Pulmonary fibrosis and gammaherpesvirus infection in horses

Pulmonary fibrosis is a devastating and often progressive condition leading to exercise intolerance and frequently the demise of the animal. Although uncommonly encountered in horses, the condition is intensely researched both in human medicine and animal models. Viral infections have long been suspected to play a part in the development of pulmonary fibrosis and neoplastic conditions in other species. In 2007, an association between equine herpesvirus 5 (EHV‐5) infection and nodular pulmonary fibrosis in horses was suggested and the name equine multinodular pulmonary fibrosis (EMPF) was introduced. Recently, the presence of EHV‐5 in equine lymphoma has also emerged. The case report by Schwarz et al. in this issue describes a horse suffering from concurrent T cell leukaemia and EMPF in association with EHV‐5. This article summarises current knowledge about EMPF and EHV‐5 infections in horses, recent developments in the understanding of pulmonary fibrosis in man and the proposed contribution of viral infections to pulmonary fibrosis and neoplastic conditions.     

Concurrent T cell leukaemia and equine multinodular pulmonary fibrosis in a Hanoverian Warmblood mare

Equine multinodular pulmonary fibrosis (EMPF) and primary leukaemia are uncommon diseases in horses. This case report describes a horse with both diseases which might be linked via the equine γ‐herpesvirus EHV‐5. In man Epstein Barr Virus (EBV), also a γ‐herpesvirus, is associated with idiopathic pulmonary fibrosis as is EHV‐5 with EMPF. Furthermore, EBV is also associated with lymphoproliferative disease. Similarly in horses, primary leukaemia might be associated with EHV‐5. This is the first report associating EHV‐5 with primary leukaemia in horses.     

Successful outcome in a case of equine multinodular pulmonary fibrosis (EMPF) treated with valacyclovir

Equine multinodular pulmonary fibrosis (EMPF) is a progressive fibrosing interstitial lung disease, which has been associated with γ‐herpesviruses. This report describes a 22‐year‐old Warmblood stallion diagnosed with EMPF on the basis of clinical examination, laboratory results, lung radiographs and ultrasound, lung biopsy and inclusion bodies found in macrophages of broncho‐alveolar lavage fluid (BALF) as well as an identification of EHV‐5 DNA by polymerase chain reaction (PCR). The horse recovered after one week of treatment with valacyclovir (40 mg/kg bwt per os q. 8 h) and is clinically healthy 2 years later. To our knowledge this is the first report describing treatment of EMPF with valacyclovir.     

Equine Multinodular Pulmonary Fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: a case report

A standardbred gelding with a history of 10 days pyrexia and lethargy was referred to the Equine Hospital at the Swedish University of Agricultural Sciences in Uppsala, Sweden.The horse had tachypnea with increased respiratory effort and was in thin body condition. Laboratory findings included leukocytosis, hyperfibrinogenemia and hypoxemia. Thoracic radiographs showed signs of pneumonia with a multifocal nodular pattern, which in combination with lung biopsy findings indicated Equine Multinodular Pulmonary Fibrosis (EMPF). EMPF is a recently described disease in adult horses with clinical signs of fever, weight loss and respiratory problems. The pathological findings include loss of functional pulmonary parenchyma due to extensive nodular interstitial fibrosis which has been related to infection with the equine herpesvirus type 5 (EHV-5). In this case, lung biopsy and tracheal wash samples tested positive for both asinine herpesvirus type 5 (AHV-5) and EHV-5 using PCR assays. The horse failed to respond to treatment and was euthanized for humane reasons. Postmortem examination confirmed the diagnosis of EMPF. This case suggests that not only EHV-5 alone should be considered in association with the development of this disease.     

Bone marrow necrosis and myelophthisis: manifestations of T‐cell lymphoma in a horse

Abstract: A 14‐year‐old spayed American Paint mare was evaluated for mild colic, anorexia, pyrexia, and pancytopenia. Physical examination revealed mild tachycardia, tachypnea, and pale mucous membranes. Serial laboratory analyses revealed progressive pancytopenia, hyperfibrinogenemia, and hyperglobulinemia. A few large atypical cells were observed in peripheral blood smears. Results of tests for equine infectious anemia and antipenicillin antibody were negative. Serum protein electrophoresis indicated a polyclonal gammopathy. Smears of bone marrow aspirates contained hypercellular particles, but cell lines could not be identified because the cells were karyolytic, with pale basophilic smudged nuclei and lack of cellular detail. A diagnosis of bone marrow necrosis was made. Treatment consisted of antimicrobials, nonsteroidal anti‐inflammatory drugs, and corticosteroids. The pyrexia resolved; however, the pancytopenia progressively worsened and petechiation and epistaxis developed. The horse was humanely euthanized. Postmortem examination revealed a diffuse round cell neoplasm infiltrating the kidneys, spleen, lymph nodes, lungs, and bone marrow. Immunophenotyping results (CD3+, CD79α−) indicated the neoplastic cells were of T‐cell lineage. Infiltration of lymphoma cells into the bone marrow appeared to have resulted in severe myelophthisis and bone marrow necrosis. Bone marrow necrosis has been associated previously with lymphoma in humans and dogs. To our knowledge, this is the first reported case of lymphoma resulting in bone marrow necrosis in a horse.     

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.     

Equine herpesvirus 1‐associated ulcerative dermatitis in a horse

This case report describes the clinical and histopathological findings of an infection caused by equine herpesvirus‐1 (EHV‐1) in a horse showing respiratory signs and a papular, crusted and ulcerative dermatitis involving mucosae. This diagnosis was supported by real‐time PCR positive for EHV‐1 on nasal swabs and tissues.     

Molecular Investigation of the Viral Kinetics of Equine Herpesvirus‐1 in Blood and Nasal Secretions of Horses after Corticosteroid‐Induced Recrudescence of Latent Infection

Background: Recrudescence of latent equine herpesvirus 1 (EHV‐1) with subsequent viral shedding via nasal secretions is a potential source of infection for susceptible horses and has been implicated in outbreaks occurring in closed populations. Objectives: To describe the viral kinetics of reactivated EHV‐1 in blood and nasal secretions from latently infected horses after administration of corticosteroids, and to study the infectious nature of reactivated EHV‐1 to sentinel horses. Animals: Eight healthy horses. Methods: Four horses infected 4 months previously with EHV‐1 received dexamethasone on 5 consecutive days. Four seronegative horses served as sentinels and had direct contact with the latently infected horses. All horses were monitored daily for development of clinical signs. Whole blood and nasal secretions were collected daily for molecular detection and cell culture of EHV‐1. Serum was collected weekly for the detection of antibodies against EHV‐1. Results: All horses in the latently infected group showed transient molecular detection of EHV‐1 in blood and nasal secretions, but only 1 horse developed fever. Three latently infected horses developed an increase in antibody concentrations against EHV‐l. Viral cultures remained negative for all latently infected horses after corticosteroid administration. None of the sentinel horses developed clinical signs, viremia, viral shedding, or seroconversion. Conclusions and Clinical Importance: EHV‐1 was successfully reactivated after corticosteroid administration in latently infected horses. However, transmission of reactivated virus to sentinel horses was unsuccessful. Failure to effectively transmit EHV‐1 to susceptible horses may have resulted from the low level and short period of viral shedding in latently infected horses.     

Multinodular pulmonary fibrosis in five horses

CASE DESCRIPTION: 5 horses were evaluated because of decreased appetite, weight loss, fever, cough, tachypnea, and respiratory distress. CLINICAL FINDINGS: Tachycardia, tachypnea, increased respiratory effort, lethargy, fever, poor body condition, and nasal discharge were detected in various combinations on initial physical examination. Evaluation of the lower portion of the respiratory tract via radiography and ultrasonography revealed a severe nodular interstitial pattern. Histologic examination of lung tissue revealed interstitial expansion of alveolar parenchyma with collagen, intraluminal accumulation of neutrophils and macrophages within the alveoli, and occasional intranuclear inclusion bodies within alveolar macrophages. Equine herpesvirus type 5 was detected in samples of lung tissue, bronchoalveolar lavage fluid, or both via polymerase chain reaction assay in all cases. A diagnosis of equine multinodular pulmonary fibrosis (EMPF) was established. TREATMENT AND OUTCOME: Horses were provided supportive treatment and were administered a variety of medications including corticosteroids and acyclovir. Two horses survived and returned to their previous level of activity. Three horses were euthanized because of either deterioration of clinical condition (n=2) or failure to improve within 4 weeks of initiation of treatment (1). CLINICAL RELEVANCE: EMPF should be considered as a differential diagnosis for adult horses with interstitial pneumonia and should be suspected on the basis of characteristic radiographic, ultrasonographic, and histopathologic findings. Equine herpesvirus type 5 is found in association with EMPF; although the exact pathogenic role this virus plays in EMPF is unknown, equine herpesvirus type 5 may be an etiologic agent or cofactor in the development of EMPF.     

应用多重PCR检测和区分3个型的马疱疹病毒

针对马疱疹病毒（EHV）的EHV-1、EHV-2和EHV-4糖蛋白B基因序列，设计、合成了3对特异性引物进行多重PCR，不仅可以在数小时内分别检测这3个型的EHV，而且在同一反应系统内可以清晰地区分EHV-1、EHV-2和EHV-4，其PCR产物大小分别为226、333、570bp，符合预期的片段大小，序列分析证实与已发表的序列一致；该检测方法的灵敏度达到10^3 TCID50；分别从血清学阳性但病毒分离为阴性的1匹进口马组织样品和一些出口前检疫马的鼻咽样品检测到EHV-1和EHV-4特异性核酸。     

Suspected case of hypersplenism as a cause of anaemia,thrombocytopenia and leucopenia in a Miniature Horse gelding

A 23-year-old Miniature Horse gelding was presented to the Lloyd Veterinary Medical Center with a 3-week history of decreased appetite, lethargy and mild intermittent colic. A complete blood count revealed leucopenia, characterised by neutropenia and lymphopenia, as well as anaemia, thrombocytopenia and hyperproteinaemia whereas hypertriglyceridaemia was noted on serum biochemistry profile. Bone marrow evaluation was nondiagnostic and the horse was negative for antiplatelet antibody testing, Coombs test, equine infectious anaemia virus and Anaplasma phagocytophilum. The horse was hospitalised for 36 days and received supportive care, antibiotics, corticosteroids, dextrose-containing fluids and a whole blood transfusion. Following initial improvement and stabilisation, the horse became severely anaemic and acutely recumbent on Day 36 and was subjected to euthanasia. Post-mortem examination provided a diagnosis of hypersplenism with a markedly enlarged spleen along with histiocytic phagocytosis of erythrocytes and platelets. Examination of bone marrow showed appropriate erythroid hyperplasia and no evidence of myelopthisis or neoplasia. This report describes the first presumptive case of primary hypersplenism in an equid as a cause of pancytopenia.     

Molecular pathogenesis of equine coital exanthema: temperature-sensitive function(s) in cells infected with equine herpesviruses

Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 degrees C. The efficiencies of plating (EOP) observed were 10(-2) for both EHV 1 and 2, and 1 X 10(-6) for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34 degrees C and 39 degrees C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 degrees C, reflected those EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34 degrees C and 37 degrees C. Temperature shift experiments with EHV 3-infected cultures revealed that a temperature-sensitive function(s) responsible for the reduction in titer appeared to be a late function(s). All strains examined appeared to incorporate H3-thymidine into viral-density DNA at the non-permissive temperature of 39 degrees C. Electron microscopy of EHV 3-infected cell cultures, incubated continuously at the non-permissive temperature and examined at 18 h after infection, revealed structures consistent with the accumulation of nucleocapsids within the nucleus. The evidence presented is consistent with the hypothesis that in equine dermal cells infected with a plaque-purified wild-type strain of EHV 3 (1118LP), a function needed for the egress of nucleocapsids from the nucleus is absent at 39 degrees C. The significance of these findings relative to the pathogenicity of the disease (equine coital exanthema) caused by this virus is discussed.     

The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989

The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Bam HI fingerprints the commonest virus identified in our study was EHV1.IP (P is for prototype strain). There was a single notable exception in that the Bam HI fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.IB that was identified as a cause of abortion in Central Kentucky in 1970 to 1974. We present evidence that EHV1.IB caused abortion in California in 1964 and has remained unaltered in its Bam HI restriction pattern. No antigenic differences were found among 4 distantly related EHV1 isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHV1 isolates is further evidence for a recent origin for EHV1 and may help to explain the natural history of this virus in the horse in which it seems to be a cause of serious epidemics of abortion and perinatal mortality, and less commonly of encephalitis.     

Myelophthisic pancytopenia in a pony mare

Myelophthisic pancytopenia was diagnosed in a 10-year-old pony mare with a history of recurring colic and anemia. Physical findings were unremarkable, with the exception of pale mucous membranes. Hematologic analysis revealed nonregenerative pancytopenia. Testing for equine infectious anemia and antiglobulin (Coombs) yielded negative results. The mare was treated with antibiotics, boldenone undecylenate, and corticosteroids, but a regenerative bone marrow response was not seen. Postmortem examination revealed severe myelofibrosis and multiple sites of extramedullary hematopoiesis. Myelophthisic pancytopenia develops when a space-occupying lesion destroys sufficient bone marrow or disturbs marrow architecture, resulting in decreased production capacity. Pancytopenia in the pony of this report resulted from inadequate production of blood cellular components secondary to replacement of the bone marrow by myelofibrosis. Cause of the myelofibrosis was not identified.     

Use of antiviral medications against equine herpes virus associated disorders

Numerous types of equine herpesviruses (EHV) continue to afflict horses resulting in a variety of clinical manifestations. While many of the clinical manifestations of EHV are self‐limiting or require only supportive care, some clinical expressions of EHV infections cause severe risk to the horse's overall health and can result in abortion, long‐term deficits or death. Antiviral medications are infrequently utilised therapeutics in equine medicine and their exact efficacy is largely unknown. However, the use of antiviral medications may potentially decrease convalescent time and improve outcome in horses with EHV‐related diseases. Thus, equine practitioners should consider the potential use of antiviral medications in the future. The purpose of this article is to familiarise the equine practitioner with current information in regard to antiviral medications and their potential uses in equine medicine.     

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.     

EQUINE HERPESVIRUSES

The immunological and virological status of 3 foals in respect of equine herpesviruses (EHV) was established and the foals were sequentially infected with EHV2, EHV3 and EHV1. Following experimental infection with EHV2, no clinical signs of disease were observed in any foal. The inoculation of EHV3 into the genital tract resulted in lesions of the mucous membrane and perineal skin that were considered typical of equine coital exanthema. Following intransal inoculation of EHV3 extensive ulceration and pustule formation on the nasal mucosa was observed by day 5 accompanied at day 7 by a profuse, mucopurulent nasal discharge; on day 8 pustular lesions of the skin about the external nares appeared. Signs of disease after inoculation with EHV1 were milder than expected in 2 of the 3 foals. The possibility that recent, prior infection with EHV2 and/or 3 may protect against EHV1 was considered.     

Pharmacokinetics of ganciclovir and valganciclovir in the adult horse

Equine herpes myeloencephalopathy, resulting from equine herpes virus type 1 (EHV‐1) infection, is associated with substantial morbidity and mortality in the horse. As compared to other antiviral drugs, such as acyclovir, ganciclovir has enhanced potency against EHV‐1. This study investigated the pharmacokinetics of ganciclovir and its oral prodrug, valganciclovir, in six adult horses in a randomized cross‐over design. Ganciclovir sodium was administered intravenously as a slow bolus at a dose of 2.5 mg/kg, and valganciclovir was administered orally at a dose of 1800 mg per horse. Intravenously administered ganciclovir disposition was best described by a three‐compartment model with a prolonged terminal half‐life of 72 ± 9 h. Following the oral administration of valganciclovir, the mean observed maximum serum ganciclovir concentration was 0.58 ± 0.37 μg/mL, and bioavailability of ganciclovir from oral valganciclovir was 41 ± 20%. Superposition predicted that oral dosing of 1800‐mg valganciclovir two times daily would fail to produce and maintain effective plasma concentrations of ganciclovir. However, superposition suggested that i.v. administration of ganciclovir at 2.5 mg/kg every 8 h for 24 h followed by maintenance dosing of 2.5 mg/kg every 12 h would maintain effective ganciclovir serum concentrations in most horses throughout the dosing interval.     

Equine herpesvirus 1 and 4 infections: an update

Equine herpesvirus 1 (EHV1) and equine herpesvirus 4 (EHV4) are important ubiquitous equine viral pathogens, causing much damage to the horse industry. EHV1 strains are associated with respiratory disease, abortion, and paresis/paralysis, whereas EHV4 strains are predominantly associated with respiratory disease. In the past decades much research effort has been put into improving knowledge about these viruses. In this paper the current state of knowledge of these viruses and the most important aspects of these virus infections, e.g. epidemiology, clinical aspects, pathogenesis and pathology, immunity, diagnosis, preventive management and management in the course of an outbreak and vaccination, is reviewed. Because we performed some research ourselves in the areas of diagnosis, epidemiology and vaccinology these aspects are reviewed in more depth than the other aspects. Still many questions have remained and new questions have risen. Consequently, research priorities should be made in an attempt to answer these questions. Therefore, this review ends with some personal recommendations for important priorities for future research.