copyright 2009 Elsevier B.V. All rights reserved.)

Wildlife serves as a source and a target for some infectious diseases of cattle. This article covers characteristics of the pathogens, Brucella abortus, Mycobacterium bovis, and bovine viral diarrhea virus; host species behavior; and external factors that influence the introduction, maintenance, and spread of infectious agents between wildlife and cattle., (Copyright 2010 Elsevier Inc. All rights reserved.)

The consequences of viral infection during pregnancy include impact on fetal and maternal immune responses and on fetal development. Transplacental infection in cattle with noncytopathic bovine viral diarrhea virus (ncpBVDV) during early gestation results in persistently infected (PI) fetuses with life-long viremia and susceptibility to infections. Infection of the fetus during the third trimester or after birth leads to a transient infection cleared by a competent immune system. We hypothesized that ncpBVDV infection and presence of an infected fetus would alter immune response and lead to downregulation of proinflammatory processes in pregnant dams. Naïve pregnant heifers were challenged with ncpBVDV2 on day 75 (PI fetus) and day 175 [transiently infected (TI) fetus] or kept uninfected (healthy control fetus). Maternal blood samples were collected up to day 190 of gestation. Genome-wide microarray analysis of gene expression in maternal peripheral white blood cells, performed on days 160 and 190 of gestation, revealed multiple signal transduction pathways affected by ncpBVDV infection. Acute infection and presence of a TI fetus caused upregulation of the type I interferon (IFN) pathway genes, including dsRNA sensors and IFN-stimulated genes. The presence of a PI fetus caused prolonged downregulation of chemokine receptor 4 (CXCR4) and T cell receptor (TCR) signaling in maternal blood cells. We conclude that: 1) infection with ncpBVDV induces a vigorous type I IFN response, and 2) presence of a PI fetus causes downregulation of important signaling pathways in the blood of the dam, which could have deleterious consequences on fetal development and the immune response.

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.

Bovine viral diarrhea virus (BVDV) infection occurs in the cattle population worldwide. Non-cytopathic (ncp) BVDV strains cause transient infection (TI) or persistent infection (PI) depending on the host's immune status. Immunocompetent adult animals and fetuses in late gestation resolve the infection. Fetal infection in early gestation results in PI with chronic viremia and life-long viral shedding, ensuring virus perpetuation in the population. Eighteen pregnant heifers, divided into three groups, were intranasally inoculated with ncp BVDV2 virus early (day 75) and late (day 175) in gestation, or kept BVDV-naïve. Fetuses were retrieved on day 190. Antiviral activity in blood of dams and fetuses, maternal expression of interferon (IFN) stimulated gene 15kDa (ISG15), virological and serological status of heifers and fetuses, and fetal growth were studied. A pronounced antiviral activity in blood of heifers and TI fetuses during acute BVDV infection was accompanied by drastic up-regulation of ISG15 mRNA in maternal blood. Only one PI fetus expressed low IFN response 115 days post inoculation despite high BVDV antigen and RNA levels. PI fetuses presented with growth retardation. Infection of pregnant heifers with ncp BVDV2 early in gestation adversely affects fetal development and antiviral responses, despite protective immune responses in the dam.

Objective: To assess the likelihood of an introduction of foot-and-mouth disease (FMD) into the Malaysia-Thailand-Myanmar (MTM) peninsula through terrestrial movement of livestock., Animals: 89,294 cattle and buffalo legally moved into the MTM peninsula., Procedures: A quantitative risk assessment was conducted by use of a stochastic simulation. Patterns of livestock movement were ascertained through review of relevant governmental records and regulations and by interviewing farmers, traders, and local officers when the records did not exist. Parameters identified in the process were the probabilities of livestock having FMD and of FMD infection going undetected during import processes. The probability of an animal accepted for import having FMD was also assessed. Sensitivity analysis was performed to determine the effects that each parameter had on the model., Results: The simulation yielded an average consignment prevalence of 10.95%. Typically, each animal in a quarantine facility had a 2.7% chance of having an inapparent form of FMD infection; hence, it was likely an animal would not be identified as infected. Findings revealed that the mean probability of an animal accepted for import having FMD was 2.9%, and the risk was as high as 11%., Conclusions and Clinical Relevance: Results of the model allowed for the evaluation of movement regulations currently imposed in the MTM peninsula. Evidence from the study suggested that current practices in animal movement were far from efficient in preventing introduction of FMD-infected animals into the MTM region, and additional measures will be necessary.

Objective: To assess the impacts of the introduction of foot-and-mouth disease (FMD) and various FMD control programs in southern Thailand., Animals: A native population of 562,910 cattle and 33,088 buffalo as well as 89,294 animals legally transported into southern Thailand., Procedures: A quantitative risk assessment was used to ascertain the probability of FMD introduction, and an intrinsic dynamic model was used to assess impacts. Value for the transmission rate (beta) was estimated. Five scenarios created to assess the impacts of nonstructural protein (NSP) testing, mass vaccination, and culling were examined. Impacts were assessed through an examination of the estimated annual cumulative incidence (ACI) of FMD. The ACIs of various scenarios were compared by use of the Tukey Studentized range technique., Results: beta was estimated at 0.115. Approximately 35,000 cases of FMD would be expected from the baseline situation. A 30% reduction of ACI was detected with the introduction of NSP antibody testing. Prophylactic vaccination resulted in an 85% reduction of ACI. Concurrent use of NSP antibody testing and vaccination reduced the ACI by 96%, and the addition of an eradication policy resulted in a slightly greater decrease in the ACI (98%)., Conclusions and Clinical Relevance: The study used epidemiologic models to investigate FMD control interventions. Results suggested that vaccination has more impact than the use of NSP testing. Use of the NSP test reduced ACI during peak seasons, whereas vaccination diminished the underlying incidence. The best mitigation plan was an integrated and strategic use of multiple control techniques.

Malignant catarrhal fever (MCF) was diagnosed in four free-ranging mule deer (Odocoileus hemionus) in January and February of 2003. Diagnosis was based on typical histologic lesions of lymphocytic vasculitis and PCR identification of ovine herpesvirus-2 (OHV-2) viral genetic sequences in formalin-fixed tissues. The animals were from the Uncompahgre Plateau of southwestern Colorado. Deer from these herds occasionally resided in close proximity to domestic sheep (Ovis aries), the reservoir host of OHV-2, in agricultural valleys adjacent to their winter range. These cases indicate that fatal OHV-2 associated MCF can occur in free-ranging mule deer exposed to domestic sheep that overlap their range.

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.

Objective: To describe the prevalence of West Nile virus (WNV) infection and evaluate factors associated with positive IgM capture ELISA results in equids with clinical signs compatible with WNV infection., Design: Retrospective case series., Sample Population: Laboratory submission forms from 1,104 equids tested for WNV in Colorado in 2003., Procedures: Submission forms accompanying samples submitted for detection of WNV via IgM capture ELISA were obtained from the Colorado state veterinarian and diagnostic laboratories performing the tests. Data on signalment, clinical signs, history of vaccination against WNV, and assay results were collected from laboratory submission forms. Equids with clinical signs compatible with WNV infection in which IgM capture ELISA results were positive were considered as case equids., Results: 1,104 equids were tested for WNV; 1,017 (92.1%) had clinical signs compatible with WNV infection. Among equids with clinical signs compatible with WNV infection, the odds of testing positive for WNV via IgM capture ELISA were lower in males and in vaccinated equids and higher in equids with moderate and severe illness, compared with females, unvaccinated equids, and equids with mild illness., Conclusions and Clinical Relevance: Among equids with clinical signs compatible with WNV infection, vaccination against WNV, severity of clinical signs, duration of illness, and region in Colorado were associated with increased risk of having a positive IgM capture ELISA result.

We used a Bayesian classification approach to predict the bovine viral-diarrhoea-virus infection status of a herd when the prevalence of persistently infected animals in such herds is very small (e.g. <1%). An example of the approach is presented using data on beef herds in Wyoming, USA. The approach uses past covariate information (serum-neutralization titres collected on animals in 16 herds) within a predictive model for classification of a future observable herd. Simulations to estimate misclassification probabilities for different misclassification costs and prevalences of infected herds can be used as a guide to the sample size needed for classification of a future herd.

Objective: To develop a method for percutaneous collection of fetal fluid from cattle in the late stages of gestation and determine whether bovine viral diarrhea virus (BVDV) can be isolated from such fluids., Design: Case series., Animals: 169 pregnant beef cattle., Procedure: Animals were restrained in a squeeze chute, and hair was clipped from a region of the right flank. Pregnancy was confirmed, and fetal fluids were identified by means of abdominal ultrasonography. Fetal fluid was collected with a spinal needle. Virus isolation was performed on fetal fluids, WBC lysates from 160 live calves, and tissues from 12 calves that died or were aborted. Blood samples collected from adult cattle were assayed with an immunoperoxidase monolayer assay., Results: Fourteen animals aborted or delivered premature calves within 3 weeks after fetal fluid collection; however, it could not be determined whether this was a complication of the procedure or attributable to other factors. Results of BVDV isolation from fetal fluid samples were negative for 168 animals. However, a noncytopathic BVDV was isolated from fetal fluid obtained from a 2-year-old heifer; results of the immunoperoxidase assay of serum from this heifer were also positive, and a noncytopathic BVDV was isolated from tissue specimens from a stillborn calf produced by this heifer., Conclusions and Clinical Relevance: Results suggest that fetal fluids can be collected percutaneously from cattle in the late stages of gestation and that virus isolation performed on fetal fluids can be used to identify fetuses infected with BVDV in utero. However, safety of the procedure could not be evaluated.