Gillian A. Perkins

A point mutation in a herpesvirus polymerase determines neuropathogenicity

Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission.

Plasma adrenocorticotropin (ACTH) concentrations and clinical response in horses treated for equine Cushing's disease with cyproheptadine or pergolide

Plasma ACTH levels have been variable in horses with a positive clinical response for therapy for equine Cushings Disease (ECD). Therefore, our purpose was to determine the value of monitoring plasma adrenocorticotropin (ACTH) levels during treatment of equine Cushings disease (ECD) with either cyproheptadine (n = 32) or pergolide (n = 10). First, we validated the chemiluminescent ACTH assay (specificity, precision, accuracy, intra-assay and interassay variations) and tested methods of handling the whole blood from the time of collection to when the ACTH was assayed. The sensitivity and specificity of high plasma ACTH levels for detecting ECD was determined in a retrospective study on hospitalised horses (n = 68). Surveys were sent to veterinarians who submitted equine ACTH levels that were high initially and had at least 2 ACTH samples to determine the value of monitoring ACTH levels during therapy of ECD. The ACTH chemiluminescent assay was valid. The ACTH was stable when whole blood was collected and held in plastic tubes for 8 h before separating the plasma. The sensitivity and specificity of plasma ACTH levels for detecting ECD were 84% (n = 19,95% CI 60,97) and 78% (n = 49,95% CI 63,88), respectively. Treated horses generally showed a decrease in plasma ACTH. Plasma ACTH levels may be helpful when monitoring therapy of ECD, although improvement in clinical signs should be considered most important. There were no differences between cyproheptadine and pergolide in terms of improvements in any of the clinical signs.

A single-nucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease.

Epidemiological studies have shown that a single-nucleotide polymorphism in the equid herpesvirus type 1 DNA polymerase gene is associated with outbreaks of highly lethal neurological disease in horses. Reverse genetics experiments further demonstrated that a G(2254) A(2254) nucleotide mutation introduced in neurovirulent strain Ab4, which resulted in an asparagine for aspartic acid substitution (D(752) N(752)), rendered the virus nonneurovirulent in the equine. Here, we report that the nonneurovirulent strain equid herpesvirus type 1 strain NY03 caused lethal neurological disease in horses after mutation of A(2254) G(2254) (N(752) D(752)), thereby providing final proof that the D(752) allele in the viral DNA polymerase is necessary and sufficient for expression of the lethal neurovirulent phenotype in the natural host. Although virus shedding was comparable between the N(752) and D(752) variants, infection with the latter was accompanied by efficient establishment of prolonged cell-associated viremia in peripheral blood mononuclear cells and neurological disease in 2 of 6 animals.

Clostridial myonecrosis in horses (37 cases 1985–2000)

REASONS FOR PERFORMING STUDY Previous reports of clostridial myonecrosis have either focused on individual case reports or have been small retrospective studies reporting very high mortality rates. OBJECTIVES The objective of this study was to describe the outcome of cases of clostridial myonecrosis submitted to 2 referral equine hospitals in the United States over a 15 year period. METHODS A retrospective study of case material selected on the basis of positive Clostridium spp. culture or the identification of Clostridium spp. by specific fluorescent antibody testing from soft tissue wounds was performed at Cornell and Wisconsin. RESULTS 37 cases of clostridial myonecrosis were documented. Twenty-seven horses survived, 8 were subjected to euthanasia and 2 died during treatment for an overall survival rate of 73%. Twenty-five cases (68%) were associated with Clostridium perfringens alone, 6 cases (16%) with Cl. septicum alone, 4 cases with mixed clostridial infections (11%), 1 case with Cl. sporogenes and 1 with an unspeciated Clostridium spp. The highest survival rate of 81% was documented for those cases from which Cl. perfringens alone was isolated. The most common antecedent condition prior to referral was colic. The myonecrotic lesion occurred within 6-72 h of a soft tissue injection in 34 cases but was associated with a wound or laceration in the remaining 3 cases. Of the 34 cases associated with recent injections, 24 were associated with i.m. injections in the cervical region, 4 in the semimembranosus/semitendinosus region, 3 in the gluteal region, 2 with perivascular leakage of drugs administered into the jugular vein and 1 case developed simultaneously in the gluteal and neck region following injections at both sites. CONCLUSIONS Clostridial myonecrosis can occur following the i.m. or inadvertent perivascular administration of a wide variety of commonly administered drugs. It is most common in the neck musculature. Aggressive treatment can be associated with survival rates of up to 81% for cases due to Cl. perfringens alone. Survival rates for other Clostridial spp. tend to be lower. POTENTIAL RELEVANCE A combination of high dose i.v. antibiotic therapy and surgical fenestration/debridement is the best approach to cases of clostridial myonecrosis. With rapid diagnosis and therapeutic intervention, horses may have up to an 81% chance of survival.

Detection of Equine Herpesvirus-1 in Nasal Swabs of Horses by Quantitative Real-Time PCR

BACKGROUND Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples. OBJECTIVES To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses. ANIMALS Fifteen horses experimentally infected with EHV-1. METHODS Experimental study: Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Mareks disease virus as an internal control. qPCR was performed. RESULTS The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI (P< .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86-100) and 27% (95% CI: 20-35). CONCLUSIONS AND CLINICAL IMPORTANCE We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C(T) values are provided as well as justification of a minimum 10-day quarantine period.

Effective Treatment of Respiratory Alphaherpesvirus Infection Using RNA Interference

Background Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNAs), delivered intranasally, could prove a valuable alternative for infection control. siRNAs against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrease EHV-1 infection in a mouse model. Methodology/Principal Fndings siRNA therapy in vitro significantly reduced virus production and plaque size. Viral titers were reduced 80-fold with 37.5 pmol of a single siRNA or with as little as 6.25 pmol of each siRNA when used in combination. siRNA therapy in vivo significantly reduced viral replication and clinical signs. Intranasal treatment did not require a transport vehicle and proved effective when given up to 12 h before or after infection. Conclusions/Significance siRNA treatment has potential for both prevention and early treatment of EHV-1 infections.

The Effect of Adding Oral Dexamethasone to Feed Alterations on the Airway Cell Inflammatory Gene Expression in Stabled Horses Affected with Recurrent Airway Obstruction

BACKGROUND Chemokine expression in airway epithelium and bronchoalveolar lavage fluid (BALF) cells of horses with recurrent airway obstruction (RAO) is increased. HYPOTHESIS For RAO-affected horses that are stabled and fed a pelleted ration, the addition of oral dexamethasone further improves pulmonary function and reduces inflammatory gene expression in pulmonary cells. ANIMALS Twelve RAO-affected horses. METHODS In a randomized cross-over experiment, the effect of feeding pellets in lieu of hay to stabled, RAO-affected horses was compared with the effect of feeding pellets and administering a 21-day decreasing dose regimen of oral dexamethasone on the expression (by kinetic polymerase chain reaction) of interleukin-8 (IL-8), chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, IL-6, and beta-actin in the BALF cells and of IL-8, CXCL2, 2 IL-1 receptor (IL-1R2), Toll-like receptor 4 (TLR4), and glyceraldehyde 3-phosphate dehydrogenase in the bronchial epithelium 2 days after the final dose. RESULTS Both treatments reduced airway neutrophilia and breathing efforts but the addition of dexamethasone was associated with fewer treatment failures. Compared with feed changes alone, dexamethasone administration further reduced the expression of IL-8, CXCL2, and IL-1beta in the BALF cells 3.3-, 2.5-, and 4.7-fold, respectively. In the airway epithelium, both treatments were equally efficacious in reducing the expression of IL-8 and CXCL2 expression relative to pretreatment values, but either treatment failed to alter the expression of IL-1R2 and TLR4. CONCLUSIONS AND CLINICAL IMPORTANCE For a rapid and consistent improvement in pulmonary function and a reduction in inflammatory gene expression of the BALF cells, a decreasing dose of oral dexamethasone in combination with feed alterations is more efficacious for horses that must remain stabled.

The development of equine immunity: Current knowledge on immunology in the young horse

The development of equine immunity from the fetus to adulthood is complex. The foals immune response and the immune mechanisms that they are equipped with, along with changes over the first months of life until the immune system becomes adult-like, are only partially understood. While several innate immune responses seem to be fully functional from birth, the onset of adaptive immune response is delayed. For some adaptive immune parameters, such as immunoglobin (Ig)G1, IgG3, IgG5 and IgA antibodies, the immune response starts before or at birth and matures within 3 months of life. Other antibody responses, such as IgG4, IgG7 and IgE production, slowly develop within the first year of life until they reach adult levels. Similar differences have been observed for adaptive T cell responses. Interferon-gamma (IFN-γ) production by T helper 1 (Th1)-cells and cytotoxic T cells starts shortly after birth with low level production that gradually increases during the first year of life. In contrast, interleukin-4 (IL-4) produced by Th2-cells is almost undetectable in the first 3 months of life. These findings offer some explanation for the increased susceptibility of foals to certain pathogens such as Rhodococcus equi. The delay in Th-cell development and in particular Th2 immunity during the first months of life also provides an explanation for the reduced responsiveness of young horses to most traditional vaccines. In summary, all immune components of adult horses seem to exist in foals but the orchestrating and regulation of the immune response in immature horses is strikingly different. Young foals are fully competent and can perform certain immune responses but many mechanisms have yet to mature. Additional work is needed to improve our understanding of immunity and immune regulation in young horses, to identify the preferred immune pathways that they are using and ultimately provide new preventive strategies to protect against infectious disease.

Report of the third Havemeyer workshop on infection control in equine populations

Infectious diseases are an ever present threat to the health of individual horses, local, regional and national herds and the equine industry as a whole. Treating infectious disease has always been one of the foundations of veterinary medicine. However, infectious disease prevention is becoming increasingly important because of the high visibility of recent outbreaks of infectious disease, the increasing frequency and ease of national and international horse movement and emergence and re-emergence of equine pathogens. This is especially true in large, transient horse populations. For many veterinarians and facility managers, infection control remains a subject they are reluctant to discuss for fear of it reflecting poorly on them, yet management of infectious diseases is an unavoidable issue that must be addressed by all equine practitioners regarding every equine population under their care. The field of veterinary infection control, although still young compared with equivalent efforts in human medicine, has advanced considerably in the last decade thanks to those that have willingly and openly shared their experiences – both good and bad – with regard to hospital and field-based outbreaks of infectious diseases and subsequent mitigation efforts. In veterinary hospitals, infection control is often not considered until after individual patient care is addressed and thus prevention of infectious disease transmission can become a secondary activity relative to the treatment of individual horses and a tertiary activity relative to the care of the larger hospital population. Thus infection control has often been largely reactive rather than proactive in many equine facilities. Control efforts are sometimes hurriedly implemented after a disease outbreak is well underway, rather than being used to prevent sporadic cases from escalating into an outbreak. It is clear that outbreaks of infectious disease can occur with alarming frequency, even in highly controlled environments such as veterinary teaching hospitals. A survey of personnel responsible for infection control at 38 American Veterinary Medical Association (AVMA) accredited veterinary teaching hospitals found that 82% had identified at least one outbreak of hospital-associated disease in the previous 5 years with 32% reporting outbreaks so significant that hospital closure was utilised to aid in mitigation efforts [1]. Outbreaks are undoubtedly under-reported in the scientific literature but a variety of organisms have been previously documented as causes of epidemic disease in equine hospitals, including Salmonella enterica [2–5], methicillin-resistant Staphylococcus aureus (MRSA) [6–8], equine herpesvirus type 1 [9–11] and Cryptosporidium [12]. However, equine infectious diseases are not solely associated with veterinary hospitals. The last decade has seen the epidemic spread of West Nile virus in North America, a major equine influenza epidemic in Australia, outbreaks of contagious equine metritis in the USA and South Africa, the re-emergence of piroplasmosis in the USA and dramatically increasing concerns regarding equine herpesvirus myeloencephalopathy and multidrug resistant bacterial pathogens (e.g. MRSA). There are several significant differences with regard to the practice of infection control in equine populations outside of veterinary hospitals (i.e. ‘in the field’). The focus in field situations is likely to be the protection of a relatively healthy population of horses from disease incursion, rather than protection of hospitalised patients with varying degrees of compromised health. The differences are similar to those between protection of public health in the community and infection control in human hospitals. Infection control in the field has often focused heavily on vaccination; however, effective vaccines cannot fully protect all horses as vaccines are not available for many diseases and vaccination cannot be used to control emerging diseases. A fundamental shift in the mindset of clinicians and horse owners must occur whereby vaccination is considered a last line of defence and overall infection prevention emphasises implementation of other control measures. This is particularly critical in large, transient horse populations that gather for specific events due to the potential for serious and widespread repercussions when these populations disperse, as was seen following the National Cutting Horse Association Western National Championship in Ogden, Utah, in May 2011. It is likely that exposure to a single horse shedding equine herpesvirus-1 (EHV-1) at this event resulted in over 165 horses developing clinical disease that was known or suspected to be caused by EHV-1 and at least 13 of these horses were subjected to euthanasia. The outbreak spanned at least 10 western US states and 2 Canadian provinces ([13], P.S. Morley, personal communication 2012). Clearly infectious diseases have a tremendous impact on equine populations and efforts to control and prevent spread are critical to the well-being of individual horses and horse populations. The Dorothy Russell Havemeyer Foundation Inc. (http://www.havemeyerfoundation. org) is a private foundation that supports scientific efforts to improve the health and welfare of horses. To this end, the foundation conducts workshops in a variety of different subject areas, including the control of infectious diseases. The most recent Havemeyer workshop conducted on infection control in equine populations was held in September 2010, bringing together a diverse group of internationally recognised experts in fields related to infection control. The overarching goal of the workshop was to advance the discipline by providing guidance and inspiration to those currently involved in equine infection control and insight to those who may still be on its periphery. More specifically, the objective of this workshop was to identify the most urgent and critical priorities in equine infection control so that these might be targeted for research and resource development. In this report, we summarise the consensus opinions, major ideas and recommendations developed during the workshop. In working to summarise the discussion and major conclusions of this workshop, several common themes became apparent that were deemed essential to the future progress of the discipline of infection control. Specifically, these included ethical obligations and standards of practice regarding infection control, improvements and standardisation for surveillance methods, developing better education and training programmes, standardisation and development of new diagnostic tests for important contagious pathogens, objectively quantifying the costs and benefits of infection control programmes, expanding knowledge necessary for design of control programmes for important contagious pathogens, developing and promoting multicentre studies and promoting judicious antimicrobial use practices. Approaches that can be used for development of infection control programmes (the ‘how to’ of infection control) were discussed in previous Havemeyer workshops and have been reviewed in detail elsewhere [14–17]. Issues regarding the detection and control of specific agents were discussed and were used as examples during the meeting but the focus of this document is on the future needs of infection control in a broader context, not in relation to specific diseases.

Clinical Signs, Computed Tomographic Imaging, and Management of Chronic Otitis Media/Interna in Dairy Calves

A31⁄2-week-old Holstein heifer calf was presented to Cornell University Hospital for Animals (CUHA) with a 2-week history of intermittent fever, coughing, and decreased growth and a 1-week history of drooling and droopy ears. The calf had been treated with several antibiotics without marked improvement. On physical examination, the calf was depressed, underweight, bradycardic (heart rate 60 bpm) and febrile (103.48F, 39.78C). Clinical signs attributable to cranioventral pneumonia included bilateral mucopurulent nasal discharge, coughing, and abnormal bronchial tones. Intermittently, there were food particles in the nasal discharge, ptyalism, difficult prehension and mastication of food, and regurgitation of green material from both the nose and mouth. Neurologic examination revealed depression, bilateral facial nerve paresis (decreased lip, eyelid, and ear tone; absent palpebral or menace reflex), and bilateral vestibular disease (balance loss to either side, no head tilt, and loss of physiologic vestibular nystagmus). No abnormal nystagmus was observed, but the eyeballs did not show the physiologic vestibular eyedrop on head and neck extension. The gait was normal. The diagnosis of bilateral cranial nerve (CN) VII and VIII disease suggested bilateral otitis media/interna. Deep palpation of the base of the ears elicited a pain response and nonodorous otorrhea was present, indicating a component of otitis externa. Upper airway and esophageal endoscopy was consistent with dysphagia and disturbed esophageal motility and showed tracheal mucopus, nasopharyngeal collapse, dorsal displacement of the soft palate, and esophageal dilatation with few contractions. Thoracic radiography and transtracheal aspirate cytology (many degenerate neutrophils, few macrophages, large amounts of mucus, no bacteria) confirmed the presence of bronchopneumonia. The transtracheal aspirate yielded Arcanobacter pyogenes and Mycoplasma spp., but fluorescent antibody testing for common respiratory viruses was negative. CBC results were normal. Lumbosacral cerebrospinal fluid (CSF) had a slightly high nucleated cell count (9 nucleated cells/mL, normal ,5 cells/mL; total protein concentration 17 mg/dL, normal ,70 mg/dL). Lateral skull radiographs did not show abnormalities, but computed tomography (CT) imaging indicated that both tympanic bullae and the right petrous temporal bone were enlarged,