THE ROLE OF THE EQUINE PRACTITIONER IN BIOSECURITY

Dr. Nicola Pusterla, PhD, Diplomate ACVIM

Professor Equine Internal Medicine

Department of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis,

Davis, California

Objectives of the Presentation

Review basic principles of biosecurity

Discuss practical applications of biosecurity to prevent transmission of contagious enteric and

respiratory pathogens

Take Home Message

Biosecurity and infection control are important aspects of the day-to-day operation of any

equine facility and are especially important for equine hospitals.

It is essential that the people in charge, such as clinicians, owners, and trainers observe the

implemented infection control measures.

Leadership by example is the best way to ensure compliance of all personnel.

In addition, educational efforts should be undertaken to make sure all workers understand the

importance of biosecurity and their role in maintaining the facility as a safe place for horses and

their human caregivers.

Introduction

Application of the concepts of biosecurity and bio-containment is important not only in

veterinary hospitals, but also for ambulatory practices, equine breeding facilities, training

facilities and other facilities that house horse populations. A passive attitude towards infection

control may have detrimental consequences, including a large financial impact. Outbreaks at

equine veterinary hospitals are often associated with public relations issues, such as loss of

confidence and business of those who would bring their animals to the premise. Further, some

contagious diseases can lead to severe disease with possible death in infected animals. This may

potentially lead to litigation issues. Last but not least, outbreaks diminish morale of hospital staff

and clinicians.

Basic Principles of Biosecurity

Properly implemented biosecurity measures may significantly decrease the risks for disease

introduction and spread of infectious pathogens. Infectious disease control relies on several basic

principles, which include understanding the biology of infectious pathogens and route of

pathogen transmission, housing horses based on exposure risk, daily monitoring for signs

associated with infectious diseases, implementing proper hygiene and cleanliness protocols,

educating the horse community and having a contingency plan in place in case of an outbreak.

Most of these steps will prevent and minimize exposure to infectious pathogens at an individual

and population level.

Farm-based infectious disease control measures should include the segregation of horses into

small groups based on age, use and gestational time. This measure may not eliminate infectious

diseases in horses but, hopefully, may limit the severity of the problem by minimizing the

number of affected animals. Housing at boarding facilities or horse event may represent a true

challenge due to the high traffic and horse density. However, in order to minimize risk of disease

outbreak, each horse should be considered at risk and handled like a single unit. Ideally such

horses should be kept in individual stalls with no direct contact to other horses. The health status

of such horses needs to be assessed and possibly recorded daily. Further, reducing unnecessary

movement of animals and humans is an effective way to minimize spread. If equipment

(grooming, cleaning and tack equipment) is shared between horses, it should be cleaned and

disinfected after every individual use. Specific housing measures apply to hospitalized patients.

This means that all patients are to be screened before admission to the hospital for signs of

contagious disease (physical examination and accurate history). Patients should be hospitalized

in dedicated stalls/barns based on their infectious status. Isolation wards should be available for

patients with confirmed and/or suspected contagious diseases. It is important to maintain hygiene

of personal and facility so that hospitalized horses and their bodily fluids and excrements stay

separated from other horses. It is important to monitor patients daily for the occurrence of

infectious diseases through observation of clinical signs (fever, diarrhea, nasal discharge,

coughing) and through strategic testing of biological samples. For example, feces may be

collected for culture or PCR to detect Salmonella spp. in high risk patients on admission and at

regular intervals thereafter during the entire hospitalization time. Even with well-established

infectious control protocols, it may not be possible to virtually eliminate all risks of nosocomial

infections.

Probably one of the most underutilized principles of biosecurity is the daily monitoring off at

risk horses. The idea behind the assessment of daily health is to recognize early clinical signs and

to take proper action to prevent disease spread. Daily monitoring by owners, trainers and care

takers should include the assessment of attitude, appetite and rectal temperature. Additional signs

such as nasal discharge, coughing, changes in fecal character and acute onset of neurological

signs should also be recognized and reported to a health care provider.

Of all the possible measures that can be taken to reduce nosocomial and zoonotic infections,

hand hygiene is perhaps the most important and cost-effective, easiest to use but also most

underused measure. Hands should be washed before and after attending each individual animal.

In addition to soap and water, alcohol-based hand sanitizers can be a useful adjunct to hand

washing in veterinary hospitals and can provide a practical option for improving hand hygiene

for ambulatory practitioners.

One additional means to prevent exposure to zoonotic pathogens and prevent transmission via

contaminated hands and clothing is the use of personal protective equipment (PPE). Standard

outerwear should be clean and should be changed if contamination occurs. Also dedicated

clothing and footwear should be worn when working with infectious patients and in high-risk

areas such as intensive care unit, isolation, foaling facility, quarantined barns, etc. Minimal PPE

when working with infectious pathogens should include designated scrubs/coveralls/lab coats,

disposable gloves and shoe covers/dedicated shoes/boots. Contamination of personal items such

as stethoscope, thermometer, pencil, phone, pager, etc. occurs routinely when working with

horses. In order to minimize exposure and transmission with infectious pathogens, one should

strongly consider disinfecting all mentioned items if such items have been used while attending

the patient.

Virtually all pathogens in equine facilities are associated with some organic matter, including

feces, urine, saliva and sweat. Experimentally, cleaning alone has been shown to decrease the

bacterial load by 90% on a concrete surface. Another 6-7% of bacteria are killed by disinfectants.

There is enough convincing evidence of the necessity to clean surfaces thoroughly before

disinfection. Even the best disinfectants are less effective in the presence of organic matter.

Housing stalls and trailers should be cleaned and disinfected between horses. Also consider

regular hosing and disinfection of aisles and high traffic areas such as wash stalls and

treatment/examination rooms.

It is important to have a contingency plan in place on what to do when dealing with a

potentially infectious animal. The plan should be known by all care takers, trainers and owners at

a boarding facility and by all staff and veterinarians at a veterinary hospital. Ideally, written

protocols should be available and regularly reviewed and updated if needed. A logical action

plan for a horse owner should include: general recommendations to what represents a trigger

point (i.e fever, acute onset of nasal discharge, coughing, ataxia, diarrhea); isolating a sick

animal (s) in previously designated areas; designate a dedicated care taker and equipment to

attend the care of the sick horse; use of PPE when attending the sick horse; close or disinfect

areas where the sick horse was housed or held; institute barrier nursing to prevent spread of

infectious pathogens (foot bath, gloves, dedicated clothing and foot wear); contact the care

provider to evaluate the horse and collect diagnostic samples; reduce overall traffic within

premise and monitor horses with possible contact to the index case.

Infection Control for Gastrointestinal Pathogens

Horses are very vulnerable to infectious enteric disorders, especially salmonellosis and

clostridial infections. Several factors including stress, transportation, changes in feed, fasting,

surgery, antimicrobial use, concurrent GI disturbances and elevated ambient temperature have

been linked to an increased susceptibility to Salmonella enterica infection. Salmonellosis and/or

clostridial infection should be considered when horses develop gastrointestinal signs (colic,

diarrhea), fever and leukopenia. Further, the previous use of antimicrobials in patients

developing any of the mentioned clinical signs is highly suggestive of an infectious

gastrointestinal disorder. Outbreaks with enteric pathogens can be devastating in any situation

(farm and hospital). To determine the magnitude of the outbreak, all horses or a representative

sample of resident horses should be screened for enteric pathogens using conventional

microbiology and/or PCR detection. Horses testing positive for an enteric pathogen (clinical or

subclinical) should always be isolated from the rest of the population to decrease the exposure

risk and environmental contamination. Establish barrier nursing in the form of footbath or mats

in front of the isolation unit and each stall. This will minimize the spread of pathogens from

stalls to clean areas. Phenolics compounds, QAC and peroxygens compounds have been shown

to retain activity in the presence of organic matter. Phenolics and peroxygen compounds are the

only disinfectant to have an activity against rotavirus. Peroxygens and high concentration of

bleach (8 oz/gallon) are effective at neutralizing clostridial spores. Phenols, QAC and bleach at 4

oz/gallon are effective against Salmonella. Care takers and owners should wear gloves,

protective clothing (coveralls, disposable gowns) and dedicated footwear. Good hand hygiene

should be instituted (faucet with warm/cold water or handsanitizer). It is very important to

control traffic and minimize contact of affected horses with the general public. Remember that

enteric pathogens such as Salmonella, Cryptosporidium and Clostridium difficile are potential

zoonotic agents and represent a greater risk for immunocompromised humans, infants, and

elderly people. Instruct caretakers/owners to handle diseased horses last and to use separate

equipment (cleaning equipment, tractor, hay wagon, wheelbarrow, etc). Hygiene should be

maximized by regular cleaning and disinfecting. Waste from positive animals should be either

removed from the premise, or composted or spread in sunlight in a place with no direct access to

horses.

Infection Control for Respiratory Pathogens

During outbreak of respiratory disease, aerosol and droplet infection can be minimize by

separating animals according to their infections status (infected, exposed versus non-exposed).

Air movement may play an important role in transmission of aerosolized virus, since viral

respiratory pathogens, such as EIV, have been shown to be transmittable over a distance of 150

feet. The -herpesviruses (EHV-1/-4) require closer contact and are generally transmitted via

nose-to-nose contact or fomites. Transmission of Streptococcus equi subsps. equi usually

requires direct physical contact between infected and susceptible horses but can also be

transmitted via fomites (hands, shared equipment). Fortunately, outbreaks of respiratory

pathogens can successfully be controlled via appropriate infectious disease control measures

(separation of infected animals, cleanliness and hygiene, restrict movement and traffic, use of

PPE and barrier nursing). Common infectious respiratory pathogens are susceptible to the

majority of commercially available disinfectants.

EHV-1 MYELOENCEPHALOPATHY – LESSONS LEARNED FROM RECENT

OUTBREAKS

Dr. Nicola Pusterla, PhD, Diplomate ACVIM

Professor Equine Internal Medicine

Department of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis,

Davis, California

Objectives of the Presentation

Understand the difference between EHV-1 genotypes (N752 versus D752)

How to recognize and diagnose EHM

How to treat a horse affected by EHM

How to prevent the occurrence of EHV-1 infection

Take Home Message

EHM is a sporadic and relatively uncommon manifestation of EHV-1 infection.

A suspected diagnosis of EHM is based on historical and clinical findings and CSF analysis.

Laboratory confirmation of EHV-1 infection is usually based on the PCR detection of EHV-1

in blood and nasal secretions.

To date, no specific treatment for EHM affected horses is available, thus management of

affected horses aims toward supportive nursing, nutritional care and reduction of CNS

inflammation.

Disease control measures such as isolation of affected horses, segregation and monitoring of

exposed horses and quarantine measures should be established in order to prevent spread of

EHV-1.

While there are several vaccines available as an aid in the prevention of both respiratory and

abortigenic forms of EHV-1, at this time there is no equine vaccine that has a label claim for

the protection against the development of EHM.

Etiology

Equine herpesvirus-1 (EHV-1) is an important, ubiquitous equine viral pathogen that exerts its

major impact by inducing abortion storms or sporadic abortions in pregnant mares, early

neonatal death in foals, respiratory disease in young horses and myeloencephalopathy. Equine

herpesvirus-1 myeloencephalopathy (EHM) is a sporadic and relatively uncommon

manifestation of EHV-1 infection; however, it can cause devastating losses and severely impact

the horse industry, as exemplified by recent outbreaks at riding schools, horse venues and

veterinary hospitals throughout North America and Europe. Clinical signs of neurologic disease

reflect a diffuse multifocal myeloencephalopathy secondary to vasculitis, hemorrhage,

thrombosis, and ischemic neuronal injury. Sudden onset and early manifestation of signs

including ataxia, paresis and urinary incontinence; involvement of multiple horses on the

premises; and a recent history of fever, abortion, or viral respiratory disease in the affected horse

or herdmates, are typical features, although there is considerable variation between outbreaks

with respect to epidemiological and clinical findings. Prevention is difficult because many

asymptomatic horses are latently infected with EHV-1, allowing the virus to circulate in a silent

cycle in horse populations, and currently available vaccines do not confer protection against the

neurologic manifestations of EHV-1 infection. Although outbreaks of EHM have been

recognized for centuries among domestic horse populations, many aspects of this disease have

remained poorly characterized.

Natural infection with EHV-1 occurs by inhalation or ingestion, after which the virus attaches

to, and rapidly replicates in cells of the nasopharyngeal epithelium and associated

lymphoreticular tissues, causing necrosis, exudation and infiltration of phagocytic cells and viral

shedding for 10-14 days post-infection, or even longer in EHM affected horses. Cell-to-cell

spread results in the presence of virus in respiratory tract lymph nodes within 24-48 hours post-

infection. A leukocyte-associated viremia is then established which is directly responsible for the

delivery of EHV-1 to other tissues; the specific leukocyte subset(s) harboring EHV-1 remain

poorly defined. The viremia can persist for at least 14 days, and is a prerequisite for EHM and

abortion as it allows for transport of the virus to the vasculature of the pregnant uterus or the

CNS where endothelial cell infection occurs. This results in damage to the microvasculature of

the CNS due to initiation of an inflammatory cascade, vasculitis, microthrombosis, and

extravasation of mononuclear cells resulting in perivascular cuffing and local hemorrhage. The

spinal cord gray and white matter are most commonly affected, with the brainstem being

infrequently affected. While viremia is a common sequel to EHV-1 infection, transfer of virus to

the CNS endothelium and development of EHM is not; typically some 10% of infected horses

develop neurological signs during EHM outbreaks.

The factors determining whether horses develop EHM after EHV-1 infection are poorly

understood. It has been proposed that the magnitude of cell-associated viremia is an important

factor for the development of EHM because infection with the DNApol D752 neuropathogenic

strain leads to a higher magnitude and duration of viremia. The hypothesis that the duration or

magnitude of viremia directly determines the occurrence of EHM is appealing, and there is new

evidence to support it. It is unlikely to be a simple relationship, as considerable differences are

observed in levels of viremia produced by different DNApol D752 neuropathogenic strains, such as

Ohio ’03 and Ab4. It is also noteworthy that in experiments in which neurological signs were

described in control horses, but not in vaccinates, the level of viremia was the same in both

groups. If the D752 strain has an increased ability to cause EHM, the mechanisms by which it

achieves this, may extend beyond simply inducing high levels of viremia.

The host and environmental factors that determine the occurrence of EHM are similarly ill-

defined. It is known that older horses are generally more susceptible to EHM. This implies a

possible role for immunological status in the pathogenesis of EHM, as older horses generally

demonstrate a greater IFN-gamma based cellular response to EHV-1. Nevertheless, at this time

our understanding of how and why EHV-1 infection leads to EHM in some horses remains

rudimentary.

Diagnostic work-up

The multifocal distribution of lesions caused by EHV-1 results in variability of clinical

presentation. This necessitates inclusion of a number of conditions in the differential diagnosis,

such as equine protozoal myeloencephalitis, cervical stenotic myelopathy and cervical vertebral

instability (wobbler syndrome), cervical vertebral fracture or other CNS trauma, neuritis of the

cauda equina, fibrocartilaginous infarction, aberrant parasite migration, degenerative

myelopathy, togaviral encephalitis (Flaviviruses and Alphaviruses), rabies, botulism, CNS

abscess, and a variety of plant and chemical intoxications. Sudden onset of neurologic signs

including ataxia, paresis and urinary incontinence; involvement of multiple horses on the

premises; and a recent history of fever, abortion, or viral respiratory disease in the affected horse

or herdmates is sufficient to make a tentative diagnosis of EHM. Pyrexia has consistently been

reported as the major clinical sign before the onset of neurological disease but is frequently not

present by the time neurologic deficits become apparent.

Hematological abnormalities in horses with EHM are inconsistent and may include mild

anemia and lymphopenia in the early stage, followed a few days later by mild

hyperfibrinogenemia. Azotemia and hyperbilirubinemia may occur secondary to dehydration and

anorexia, respectively.

Cerebrospinal fluid analysis typically, although by no means always, reveals xanthochromia,

an increased protein concentration (100 to 500 mg/dL), and increased albumin quotient (ratio of

CSF to serum albumin concentration), reflecting vasculitis and protein leakage into CSF. The

nucleated cell count in CSF is usually normal (0 to 5 cells/µL) but is occasionally increased.

Rapid laboratory tests for detection of EHV-1 are most useful in potential epidemic situations,

because rapid identification of the causative agent is often critical for guiding management

strategies. Polymerase chain reaction (PCR) has become the diagnostic test of choice due to its

high analytical sensitivity and specificity. PCR is an enzymatic exponential DNA amplification

technique, that under optimal condition is capable of detecting a small number of target viral

genes. PCR detection of EHV-1 is routinely performed in respiratory secretions from a nasal or

nasopharyngeal swab and uncoagulated blood samples collected into EDTA tubes. Many

conventional PCR protocols (single or nested PCR) targeting specific genes of EHV-1 have been

published in recent years for the molecular detection of EHV-1. Although considerable progress

has been made in developing PCR assays, the lack of protocol standardization between

laboratories and the variability in use of contamination and quality assurance controls remain an

ongoing challenge. The increasing application of PCR for the molecular detection of EHV-1 in

practice settings has presented new dilemmas with regard to how test results are interpreted and

used by both, equine practitioners and regulatory veterinarians, since routine PCR assays are

unable to differentiate between replicating (lytic), non-replicating or latent virus. Advances in

technology and the use of novel PCR platforms, such as real-time PCR, allow calculation of viral

loads for equine herpesviruses. Viral load testing, although not routinely offered by many

veterinary laboratories, represents a major improvement in the interpretation of PCR results in

the EHV-1 field, allowing better characterization of disease stage, better assessment of risk of

exposure to other horses, and better monitoring of response to therapy. Real-time PCR has

recently been used to document differences in EHV-1 viral loads in blood and nasal secretions

between horses in the febrile and neurological stages of disease and between clinically affected

and subclinically infected horses. The finding of high viral loads in the nasal secretions of

neurological horses confirms their importance as a potential source of contagion for other horses

and highlights the need for imposition of strict biosecurity measures when faced with horses with

suspected or confirmed EHM. However, the random testing of normal horses for EHV-1 by PCR

should be avoided, since practicing veterinarians and regulatory officials who receive positive

PCR test results on samples they submit may be unaware of the complexities involved in test

interpretation, leading them to make inappropriate decisions regarding quarantine of equine

facilities or cancellation of competitions. The situation is likely different when healthy horses

determined to be at high risk of exposure are tested for surveillance purposes during active

outbreaks of clinical EHV-1 infection. Under such circumstances, horses that test positive by

PCR on nasal or nasopharyngeal secretions should be isolated and closely monitored for the

development of clinical signs, since the viral load pattern of infected horses during the early

incubation period is similar to that of subclinical carriers. Follow-up assessment of viral loads in

blood and nasal or nasopharyngeal secretions can be used to help guide modification of

infectious disease control measures, including lifting of quarantine, for individual horses that test

negative on a subsequent sample. It should be noted that exposed but latently infected horses

may test positive for EHV-1 but never exhibit signs of active clinical disease due to their

exposure to an index case. Such latently infected horses may continue to test positive for

extended periods of time, thus further confusing their clinical evaluation and confounding

control procedures for all exposed horses.

Research groups have recently identified a region of variation in the genome of different EHV-

1 strains, that correlates directly with the likelihood that they will cause neurologic disease. The

sequence variation occurs in the DNA polymerase gene (ORF 30) of the virus, which is involved

in initial viral replication within infected cells and may also be involved in establishment of

latency and reactivation. PCR assays based on ORF 30 have recently been developed and used to

differentiate between so-called neuropathogenic and non-neuropathogenic strains in samples

submitted from field outbreaks. Results are typically reported as positive or negative for the

neuropathogenic strain of EHV-1 resulting in either increased concern or panic when a positive

result is obtained or, conversely, complacency when a result is reported as negative for the

neuropathogenic strain. The latter approach ignores the fact that approximately 14% of EHV-1

isolates from horses with EHM do not have the neuropathogenic marker. Therefore, it is the

author’s opinion, that such assay should be carefully used, and the results always interpreted in

the context of clinical presentation.

Serologic testing which demonstrates a fourfold or greater increase in serum antibody titer,

using serum-neutralizing (SN) or complement fixation (CF) tests, on acute and convalescent

samples collected 7 to 21 days apart provides presumptive evidence of infection. Many horses

with EHM, however, do not show a four-fold rise in SN titer. This may be explained by the

finding that, when antibody titers rise, they do so rapidly within 6 to 10 days of infection and

already may have peaked by the time neurologic signs appear. Although serologic testing has

limitations in confirming a diagnosis of EHM in an individual horse, testing of paired serum

samples from in-contact horses is recommended because a significant proportion of both affected

and unaffected in-contact horses seroconvert, providing indirect evidence that EHV-1 is the

etiologic agent. Interpretation of the results of serologic tests is complicated by the fact that the

serum-neutralizing, complement-fixation, and ELISA tests in use at most diagnostic laboratories

do not distinguish between antibodies to EHV-1 and EHV-4 because of cross-reaction between

these viruses. A specific ELISA test based on the C-terminal portion of glycoprotein G of both

viruses has been developed and should prove valuable in the investigation and management of

disease outbreaks in the future. However, this assay is not yet commercially available in North

America.

Treatment

The treatment of horses with EHM involves empiric supportive care, including nursing and

supportive care in cases of recumbency, maintenance of hydration and nutrition, and frequent

bladder and rectal evacuation. Nonsteroidal anti-inflammatory therapy is frequently used as an

adjunctive therapy, although its capacity to affect the development of the pathological changes of

EHM is unknown. Similarly, corticosteroids and more recently immunomodulators are both

employed in EHM treatment, although the justification is theoretical, as no evidence based study

has demonstrated efficacy of either drug class for EHM. Similarly, anti-viral drugs are also

unproven in terms of their value to treat EHM, although their theoretical appeal has led to their

increasing use against a background of an improved understanding of their pharmacodynamics.

Corticosteroids are immunosuppressive drugs and could aid in the control or prevention of the

cellular response adjacent to infection of CNS endothelial cells, thereby potentially reducing

vasculitis, thrombosis and the resultant neural injury. This theoretical benefit of corticosteroid

treatment of EHM has never been demonstrated in a clinical setting. Possible outcomes could

include a positive effect through reduction of hypersensitivity disease associated with infection,

or a deleterious effect due to a reduced immunological control of EHV-1 infection. Given our

poor understanding of their pharmacodynamics in EHM, the use of corticosteroids is currently

reserved for EHM cases presenting in recumbency or with severe ataxia, in which the prognosis

is guarded for survival.

The value of immunostimulants treatment for prevention of EHV-1 infection is similarly hard

to assess currently. Immunostimulants could be administered to a horse before a stressor (e.g.

transportation, performance, changes in the environment, exposure to new horses). In this case,

the activation of the immune system could theoretically prevent viral reactivation or replication.

One study evaluated the efficacy of inactivated Parapox ovis virus in young horses subjected to

stress (weaning, transport, and commingling with yearlings) and their susceptibility to clinical

respiratory disease during natural EHV-1 exposure; there was some evidence of a reduction in

clinical signs of respiratory disease. No other published studies are available describing the use

of immunomodulators for treatment or prevention of EHV-1 infection, and consequently it is

impossible at this time to determine whether they have any value. Overall, our understanding of

the value of immunomodulators for EHV-1 treatment remains rudimentary.

Antiviral drugs, and specifically virustatics, are of theoretic value for the treatment of EHV-1

and have demonstrated in vitro efficacy against EHV-1. The thymidine kinase inhibitor acyclovir

is a synthetic purine nucleoside analog that selectively inhibits the replication of herpesviruses.

The drug is phosphorylated initially by herpesvirus viral thymidine kinase, followed by 2 other

phosphorylations by host cell kinases. The triphosphate acyclovir compound binds to and

inhibits the viral DNA polymerase for the formation of viral DNA. Pharmacokinetic of acyclovir

after single oral administration (10 and 20 mg/kg) to adult horses has been associated with high

variability in serum acyclovir-time profiles and poor bioavailability below the levels required for

viral inhibition. A single 10 mg/kg intravenous infusion results in a greater peak serum

concentration. Controlled clinical studies of intravenous acyclovir in EHV-1 infected horses have

yet to be performed, and it seems likely that there are better choices than acyclovir for EHV-1

treatment.

Another nucleoside analog, valacyclovir shows greater promise based on pharmacokinetic

data. The bioavailability of the prodrug valacyclovir at 30 mg/kg orally twice a day is in the

order of 35 to 40%. The recommended dose of valacyclovir is 30 mg/kg PO two to three times

daily for the first 48 hours, decreased to 20 mg/kg PO twice daily. Currently, the effects of

timing of valacyclovir administration relative to the onset of EHV-1 infection or EHM

development on treatment outcome are unknown.

Prevention

In the wake of recent outbreaks of EHM in populations of horses in several regions of North

America, many racing jurisdictions and managers of equine facilities and events have imposed

EHV-1 vaccination requirements for incoming and resident horses in the hope that EHV-1

infection and development of EHM can be prevented. The efficacy of this approach remains to

be proven. In fact, frequent revaccination of mature horses to prevent the neurological form of

EHV-1 is not clearly justified in most circumstances because EHM is a relatively rare disease

from a population standpoint, most mature horses have previously been infected with EHV-1 and

are latent carriers, and none of the currently available EHV-1 or EHV-4 vaccines carry a claim

that they prevent EHM. Currently available vaccines do not reliably block infection, the

development of viremia, or establishment of latency and EHM has been observed in horses

vaccinated against EHV-1 regularly at 3- to 5-month intervals with inactivated or modified live

vaccines. Furthermore, vaccination has been cited by some as a potential risk factor for

development of neurological EHV-1, although evidence to support this opinion is far from

conclusive. In contrast, field experience in North America strongly suggests that regular

revaccination of pregnant mares and other horses on breeding farms reduces the risk of EHV-1

induced abortion and is well justified.

There is no known method to reliably prevent the neurologic form of EHV-1 infection;

however, implementation of routine management practices aimed at reducing the likelihood of

introducing and disseminating infection is justified. New arrivals should be isolated for at least 3

weeks before joining the herd, distinct herd groups should be maintained based on the age and

use of horses, and care should be taken to minimize or eliminate commingling of resident horses

with visiting or transient horses. In particular, pregnant broodmares should be maintained in

groups separate from the remaining farm population. In addition, it is prudent to minimize stress

associated with overcrowding and handling procedures in an attempt to reduce recrudescence of

latent EHV-1 infection.

As with other herpesviruses, the ability of EHV-1 to infect horses and establish a long-term

latent carrier state in the face of host immune responses assures indefinite persistence of endemic

EHV-1 infection in the equine population. Resistance to re-infection resulting from recovery

from field infection with EHV-1 is short-lived, lasting only a few weeks to a few months. After

infecting the horse via the respiratory tract, EHV-1 rapidly becomes internalized by cells,

including circulating lymphocytes, and is then passed directly from cell to cell without an

extracellular phase during which the virus could otherwise be exposed to neutralizing antibodies

and other immune effectors. In order to be highly effective, it would therefore be necessary for

EHV-1 vaccines to satisfy a challenging set of demands that would exceed those invoked in the

immune response resulting from natural infection. The ideal EHV-1 vaccine would not only be

safe and lend itself to efficient delivery, it would also invoke strong and persistent local humoral

(virus-neutralizing antibody) and cellular (cytotoxic T-lymphocyte, CTL) responses at the level

of the mucosal lining of the respiratory tract in order to block infection. In addition, it would

induce durable systemic humoral and CTL responses to rapidly clear free virus and destroy

virus-infected cells in the event that the mucosal response was not successful in blocking

infection. Beyond that, the ideal vaccine would need to be capable of inducing this broad

spectrum of immune responses in foals at a young age to protect them against the field challenge

that inevitably occurs during the first year or two of life and leads to a chronic latent-carrier state.

Not surprisingly, available vaccines fall short of these ideals.

Laboratory contact

Quantitative Real-Time PCR

Real-time PCR Research and Diagnostics Core Facility, School of Veterinary Medicine,

Department of Medicine and Epidemiology, 3110 Tupper Hall, University of California, One

Shields Avenue, Davis, CA 95616. Phone: (530) 752-7991; Fax: (530) 754-6862; Email:

carjohnson@ucdavis.edu; Website: www.vetmed.ucdavis.edu/vme/taqmanservice/

diagnosics.html.

UNDERSTANDING MOLECULAR DIAGNOSTIC TESTS FOR THE

DETECTION OF EQUINE INFECTIOUS PATHOGENS

Dr. Nicola Pusterla, PhD, Diplomate ACVIM

Professor Equine Internal Medicine

Department of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis,

Davis, California

Objectives of the Presentation

Review the basic understanding of PCR

Understanding the advantages and pitfalls of PCR

Discuss PCR applications for respiratory, neurologic and enteric pathogens

Take Home Message

Polymerase chain reaction or PCR is a nucleic acid-based amplification technique that has been

described as genetic photocopying. PCR allows the detection of infectious pathogens in host

tissues even when only a small amount of pathogen is present.

PCR has distinct advantages as a diagnostic tool and is best used in clinical situations requiring

rapid, sensitive and specific answers, which is often the case when dealing with highly infectious

organisms.

Whenever using molecular detection assays, one must keep in mind the biology of the

suspected pathogen to be detected, the disease stage of the animal, the adequate specimen to be

used and test to be requested. The use of a panel strategy will truly facilitate the choice of assay

requested by widening the spectrum of common pathogens involved with a specific syndrome.

PCR should always be interpreted within the clinical context. If the results don’t match your

clinical impression, either repeat the test or investigate the laboratory’s performance.

Introduction

The focus of rapid diagnosis of infectious disease of horses in the last decade has shifted from

the conventional laboratory techniques of antigen detection, microscopy and culture to molecular

diagnosis of infectious agents. Equine practitioners must be able to interpret the use, limitations

and results of molecular diagnostic techniques as they are increasingly integrated into routine

microbiology laboratory protocols. Polymerase chain reaction (PCR) is the best known and most

successfully implemented diagnostic molecular technology to date. It can detect slow-growing,

difficult-to-cultivate or uncultivable microorganisms and can be used in situations in which

clinical microbiology diagnostic procedures are inadequate, time-consuming, difficult, expensive

or hazardous to laboratory staff. Inherent technical limitations of PCR are present, but they are

reduced in laboratories that use standardized protocols, conduct rigid validation protocols and

adhere to appropriate quality control procedures. Improvements in PCR, especially probe-based

real-time PCR, have broadened its diagnostic capabilities in clinical infectious diseases to

complement and even surpass traditional methods in some situations. Automation of all

components of PCR is now possible, which will decrease the risk of generating false positive

results due to contamination. The diagnostic PCR applications most relevant for equine practice

are presented below along with their advantages and potential pitfalls.

Respiratory Pathogens

Respiratory pathogens are often contagious, and these infections must be diagnosed rapidly in

order to prevent a disease outbreak and to institute the appropriate management plan. The short

turn-around time and reliability of PCR makes this technology an ideal tool for the diagnosis of

respiratory pathogens.

Equine influenza is commonly diagnosed by virus isolation or detection from nasopharyngeal

swabs collected from horses during the early febrile stage of the disease. Although isolation of

the virus is essential to allow antigenic and genetic characterization of the strain, this technology

is time-consuming and successful isolation is, to be expected at best in about 50% of the cases.

In recent years new methods for virus detection, such as antigen detection ELISAs and PCR,

have been described allowing a rapid diagnosis in the acute phase of infection. PCR based assays

have been described for the identification of equine influenza virus directly from clinical samples

with higher sensitivity than virus isolation and antigen-capture ELISA. Amplification of the

single stranded RNA of equine influenza viruses is performed by reverse transcription-PCR (RT-

PCR) technology, using either a one-step, nested or real-time approach. The hemagglutinin,

nucleoprotein and matrix genes are the common target for these PCR assays. Unfortunately,

comparison of the different PCR assays is precluded by the use of different technologies, the lack

of standardization among the assays, and variation in targeted genes. Nucleotide and deduced

amino-acid sequences of portions of the hemagglutinin gene are now routinely used for

phylogenetic characterization of outbreak strains. Further, novel real-time PCR assays can be

used as a viable replacement for the more traditional methods of quantifying equine influenza

virus in vaccine efficacy studies. Another advantage of PCR is the ability to detect non-viable

virus, a situation which may occur when nasopharyngeal samples are frozen or not adequately

stored and/or shipped to a diagnostic laboratory.

Equine herpesvirus (EHV)-1 and EHV-4 are important, ubiquitous equine viral pathogens, that

cause important economic losses in the equine industry. Both are double-stranded DNA -

herpesviruses that affect the equine respiratory tract and can establish life-long latent infection

after primary exposure. Traditionally, virus isolation has been the gold standard for diagnosing

EHV-1 and EHV-4 infections using blood and nasal secretions. Virus isolation is often hampered

by the fragility of the virus, intermittent viral shedding and the interference with local antibodies.

PCR offers an alternative to virus isolation and has proven to be a sensitive method of detecting

EHV-1/4 in respiratory secretions, peripheral blood lymphocytes and other tissues. Many

conventional PCR assays have been established to study the pathophysiology and improve the

diagnosis of these viruses. Conventional one-step or nested PCR assays do have inherent risks of

carry-over contamination due to post-amplification steps required to detect the PCR products.

Novel molecular platforms such as the real-time PCR have strongly reduced the risk of

contamination. PCR assays used in routine diagnostics are based on the detection of viral

genomic DNA and are therefore unable to distinguish between lytic, non-replicating or latent

virus. Alternative molecular approaches have recently been established using the real-time PCR

to allow discrimination between the different viral states in horses naturally infected with EHV.

Streptococcus equi subsp. equi (S. equi) infection rarely is associated with detection difficulties

when using conventional culture in clinically affected horses. Culture of nasal swabs, nasal or

guttural pouch washes or exudates aspirated from an abscess remains the gold standard for the

detection of S. equi. Culture however may be unsuccessful during the incubation and early

clinical phase of infection and the presence of other -hemolytic streptococci, especially S. equi

subsp. zooepidemicus, may complicate interpretation of cultures. Available PCR assays are

designed to detect the DNA sequence of the S. equi M protein (SeM) gene, the gene for the

antiphagocytic M protein of S. equi. This gene offers enough nucleotide variations between the

two S. equi subspecies to allow full discrimination in clinical specimens. The test can be

completed in a few hours and results may be available on the same day samples are taken. One

of the pitfalls of PCR has been its inability to distinguish between dead and live organisms,

therefore, positive results have in the past been considered presumptive until confirmed by

culture. Nowadays, the viability issue can be addressed by quantitation of the SeM gene or

detection of transcriptional activity of the SeM gene at the RNA level. In several studies, PCR

proved to be up to 3 times more sensitive than culture. PCR accompanying culture on a nasal

swab or guttural pouch lavage may be used in a control program to select possible carrier

animals. PCR should be considered to detect asymptomatic carriers, establish the S. equi

infection status of asymptomatic horses and determine the success of S. equi elimination from

the guttural pouch. A particular problem in the management of strangles outbreaks is the lack of

a suitable PCR assay to differentiate between wild-type and vaccine S. equi strains.

Rhodococcus equi is an important cause of chronic suppurative bronchopneumonia with

extensive abscessation in foals 3 weeks to 6 months of age. Culture of the organism from

tracheal wash (TW) fluid currently is considered the gold standard for diagnosis. However, it can

be difficult to reliably grow R. equi from a single TW sample, possibly because of prior

antimicrobial administration or overgrowth by multiple pathogenic bacterial species. PCR has

been evaluated in order to increase the diagnostic sensitivity of TW fluid samples. Strains of R.

equi isolated from sick foals uniformly contain an 85- to 90-kb plasmid that carries the gene

responsible for expression of a 15- to 17-kDa antigen (vapA) of undetermined function.

Environmental strains of R. equi not associated with disease do not contain this plasmid.

Therefore, detection of the vapA gene of R. equi in a TW fluid sample from a foal with

pneumonia can be considered diagnostic. Both culture and PCR, however, may detect

environmental contaminants of R. equi in TW fluid, but PCR has the ability to distinguish

between virulent and avirulent strains. PCR should be used in conjunction with standard culture

because of the possibility that multiple bacterial pathogens are present in the lower airways and

the inability of PCR to determine antimicrobial sensitivity of R. equi. PCR with its higher

sensitivity and specificity may be useful to rule out R. equi pneumonia in culture-negative foals

that have failed to improve with standard antimicrobial therapy and have clinical signs consistent

with R. equi pneumonia. In clinical situations where the severity of the respiratory signs of the

patient prevents the collection of TW fluid, feces have been shown to be a sensitive surrogate

specimen for the molecular detection of R. equi.

Equine rhinitis A and B virus and equine arteritis virus, although less commonly associated

with infectious upper respiratory tract diseases (IURD), are routinely detected via PCR. The role

of EHV-2 and EHV-5 in nasal secretions of horses with IURD is still unclear. Due to their high

prevalence in horse population and in order to avoid dilemmas with the interpretation of PCR

results, the testing of gamma herpesviruses is at this time not recommended.

Neurologic Pathogens

Although highly sensitive and specific PCR assays have been developed for the detection of

viral and protozoal pathogens in the cerebrospinal fluid (CSF) of neurologic patients, these

methods often are of limited value in the routine diagnosis of these diseases because viremia is

often very short-lived or the pathogen has no affinity to the cells of the CSF. Consequently,

pathogens are usually no longer detectable at the onset of systemic or CNS signs.

Equine protozoal myeloencephalitis (EPM), caused by the protozoal apicomplexa parasites

Sarcocystis neurona and Neospora hughesi, represents one of the greatest diagnostic challenges

for equine practitioners. Molecular diagnostics have also been investigated but their sensitivity

was found to be low. Apparently, intact merozoites rarely enter CSF and free parasite DNA is

destroyed rapidly by enzymatic action. Based on its low sensitivity, PCR testing of CSF should

not be recommended for routine diagnosis of EPM. In contrast, PCR testing of neural tissue has

been shown to be useful as a postmortem test.

Diagnosis of West Nile virus (WNV) encephalitis in horses currently is based on observation

of compatible clinical signs and the detection of serum IgM antibody to WNV by IgM-capture

ELISA. Given the non-specificity of the IgM ELISA (i.e. does not differentiate between disease

and exposure) and the time required to serologically confirm WNV infection, alternative tests

able to rapidly detect WNV in clinical specimens are important. RT-PCR using either a one-step,

nested or real-time approach has been evaluated to investigate ante-mortem cases of suspected

WNV encephalitis in horses and humans using blood. The diagnostic sensitivity of WNV RT-

PCR using either serum or whole blood was very low. However, 57 to 70% of CSF samples from

human beings with serologically confirmed WNV infection tested positive by real-time RT-PCR.

The reduced ability to detect WNV in CSF or serum from equine patients with serologically

confirmed WNV infection is likely due to the short-lived viremia in dead-end hosts, and

emphasizes the fact that in order to detect WNV in blood or CSF, the sample should be collected

early during the disease process. Investigation of the sensitivity of RT-PCR on CSF from horses

with WNV encephalitis has not yet been reported.

EHM is supported by historical and clinical findings, the presence of xanthochromia and

elevated total protein concentration in CSF and the laboratory detection of EHV-1 in blood

and/or nasal secretions by PCR. Because affected horses can shed the virus in nasal secretions

and thus represent a risk of infection for unaffected in contact horses, it is imperative to

determine the risk of shedding in a suspected horse in order to initiate an appropriate infectious

disease control protocol. The dilemma as to whether the virus is in a lytic, non-replicating or

latent state can be addressed by using absolute quantitation or transcriptional activity of the

target gene similar to the approach used for EHV-4. Research groups have recently identified

regions of variation in the genome of different EHV-1 strains (neuropathogenic versus non-

neuropathogenic). A single nucleotide polymorphism at position 2254 of the DNA polymerase

gene (ORF 30) has been associated with a higher risk of EHM development. Rapid PCR assays

have been established to allow differentiation between neuropathogenic and non-

neuropathogenic strains. However, such assays have moderate specificity, since 74 to 87% of

EHV-1 stains associated with EHM have been shown to be of the neuropathogenic genotype.

Therefore, these assays should be used judiciously and the results should always be interpreted in

the context of clinical presentation. Further, these assays should be coupled with additional

assays targeting conserved regions of the EHV-1 genome.

Gastrointestinal Pathogens

The detection of equine gastrointestinal pathogens using conventional tests can be challenging

because these pathogens are sometimes difficult to grow. The use of fecal material for molecular

diagnostics of selected pathogens has been associated with false negative results due to the

presence of inhibitory substances in the feces that can interfere with nucleic acid extraction or

amplification.

Salmonella enterica can cause enterocolitis in susceptible horses; however, infection can also

be present without clinical disease in 1 to 5% of healthy horses and such horses are transient

subclinical shedders. Several factors, including transportation, surgery, antimicrobial treatment,

changes in diet, elevated ambient temperatures and pre-existing gastrointestinal disease, have

been associated with the development of clinical salmonellosis in susceptible horses. Because

these factors are often common among hospitalized horses and because the contamination of the

environment by subclinical shedders poses a risk to the health of hospitalized patients and

personnel, the rapid identification of horses infected with Salmonella enterica is of considerable

importance and allows implementation of appropriate infectious disease control measures.

Microbiologic culture of feces is considered the gold standard in the detection of horses shedding

Salmonella enterica. Time to microbiological culture and positive identification of Salmonella

from feces by clinical laboratories requires at least 48 hours. When small numbers of Salmonella

are present in feces, enrichment steps using selective broth are required, which prolongs the

detection time even further. In recent years, PCR assays have been evaluated for the detection of

Salmonella enterica in feces from horses admitted to veterinary hospitals. Collectively, these

studies have shown that significantly more fecal samples tested positive by PCR than by

microbiological culture. Today’s modern approach to screen environmental samples and feces is

the PCR testing of samples following a selective enrichment step (20 hr) coupled with

conventional microbiological identification. PCR has the advantage of having a very short turn-

around-time and results can be available within 4-6 hours of having completed the selective

enrichment step, which is still 2.5 days shorter than identification through conventional culture.

However, conventional culture will remain the only diagnostic tool allowing serotyping and MIC

testing if needed.

Neorickettsia risticii is the rickettsial agent responsible for Equine Neorickettsiosis (EN) or

Potomac horse fever, a serious enterocolitis of horses. A provisional diagnosis of EN is often

based on the presence of typical clinical signs and the seasonal and geographical occurrence of

the disease. A definitive diagnosis of EN, however, should be based on isolation or detection of

N. risticii from blood or feces of infected horses. Isolation of the agent in cell culture, although

possible, is time-consuming and not routinely available in many diagnostic laboratories. The

recent development of N. risticii-specific PCR assays has greatly facilitated the diagnosis of EN.

Nucleic acid of N. risticii can be detected in the blood and feces of naturally or experimentally

infected horses, but the detection period does not necessarily coincide between the two sample

types. Based on these results, it is recommended to analyze both blood and feces from suspected

horses in order to enhance the chance of molecular detection of N. risticii.

An emerging equine gastrointestinal pathogen, Lawsonia intracellularis, has been described in

young horses. This obligate intracellular bacterium is the causative agent of proliferative

enteropathy (PE), a transmissible enteropathy known to affect a wide range of domestic and wild

animal species. This disease has a worldwide distribution and likely is under-recognized in

horses. Antemortem diagnosis can be challenging and is based on interpreting clinical signs,

clinicopathologic results, ultrasonographic findings and excluding other causes of similar

gastrointestinal findings. Currently, culture of the organism is difficult and is not routinely

offered by laboratories. Antemortem diagnosis relies on serology and PCR, but these tests have

not been systematically evaluated in horses. The combination of both tests as well as repeated

fecal sampling for PCR from target animals will increase the chance of diagnosing the disease.

Novel PCR assays, such as the real-time PCR, have increased the sensitivity of molecular

detection, compared to initial conventional assays. PCR has the advantage of being fast and can

yield positive results in the early stage of disease, when antibodies are not yet measurable.

Furthermore, the molecular assay can be used to monitor treatment success and study the

epidemiology of this pathogen.

The detection of equine coronavirus (ECoV) by PCR in the feces of foals with fever and

diarrhea is difficult to interpret because ECoV has also been detected in the feces of healthy

foals. Healthy foals have been found to be infected mostly by ECoV in a single infection without

any other coinfecting agents, whereas ECoV was found exclusively in association with other

coinfecting agents in sick foals. This is in agreement with coronaviruses in other species, where

the virus may not have enough pathogenic potential to cause disease, but causes local immune

suppression allowing secondary infections to take place more efficiently. In adult horses ECoV

causes a self-limiting disease characterized by depression, anorexia, fever and less frequently

changes in fecal character and colic. More epidemiological studies are needed to better

understand the impact of this emerging disease.

Conclusions

The number of commercially available PCR assays continues to expand and many molecular

assays continue to be developed in the research setting. In the meanwhile, efforts should continue

to increase understanding of the strengths and limitations of these new assays. Molecular

diagnostic tests may enhance diagnostic capabilities, but they should be interpreted within

clinical context and on the basis of individual laboratory performance. Extensive clinical

research and strict adherence to guidelines for method validation are necessary to compare new

molecular diagnostic techniques with existing methodologies, to validate new technology when

comparable conventional techniques are unavailable, and to determine a method’s clinical utility.

Probe-based real-time PCR is an established research tool to quantify infectious agents during

disease and after vaccination or therapy. Adaptations of these research applications will continue

to impact testing in clinical laboratories

RELEVANCE AND IMPACT OF COMMON AND LESSER

CHARACTERIZED RESPIRATORY VIRUSES ASSOCIATED WITH

UPPER AIRWAY DISEASE

Dr. Nicola Pusterla, PhD, Diplomate ACVIM

Professor Equine Internal Medicine

Department of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis,

Davis, California

Objectives of the Presentation

Review the relevance and diagnostic approach of infectious upper respiratory tract diseases

(IURD)

Present epidemiological information on biosurveillance submissions targeting horses with

IURD

Review well characterized and less characterized equine pathogens associated with IURD

Take Home Message

Infectious diseases involving the respiratory tract of horses have been identified as one of the

most common medical entities encountered by ambulatory practitioners or equine internists

nationwide.

Biosurveilance results from the past six years have shown that EHV-4 is the most common

pathogen detected from horses with clinical signs of IURD, followed by EIV, S. equi subsp. equi

and EHV-1.

EIV is being increasingly detected in adult horses with current vaccine history.

Lesser characterized infectious respiratory pathogens such as EHV-2, EHV-5, ERAV and

ERBV may be involved with IURD.

Introduction

Infectious diseases involving the upper respiratory tract of horses are a common medical entity.

Equine herpesvirus-1 (EHV-1), EHV-4, equine influenza virus (EIV) and S. equi subsp. equi are

among the most common pathogens recognized with infectious upper respiratory tract disease

(IURD). Lesser characterized respiratory pathogens such as EHV-2, EHV-5 and equine rhinitis A

and B virus (ERAV, ERBV) have also been associated with IURD. All these respiratory

pathogens spread rapidly due their short incubation time and transmission occurs via fomites,

droplets and aerosols. Although outbreaks can occur at any time of the year, they are most

commonly seen in the late fall, winter and spring, because of the high concentration of young

susceptible horses. The morbidity to infectious respiratory pathogens can reach 100% in a

population of susceptible horses, while the mortality is generally low. The severity of clinical

signs depends mostly on the age and the immune status of the infected horse.

Diagnostic work-up

The diagnosis of IURD relies on the combination of historical information, physical findings,

blood work and antigen detection in nasal secretions. In the case of a viral disease, the blood

work may show mild anemia and lymphopenia during the acute phase, while bacterial diseases

such as strangles are generally characterized by elevated fibrinogen and leukocyte count at the

time the patient displays nasal discharge. Polymerase chain reaction (PCR) has supplanted

conventional culture-based detection methods for the diagnosis of IURD. PCR is fast, reliable,

cost-effective and more sensitive than conventional detection methods.

Respiratory pathogens associated with IURD

Equine Influenza virus

EIV is a single stranded RNA orthomyxovirus of the influenza A type. EIV is classified into

subtypes based on two glycoproteins (hemagglutinin and neuraminidase) projected from the

envelope. These two surface glycoproteins appear to be the most important antigens in terms of

stimulation of protective immunity in the host. The only contemporary subtype of EIV

circulating amongst horse population is the H3N8 (A/equi 2/ prototype Miami/63). Since around

1987 evolution of European and American isolates has apparently diverged, giving rise to two

families of viruses. Consistent with the international movement of horses, American-like viruses

have been isolated in many European countries and to a lesser extent European-like viruses have

been isolated in North America. The American lineage further evolved during the 1990s into the

Kentucky, Florida and South America sublineages. For the Florida sublineage, there are two

clades identified, Clade I with the representatives being Ohio 03 and South Africa 03 and Clade

2 with Richmond 07 as the main representative. According to the OIE surveillance panel, only

representatives of the Florida sublineage circulate amongst horse population with Clade I strains

being present in North America, Europe, South Africa and Japan, and Clade II representatives

being present in Europe, India and China.

Equine herpesvirus-1/-4

EHV-1 and EHV-4 are important, ubiquitous equine viral pathogens affecting the upper

respiratory tract of horses and causing significant economic losses to the equine industry.

Both are double stranded DNA -herpesviruses. Exposure to these viruses and clinical disease

are commonly manifested in foals, weanlings, and yearlings, although clinical signs can be seen

in all age groups. In many countries the seroprevalence for EHV-1/-4 is high, reaching 100% for

EHV-4 and up to 30% for EHV-1. Like other -herpesviruses life-long latent infection can

establish after natural exposure to EHV-1 and EHV-4. The most important sites of latency are

lymphoid tissues, peripheral leukocytes and trigeminal ganglia. Reactivation and shedding of

EHV-1/-4 creates the opportunity for transmission to other horses, which is considered important

in the epidemiology of these viruses and might explain why outbreaks can occur in closed

populations.

Equine herpesvirus-2/-5

It is difficult to determine the exact role -herpesviruses play in the development of IURD.

EHV-2 and EHV-5 are wide spread in horse populations; hence the detection of any of these

viruses can occur in healthy but also in sick animals. These viruses are optimally adapted to their

host, which means that significant clinical expression of infection is rarely encountered. These

viruses contribute to various forms of clinical disease in young horses, including upper

respiratory tract signs, fever, pharyngitis and enlarged lymph nodes. Recent work has shown that

different genetic variants of these viruses circulate amongst healthy horses, hypothesizing that

viral re-infection or re-activation may be the origin of clinical disease. Another characteristic of

the -herpesviruses is their ability to immunomodulate the immune system. This characteristic

makes EHV-2/-5 potential co-factors of infection or disease.

Equine rhinitis A and B virus

Equine rhinitis viruses (ERV) have been given little attention by practitioners compared to

other respiratory viruses, mainly because of the lack of diagnostic modalities. These viruses are

common in horse populations but knowledge of their epidemiology, pathogenesis and association

with disease is poor. There are two ERVs, namely ERAV formerly known as equine rhinovirus 1

and ERBV formerly known as equine rhinovirus 2. These important pathogens are capable of

infecting both the lower and the upper airways. Both natural and experimental infection of

seronegative horses with ERAV has been associated with fever, anorexia, seromucoid nasal

discharge, coughing, lymphadenopathy and occasionally lower limb swelling. Recent outbreaks

of ERAV in Ontario have shown that up to 60% of young horses in race training developed

clinical signs. The diagnosis has remained a true challenge for ERVs until recently. The infection

may be diagnosed by virus isolation, detection of viruses by PCR or demonstration of rising

antibody titers to ERAV or ERBV through virus neutralization using an acute and a convalescent

serum samples. One of the diagnostic challenges with ERAV is that shedding time is very short

following the development of clinical signs. Further, prolonged excretion of ERAV in urine in

racehorses is common, particularly in 2-and 3-year-old and is probably an important source of

infection for other susceptible horses.

Streptococcus equi subsp. zooepidemicus

Streptococcus equi subsp. equi is an obligate pathogen that causes strangles. The closely-

related S. equi subsp. zooepidemicus is an opportunistic pathogen and a common cause of

secondary bacterial bronchopneumonia in young and adult horses. Streptococcus equi subsp.

zooepidemicus has recently been associated with outbreaks of IURD. The clinical signs are

undistinguishable from S. equi subsp. equi infection and include depression, anorexia, elevated

rectal temperature, serous to purulent nasal discharge, coughing and lymphadenopathy with

abscessation. The diagnosis is based on the microbiological or molecular detection of S. equi

subsp. zooepidemicus from draining lymph nodes and ruling out other pathogens causing IURD.

RECENT INSIGHTS INTO EQUINE PROTOZOAL MYELOENCEPHALITIS

Dr. Nicola Pusterla, PhD, Diplomate ACVIM

Professor Equine Internal Medicine

Department of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis,

Davis, California

Objectives of the Presentation

Understand the epidemiology of Sarcocystis neurona and Neospora hughesi

How to recognize and diagnose EPM

How to treat horses with EPM

How to prevent the occurrence of EPM

Take Home Message

EPM is a commonly diagnosed neurological disease of horses in North America caused by S.

neurona and N. hughesi

EPM is often a progressively debilitating disease and clinical signs are dependent on the area

of the CNS parasitized

A presumptive diagnosis of EPM is based on the presence of compatible neurologic signs, the

exclusion of other potential diseases, the presence of specific antibodies to S. neurona and/or

N. hughesi in serum and/or CSF and the response to antiprotozoal therapy

Treatment consist in supportive and medical treatment including anti-inflammatory and anti-

protozoal drugs

Prevention of EPM can be achieved by decreasing stress and exposure to scat from opossums

by feeding off the ground, offering fresh water and preventing wildlife access to horse

pasture/paddocks

Etiology

Equine protozoal myeloencephalitis (EPM) is a commonly diagnosed neurological disease of

horses in North America. The most common cause is Sarcocystis neurona, although other

protozoal pathogens, such as Neospora hughesi, have also been identified in the CNS of horses

with EPM. In the live horse, EPM is a presumptive diagnosis, since antigen detection requires

post-mortem examination. Several serological tests have been developed to aid in the

presumptive diagnosis of EPM, but test interpretation can be difficult, since many horses develop

antibodies to the apicomplexan protozoal organisms in the absence of neurological disease.

Members of the Sarcosystis genus have a 2-host life cycle. The definitive horse, generally a

carnivorous animal, becomes infected via ingestion of sarcocyst infested flesh of an intermediate

host. Following sexual reproduction of the parasite in the wall of the small intestine, oocysts,

which are immediately infectious, are shed via feces. Intermediate hosts become infected by the

accidental ingestion of sporocysts. Within the intestine of the intermediate host, the sporocysts

encyst, releasing the sporozoites, which after penetrating epithelial cells undergo asexual

proliferation by shizogony to produce shizonts/merozoites. Shizogony may take place in any

tissue. The final stage of differentiation in the intermediate host is the formation of sarcocysts,

usually in skelettal muscles. For S. neurona, the Virginian opossum is the definitive host.

Because of the geographic distribution of the definitive host, EPM has only been reported from

the Americas (USA, Canada, Brazil, Panama). The percentage of opossum infected with S.

neurona reported in the literature ranges from 4.5% to 18%. Raccoon, cat, skunk, the nine-

banded armadillo and brown-headed cowbirds have been recognized as intermediate hosts.

Horses acquire the parasite by ingestion of feed or water contaminated by opossum feces. Horses

have been classified as aberrant or accidental hosts because the terminal asexual sarcocyst stage

that is required to the definitive host has not been found in their tissue, despite extensive efforts

(only schizont/merozoites stage in CNS). However, a recent case report involving a 4-month old

filly with neurologic signs, demonstrated shizonts in the brain and spinal cord and mature

sarcocysts in the tongue and skeletal muscle, both genetically and morphologically similar to S.

neurona. This would imply that horses can act as intermediate host as well.

The life cycle of N. hughesi is mainly uncharacterized. However, it must have some similarity

to the close related N. caninum. The definitive host (dog, coyote) for N. caninum eats material

(aborted fetus, placenta or raw infected carcasses) containing N. caninum tissue cysts. Sexual

multiplication takes place in the intestine of the definitive host and oocysts are produced. N.

caninum oocysts are excreted with the feces of the definitive host. Cattle, the intermediate host,

become infected by ingesting food contaminated by infected dog feces containing N. caninum

oocysts. In the gut sporozoites are released from the oocysts and penetrate the epithelium of the

small intestine. The sporozoites then enter different tissues but are mostly found in the reticulo-

endothelial system. Here they further differentiate into tachyzoites. Tachyzoites reproduce

rapidly asexually and invade different tissues and in pregnant animals the fetus via the placenta.

With the onset of the bovine immune response, tachyzoites revert to dormant bradyzoites within

tissue cysts. During future pregnancies, in infected animals bradyzoites are reactivated and

differentiate into tachyzoites that invade the fetus via the placenta. The congenital route of

transfer of N. caninum is the main transmission route in cattle. Although abortion can occur after

tachyzoites infect the fetus, many calves are born with no clinical sign of neosporosis. These

calves are capable to transmit the parasite to their offspring. We recently documented the

transplacental transmission of N. hughesi in broodmares with persistent N. hughesi infection and

were able this year to show for the first time that transmission occurs across two generations.

Epidemiology

Although the incidence of EPM is low (national average of 0.014%), exposure to S. neurona is

defined by the geographic range of the opossum, the definitive host for S. neurona, and ranges

from 0 to 50%, depending on the State and group of horses tested. Geographic distribution and

exposure to N. hughesi has remained poorly investigated. Our study group recently evaluated all

laboratory submissions to the Immunology Laboratory at the William R. Pritchard Veterinary

Medical Teaching Hospital, School of Veterinary Medicine, University of California at Davis

submitted for the detection of specific antibodies to S. neurona and N. hughesi during the time

period from 12/1/2010 to 11/30/2011. Based on the serological results using the NeoFluor and

SarcoFluor, EPM suspected horses were allocated to one of 4 groups: Neospora seropositive

only group; S. neurona seropositive only group; N. hughesi and S. neurona seropositive group;

and N. hughesi and S. neurona seronegative group. A total of 3,123 of the 4,250 (73.5%) serum

samples submitted over the 12 months study period were retained for the study evaluation. The

submissions originated from 49 States . Thirty-eight horses (1.2%) from 21 States were in the

Neospora only group, 840 horses (26.9%) from 40 States were in the Sarcocystis only group, 25

horses (0.8%) from 14 States were in the Neospora and Sarcocystis group, and 2,220 horses

(71.1%) from 49 States were in the seronegative group. The results of this retrospective study

showed that N. hughesi is, alone or in combination with S. neurona, associated with EPM cases.

The wide geographic origin of N. hughesi seropositive horses, including Eastern, Southern,

Midwestern and Western States highlights the need to test for both protozoal pathogens in

neurologically affected horses with suspected EPM.

Clinical Signs

S. neurona and N. hughesi can parasitize any region of the CNS from the anterior part of the

cerebrum to the end of the spinal cord. EPM is often a progressively debilitating disease and

clinical signs are dependent on the area of the CNS parasitized. The most common clinical signs

include asymmetrical weakness, ataxia, spasticity involving all 4 limbs and focal muscle atrophy.

Less frequent signs include depression, behavioral changes, cranial nerve paralysis (facial nerve

paralysis, tongue paralysis, dysphagia), head tilt, seizure and gait abnormality.

Diagnosis

Today’s available serological tests are based on the detection of a specific IgG response to the

causative parasite. The different assays (Western Blot (WB), immunofluorescent antibody test

(IFAT), enzyme linked immunosorbent assay (ELISA)) are distinguishable by the strain of

parasite used as antigen source, assay format, validation samples, recommended diagnostic

sample and result interpretation.

The WB is the oldest test, allowing the semi-quantitative detection of immunoreactive bands to

S. neurona. This method is more sensitive to blood contamination in CSF samples, which could

cause false positive results. The original standard WB was well validated with 295 neurologic

horses. Results for WB on serum yielded a sensitivity of 89% and specificity of 71%, while WB

on CSF yielded sensitivity and specificity of 89%. The original WB was modified by adding a

blocking step and test sensitivity and specificity were estimated at 100% and 98%, respectively,

using 6 confirmed positive horses and 57 true negative horses.

The IFAT is a visual test that interprets the fluorescence of whole pathogens (S. neurona or N.

hughesi). Parasites are immobilized on glass slides and the IgG titers of a sample are determined

by visual assessment of indirect detection of surface immunofluorescence. It was validated using

paired sera and CSFs from a set of 110 general necropsy cases including 8 EPM positive cases

with reported sensitivities of 83% (serum) and 100% (CSF) and specificities of 97% (serum) and

99% (CSF). A subsequent paper included samples from 182 horses - many with multiple

collections - with either natural infections, experimental infections or enrolled in a vaccine study

to examine persistence of antibody. The analysis of this entire set (102 paired sera & CSFs, 326

serum only and 253 CSF only) was used to generate theoretical probabilities of clinical EPM

based on mathematical modeling/simulation.

The ELISA is a simple, quantitative method which generates titers. Depending on the antigen

used, the actual titer values and their significance vary. The immunodominant surface antigen

designated 1 (SAG1) ELISA was validated (serum and CSF) using 6 experimentally infected

horses. Based on this limited set, the sensitivities were reported as 94% (serum) and 100% (CSF)

and specificities as 86% (serum) and 94% (CSF). It is now well documented that a number of S.

neurona isolates lack the SAG1 gene and will test as false negative. The surface antigens 2, 3

and 4 are the basis of the newest EPM test. They were validated with paired sera and CSF

samples from 66 necropsy (neurologic) cases and 200 well characterized field cases. While

individual serum and CSF titers can be determined, the ratio of serum:CSF titers is very

predictive of an EPM diagnosis. As intrathecal IgG production increases, the titer ratio decreases

and ratios of <100 strongly correlate with EPM. A <100 ratio has a sensitivity of 83% and a

specificity of 97%.

Comparative studies are lacking and for this reason we recently evaluated the performance of

the SarcoFluor/NeoFluor (IFAT) with the Sn SAG 2,4,3/Neospora rNhSAG1 ELISA using gold

standard necropsied horses. Both tests performed similarly with a sensitivity of 83.3 and 100%

for the IFAT and ELISA, respectively and a specificity of 82.4 and 100% for the IFAT and

ELISA, respectively. Although both tests performed similarly, the IFAT has two undisputable

advantages: (i) Serum can be used as a stand-alone sample for screening neurological horses with

suspected EPM and calculated likelihood of disease does not require CSF analysis; (ii) The

SarcoFluor and NeoFluor are the only commercially available tests that do test for both protozoal

pathogens (S. neurona and N. hughesi).

Treatment

Several anti-protozoal drugs have been used for the treatment of EPM (dihydrofolate reductase

inhibitors such as potentiated sulfonamides and pyrimethamine; anti-protozoal drugs such as

diclazuril, toltrazuril, ponazuril and nitazoxanide). The clinical improvement rates for the

different drugs is similar and ranges from 57.1 to 61.5% (mean 58.9%). Data show that

approximately 10% of treated horses recover completely during the periods of evaluation.

Additional drugs that have been used in conjunction to anti-protozoal drugs include NSAIDs

(flunixin meglumine), vitamin E and DMSO.

Prevention

Prevention of EPM can be achieved by decreasing stress and exposure to scat from opossums

by feeding off the ground, offering fresh water and preventing wildlife access to horse

pasture/paddocks. No vaccine is actually available on the market.

Prophylactic treatment with coccidiostatic and coccidiocidal drugs is at this stage empiric and

not based on any scientific data. Anecdotally, ponazuril has been given at an interval of 1

week/month. A recent study from Florida showed that the oral administration of ponazuril

significantly decreased the intra-thecal anti-S. neurona antibody response in horses inoculated

with S. neurona sporocysts.

Laboratory Contact

IFAT (SarcoFluor and NeoFluor)

Immunology Laboratory, William R. Pritchard Veterinary Medical Teaching Hospital, 1

Garrod Drive, Room 1023, Davis, CA 95616. Phone: (530) 752-7373; Fax: (530) 754-9007;

Website:www.vetmed.ucdavis.edu/vmth/small_animal/laboratory/lab_pages/immunology/For

ms.cfm.

SAG 2,4/3 ELISA

Equine Diagnostic Solutions, LLC, 1501 Bull Lea Rd. Suite 104, Lexington, KY 40511.

Phone: (859) 288-5255; Website: www.edslabky.com/home.html.

EMERGING ENTERIC PATHOGENS - EQUINE CORONAVIRUS AND

LAWSONIA INTRACELLULARIS

Dr. Nicola Pusterla, PhD, Diplomate ACVIM

Professor Equine Internal Medicine

Department of Medicine and Epidemiology, School of Veterinary Medicine, UC Davis,

Davis, California

Objectives of the Presentation

Understand the epidemiology of equine coronavirus (ECoV) and of Lawsonia intracellularis

How to recognize and diagnose ECoV and Lawsonia intracellularis infection

How to treat animals with ECoV and equine proliferative enteropathy

How to monitor and prevent the occurrence of emerging enteric pathogens

Take Home Message

ECoV causes a self-limiting disease in adult horses characterized by fever, depression,

anorexia and less commonly diarrhea, colic and encephalopathy. Leukopenia due to

lymphopenia and/or neutropenia are consistent hematological abnormalities associated with

ECoV infection.

The diagnosis of ECoV infection is based on the molecular detection of ECoV in feces.

EPE is a disease of foals and is characterized by fever, depression/anorexia, edema, diarrhea,

colic or weight loss. Hypoproteinemia due to hypoalbuminemia is a consistent feature of EPE.

Diagnosis of EPE is based on the presence of clinical signs in conjunction with serology and

PCR.

Treatment consist in supportive and medical treatment.

Monitoring strategies combine daily physical evaluation of foals in an attempt to detect subtle

clinical signs with the regular measurement of serum protein/albumin concentration and

antibody detection of L. intracellularis.

Equine Coronavirus

Equine coronavirus (ECoV) is classified within the Betacoronavirus genus, along with bovine

coronavirus (BCoV), porcine hemagglutinating encephalomyelitis virus, mouse hepatitis virus,

rat coronavirus (sialodacryoadenitis virus), and certain human coronaviruses such as OC43 and

HKU1, and also with Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), and

Middle East Respiratory Syndrome coronavirus (MERS-CoV), that have caused epidemic

outbreaks of respiratory disease in human beings in the last decade. ECoV has been recently

associated clinically and epidemiologically with emerging outbreaks of pyrogenic and enteric

disease in adult horses in Japan, and anorexia, lethargy and fever in the United States

Coronavirus infection typically begins in the proximal small intestine and subsequently spreads

to the colonic crypt cells, leading to blunting of the villi and subsequent villous atrophy. It is the

loss of epithelial cells that results in malabsorption and maldigestion of nutrients and acute

diarrhea. Following a short incubation period of 48-72 hours, adult horsed develop fever,

anorexia and depression. Changes in fecal character, ranging from soft-formed to watery

consistency, and colic are seen in less than 20% of affected horses. A small number of horses

develop acute neurologic signs consistent with encephalopathy and characterized by severe

depression, headpressing, ataxia, proprioceptive deficits, recumbency, nysthagmus and seizure.

Common hematological abnormalities are leucopenia due to neutropenia and/or lymphopenia.

ECoV infection generally resolves within 1-4 days with supportive care consisting in the

administration of anti-inflammatory drugs and oral or intravenous fluids. Fatalities have been

associated with septicemia, endotoxemia as well as metabolic derangements leading to

encephalopathy (hyperammonemia).

Historically, the detection of ECoV has relied on either electron microscopy, antigen-capture

ELISA or viral isolation from the feces. All these detection modalities lack sensitivity, especially

if viral particles are not present in sufficient numbers. Real-time PCR for the detection of ECoV

has supplanted many conventional virological assays, mainly due to its short turn-around-time,

high throughput capability and increased analytical sensitivity and specificity. The overall

agreement between clinical status and PCR results for ECoV is over 90%.

Infected horses can shed ECoV up to 14 days. Due to the feco-oral transmission route and the

high infectious nature of ECoV, common sense biosecurity protocols should be instituted during

an outbreak of ECoV. ECoV PCR positive horses (clinical or subclinical) should always be

isolated from the rest of the population to decrease the exposure risk and environmental

contamination. Potentially exposed horses should not be moved until their definitive status has

been determined. For isolation purposes use an empty barn or an isolation unit. In a barn

situation, close one end of the barn and use it as isolation area. Caretakers and owner should

wear gloves, protective clothing (coveralls, disposable gowns) and dedicated footwear.

Good hand hygiene should be instituted (faucet with warm/cold water or hand sanitizer).

Establish barrier nursing in the form of footbath or mats in front of the isolation unit and each

stall. This will minimize the spread of pathogens from stalls to clean areas. It is very important to

control traffic and minimize contact of affected horses with the general public. Hygiene should

be maximized by regular cleaning and disinfecting.

Lawsonia intracellularis

Lawsonia intracellularis is the etiologic agent of the recently recognized and emerging

intestinal disease in horses, called equine proliferative enteropathy (EPE). L. intracellularis is an

obligate intracellular, curved, gram-negative bacterium that resides freely within the apical

cytoplasm of infected intestinal enterocytes. It causes proliferation of the affected enterocytes,

resulting in a thickened small and sometimes large intestine. L. intracellularis can only be grown

in vitro in cell culture and requires a specific atmosphere for growth. Besides horses, L.

intracellularis infects many species of domestic and wild animals, including pigs, hamsters,

rabbits, fox, deer, ferrets, ostriches, and non-human primates. In the last few years, reported

cases of EPE have been increasing, primarily in post-weaning foals and occasionally in adult

horses. The disease has almost reached a worldwide occurrence and has been reported in the

USA, Canada, Europe, South Africa, South America, Japan and Australia.

Predisposing factors such as the stress of weaning and parasitism have been suggested in the

development of EPE in foals. A feco-oral infection route has been established for foals via

contaminated feed and/or water. In pigs, the incubation period is 2-3 weeks following exposure,

however, this period has not been determined for horses. Because of the wide host range of PE,

numerous potential reservoir hosts exist. L. intracellularis has recently been detected by PCR

from the feces of a variety of domestic and wild animals. A preliminary investigation into the

epidemiological relationships between L. intracellularis isolates from pigs and horses suggests

that they represent different strains and therefore may be host-specific.

There are characteristic signalment, seasonality, clinical signs and blood work abnormalities

associated with EPE. The disease is generally manifested in foals 2-8 months of age and in North

America is often seen between August and February. Lethargy, anorexia, fever, peripheral

edema, weight loss, colic and diarrhea are amongst the most common clinical findings in

affected foals. Although diarrhea is commonly seen in affected foals and can vary from cow pie

to watery, some affected foals may have normal fecal character. Foals with EPE may also have

concurrent disorders such as respiratory tract infections, gastric ulcerations and intestinal

parasitism.

The most consistent laboratory finding is hypoproteinemia due to hypoalbuminemia. Total

protein is generally less than 5.0 g/dl and albumin is usually less than 2.0 g/dl. Affected foals may

also demonstrate non-specific blood abnormalities such as anemia or hemoconcentration,

leukocytosis or neutropenia, hyperfibrinogenemia, increased activity of muscle enzymes and

electrolyte abnormalities.

A presumptive diagnosis of EPE is generally made based on age of the affected animal, clinical

signs, hypoproteinemia/hypoalbuminemia, presence of thickened small intestinal loops on

ultrasonographic evaluation and ruling out other causes of enteropathy and protein losses. An

ante-mortem diagnosis is generally confirmed via PCR detection of L. intracellularis in feces or

rectal swab and/or serology. It is essential to combine both molecular and serologic diagnostic

testing, since these modalities have high analytical specificity but variable sensitivity depending

on the situation. Negative PCR results can be expected if the fecal samples are collected from

foals with prior antimicrobial treatment or during advanced disease stage, when L. intracellularis

organisms are no longer expected in the feces. Negative serological results can be expected in the

early stage of the disease, when humoral immune responses are not yet strong enough to be

detectable by serology.

It is important to treat affected animals early, before lesions become advanced and result in

marked weight loss and critically low serum protein values. Treatment of EPE in horses involves

the use of antimicrobials such as macrolides, alone or in combination with rifampin,

chloramphenicol, oxytetracycline, or doxycycline administered for 2-3 weeks. The choice of

antimicrobial in the treatment of EPE should take into account the risk of inducing disturbance of

the gastrointestinal flora and renal toxicity. This is especially a concern when treating older foals

with severe hypoalbuminemia. In addition, supportive care such as intravenous fluids, plasma

transfusion, parenteral nutrients and anti-ulcer drugs are commonly used to treat affected foals.

Concurrent medical conditions should also be addressed. Rapid clinical improvement following

treatment is to be expected; however, it may take weeks for the hypoproteinemia to resolve.

Spontaneous recovery of clinically affected foals has not been documented, and treated foals

usually survive the disease. Long-term sequelae have not been reported; however, clinically

affected and successfully treated foals sell for an average of 68% of the average price of

unaffected foals by the same stallion.

Early recognition of clinical cases and separating them from the rest of the susceptible foals

until full recovery or cessation of fecal shedding appears to be a logical biosecurity measure to

prevent spread and environmental contamination. The monitoring of a herd with endemic EPE

status includes the regular physical evaluation of resident foals and the monthly or bimonthly

assessment of total protein concentration and monthly serological status. Monitoring for

exposure to L. intracellularis and hypoproteinemia/hypoalbuminemia should begin at least 4

weeks prior to the historical first detection of clinical cases.

Prevention strategies have been best described in pigs using in-feed antimicrobials and a

commercially available L. intracellularis vaccine. Recent work has shown that detectable

humoral and cellular responses can be measured in foals administered an avirulent live L.

intracellularis vaccine. The L. intracellularis vaccine has been shown to be safe and the

administration well tolerated by the foals. Under experimental conditions, weanling foals

vaccinated intra-rectally with an avirulent live vaccine against L. intracellularis were protected

against clinical and subclinical EPE following challenge exposure with a virulent L.

intracellularis isolate of equine origin. The extra-label use of the L. intracellularis vaccine

should be considered on naïve and endemic farms in an attempt to reduce or prevent EPE.

Timing of vaccine administration should again be synchronized with historical disease

occurrence. Further, routine monitoring for clinical signs and hypoproteinemia/hypoalbuminemia

is still recommended even when vaccine prophylaxis is used.