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Keywords:

  • Equine herpesvirus type 1-associated myelopathy;
  • Equine herpesvirus type 1 gG ELISA;
  • Herpesvirus;
  • Horse;
  • Nasal shedding;
  • Polymerase chain reaction

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Background: Because of the serious disease sequelae associated with equine herpesvirus type 1 (EHV-1) infections, awareness and control measures used to control outbreaks are important issues for all horse populations.

Objectives: Describe the occurrence and management of an outbreak of EHV-1 infection at a veterinary hospital.

Animals: Horses hospitalized at a referral veterinary hospital.

Methods: A horse with myeloencephalopathy associated with EHV-1 infection (EHM) was admitted for diagnostic evaluation and treatment under strict infection control procedures. We describe the occurrence and management of a nosocomial outbreak of EHV-1 infections associated with admission of this patient.

Results: Despite institution of rigorous biosecurity precautions at the time of admission of the index case, EHV-1 infections spread to 6 other horses that were hospitalized at the James L. Voss Veterinary Teaching Hopsital, including 2 that served as sources of infection for horses on their home premises after discharge. Infection with EHV-1 was confirmed by polymerase chain reaction (PCR) and by seroconversion documented by glycoprotein G ELISA. A voluntary quarantine was imposed and admissions were restricted to prevent additional horses from being exposed. Quarantine duration was abbreviated by serial testing of all horses with PCR.

Conclusions and Clinical Importance: These findings illustrate the contagious disease risk that can accompany management of horses with EHM. Horses with active nasal EHV-1 shedding should be isolated in an airspace that is separate from other horses by strictly enforced biosecurity and isolation procedures. Serial testing with PCR may be a useful adjunct to determine when the risk of transmission has been minimized.

Abbreviations:
convPCR

conventional (gel-based) PCR for EHV-1

CSF

cerebrospinal fluid

CSU

Colorado State University

CSU-VDL

Colorado State University Veterinary Diagnostic Laboratory

EHM

equine herpesvirus-associated myelopathy

EHV-1

equine (or equid) herpesvirus type 1

gB-EHV1

EHV-1 specific glycoprotein B genome sequence

gG ELISA

EHV-1 specific glycoproteinG-based ELISA

IHC

immunohistochemistry

JLV-VTH

James L. Voss Veterinary Teaching Hopsital

PBMC

peripheral blood mononuclear cells

qPCR

quantitative polymerase chain reaction for EHV-1

SN(EHV)

cross-reactive EHV-1/4 serum neutralization assay

Despite the high seroprevalence of equine herpesvirus type 1 (EHV-1) in horse populations worldwide, there are few reports about outbreaks of equine herpesvirus type 1 associated myeloencephalopathy (EHM) in the veterinary literature. Those reported are either characterized by high case fatality rates,1–3 occurred on renowned horse operations,4 or were the first described outbreak in a country or continent.5 EHM probably is a rare disease, but EHM outbreaks are likely underreported. Few publications describe the sequence of events after admission of EHM-affected horses into an equine referral hospital of veterinary college.3,6,7 Horses with acute clinical signs of EHM still may shed virus through nasal secretions into the environment. Therefore, barrier precautions and isolation from other horses are strongly advised when a horse with EHM is admitted into any equine hospital. However, there are still many unanswered questions: What is the duration of viral nasal shedding? What are the most appropriate diagnostic tests? What environmental control measures should be in place? What is the appropriate duration of quarantine?

This report describes the events that occurred during an outbreak of EHV-1 infection associated with EHM that occurred at a referral hospital and summarizes the measures that were taken to mitigate this disease.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Floor Plan and Description of the Premises

The equine hospital of the James L. Voss Veterinary Teaching Hopsital (JLV-VTH) contains 4 wings, positioned in the shape of an “H” (Fig 1). Each wing is named after its geographical orientation, and has specific patient assignments. The Northwest aisle (NW) is used to house patients admitted by the medicine service (14 stall units); the Northeast (NE) aisle is used to house horses that present with gastrointestinal problems other than colitis, fever, and leukopenia (10 stalls); the Southeast (SE) aisle is reserved for orthopedic cases (14 units); and the Southwest (SW) aisle is used to house patients from a variety of services (13 units). An approximately 3 m high solid wall separates the eastern from the western wings. However, there is a 0.2 m space between the top of this wall and the roof. There is limited possibility for air flow from one aisle to the other.

image

Figure 1.  Equine barn floor plan of the James L. Voss Veterinary Teaching Hospital. NE, SE, SW, NW are the 4 wings of the equine barn. Numbers represent individual stall units. Occupied units on the index case (!) admission day are indicated by a letter A–G, or X. Areas of routinely heightened biosecurity precautions (NE, colic aisle) or ad hoc implemented heightened biosecurity precautions (index case admission site) are lightly shaded.

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A separate isolation facility is located 50 m north of the main hospital building, and provides 5 equine stall units. Additionally, when needed, there are 2 stall units available in the livestock animal (LA) hospital. The LA hospital has construction similar to the equine hospital. However, it is separated by a wall and solid door that is typically kept closed. The LA hospital patient entrance is separate from the equine entrance, and there is very limited personnel or equipment traffic between the 2 areas of the LA hospital.

The equine hospital connects to an enclosed breezeway that is used for outpatient examinations and connects to other areas of the main hospital. A direct northern access to the NW wing usually is closed by a solid door. This entrance is used only for patients when a recumbent horse is admitted into the stall unit used for neurological or recumbent horses. This unit has padded walls and a central hoist (combined units 12 and 13, Fig 1).

A fenced paddock located to the east of the LA hospital houses a small herd of horses (n = 10) used for teaching. On the day the index case was presented (Day 1) this herd included 9 male horses and 1 female horse, and the group composition had been stable for approximately 12 months.

Routine Biosecurity Measures in the Equine Hospital of the JLV-VTH

Infection control and biosecurity policies and procedures are described in detail and available online to all personnel, and all students, staff, and house officers received formal orientation before working with patients.8 It was a stated expectation that all personnel working in the JLV-VTH know and comply with all aspects of these policies and procedures.

Briefly, all inpatients in the equine hospital were housed in individual stalls with solid side and rear walls and a slatted entry gate. Footmatsa containing a peroxymonosulfate disinfectantb were placed at the entry and exit points for the aisles leading to different sections of the equine hospital, at the gate for all colic recovery stalls, at the entry of all isolation stalls, and at the entrance to office and meeting areas (eg, rounds rooms, technicians' offices). Standard cleaning and disinfection procedures included removal of gross contamination, scrubbing surfaces with detergent and sodium hypochlorite (1 : 32), rinsing, and application of quaternary ammonium disinfectant (1 : 256).c,8 Additional measures are used when cleaning and disinfecting stalls and other areas associated with high risk patients (eg, colic aisle, isolation). These included an additional application of sodium hypochlorite solution (1 : 12), and high-pressure, high-temperature rinsing as a final step. Additionally, a 4 times normal concentration (ie, 4%) solution of peroxymonosulfate disinfectant was applied periodically as a mist to all surfaces in the LA Hospital as described previously.9

All wings of the equine hospital have hand wash stations and dispensers for alcohol-based hand sanitizer. Hand sanitizing (washing or use of sanitizer) is required before personnel contact animals. Dispensers for disposable gloves are located throughout the 4 wings of the equine hospital. Limited barrier precautions are required when working in the outpatient and general inpatient areas of the equine hospital (eg, clean protective outer garments). Increased barrier precautions are used when working in the NE (colic) aisle. Rubber overboots, fabric gowns, and gloves are required to be worn when entering this area. Additionally, disposable plastic gowns (worn over the cloth gowns) and disposable gloves are required to be worn when working with patients. Grooming tools, thermometer and stethoscope are assigned to each patient at admission. Additional isolation and barrier precautions are used when managing patients in the isolation building as described.8

Personnel

At the time of admission of the index case (Day 1), 2 faculty clinicians (an equine medicine clinician and a critical care clinician) together with 2 equine medicine residents were assigned to NW and NE wings. There were up to 4 final-year (senior) veterinary students on a 2-week rotation with both services. Daytime technical support for the entire equine hospital area consisted of 3 veterinary technicians. One technician was assigned to work solely in the NE wing while the others provided service throughout the equine hospital. Two senior students, together with a veterinary technician and a clinician received emergencies through the evening and night hours, and were responsible for monitoring and treating inpatients in all areas including the isolation facility. Daily stall cleaning duties was conducted by professional staff. If increased biosecurity precautions were being used in an area or a stall, these measures also were followed by cleaning personnel. Critically ill patients were fed by the senior student assigned to the case, in consultation with the veterinarian responsible for that patient.

Diagnostic Tests

Polymerase chain reaction (PCR). For nasal swab sample analysis, 2 different PCR assays for EHV-1 specific glycoprotein B sequence (gB-EHV1) detection were used. The 1st assay was the conventional (gel-based) PCR for EHV-1 (convPCR) assay that is routinely used by Colorado State University Veterinary Diagnostic Laboratory (CSU-VDL)10 and validated by the American Association of Veterinary Laboratory Diagnostics (AAVLD). The qPCR assay was a quantitative real-time PCR assay designed as a research diagnostic tool in the laboratory of two of the authors, and has been validated.11

Serology. Seroconversion was evaluated with 2 assays, a serum neutralization (SN) test (recommended by Office International des Epizooties [OIE], http://www.oie.int/eng/normes/mmanual/2008/pdf/2.05.09_EQUINE_RHINO.pdf), and an ELISA that detects antibodies produced against the EHV-1 specific glycoprotein G (gG ELISA).d The gG ELISA was performed according to the manufacturer's description. For the SN test, sera were serially diluted (1 : 2) and added to a standardized amount of a reference strain of EHV-1 (strain: NVSL Ames). After a short incubation, the dilutions were inoculated onto Kentucky equine dermal cells. After 48 hours of incubation, the cells in each serum dilution were evaluated for cytopathic effect (CPE) indicating virus growth. The highest dilution without CPE was reported as a titer.

Cerebrospinal Fluid (CSF) Analysis. For routine cell count, a Fuchs-Rosenthal cytometer was used, and total protein in CSF was measured by a turbimetric benzethonium chloride precipitation method. Cytospin preparations were made for cytological evaluation after routine Wright's staining.

Histopathology and Immunohistochemistry (IHC). Complete necropsy and histopathological examination of the index case was performed after euthanasia. Immunohistochemical staining of sections from the brain and spinal cord was performed by an avidin-biotin peroxidase method with a combination of 3 monoclonal antibodies that are cross-reactive for EHV-1 and EHV-4.12

Clinical Definitions

Fever in a horse was defined as a rectal temperature ≥38.5°C (101.3°F). Neurological gait abnormalities were described in grades of ataxia, dysmetria, and weakness (0 = absent, 4 = recumbent and unable to rise).13

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Admission of the Index Case

An 8-year-old Quarter Horse mare was admitted to the JLV-VTH on a Monday in mid-October. The admission day of the index case was designated “Day 1” (all other time events were expressed relative to this date [negative days for events that occurred before admission of the index case (Day −x), or positive days for days after admission of the index case (Day x), respectively]. The mare had been at a competition 1 week before admission, and presented to the referring veterinarian on Day −4 with mild signs of discomfort or colic. By Day −2 mild ataxia of the hind limbs was noticed, which became moderate in severity by Day −1. On presentation, the horse was tetraplegic with some forelimb activity. Because of the suggestive history and presentation, EHM was considered the most likely diagnosis. The horse was transported to the north entrance of the NE aisle next to the padded stall that is maintained for neurological or recumbent horses. Because the horse was fractious when manipulated, general anesthesia was induced with injectable agents to allow transport of the horse from the trailer into the stall using a hoist.

Hospital Occupation of Other Horses on Day 1

In addition to the index case, there were 3 inpatient horses housed in the NE aisle, 2 in the NW aisle, 6 in the SE aisle, and 7 in the SW aisle (including a mare in the 7th month of gestation; Fig 1). Horses housed in the NE aisle included a 6-month-old Quarter Horse weanling and a postsurgical colic case that had been febrile on Day −3. Other horses were of various breeds, and were 4–25 years old.

Heightened Biosecurity and Barrier Precautions

The empty stall units 9, 10, 11, and 14, those that are adjacent to the stall where the index case was hospitalized (Fig 1), were modified to serve as a temporary staging area where patient-assigned materials were stored and where barrier clothing was doffed and donned. This space also served as a barrier to separate the area with heightened biosecurity from the rest of the equine hospital. Before entering the patient's stall, personnel donned disposable exam gloves and a disposable plastic barrier gown over the coveralls. A disinfectanta footbath was placed outside the patient's stall. The barrier gown was stored stall-side after personnel had exited the stall and gloves were discarded. Disinfectanta-soaked foot mats were placed the outside buffer zone and personnel were required to step on them when entering or leaving the area. Alcohol-based hand sanitizer was available and there was a hand wash station located outside the biosecurity area between stall units 14 and 15 (Fig 1). Personnel were required to wash their hands when leaving this area. A small stock of materials was assigned for dedicated use with this horse and also was stored in this staging area. These materials were either discarded after use or cleaned and disinfected before being used with other patients. This included stall cleaning implements. Both daytime and nighttime personnel had other in-patients and outpatients under their care, although every effort was made to minimize contacts when possible.

Additionally, the primary clinician notified biosecurity personnel that EHM was the primary differential diagnosis for this horse when it was admitted (in accordance with established biosecurity protocols for the JLV-VTH).8 Because the occurrence of fevers in multiple horses was one of the first signs indicating nosocomial spread of EHV-1 during a previous outbreak,3 an e-mail was sent on Day 1 to all faculty, residents, and staff working in the equine hospital requesting that they strictly follow standard infection control procedures when caring for other horses (especially hand hygiene practices) and that they closely monitor other hospitalized horses for signs associated with EHV-1 infections. Specifically, all hospitalized horses were required to be monitored at least twice daily for evidence of fever and other clinical signs, and to notify biosecurity personnel if any pertinent signs were noted.

Samples Collected during Admission and Results

After transportation into the stall, while the index case was anesthetized, an atlanto-occipital CSF centesis was performed. A lumbosacral sample collection also was attempted at that time, but was unsuccessful. A nasal swab sample and a whole blood sample in EDTA were submitted for EHV-1 PCR analysis. Serum collected upon admission was frozen and stored at −20°C until further analysis. ConvPCR results from the nasal swab sample and the whole blood sample were negative. qPCR, however, detected gB-EHV1 in the nasal swab sample and in the isolated peripheral blood mononuclear cell (PBMC) fraction of the EDTA sample. The results of the CSF analysis were within normal limits with a total nucleated cell count of 2 cells/μL and a protein concentration of 35 mg/dL. EHM is a reportable disease in Colorado, and thus the office of the Colorado State Veterinarian was notified of the positive PCR results as soon as they became available (Day 2).

Disease Progression in the Index Case

The index case received supportive care consisting of polyionic isotonic IV fluids (Plasmalyte,e at 2 mL/kg/h), antibiotics (ceftiofur-Na,f 4.4 mg/kg IV q12h) and dexamethasone phosphate (Dexamethasoneg 0.05–0.1 mg/kg; IV q12h for 3 days), and the horse was turned from side to side every 8–12 hours. On Day 2, the horse was tetraplegic with some forelimb activities, which resulted in heavy paddling and digging movements. The horse's tail was flaccid and the bladder was distended and flaccid on rectal palpation. Urine could be easily evacuated by gentle pressure during rectal palpation. To protect the horse from self-inflicted injury, the mare was sedated using a continuous rate infusion (CRI) of detomidineh (10 mg/h) starting in the evening of Day 2 and the CRI was discontinued on the morning of Day 6. On Day 4, sling-supporti was applied. CRI was stopped 1 hour before an attempted lift, and remaining detomidine sedative effect was antagonized by injecting atipamezole hydrochloridej (25 mg IV). The horse was unable to stand during this first attempt. The same procedure was repeated on Day 5 and Day 6. On Day 5, the horse was able to stand assisted by the sling for approximately 3–5 minutes. On Day 6, the horse tripped in the sling, and was nonweight bearing on its hind limbs for a short period of time. The sling procedure was discontinued. A few hours later, the mare developed mild colic signs, and ultrasonographic examination of the abdomen disclosed abdominal effusion. The fluid was identified as urine after abdominocentesis and determination of fluid-to-plasma creatinine concentration ratio, suggesting bladder rupture. Based on the poor prognosis, the mare was euthanized and submitted to CSU-VDL for complete necropsy.

Pathological Examination of the Index Case

Necropsy confirmed the presence of a ruptured bladder and uroperitoneum. Other findings included mild pericardial effusion and mild gastric ulceration. Microscopic examination identified moderate, multifocal vasculitis in the spinal cord that was accompanied by axonal degeneration and hemorrhage. Mild multifocal vasculitis with gliosis in the brain; moderate, multifocal, acute hepatocellular necrosis; and moderate diffuse subacute cystitis were additional findings. The microscopic lesions in the brain and spinal cord were suggestive for EHM. IHC was performed and showed the presence of EHV antigen primarily in glial cells near lesions.

Fevers in Other Hospitalized Horses

A 7-year-old QH mare (A) hospitalized in NE after celiotomy for a small colon impaction (Day −5) was found to be febrile on Day −3 and then again on Day 2 and Day 4, and remained febrile until Day 6 (Fig 1). Because this horse had been febrile before admission of the index case, subsequent febrile episodes were not immediately attributed to EHV-1 infection. Two other horses (B and C), both housed in the SE wing, were found to be febrile on Day 6. Horses D and E, which had been housed in stalls opposite from Horses B and C, were released from the hospital hours before the fevers in horses B and C were detected. Biosecurity personnel at the JLV-VTH were notified of the occurrence of fevers in horses B and C on Day 6, and increased biosecurity precautions were immediately implemented for all horses in the SE wing (ie, rubber overboots, plastic barrier gowns, and disposable exam gloves were worn when entering stalls; personnel were required to use foot baths containing peroxymonsulfate disinfectant when entering and exiting stall; and hand washing was required after leaving stalls). Nasal swab samples from horses B and C and from all horses in the SE wing were collected on Day 6 and Day 8, and submitted to CSU-VDL for analysis by convPCR. Results for these tests became available on the afternoon of Day 8 indicating that horses B and C were positive for EHV-1. Results for the other 3 horses housed in the SE wing were negative. Serum samples from all hospitalized horses were collected on Day 6, and stored frozen. Despite instructions that rectal temperatures be measured twice daily, this was not done with 2 hospital-owned teaching horses (F and G) that had been temporarily housed in the SW aisle beginning on Day 5. This oversight was detected on Day 9 and both horses were found to be febrile on that day. Nasal swabs from horses B, C, F, and G, collected during Day 6 through Day 9, were positive for gB-EHV1 using convPCR (EHV1). Swabs from other horses collected in the SE aisle were negative.

Measures Used to Prevent Further Spread of Infection at the JLV-VTH

On Day 9, the Colorado State Veterinarian was notified that additional horses in the facility were infected. Despite biosecurity precautions that had been implemented, it seemed likely that these infections occurred through nosocomial transmission. Quarantine of all 19 hospitalized horses was initiated voluntarily by the JLV-VTH in the evening of Day 8, and admission of other horses was stopped until the risk of nosocomial transmission was controlled. Owners of horses that had been admitted to the JLV-VTH as outpatients or inpatients between Day 1 and Day 9 were notified about the EHV-1 outbreak, as were all referring veterinarians. Owners were advised to take rectal temperatures of their horses twice daily, and to isolate them from other horses on their property if possible.

On Day 8, biosecurity precautions were increased for horses hospitalized in the SW and NW wings, using precautions that were already instituted for the NE and SE wings. Additionally, all personnel were required to wear coveralls and rubber boots when entering the equine inpatient areas. All horses housed in the equine hospital were considered to be exposed to the virus regardless of their clinical status and remained within their respective wings of the hospital. However, to reduce the risk of virus transmission, horses were separated if possible from one another leaving at least 1 empty stall between them. The only exceptions were a pregnant mare, which was relocated on Day 10 from SW wing to a stall in the isolation unit to minimize the risk of exposure, and horse A that was relocated to a unit in the LA hospital. A horse that had been donated to the JLV-VTH because of severe lameness that had been housed in the SE wing was euthanized on Day 11. All horses remained strictly stall-confined and were fed a hay diet only. Access to the equine hospital was restricted to necessary personnel.

Neurological Sequelae in Other Horses

By Day 10, Horse A demonstrated moderate, asymmetrical ataxia and limb weakness consistent with EHM. After the horse's immediate relocation to the LA hospital area, treatment with valacyclovir hydrochloridek (30 mg/kg PO q12h) was initiated and continued for 10 days.14 Horses D and E that were discharged from the hospital on Day 6 before detection of the fever in horses B and C were linked to propagation of disease at home. Several horses on farms D and E became febrile. In addition, 1 horse on farm E developed clinical signs consistent with EHM. Infection was confirmed in febrile horses using convPCR. Quarantine was initiated on these other premises in collaboration with the Colorado State Veterinarian.

Nasal Swab Samples Collected during Quarantine

Between Day 11 and Day 25, nasal swab samples were collected daily from all horses in the hospital. Nasal swab specimens collected between Day 22 and Day 25 were submitted for gB-EHV1 PCR analysis (convPCR). PCR results on all of these nasal swab samples were negative. Based on these results and the lack of occurrence of any new clinical cases, the voluntary quarantine of the JLV-VTH was lifted on Day 25 (in consultation with the Colorado State Veterinarian). Hospitalized patients were released with instructions to isolate them for another 2 weeks and take daily rectal temperature measurements. All nasal swab samples, collected between Day 11 and Day 25, were retested by qPCR and found to be negative for gB-EHV1.

Before admission of any new horses as outpatients or inpatients (after release of the quarantine and discharge of all horses), all Equine Hospital facilities were cleaned and disinfected by standard procedures,8 in addition to disinfection by mist application of 4 times regular strength peroxymonosulfate disinfectant solution as described previously.9 Patient admission was resumed on Day 28.

Seroconversion of Hospitalized Horses

Serum samples collected from all horses on Day 6 and Day 26 were analyzed for the presence of EHV antibodies by gG-ELISA and SN assay. Serum samples from horses D and E were not available for testing. Seroconversion (defined as a 4-fold increase in titer), based on gG-ELISA results, occurred in horses A, B, C, F, and G but did not occur in the other 12 horses that had been quarantined at the JLV-VTH. In addition, the acute serum sample (Day 6) from horse B was positive by gG-ELISA with an absorbance of 1.38 (negative < 0.5). In contrast, only horses A and G seroconverted based on SN testing. Horses B, C, and F showed only a 2- to 3-fold titer increases in SN antibodies.

Long-Term Follow-Up

The pregnant mare, relocated to the isolation unit, delivered a healthy, term foal. Horse A was readmitted to the hospital 6 weeks after dismissal with mild signs of colic, which resolved with medical therapy. At that time the horse's neurological deficits were unchanged since the time of its previous discharge.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Despite institution of rigorous biosecurity precautions at the time of admission of the Index Case, EHV-1 infections spread to 6 other horses that were hospitalized at the JLV-VTH, including 2 that served as sources of infection for horses on their home premises after discharge. These findings illustrate the contagious disease risk that can accompany management of horses with EHM. Our experience suggests that horses with active nasal EHV-1 shedding should be isolated in an airspace that is separate from other horses using strictly enforced biosecurity and isolation procedures. Although it often is not feasible to use different personnel to manage every quarantined case, all efforts should be made to avoid contact between the isolated horse and the general hospital population.

Once EHV-1 or any other contagious disease has been identified in a hospital population, recording of rectal temperatures at least twice daily should be implemented to enhance the ability to quickly identify additional cases.

Several experimental EHV-1 infection studies have shown that nasal shedding of virus post-infection can last for up to 14 days or longer in individual horses.15 Neurological disease after experimental infection has been shown to typically accompany resolution of viremia on Days 8–12 postinfection.16 Virus shedding through nasal secretions therefore may overlap with the development of EHM. Although this may not be true under natural outbreak conditions for all horses, the possibility of continued nasal shedding in EHM-affected horses should be assumed until proven otherwise to minimize the risk of virus spread to in-contact horses. The EHM outbreak at Findlay University, Ohio, demonstrated that transmission of virus to horses at other, connected premises was possible.3 Horses from the primary outbreak site were referred to the Equine Hospital, where others developed EHM, and the hospital release of a seemingly healthy horse induced EHM in a horse in its home barn.

A horse with acute neurological disease should be considered a suspect EHM case, particularly if neurological signs were preceded by fever in the patient or any in-contact horses on the farm of origin, and should only be admitted to an equine hospital under strict isolation precautions. These patients should be tested immediately by PCR for the presence of EHV-1 genomic copies in their nasal secretions and in whole blood (PBMC). Tests performed on nasal or nasopharyngeal swab samples appear to be the more sensitive when compared with tests performed on PBMC because the viral load of nasal secretions typically is higher than in PBMC.17 However, whole blood samples (PBMC) should also be submitted to determine the viral load because this may be an important predictor for the likelihood of EHM development.15 Furthermore, nasal virus shedding may cease before resolution of viremia resulting in a negative gB-EHV-1 PCR result of the nasal swab samples despite continued presence of virus in PBMC.

Optimally, results of PCR testing can be available within a few hours and can be used to guide clinical management and establishment of appropriate infection control procedures. If PCR testing is not readily available, results of CSF analysis may aid in the preliminary diagnosis of EHM. However, strict isolation should be maintained so that patient handling required for CSF collection does not increase infection risk for other patients. Additionally, although xanthochromia, absence of pleocytosis, and increase in CSF protein concentration are suggestive of EHM, these findings are not definitive. In general, better diagnostic results are achieved if CSF is obtained from the site closest to the suspected lesions.7 Because EHM typically is associated with spinal cord disease, a CSF sample collected from the lumbosacral space may be more sensitive for diagnosis of EHM than a sample collected at the atlanto-occipital site.

Strict precautions were initiated upon admission of the index case, including patient segregation, use of barrier nursing precautions, and hygiene. Moreover, these biosecurity measures were maintained for the index case even after obtaining a negative gb-EHV1PCR result on the first nasal swab sample collected on Day 1 as well as normal CSF analysis. Still, despite these precautions, the virus spread to other hospitalized horses. Interestingly, no signs of disease were detected in horses that were present in the equine hospital as outpatients.

Three clusters of infection followed admission the index case. Cluster 1 occurred in the NE wing (horse A), cluster 2 occurred in the SE wing (horses B, C, D, E), and cluster 3 occurred in the SW wing (horses F and G). Within a cluster, viral spread most likely was due to short distance transmission (1–2 m) of virus. Based on stabling and the design of the stall units, direct nose-to-nose contact was only possible between horses F and G. Although the exact mode of transmission of EHV-1 to horses in these 3 clusters is unknown, virus spread could have occurred by 1 of 3 possible routes. Firstly, aerosolized virus originating from the index case may have been dispersed into common airspace. Secondly, virus transmission may have occurred through fomites, and lastly, recrudescence and spread of virus may have occurred within a cluster and virus transmission among horses in the cluster could have been unrelated to the index case. Given the distances and barriers (walls) between the North and South wings (Fig 1), it seems likely that at least some of the transmission may have resulted from exposure to contaminated fomites, including the possibility of exposure to contaminated hands or clothing of personnel.

EHV-1 and -4 are members of the Alphaherpesvirinae subfamily. The Alphaherpesvirinae represent a group of large enveloped DNA viruses and virus spread occurs through direct horse-to-horse contact, fomites, droplet, or aerosol transmission. Although there is considerable support for a contribution of aerosol transmission to the spread of EHV-1 and 4, the debate over the importance of aerosol transmission of the Alphaherpesvirinae has been rekindled in the past few years. For instance, Mars et al demonstrated that the odds ratio for infection with Bovine Herpesvirus in calves becomes >1 when the distance between a shedding calf and a sentinel decreases to <4.4 m.18 In contrast, Pusterla et al showed that air samples collected 14.5 m away from a source of nebulized modified-live EHV-1 vaccine were still positive in qPCR-EHV1 analysis.19

Although it is possible that aerosolized virus originating from the index case crossed the incomplete wall between the NW and NE wings to infect horse A, none of the other horses housed in the NW or NE wings of the equine hospital seroconverted. Therefore, it appears more likely that virus spread between the index case and horse A occurred by fomite transmission. Because supportive care for the recumbent index patient was highly labor- and personnel-intensive, the greater human traffic may have resulted in an increased risk of fomite-associated pathogen transfer among patients in the hospital. Additionally, the number of personnel available at night for patient observation and treatments is substantially less than during the day, which increases the potential for exposure of other patients.

The third possibility, simultaneous and independent recrudescence of virus from latency, follows an interesting observation by Pusterla et al.20 The authors described the cocirculation of a neuropathogenic and a nonneuropathogenic strain of EHV-1 during an EHM outbreak at a Californian racetrack. Independent recrudescence of virus in separate clusters also may have occurred in the outbreak described here. Unfortunately, this hypothesis could not be tested, because viruses could not be isolated from positive nasal swab samples. Therefore, DNA sequencing could not be performed in order to establish the phylogenetic relationships among the isolates. However, the fever in horse A on Day −2 and on Days 2 and 4–6, and the already existing presence of EHV-1–specific antibodies (gG ELISA) by Day 6 in horses A and B, may argue in favor of independent recrudescence.

Once transmission of virus within the hospital population was identified, the implementation of strict biosecurity procedures, such as isolation and traffic control as well as the use of barrier precautions, appears to have resulted in the successful halt of further spread of virus. This is highlighted by the fact that, with the exception of the previously described clusters of infection, none of the other horses housed in the hospital in-patient areas seroconverted or had evidence of EHV-1 in nasal swab samples (convPCR and qPCR).

A novel approach taken in the control of this outbreak was the shortening of the recommended quarantine period from 28 to 15 days. A 3- to 4-week quarantine period has been recommended previously after cessation of fever in the last clinical case.21 However, these recommendations do not rely on use of testing to document the absence of shedding. In order to shorten this quarantine period, it was considered extremely unlikely that greater than average infection risk was present if all horses were negative on all samples taken on 4 consecutive days. Thus, after removing all horses with clinical signs from the asymptomatic population (the index case was euthanized on Day 6 and Horse A was moved to a separate isolation stall on Day 11), the remaining quarantined population was carefully monitored for any new signs of disease for 10 days, and all nasal swabs collected during Days 22–25 were analyzed and found to be negative using convPCR. In addition, horses were actively monitored for nasal virus shedding by daily nasal swab sampling and subsequent PCR analysis. All horses were sent to their farms of origin with continued quarantine. However, the shortening of the quarantine period allowed us to clean and disinfect the outpatient and inpatient hospital areas appropriately and to reopen the hospital on Day 28.

Seroconversion, as determined by SN (EHV) testing did not supply sufficient proof of infection. The increase of absorbance in the gG ELISA provided evidence of EHV-1 infection. Seroconversion as determined by SN may be delayed. A longer period between acute and converting samples may be necessary.22

Although there will always be a risk for EHM propagation to other horses after admitting a case of EHM into an equine or LA hospital, the following biosecurity precautions may be helpful in mitigating or preventing this disease in other horses:

  • 1
    EHM (suspect) cases should be received under strict barrier precautions with as few people as possible. Contacts and procedures with an EHM case should be minimized as much as possible.
  • 2
    EHM (suspect) cases should be housed separately in a building not connected to the main hospital structure.
  • 3
    Exclusive care providers should be assigned to the EHM (suspect) case.
  • 4
    If initial PCR testing is negative but case presentation is suggestive for EHM, daily PCR testing of nasal swabs should be continued for a minimum of 3 consecutive days. Routine CSF testing is recommended.
  • 5
    Once an EHM (suspect) case is admitted, recording rectal temperature twice daily of the general equine hospital population should be mandatory. Any additional cases of fever should be thoroughly investigated and immediately isolated from other horses.8,21

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

aFootbath Mats, Gemplers, Madison, WI

bVirkon-S, Suffolk, UK

c456N, Ecolab Inc, St Paul, MN

dSvanovir, Svanova Biotech AB, Uppsala, Sweden

ePlasmalyte, Baxter, Deerfield, IL

fNaxcel, Pfizer Animal Health, New York, NY

gDexamethasone, Vetone, MWI, Meridian, ID

hDormosedan, Pfizer Animal Health

iLarge Animal Lift Enterprises, Lone Pine, CA

jAntisedan, Pfizer Animal Health

kValtrex, GlaxoSmithKline, Philadelphia, PA

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

The authors thank Dr Jan Koeman, Utrecht University, The Netherlands, for the expertise in EHV immunohistochemistry, and Dr George Allen, Gluck Center, University of Kentucky, and his laboratory for their attempts to isolate virus from the submitted nasal swab samples.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References