University of Kentucky College of Agriculture, Food & Environment

 

Gluck Center > Equine Disease Quarterly > January 2007

 

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Commentary

Digital radiographs, ultrasound, CT/MRI imaging, DNA technologies, gene sequencing, nested polymerase chain reaction tests, IgM capture ELISA tests... With the amazing technologies now available for rapid detection, diagnosis, and study of diseases, people often think that conclusive test results and a diagnosis can be made within a day, which is an unrealistic expectation fostered by television shows depicting investigative laboratory work. The “CSI Effect” is now recognized by attorneys who have noted that deliberating juries are asking why DNA, computer fingerprint analysis, and other tests have not been performed in criminal cases, even when the resolution of the case does not depend on such analyses. The perception may be that television technology equals reality, but in fact, it just isn’t that simple

As is discussed in Dr. McCoy’s article in this issue, interpretation of antibody titers (a long-used diagnostic tool) is complex and highly dependent upon knowledge of the test procedure, sample quality, clinical signs, and vaccination history. The same can be said of any complete necropsy. Final conclusions are not delivered overnight. Neurologic cases are especially challenging, since the brain and spinal cord are well protected in the skull and vertebrae, making extraction time-consuming.  It is not practical to take a section of every single piece of the spinal cord and brain for microscopic evaluation, and a final diagnosis is sometimes difficult

Yet expectations persist. Some clients and referring veterinarians expect to have diagnosticians do DNA sequencing analysis to determine if the virus affecting their horse is “new.” And there comes the rub. It takes time (sometimes months), money, and resources to grow a virus and perform DNA sequencing (a time intensive and therefore expensive tool). These procedures may or may not yield the answer to the owners and veterinarians

For an animal with obvious disease but no significant results on standard testing, further diagnostic procedures become more complex, time consuming, and costly

Consider the first diagnosed cases of West Nile virus (WNV) infection in the United States. This viral infection was then a foreign animal disease, and a confirmatory diagnosis eventually required a multitude of resources and expertise. In addition, equine influenza virus infecting dogs (trans-species infection), development of multi-drug resistant Staphylococcus aureus in domestic species and humans (emerging disease), and other evolving diseases keep scientists and diagnosticians busy. New and emerging diseases are challenges to scientists, but studying them is critical to the health and welfare of animals and people. After all, for many human diseases, animal disease surveillance is the front-line protection

In defense of veterinarians and owners, whether one horse is sick or a full-blown outbreak is ongoing, significant concern and pressure exist to determine a diagnosis so more specific treatment and quarantine measures can be undertaken. Not all local diagnostic laboratories offer needed tests, so samples must to be mailed to other labs. and, as Murphy’s law dictates, high-profile outbreaks always happen on a Friday afternoon on a holiday weekend, further delaying laboratory results. Patience is a virtue on everyone’s part.

CONTACT:
Dr. Roberta M. Dwyer, (859) 257-4757,rmdwyer@uky.edu
Maxwell H. Gluck Equine Research Center
University of Kentucky, Lexington, Kentucky

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Third Quarter 2006

The International Collating Center, Newmarket, England and other sources reported the following disease outbreaks:

Contagious Equine Metritis (CEM) was confirmed in a non-Thoroughbred Franches- Montagne stallion at the national stud of Switzerland. Cases of Eastern Equine Encephalitis (EEE) were reported from states in the southeastern USA. The respiratory form of equine herpesvirus (EHV) was widely reported in France among several breeds of horses, and EHV-1 abortion was confirmed among Thoroughbred horses on five premises in the KwaZulu-Natal province of South Africa.

The Department of Agriculture and Food, Ireland, confirmed 25 cases of Equine Infectious Anemia (EIA) up to the end of September, primarily in the Meath/Kildare/Dublin area.  All but three of the cases have been in Thoroughbred horses. The origin of the outbreak is considered to be a result of the administration of an infected equine biological product. EIA was also confirmed in Eastern Germany among nine horses on one premise and in Italy on 16 premises.

Isolated cases of equine influenza were diagnosed in two horses in the United Kingdom that had recently been imported, one from Ireland and one from Poland. Potomac horse Fever (PHF) was diagnosed in samples submitted from 17 sick horses in Kentucky, USA, four of which died. Rhodoccocus equi infection was identified on several farms in Italy. Strangles was reported on premises in Italy, South Africa, Sweden, and Switzerland.

Vesicular stomatitis (VS) attributable to the New Jersey strain was confirmed among horses on three premises in Wyoming, USA. West Nile virus (WNV) infection was extensively reported in the western and central states of the USA, although the number of equine cases reported was less when compared to the same period in 2005. An equine case was also confirmed in the south of France.

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Current Treatments for Equine Protozoal Myeloencephalitis

Equine Protozoal Myeloencephalitis (EPM) is one of the most common treatable neurological diseases of American horses and is caused by the apicomplexan protozoan Sarcocystis neurona. This protozoan penetrates the central nervous system, producing varying levels of neurological disease. In parts of the United States virtually all horses are exposed, with a small proportion (< 0.5%) exhibiting neurological symptoms. Because S. neurona can locate anywhere in the brain and spinal cord, the disease can mimic any neurological condition, and diagnosis can be challenging.  EPM was first identified in the 1960s, with development of improved diagnostic tests in the 1990s. Since 2000, three Food and Drug Administration (FDA) approved treatments for EPM have been brought to market. 

Treatment of EPM is challenging because S. neurona is an intracellular parasite and an expert in avoiding immune system attack. The three different anti-protozoal treatment modalities currently available work on entirely different pharmacological principles.

Traditionally, EPM was treated with combinations of pyrimethamine and a sulfonamide, one of the so-called “potentiated sulfonamides” used in classic anti-malarial therapy. These drugs act “in sequence” on nucleic acid synthesis. The sulfonamide directly inhibits the incorporation of para-amino benzoic acid (PABA) into folic acid, and pyrimethamine selectively inhibits dihydrofolate reductase. When present together in the brain at effective concentrations, these drugs produce a 1 to > 2 synergistic inhibition of nucleic acid metabolism. This combination was, for many years, the only known treatment for EPM, although the duration of treatment was often prolonged. Potentiated sulfonamide combinations have long been marketed by compounding pharmacies, and more recently, Phoenix Laboratories has brought to market an FDA-approved formulation of pyrimethamine and sulfadiazine, marketed as ReBalance™.

Side effects that may be associated with pyrimethamine-sulfonamide combinations are related to inhibition of host nucleic acid metabolism.  Animals on potentiated sulfonamide treatments should optimally be monitored for inhibition of red cell formation, leukopenia, and thrombocytopenia. Some of the earlier potentiated sulfonamide preparations were associated with reports of reduced spermatogenesis in stallions.

Another treatment, Marquis™ (ponazuril), is adapted from a widely used poultry coccidiostat, toltrazuril. These drugs act by directly attacking the “apicoplast” organelle of S. neurona.  Apicoplasts are chloroplast-related organelles that were acquired by S. neurona millions of years ago. They are highly susceptible to specific attack by herbicide-related drugs such as ponazuril. Working with these drugs, University of Kentucky researchers showed that they are highly specific and effective treatments for EPM. Ponazuril is well absorbed orally, has a 4.5 day plasma half-life, and is virtually non-toxic to equines at clinically effective doses. Single daily dosing is effective, and in an extensive field study, no adverse responses could be linked to treatment.

The manufacturer’s suggested treatment period is 28 days. Because of its unique mechanism of action, Marquist™ is essentially specific for apicomplexans, and a positive response to treatment offers strong support for an EPM diagnosis. Marketed in 2001, Marquist™ was the first FDA-approved treatment for EPM.

Nitazoxanide (Navigator™) is in a novel class of anti-infective drugs and is thought to act by inhibiting pyruvate-ferridoxin oxidoreductase in susceptible organisms. It has a broad spectrum of action, acting on enteric bacteria, protozoa, and viruses. In human medicine it has been approved as a broad spectrum anthelmintic and anti-viral drug.

The oral dose is carefully calculated, and treatment starts at a half daily dose for the first five days, increasing to the full dose of 22 mg/kg for the remaining 23 days. The principal adverse response in the horse relates to the drug’s broad enteric action, which can change equine intestinal flora and produce enteric problems. The addition of rice bran or corn oil to the diet helps to reduce the incidence of intestinal problems.

Horses on any long-term therapy for EPM should be monitored daily for adverse reactions and changes in clinical signs.

Numerous adjunctive therapies for EPM are also utilized. Anti-inflammatory therapy can help reduce inflammatory responses to the protozoan and may be useful in “treatment crisis” (transient worsening of clinical signs early in treatment) reported in some severe cases receiving an anti-protozoal medication. Use of corticosteroids in EPM cases is controversial among veterinarians.

Immune stimulants have also been recommended and include products such as Propionibacterium acnes administration, mycobacterial cell wall extracts, oral levamisole, and alpha-interferon. Additionally, a commercially available chemically inactivated vaccine of merozoites of S. neurona with an adjuvant has been suggested to further stimulate cell-mediated immunity.

Clinical experience suggests that rehabilitation is facilitated by mild to moderate unmounted, controlled exercise. The exercise level is dictated by the stability of the horse and the opinions of both the examining veterinarian and the owner. Complete recovery (to neurological normalcy) may not be possible, but rehabilitation and strengthening of affected horses can maximize the clinical outcome.

Based on contributions by David Granstrom, Dan Howe, Brad Bentz, Levent Dirikolu, and Thomas Tobin at the Maxwell H. Gluck Equine Research Center.

CONTACT:
Dr. Thomas Tobin, (859) 257-4757, ttobin@uky.edu
Maxwell H. Gluck Equine Research Center
University of Kentucky, Lexington, Kentucky

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Equine Neurologic Pathology

Horses are afflicted with a number of central nervous system diseases. Diagnosis of these conditions can be challenging both in the live horse and in horses that died or were euthanized due to the neurologic disease. In a clinically affected horse, central nervous system (brain and spinal cord) disease may manifest as altered mental state or behavior, cranial nerve deficits, seizures, abnormal posture, ataxia, paresis or paralysis, muscle atrophy, incontinence, pain, recumbency, or coma. With these varied signs, a systematic neurologic examination is essential for diagnosis.

The goals of the neurologic examination are to establish whether a neurologic problem is truly present and to determine the anatomic location of the process. Likewise, the postmortem examination of a neurologic horse is important to evaluate the clinical diagnosis, establish a diagnosis in undiagnosed cases, provide information that might allow prevention or treatment in herd mates, and monitor for possible zoonotic diseases.

An approach used in the pathological examination of the brain and spinal cord (as well as other body systems) is pathologic pattern recognition. Recognition of the pattern of lesions helps to categorize the condition and allows for consideration of diseases known to produce the pattern. Using both macroscopic and microscopic examination, it can be determined if the lesions affect only a single area of the nervous system or are multifocal or diffuse.  Likewise, involvement of specific anatomic structures and symmetry of the pathology are evaluated.

In horses, one pattern of pathology exhibits variably severe degeneration of all nerve fiber tracts and grey matter in a cervical spinal cord segment (see Figure 1). Cross sections of spinal cord above and below this area show distinctive patterns of change. Above the site there is degeneration of the fiber tracts in the dorsal and dorsolateral funiculi, and below the primary site the degeneration is limited to the medial and ventromedial funiculi. This is a pattern of descending and ascending neuron degeneration.  When the axon of a neuron is damaged at some point, the portion of the fiber farthest away from the nerve cell body degenerates.  The portion near the cell body can regenerate.  The dorsal and lateral funiculi represent sensory fibers, and the ventral funiculi represent motor fibers. This pattern is seen when there is focal injury to the spinal cord. In horses the most common cause by far of this injury is cervical vertebral stenotic myelopathy. Other causes would include tumors, subluxations, and abscesses that exert pressure on the spinal cord.

Cross section of spinal cord

A second pattern of neurologic pathology is characterized by lesions affecting only certain anatomical structures. In one example of this pattern, there is symmetrical degeneration of nerve cell bodies and fibers primarily in the thoracic portion of the spinal cord (nucleus thoracicus and dorsal spinocerebellar tract fibers in the lateral funiculus). Also, there is degeneration of a particular nucleus (cuneate nuclei) in the brain stem. This pattern is seen in the disease equine degenerative myelopathy.  Another disease of horses, nigropallidal encephalomalacia, is associated with the ingestion of the plant yellow star thistle (Centaurea solstitialis). Toxicity from eating this poisonous plant causes a special pattern of pathology in which there is degeneration in certain basal nuclei in the brain stem.

Another neurologic pattern is an inflammatory pattern that consists of random, variably sized (often large), asymmetric areas of inflammation that affect the brain stem and spinal cord. Aggregates of primarily mononuclear cells surround blood vessels and infiltrate into adjacent brain or spinal cord tissue, resulting in degeneration. This pattern is consistent with the disease equine protozoal myeloencephalitis.

A final example is another inflammatory-type pathologic pattern that consists of a mild accumulation of mononuclear cells (multifocal to diffuse). These infiltrates are associated with blood vessels, and occasionally the cells invade the nerve tissue. This pattern suggests viral infection and can be seen with any viral encephalitis, including rabies. Neurologic equine herpesvirus type 1 infection varies slightly in its pattern. With EHV-1, vasculitis leads to thrombosis and degeneration of the surrounding nerve tissue.

Pathologic pattern recognition is a tool utilized by pathologists to assist in diagnosis of neurologic disease. A particular pattern can suggest a diagnosis even when the causative agent cannot be demonstrated.

CONTACT:
Dr. Neil Williams, (859) 253-0571, nmwillia@uky.edu
Livestock Disease Diagnostic Center
University of Kentucky, Lexington, Kentucky

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Interpreting Diagnostic Serology

Diagnostics based on serological analysis has long been a complex task for veterinarians and diagnosticians alike. Serologic testing is frequently chosen owing to the ease of blood sample collection and handling when compared to the alternative of isolation or identification of an etiological disease agent.  Pathogen identification generally requires specimens of whole blood or swabs, which have exceedingly more stringent sample handling requirements than serum samples and sometimes require lengthy culturing and/or expensive molecular procedures. Thus, clinicians and laboratory personnel are often asked to make what can be tenuous interpretations of serological assay results.

One notion must be recognized above all others when discussing serologic reports: a result on one serum sample drawn on a particular day is not necessarily indicative of infection or disease. Most serological tests do not discriminate between recent exposure, past exposure, or vaccination.

Serological testing on a single sample can be helpful in regulatory and surveillance efforts for a particular agent in a naive population where risk of exposure to that agent is considered absent or extremely low. In such instances, a serologic assay should, theoretically, test negative for antibody for most, if not all, of the population. Additionally, vaccination against these agents must be essentially nonexistent due to the inability of most serology tests to discriminate between vaccination and natural exposure. Serological testing for exposure to vesicular stomatitis virus (VSV) fits into this category because the majority of horses in the United States are immunologically naive with respect to the virus and because vaccination is not performed. Therefore, a positive serologic test for VSV only indicates exposure, be it past or present.

Equine herpesvirus types 1 and 4 (EHV-1, EHV-4) serology is an example of a much more complex situation. When infectious agents are intimately related antigenically, as are EHV-1 and EHV-4, serologic tests are often unable to discriminate between antibodies developed against either virus. Confounding the situation, EHV-1 and EHV-4 are ubiquitous viruses in the United States, and exposure to or vaccination against either or both viruses is essentially a certainty. These circumstances foster a situation in which virtually all horses have been exposed to the viruses, have mounted immune responses, and are, therefore, antibody positive.  Such conditions make it virtually impossible to consider a positive serologic test on a single serum sample as diagnostic for disease, regardless of the antibody titer. Additionally, since conventional serologic tests used in diagnostic laboratories have a limited capacity in differentiating between antibodies developed against EHV-1 and EHV-4, it is extremely difficult to assert whether the test result is specific for the virus in question.

Finally, regardless of the disease agent in question, serological testing of acute and convalescent serum samples is crucial in identifying a recent exposure. When clinical signs are first observed, it is imperative that a serum sample be drawn immediately and followed by a second sample two to three weeks later.  A significant difference in the antibody titer of these two samples in a side-by-side analysis must be observed to indicate an acute exposure.  Diagnostic laboratory personnel can offer information in determining whether any difference observed on the serologic tests is significant. Each testing method has specific limits of specificity and sensitivity; these affect the level at which a difference may be interpreted as being significant. A two-fold difference in titer as observed on a virus neutralization test, for example, is not considered significant due to the limited sensitivity of the test. Furthermore, with each testing method having unique specificities and sensitivities, it is virtually impossible to compare antibody titers between the methods.  For example, an equine arteritis virus (EAV) neutralizing titer of 1:4 is considered significant for exposure to the virus. In comparison, a West Nile virus (WNV) IgM capture enzyme-linked immunosorbent assay (ELISA) positive at a 1:400 dilution is considered significant for exposure to WNV.

An exception to the rule of testing both acute and convalescent sera would be the use of IgM capture ELISA for detecting a subclass of antibodies developed during an initial exposure to an antigen in a single serum specimen. Such tests may be used to identify recent exposure to an infectious agent and are extremely helpful for clinicians in making diagnoses when accompanied with clinical signs of disease.  Therefore, providing case and vaccination histories is extremely important when submitting any sample for diagnostic testing, given that proper interpretation of any serologic result requires these key factors be considered.

The development and use of more antigen specific as well as antibody class and subclass specific tests is critical for the advancement of serological diagnostics. These developments will enhance not only equine veterinary medicine, but the equine industry as a whole.

CONTACT:
Dr. Morgan H. McCoy, (859) 253-0571, morgan.mccoy@uky.edu,

Livestock Disease Diagnostic Center
University of Kentucky, Lexington, Kentucky

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Editors:
Roberta Dwyer
David Powell
Neil Williams
Staff:
Diane Furry

Correspondence should be addressed to the editors,
Department of Veterinary Science, Gluck Equine Research Center,
University of Kentucky, Lexington, KY 40546-0099;
Phone (859) 257-4757; FAX (859) 257-8542; dfurry1@uky.edu.

 

Maxwell H.Gluck Equine Research Center
Department of Veterinary Science, University of Kentucky
Lexington, Kentucky 40546-0099

Main Office (859) 257-4757
Fax (859) 257-8542