R. Frank Cook

Effects of systemic inflammation on insulin sensitivity in horses and inflammatory cytokine expression in adipose tissue

OBJECTIVE To determine whether an inflammatory challenge induces insulin resistance in horses and examine possible contributions of adipose tissue to inflammatory cytokine production. ANIMALS 15 adult mares. PROCEDURES Lipopolysaccharide (0.045 mug/kg, IV) or saline solution was administered, and insulin sensitivity was determined by means of the hyperinsulinemic, euglycemic clamp procedure or an adipose tissue biopsy was performed. Adipose tissue samples were collected, and mature adipocytes were obtained. Mature adipocytes were stimulated with lipopolysaccharide or dedifferentiated into preadipocytes and then stimulated with lipopolysaccharide. Interleukin-1, interleukin-6, and tumor necrosis factor A expression in blood, adipose tissue, and adipocytes was quantified with a real-time, reverse transcriptase- PCR assay. RESULTS Lipopolysaccharide induced a transient increase in insulin sensitivity followed by a reduction in insulin sensitivity at 24 hours. Increased cytokine expression was observed in blood and adipose tissue following administration of lipopolysaccharide, and adipocytes and preadipocytes stimulated with lipopolysaccharide stained positive for tumor necrosis factor A. Expression of interleukin-1, interleukin-6, and tumor necrosis factor A was detected in preadipocytes stimulated with lipopolysaccharide, and interleukin-6 and tumor necrosis factor A were detected in mature adipocytes stimulated with lipopolysaccharide. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that insulin resistance develops following systemic inflammation in horses and suggested that adipose tissue may contribute to this inflammatory response. Methods to regulate insulin sensitivity may improve clinical outcome in critically ill patients.

The Increased Prevalence of Neuropathogenic Strains of EHV-1 in Equine Abortions

A panel of 426 archived EHV-1 isolates collected (1951-2006) from equine abortions was analyzed using a real-time Taq-Man((R)) allelic discrimination PCR assay. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A(2254)) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G(2254)) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A(2258)-->C(2258)) in ORF30, in addition to A(2254)-->G(2254). Interestingly, the non-neuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A(2254)-->G(2254) and A(2258)-->C(2258) substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.

A Review of Techniques for the Serologic Diagnosis of Equine Infectious Anemia

History and introduction This paper will review the current serologic tests to Equine infectious anemia (EIA) has been recognized diagnose infections with EIAV, discuss limitations of as an important viral disease of members of the horse the procedures, and suggest improvements based on family since its “filterable virus” etiology was proven early in the twentieth century. Only since the 1970s EIAV and the horse have we been able to diagnose infection with equine infectious anemia virus (EIAV) accurately with the use The EIAV is a member of the lentivirus subfamily of serodiagnostic methods. Early attempts to define of retroviruses and shares morphologic, antigenic, and specific diagnostic tools for EIA failed in part because genetic characters with the other lentiviruses. 17 The of a lack of suitable in vivo laboratory animal models major core protein of EIAV is a product of the viral and in vitro techniques for virus propagation, as well gag gene, 45 has a molecular weight of 26,000 daltons as the tendency of EIAV to undergo “antigenic drift.” (p26), 37 represents about 40% of the virion mass, The recognition of the highly mutable typeor subtypeand has group and interspecies antigenic determispecific antigens of EIAV which stimulated neutraliznants. 12,30,31 The major core protein appears to be highing antibodies suggested that a widely applicable diagly conserved between EIAV isolates, 44 and the imnostic test for the disease would not be forthcoming. munodominant regions of the protein have been Complement fixation and precipitin tests were tried elucidated. 4 Antibodies to the major core protein are but held little promise as accurate and/or practical didetected in the AGID test because the p26 antigen agnostics. 21,43 Horse inoculation tests were standardpredominates in antigen preparations. Occasional reized for the diagnosis of EIA, but were expensive, actions to the pl5 protein of EIAV have been noted wasteful of equine resources and required facilities in in the AGID test. 25,47

Acute systemic inflammation transiently synchronizes clock gene expression in equine peripheral blood

Peripheral clocks receive timing signals from the master mammalian pacemaker in the suprachiasmatic nucleus (SCN) and function to adaptively anticipate daily changes that influence local physiology. Evidence suggests that peripheral immune activation may act as a resetting signal for circadian clocks in peripheral tissues. We wished to investigate whether acute systemic inflammation could synchronize clock gene expression in equine peripheral blood, a tissue that does not normally oscillate in this species. We report that in vivo administration of lipopolysaccharide (LPS) results in significant upregulation of the core clock genes Per2 and Bmal1 in equine blood, in association with an acute rise in tumor necrosis factor (TNF) alpha and core body temperature compared to vehicle-treated control animals. Furthermore, co-administration of LPS and phenylbutazone, a non-steroidal anti-inflammatory drug (NSAID) known to inhibit prostaglandin (PG) E(2) synthesis in the horse, prevents both the febrile response and the synchronized increase in clock gene expression. However, the rise in Per2 and Bmal1 expression cannot be replicated in equine peripheral blood mononuclear cells (PBMCs) ex vivo by treatment with PGE(2), LPS or a heat shock mimicking the in vivo febrile response. These results may suggest an indirect communication pathway between immune modulators and the molecular machinery of cell clocks in peripheral blood. This potential immune feedback regulation of an equine peripheral clock implies a role for the circadian system in contributing to innate immune reactions and maintaining homeostasis in a tissue that acts as the first line of defense during an infectious challenge.

Evidence of an oscillating peripheral clock in an equine fibroblast cell line and adipose tissue but not in peripheral blood

The master mammalian pacemaker in the brain controls numerous diverse physiological and behavioral processes throughout the organism. Timing information is continually transmitted from the master clock to peripheral organs to synchronize rhythmic daily oscillations of clock gene transcripts and control local physiology. To investigate the presence of peripheral clocks in the horse, quantitative real-time RT-PCR assays were designed to detect levels of equine clock genes. Expression profiles for Per2, Bmal1 and Cry1 were first determined in a synchronized equine cell line. Subsequently, expression in equine whole blood and adipose tissue was assessed. Robust circadian oscillations of Per2, Bmal1 and Cry1 were observed in vitro. A synchronized molecular clock was also demonstrated in equine adipose tissue although oscillation of Bmal1 was less robust than that of Per2 and Cry1. In contrast to previous studies in humans and rats however, there was no evidence of synchronized clock gene expression in equine peripheral blood. These studies suggest that synchronous control of clock gene oscillation in equine peripheral blood is not as tightly regulated as in other species and may reflect the influence of different evolutionary challenges modifying the function of a peripheral clock.

Assessment of correlation between in vitro CD3+ T cell susceptibility to EAV infection and clinical outcome following experimental infection.

In a recent study, we demonstrated that the virulent Bucyrus strain (VBS) of EAV could infect in vitro a small population of CD3(+) T lymphocytes from some but not all horses. Furthermore, we have shown that a common haplotype is associated with this in vitro CD3(+) T cell susceptibility/resistance phenotype to EAV infection. In this study, we investigated whether the differences in the susceptibility or resistance of CD3(+) T cells in vitro correlate with the outcome and severity of clinical signs in vivo. Thus, horses were divided into two groups based on their CD3(+) T cell susceptible or resistant phenotype. Following experimental inoculation with the recombinant VBS of EAV, horses were assessed for presence and severity of clinical signs, duration and magnitude of virus shedding, as well as production of proinflammatory and immunomodulatory cytokines in peripheral blood mononuclear cells using real-time quantitative RT-PCR. The data showed that there was a significant difference between the two groups of horses in terms of cytokine mRNA expression and evidence of increased clinical signs in horses possessing the in vitro CD3(+) T cell resistant phenotype. This is the first study to provide direct evidence for a correlation between variation in host genotype and phenotypic differences in terms of the extent of viral replication, presence and severity of clinical signs and cytokine gene expression caused by infection with virulent EAV.

New Real-Time PCR Assay Using Allelic Discrimination for Detection and Differentiation of Equine Herpesvirus-1 Strains with A2254 and G2254 Polymorphisms

ABSTRACT A single-nucleotide polymorphism (A2254 or G2254) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E2) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69–72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E1) was developed by redesigning primers and probes specific to ORF30. The E1 and E2 rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A2254 or G2254) in all archived isolates plus 168 of the clinical samples. The E1 assay was 10 times more sensitive than E2, with a lower detection limit of 10 infectious virus particles. Furthermore, all A2254 and G2254 genotypes along with samples from three cases of dual infection (A2254+G2254) were correctly identified by E1, whereas E2 produced 20 false dual positive results with only one actual mixed A2254+G2254 genotype confirmed. Based on these findings, E1 offers greater sensitivity and accuracy for the detection and A/G2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

Equine Infectious Anemia in 2014: Live with It or Eradicate It?

In the absence of an effective vaccine, the success of the test and removal approach for the control of equine infectious anemia (EIA) cannot be overstated, at least in those areas where testing has been traditionally routine. This article addresses 4 main aspects: what has been learned about EIA virus, host control of its replication, and inapparent carriers; international status regarding the control of EIA; diagnostic and laboratory investigation; and reducing the spread of blood-borne infections by veterinarians. An attempt is made to put these issues into practical contemporary perspectives for the equine practitioner.

Geographic structuring of global EIAV isolates: a single origin for New World strains?

Equine infectious anaemia virus (EIAV) is classified within the Retroviridae and, like other lentivirus, has the propensity for considerable antigenic variation. An extensive phylogenetic analysis in Bayesian fashion, with significant amounts of new EIAV gag sequence information, revealed a strong geographic compartmentalization clearly related to the phylogeographic history of modern horses, pointing out that New World EIAV strains form a distinct group with a potentially common origin. This evidence suggests that a single founder event may have occurred during the reintroduction of horses to the Americas by European colonists in the 15th century, a possibility that raises many interesting scenarios with implications for all evolutionary and ecological studies.

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8+ T and CD21+ B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract

ABSTRACT Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation. IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallions reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.