Impact of the host immune response on the development of equine herpesvirus myeloencephalopathy in horses.

Herpesviruses establish a well-adapted balance with their host's immune system. Despite this co-evolutionary balance, infections can lead to severe disease including neurological disorders in their natural host. In horses, equine herpesvirus 1 (EHV-1) causes respiratory disease, abortions, neonatal foal death and myeloencephalopathy (EHM) in ~10 % of acute infections worldwide. Many aspects of EHM pathogenesis and protection from EHM are still poorly understood. However, it has been shown that the incidence of EHM increases to >70 % in female horses >20 years of age. In this study we used old mares as an experimental equine EHV-1 model of EHM to identify host-specific factors contributing to EHM. Following experimental infection with the neuropathogenic strain EHV-1 Ab4, old mares and yearling horses were studied for 21 days post-infection. Nasal viral shedding and cell-associated viremia were assessed by quantitative PCR. Cytokine/chemokine responses were evaluated in nasal secretions and cerebrospinal fluid (CSF) by Luminex assay and in whole blood by quantitative real-time PCR. EHV-1-specific IgG sub-isotype responses were measured by ELISA. All young horses developed respiratory disease and a bi-phasic fever post-infection, but only 1/9 horses exhibited ataxia. In contrast, respiratory disease was absent in old mares, but all old mares developed EHM that resulted in euthanasia in 6/9 old mares. Old mares also presented significantly decreased nasal viral shedding but higher viremia coinciding with a single fever peak at the onset of viremia. According to clinical disease manifestation, horses were sorted into an EHM group (nine old horses and one young horse) and a non-EHM group (eight young horses) for assessment of host immune responses. Non-EHM horses showed an early upregulation of IFN-α (nasal secretions), IRF7/IRF9, IL-1ß, CXCL10 and TBET (blood) in addition to an IFN-γ upregulation during viremia (blood). In contrast, IFN-α levels in nasal secretions of EHM horses were low and peak levels of IRF7, IRF9, CXCL10 and TGF-ß (blood) coincided with viremia. Moreover, EHM horses showed significantly higher IL-10 levels in nasal secretions, peripheral blood mononuclear cells and CSF and higher serum IgG3/5 antibody titres compared to non-EHM horses. These results suggest that protection from EHM depends on timely induction of type 1 IFN and upregulation cytokines and chemokines that are representative of cellular immunity. In contrast, induction of regulatory or TH-2 type immunity appeared to correlate with an increased risk for EHM. It is likely that future vaccine development for protection from EHM must target shifting this 'at-risk' immunophenotype.

Anti-inflammatory drugs decrease infection of brain endothelial cells with EHV-1 in vitro.

BACKGROUND: Equine herpesvirus-associated myeloencephalopathy is the result of endothelial cell infection of the spinal cord vasculature with equine herpesvirus-1 (EHV-1) during cell-associated viraemia. Endothelial cell infection requires contact between infected peripheral blood mononuclear and endothelial cells. Inflammation generated during viraemia likely upregulates adhesion molecule expression on both cell types increasing contact and facilitating endothelial cell infection. OBJECTIVES: Evaluating the role of anti-inflammatory drugs in decreasing endothelial cell infection with EHV-1. STUDY DESIGN: In vitro assay, crossover design, multiple drug testing. METHODS: In vitro modified infectious centre assay using immortalised carotid artery endothelial cells or primary brain endothelial cells with plaque counts per well as outcome. Cells were either anti-inflammatory drug treated or left untreated. RESULTS: Significant reduction of plaque count when cells were treated compared with untreated cells. No dose-dependent effect when drug concentrations were increased to 10× dose. Treatment of both peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) is required for significant plaque count reduction. MAIN LIMITATIONS: In vitro study. CONCLUSIONS: Anti-inflammatory drugs decrease infection of endothelial cells likely by reducing contact between EHV-1 infected PBMC and endothelial cells in vitro. The role of adhesion molecules in this process needs further investigation. In vitro results suggest anti-inflammatory drug therapy during EHV-1 infection and viraemia in horses could be clinically relevant.

Plasma D-dimer concentrations during experimental EHV-1 infection of horses.

BACKGROUND: Central nervous system blood vessel thrombosis is a part of the pathogenesis of equid herpesvirus-associated myeloencephalopathy (EHM). D-dimers (DD) are stable breakdown products of cross-linked fibrin, and increased DD-plasma concentrations could reflect the degree of systemic coagulation during EHV-1 infection. HYPOTHESIS: We hypothesized that blood DD concentrations will be increased during periods of EHV-1 fever and viremia, reflecting an activated coagulation cascade with fibrinolysis. ANIMALS: Twenty-eight equids were infected with EHV-1 in 3 experimental infection studies. Three (uninfected) horses were included in a separate study to evaluate methodology for DD concentration measurements. METHODS: Clinical data and quantitative viremia were evaluated, and DD concentrations were measured in blood samples on the day before the infection and during days 1-12 postchallenge. Uninfected horses were sampled every 3 hours for 48 hours. Logistic and linear regression was used to investigate the potential association between the fever and viremia with the presence or absence of DD concentrations in peripheral blood. RESULTS: DD concentrations were increased for 1-8 days in the majority of infected animals. Both viremia (odds ratio [OR] 6.3; 95% confidence interval [CI] 3.4-11.8; P = .0013) and fever (OR 4.9; CI 2.3-10.1; P = .001) were strongly associated with the likelihood of detecting DD in peripheral blood. CONCLUSIONS AND CLINICAL IMPORTANCE: EHV-1 viremia is associated with increases in DD concentration in horses and ponies. This indicates that EHV-1 viremia can lead to an activation of coagulation and fibrinolysis.

Infection of central nervous system endothelial cells by cell-associated EHV-1.

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia.

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for ß-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.

Detection and management of an outbreak of equine herpesvirus type 1 infection and associated neurological disease in a veterinary teaching hospital.

BACKGROUND: Because of the serious disease sequelae associated with equine herpesvirus type 1 (EHV-1) infections, awareness and control measures used to control outbreaks are important issues for all horse populations. OBJECTIVES: Describe the occurrence and management of an outbreak of EHV-1 infection at a veterinary hospital. ANIMALS: Horses hospitalized at a referral veterinary hospital. METHODS: A horse with myeloencephalopathy associated with EHV-1 infection (EHM) was admitted for diagnostic evaluation and treatment under strict infection control procedures. We describe the occurrence and management of a nosocomial outbreak of EHV-1 infections associated with admission of this patient. RESULTS: Despite institution of rigorous biosecurity precautions at the time of admission of the index case, EHV-1 infections spread to 6 other horses that were hospitalized at the James L. Voss Veterinary Teaching Hopsital, including 2 that served as sources of infection for horses on their home premises after discharge. Infection with EHV-1 was confirmed by polymerase chain reaction (PCR) and by seroconversion documented by glycoprotein G ELISA. A voluntary quarantine was imposed and admissions were restricted to prevent additional horses from being exposed. Quarantine duration was abbreviated by serial testing of all horses with PCR. CONCLUSIONS AND CLINICAL IMPORTANCE: These findings illustrate the contagious disease risk that can accompany management of horses with EHM. Horses with active nasal EHV-1 shedding should be isolated in an airspace that is separate from other horses by strictly enforced biosecurity and isolation procedures. Serial testing with PCR may be a useful adjunct to determine when the risk of transmission has been minimized.

Control of EHV-1 viremia and nasal shedding by commercial vaccines.

Equine herpesvirus-1 is a cause of outbreaks of abortion and neurological disease. The pathogenesis of both these diseases depends on establishment of viremia. An experiment was performed to determine the protective efficacy of two commercially available vaccines used with an optimized 3-dose vaccination regime: a modified-live viral (MLV) and a high antigen load killed vaccine licensed for abortion control. The study design was a blinded, randomized challenge trial. Three groups of 8 yearling ponies received one of three treatments: MLV vaccine (Rhinomune, Boehringer Ingelheim Vetmedica, Inc.); killed vaccine (Pneumabort-K, Pfizer Animal Health); or a placebo (control group). Three vaccinations were administered at intervals of 27 and 70 days followed by challenge infection 24 days later. Clinical disease after challenge was significantly reduced in both vaccine groups; the reduction was greater in the MLV vaccine group. Nasal shedding was reduced by at least 1-2 logs in both vaccine groups. The number of days of viremia was significantly reduced in the killed vaccine group only. This study demonstrated that both commercial vaccines significantly suppressed EHV-1 disease and nasal viral shedding, and one vaccine suppressed days of viremia.

Evaluation of nephelometry for albumin measurement in serum and cerebrospinal fluid: experiences with an indwelling subarachnoidal catheter system for repetitive cerebrospinal fluid collection in horses.

The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive CSF samples is a novel technique, routine cytological CSF analysis was performed along with daily clinical evaluation to ascertain the well-being of the horses. The catheters were placed in 2 horses for periods of 14 and 17 days. One horse exhibited pleocytosis on cytological evaluation of CSF on 2 occasions for a 1-2-day duration; however, the AQ showed a significant increase on only 1 occasion. The other horse had a normal cell count in CSF but showed 2 sudden changes in the AQ value; however, these values remained within the 95% confidence interval for AQ in horses. Albumin quotient values of the second horse were consistently below the lower range of the confidence interval. Results from this study indicate that nephelometry can be used for albumin determination in serum and CSF samples from horses. Furthermore, an indwelling subarachnoidal catheter system can provide serial CSF samples in horses, thus obviating the need for repetitive centesis for serial CSF sampling.

Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004.

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our understanding of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field.

Neurological syndromes among horses in The Netherlands. A 5 year retrospective survey (1999-2004).

The presence of toxins or infectious agents combined with environmental factors in combination with a susceptible host can be the cause for neurological disease in groups of horses. During a 5 year observational period outbreaks of neurological diseases among horses were evaluated. Causes of occurring neurological diseases were equine botulism, lolitrem intoxications, equine herpesvirus type 1-associated myelo(encephalo)pathy, and encephalitis caused by (disseminated) Streptococcus equi subspecies equi infection. This article focuses on the first three syndromes because of their predominant influence on locomotion. The pathogenesis of each disease is presented, followed by a description of a general presentation of the diseases as encountered under Dutch circumstances.

Brain abscesses as a metastatic manifestation of strangles: symptomatology and the use of magnetic resonance imaging as a diagnostic aid.

REASONS FOR PERFORMING STUDY: The occurrence of unexpectedly high numbers of horses with neurological signs during two outbreaks of strangles required prompt in-depth researching of these cases, including the exploration of magnetic resonance imaging (MRI) as a possible diagnostic technique. OBJECTIVES: To describe the case series and assess the usefulness of MRI as an imaging modality for cases suspected of space-occupying lesions in the cerebral cavity. METHODS: Four cases suspected of suffering from cerebral damage due to Streptococcus equi subsp. equi infection were examined clinically, pathologically, bacteriologically, by clinical chemistry (3 cases) and MRI (2 cases). In one case, MRI findings were compared to images acquired using computer tomography (CT). RESULTS: In all cases, cerebral abscesses positive for Streptococcus equi subsp. equi were found, which explained the clinical signs. Although the lesions could be visualised with CT, MRI images were superior in representing the exact anatomic reality of the soft tissue lesions. CONCLUSIONS: The diagnosis of bastard strangles characterised by metastatic brain abscesses was confirmed. MRI appeared to be an excellent tool for the imaging of cerebral lesions in the horse. POTENTIAL CLINICAL RELEVANCE: The high incidence of neurological complications could not be explained but possibly indicated a change in virulence of certain strains of Streptococcus equi subsp. equi. MRI images were very detailed, permitting visualisation of much smaller lesions than demonstrated in this study and this could allow prompt clinical intervention in less advanced cases with a better prognosis. Further, MRI could assist in the surgical treatment of brain abscesses, as has been described earlier for CT.