Transcriptome analysis of immune genes in peripheral blood mononuclear cells of young foals and adult horses.

During the neonatal period, the ability to generate immune effector and memory responses to vaccines or pathogens is often questioned. This study was undertaken to obtain a global view of the natural differences in the expression of immune genes early in life. Our hypothesis was that transcriptome analyses of peripheral blood mononuclear cells (PBMCs) of foals (on day 1 and day 42 after birth) and adult horses would show differential gene expression profiles that characterize natural immune processes. Gene ontology enrichment analysis provided assessment of biological processes affected by age, and a list of 897 genes with ≥2 fold higher (p<0.01) expression in day 42 when compared to day 1 foal samples. Up-regulated genes included B cell and T cell receptor diversity genes; DNA replication enzymes; natural killer cell receptors; granzyme B and perforin; complement receptors; immunomodulatory receptors; cell adhesion molecules; and cytokines/chemokines and their receptors. The list of 1,383 genes that had higher (p<0.01) expression on day 1 when compared to day 42 foal samples was populated by genes with roles in innate immunity such as antimicrobial proteins; pathogen recognition receptors; cytokines/chemokines and their receptors; cell adhesion molecules; co-stimulatory molecules; and T cell receptor delta chain. Within the 742 genes with increased expression between day 42 foal and adult samples, B cell immunity was the main biological process (p = 2.4E-04). Novel data on markedly low (p<0.0001) TLR3 gene expression, and high (p≤0.01) expression of IL27, IL13RA1, IREM-1, SIRL-1, and SIRPα on day 1 compared to day 42 foal samples point out potential mechanisms of increased susceptibility to pathogens in early life. The results portray a progression from innate immune gene expression predominance early in life to adaptive immune gene expression increasing with age with a putative overlay of immune suppressing genes in the neonatal phase. These results provide insight to the unique attributes of the equine neonatal and young immune system, and offer many avenues of future investigation

Effect of a Histone Demethylase Inhibitor on Equine Herpesvirus-1 Activity In Vitro

Equine herpesvirus type 1 (EHV-1) is a ubiquitous and highly contagious pathogen that causes a range of disease severities with outbreaks of notable economic impact. Given the limitations in immune protection of current vaccines and the limited effectiveness of antiviral drugs on EHV-1 infections in vivo, improved treatment measures are needed to control disease. The use of drugs that alter the epigenetic state of herpes simplex virus genome has been shown to limit viral primary infection and reactivation both in vitro and in vivo. Therefore, we tested the hypothesis that maintaining a repressive epigenetic state on the EHV-1 genome in the host equine cell would decrease viral load during lytic infection. Equine fetal kidney cells (EFKCs) or isolated peripheral blood leukocytes were treated in vitro with (a) the nucleoside analog ganciclovir; (b) the histone demethylase inhibitor OG-L002; (c) both ganciclovir and OG-L002; or (d) dimethyl sulfoxide (DMSO, vehicle control); and then infected with a clinical EHV-1 isolate. Treatment of EFKCs with ganciclovir (mean 22.3 DNA copies per cell, p = 0.0005), OG-L002 (mean 25.6, p = 0.005) or both ganciclovir and OG-L002 (mean 7.1, p = 0.0001) resulted in decreased EHV-1 viral load at 24 h post-infection (hpi) in comparison with DMSO (mean 42.0), with greater impact using the combined treatment. Further, EHV-1 gene expression at 3 hpi decreased when EFKCs were infected in the presence of ganciclovir (p = 0.04) and combined treatment of ganciclovir and OG-L002 (p = 0.0003). In contrast, under similar conditions, neither ganciclovir nor OG-L002 suppressed EHV-1 infection in leukocytes. Differences between cell types, drug penetrance, or drug turnover, may have contributed to the distinct effects observed in this study

Decreased Infectivity of a Neutralization-Resistant Equine Infectious Anemia Virus Variant Can Be Overcome by Efficient Cell-to-Cell Spread

Two variants of equine infectious anemia virus (EIAV) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. No differences were observed in the growth curves and relative fitness scores of EIAVs of principal neutralizing domain variants of groups 1 (EIAV PND-1 ) and 5 (EIAV PND-5 ), respectively; however, the neutralization-resistant EIAV PND-5 variant was less infectious in single-round replication assays. Infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000-fold more efficient than cell-free infectivity. These data indicate that efficient cell-to-cell spread can overcome the decreased infectivity that may accompany immune escape and should be considered in studies assessing the relative levels of fitness among lentivirus variants, including HIV-1

Antigen-specific immunoglobulin variable region sequencing measures humoral immune response to vaccination in the equine neonate

<div><p>The value of prophylactic neonatal vaccination is challenged by the interference of passively transferred maternal antibodies and immune competence at birth. Taken our previous studies on equine B cell ontogeny, we hypothesized that the equine neonate generates a diverse immunoglobulin repertoire in response to vaccination, independently of circulating maternal antibodies. In this study, equine neonates were vaccinated with 3 doses of keyhole limpet hemocyanin (KLH) or equine influenza vaccine, and humoral immune responses were assessed using antigen-specific serum antibodies and B cell Ig variable region sequencing. An increase (p<0.0001) in serum KLH-specific IgG level was measured between days 21 and days 28, 35 and 42 in vaccinated foals from non-vaccinated mares. In vaccinated foals from vaccinated mares, serum KLH-specific IgG levels tended to increase at day 42 (p = 0.07). In contrast, serum influenza-specific IgG levels rapidly decreased (p≤0.05) in vaccinated foals from vaccinated mares within the study period. Nevertheless, IGHM and IGHG sequences were detected in KLH- and influenza- sorted B cells of vaccinated foals, independently of maternal vaccination status. Immunoglobulin nucleotide germline identity, IGHV gene usage and CDR length of antigen-specific IGHG sequences in B cells of vaccinated foals revealed a diverse immunoglobulin repertoire with isotype switching that was comparable between groups and to vaccinated mares. The low expression of CD27 memory marker in antigen-specific B cells, and of cytokines in peripheral blood mononuclear cells upon <i>in vitro</i> immunogen stimulation indicated limited lymphocyte population expansion in response to vaccine during the study period.</p></div

Inhibition monitoring in veterinary molecular testing

Many of the sample matrices typically used for veterinary molecular testing contain inhibitory factors that can potentially reduce analytic sensitivity or produce false-negative results by masking the signal produced by the nucleic acid target. Inclusion of internal controls in PCR-based assays is a valuable strategy not only for monitoring for PCR inhibitors, but also for monitoring nucleic acid extraction efficiency, and for identifying technology errors that may interfere with the ability of an assay to detect the intended target. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians reviewed the different types of internal controls related to monitoring inhibition of PCR-based assays, and provides information here to encourage veterinary diagnostic laboratories to incorporate PCR internal control strategies as a routine quality management component of their molecular testing

Serum KLH- and influenza-specific IgG levels in vaccinated and non-vaccinated mares.

<p>Mares were vaccinated or non-vaccinated at 6 and 3 weeks prior to their expected foaling date, and antigen-specific IgG levels were measured in serum on day 3 after foaling. A) Serum KLH-specific IgG in vaccinated mares (filled circles) trended toward statistically significant greater levels (p = 0.05) in comparison to non-vaccinated mares (open circles). An outlier result is shown in the non-vaccinated group. B) There was no statistical difference (p>0.05) in the serum influenza-specific IgG levels between the vaccinated (filled circles) and non-vaccinated (open circles) mare groups. Note the presence of pre-existing influenza antibodies in the group of non-vaccinated mares despite lack of vaccination with influenza vaccine in the previous 2 years. The horizontal lines indicate median values.</p

Serum KLH-specific or influenza-specific IgG levels.

<p>Linear mixed model analysis was used to compare antigen-specific IgG levels at each time point for the groups: vaccinated foals from non-vaccinated mares (continuous lines; KLH Group A in filled circles; influenza Group C in open circles), vaccinated foals from vaccinated mares (dashed lines; KLH Group B in filled squares; influenza Group D in open squares); non-vaccinated foals from non-vaccinated mares (dotted lines; KLH Group E in filled triangles, influenza Group E in open triangles). Each symbol represents the least square means estimate generated from the data. Significant differences in serum KLH-specific IgG levels were found on days 28, 35, and 42 between Group A and Group E (filled circles versus filled triangles, p<0.0001, denoted by ***). A trend of increased (p = 0.07) serum KLH-specific IgG levels was also found between KLH-vaccinated foals from vaccinated mares (Group B, filled squares) versus non-vaccinated foals from non-vaccinated mares (Group E, filled triangles) on day 42. For influenza, there were no statistical differences (p>0.05) between vaccinated foals (Groups C (open circles) or D (open squares)) and non-vaccinated foals from non-vaccinated mares (Group E, open triangles) at all time points.</p

Germline identity of KLH-specific IGHM sequences.

<p>The percent of germline nucleotide identity of KLH-specific IGHM sequences for each group is presented for each time point. The value for each cloned PCR product is plotted, and median values are indicated by horizontal lines. No statistically significant differences (p>0.05) were identified over time within a Group or between Groups.</p

Germline identity of influenza-specific IGHM sequences.

<p>The percent of germline nucleotide identity of influenza-specific IGHM sequences for each group is presented for day 42. The value for each cloned PCR product is plotted, and the median values are indicated by horizontal lines. The germline identity values of influenza-specific IGHM transcripts from non-vaccinated foals (Group E) were significantly higher than those from influenza-vaccinated foals (Groups C, p = 0.006, and D, p = 0.04).</p