- Email: [email protected]
Nasal Shedding of Equid Herpesvirus Type 1 and Type 4 in Hospitalized, Febrile Horses
Journal of Equine Veterinary Science 33 (2013) 756-759
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Nasal Shedding of Equid Herpesvirus Type 1 and Type 4 in Hospitalized, Febrile Horses Jennifer M. Sonis DVM, MS, Lutz S. Goehring DVM, MS, PhD Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2012 Received in revised form 5 November 2012 Accepted 20 November 2012 Available online 15 March 2013
The objective of this study was to determine the prevalence of shedding of equid herpesvirus 1 (EHV-1) or EHV-4 in nasal swab samples from any febrile, hospitalized horses during a 1-year period. It was hypothesized that some fevers in horses are associated with viral replication following recrudescence of latent virus or following a horizontal viral infection prior to or during admission to a referral hospital. During the observational period, nasal swab samples were collected from 64 febrile and 10 nonfebrile hospitalized horses. Routine DNA extraction was performed, and a validated quantitative polymerase chain reaction (qPCR) assay was used to detect and quantify genomic EHV-1 and -4 DNA. Genomic DNA of EHV-4 was detected in the nasal swab specimen of 1 of 64 febrile horses. EHV-1 DNA was not detected in any of the febrile horses. Samples from all nonfebrile horses were negative for both viruses. Considering the known association between fever and shedding of EHV-1 and EHV-4, we anticipated finding a higher percentage of PCR-positive samples from febrile patients. Fevers detected were likely a result of active disease processes for which the horses were hospitalized; concurrent other diseases appeared not to affect viral recrudescence. Further studies are warranted to examine frequency and factors of EHV latency and reactivation. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Detection Equid herpesvirus Fever Latency recrudescence Nasal swab
1. Introduction Equid herpesvirus 1 (EHV-1) and EHV-4 are ubiquitous equine pathogens that typically lead to primary respiratory disease and, less frequently, to secondary complications such as abortion and myeloencephalopathy, specifically with EHV-1 infection and infrequently with EHV-4 infection [1]. EHV-1 and -4 maintain a presence in equine populations via two mechanisms. One mechanism is latency, which is a nonreplicative, nonimmunogenic stage in a horse. Latency is established after initial replicative
Corresponding author at: Lutz S. Goehring, DVM, MS, PhD, Department of Clinical Sciences, Colorado State University, 300 W. Drake Rd., Fort Collins, CO 80523. E-mail address: [email protected] (L.S. Goehring). 0737-0806/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2012.11.002
infection. Recrudescence from latency will cause viral respiratory tract replication, which may then start further horizontal spread to other in-contact horses. This horizontal spread through respiratory droplet infection or fomite transmission, but also contact with an aborted fetus or fetal fluids or membranes, is considered the second mechanism by which EHV-1 or -4 maintains presence in equine populations. Mechanisms that lead to recrudescence from latency are poorly understood, and longdistance transportation, stress, and participation at a (horse) show event are often cited [2]. Over the last decade, there have been several reports on nosocomial spread of EHV-1 infection in hospitalized horses, including development of equid herpesvirusassociated myeloencephalopathy (EHM) [3-5]. These infections have led to fatal losses of patients and voluntary quarantine of equine hospitals, with substantial financial losses and were associated with significant loss
J.M. Sonis and L.S. Goehring / Journal of Equine Veterinary Science 33 (2013) 756-759
of image for the affected institutions, as perceived by the public. Arguably the most commonly observed clinical signs during outbreaks of EHV-1 or -4 infection are pyrexia and (mandibular) lymphadenopathy [5,6]. Fever was also the main clinical sign in EHV-1-positive horses in a surveillance study that determined infectious agents in respiratory conditions in horses [7]. However, infections with EHV-1 or -4 are just one of numerous causes for development of a fever in hospitalized horses. Fever is a systemic response to inflammation whether it is caused by an infectious agent, an immune-mediated disease, neoplasia, or the result of tissue destruction and necrosis. It is not uncommon for horses in a hospitalized setting to be febrile. However, from a biosecurity standpoint, a horse shedding EHV-1 or -4 in a shared environment must be detected as quickly as possible to limit the risk of nosocomial spread. Fevers during experimental EHV-1 infections are detected during two distinct events following the infection. A fever can be expected during early upper respiratory tract replication of virus, which typically lasts from 36-48 hours following nasopharyngeal instillation of a viral inoculum. A secondary fever is then noticed between days 5 and 10 following the infection and is due to a cell-associated viremia in peripheral blood mononuclear cells. EHM may develop shortly after cessation of viremia. A number of experimental infection studies have shown that viral replication in the nasopharynx is continuous and spans the viremic phase in most cases, which has been shown by using polymerase chain reaction (PCR) assays or viral culture techniques [8]. During a recent EHM outbreak, significant nasal shedding, detected by quantitative PCR (qPCR) methods, continued in horses beyond a secondary fever and beyond the onset of EHM [5]. The objective of this study was to determine the prevalence of nasal shedding of EHV-1 or EHV-4 in nasal swab samples collected from horses with a fever while hospitalized at a university referral center. Because approximately 14% of febrile cases in a field prevalence study were attributed to either EHV-1 or EHV-4 infection, it was hypothesized that >10% of fevers in hospitalized patients would result from EHV-1 or EHV-4 replication in the respiratory tract following either recrudescence of virus from latency or following horizontal virus transmission prior to or during admission into an equine hospital [7]. 2. Materials and Methods Seventy-four horses were enrolled in the study between February 1, 2009, and March 1, 2010. During that time span, horses that developed a rectal temperature higher than 101.3 F (38.5 C) during hospitalization at the Colorado State University Veterinary Teaching Hospital, Fort Collins, CO, were sampled after client consent was obtained. Sixtyfour febrile horses were enrolled. An additional 10 afebrile horses from within the hospital or an out-patient population were enrolled (approximately 1 horse per month) after client consent was obtained. The study was approved by the Colorado State University Animal Care and Use Committee.
757
For the febrile population, swabs were collected within 6 hours after a fever was detected. Two rayon-tipped swabs (synthetic-tipped applicators; Fischer Scientific, Pittsburgh, PA) were inserted into the left or right ventral nasal meatus of each horse. Swabs were gently moved in and out, rubbing over the nasal mucosa for 3-5 seconds. Halter and lead rope were sufficient methods of restraint, and the investigator was wearing disposable gloves during sample collection. All nasal swabs were immediately immersed in 1 mL of a virus transportation medium and stored frozen at 80 C. Following collection of all samples, no more than 10 swab samples at a time were allowed to thaw. The laboratory technician was blinded to the clinical presentation of the horse associated with each sample. Manual routine DNA extraction was performed using a commercially available kit (QIAamp DNA blood mini-kit; Qiagen, Inc., Valencia, CA), and a previously validated qPCR assay using different primer and probe sets was used to detect and quantify a section of viral genomic DNA (glycoprotein B [gB]) of either EHV-1 or -4, as previously described [9]. 3. Results Of the 74 horses enrolled in the study, there were 32 geldings, 11 stallions, and 30 mares. Horses ranged in age from 1-29 years old, with a median age of 6 years old. The horses belonged to several breeds, including Quarter horse (42), Thoroughbred (6), foreign warmblood (7), Tennessee Walker (5), draft (4), Arabian (2), Paint (2), pony (2), Andalusian (1), Appaloosa (1), Missouri Fox Trotter (1), and Spanish Mustang (1). Various organ systems were associated with the primary reason for hospitalization of the horses, including gastrointestinal (36), respiratory (12), musculoskeletal (11), reproductive (3), cardiovascular (1), hepatic (1), and ocular (1). Nine horses were hospitalized as companions to other horses. In total, 1 of 64 of the febrile horses was PCR-positive for EHV-4 infection. None of the febrile horses was PCRpositive for EHV-1 in nasal secretions. None of the afebrile horses was PCR-positive for EHV-1 or -4 in nasal secretions. The PCR-positive horse was a 5-year-old, postpartum Quarter horse mare approximately 10 months in foal. The mare was presented to the hospital for a uterine torsion, which was confirmed and corrected surgically and without noticeable complications. The mare was discharged within 24 hours after the procedure; however, the mare was presented again 10 days later for signs of abdominal pain of approximately 4 hours’ duration. She was treated medically for suspected gas colic. Following resolution of colic signs, the mare remained hospitalized for monitoring and care during the last stages of gestation and during parturition. Normal parturition occurred on the sixth day of hospitalization. On the eighth day of hospitalization, she developed a temperature of 102.5 F in addition to vaginal discharge. The EHV-4-positive swab was collected at that point. The fever and vaginal discharge resolved with medical therapy, which included uterine lavages, antimicrobial drugs, and nonsteroidal antiinflammatory drugs. A specimen of vaginal discharge was not examined for viral presence.
758
J.M. Sonis and L.S. Goehring / Journal of Equine Veterinary Science 33 (2013) 756-759
4. Discussion This study was designed to gain understanding of the frequency of EHV-1 or -4 nasal shedding in the febrile equine population of a veterinary teaching hospital, with the potential risk of spreading into other hospitalized horses. Our results indicate that nasal shedding of EHV-1 or EHV-4 in febrile, hospitalized horses may be a rare event. Based on a previous prevalence study, where investigators found approximately 14% of febrile horses with nasal swabs PCR-positive for either EHV-1 or -4, we anticipated finding a percentage of PCR-positive nasal swab samples collected from hospitalized horses with concurrent fevers that was at least similar [7]. However, the main difference from the above-cited study is that it focused on horses with acute febrile, respiratory tract disease or a fever that was associated with neurological disease. Although we elected to collect nasal swab samples from any horse with a fever, not only from horses with respiratory tract disease, we still assumed we would find a similarly high PCR-positive nasal swab percentage because of two principles that are intricately linked to EHV-1 or -4 pathogenesis. These principles are recrudescence of virus infection from latency because of “stress,” and, potentially, a heightened susceptibility of hospitalized horses to infectious agents. Both the transportation of a horse and the entrance into a hospital environment are generally associated with stress, and admission to an equine hospital is often because of “other” disease. Both factors were thought to facilitate recrudescence of latently present virus, resulting in active respiratory tract replication which results in a concurrent fever. Also, an equine hospital environment presents an increased risk of direct or indirect pathogen transmission among horses. Our study shows the rare event of EHV detection in nasal swabs among febrile and hospitalized horses during a 1-year observational period. However, caution is warranted in interpreting these results. Year-to-year variation of specific pathogens that circulate in horse populations may have been the reason for a low detection frequency of EHV-1 or -4 in this study. Also, despite the fact that high shedding during EHV-1 or -4 infection is associated with especially febrile periods, the single nasal swab sampling technique, which we used, may have been associated with an occasional false-negative result. Detection frequency and sensitivity could have been overcome by sampling more often, for example, on 2-3 consecutive days during or following a fever. However, the PCR-assay used for the analysis of the collected nasal swabs has an excellent track record in sensitivity and specificity [9]. Furthermore, as rapid horizontal transmission is a feature of EHV-1 or -4 infection, none of the febrile horses sampled in this study was associated with clusters of newly febrile cases. This may indicate that either fevers in hospitalized horses are more likely to be of a noncontagious cause or febrile horses were sufficiently contained to avoid transmission of a contagious agent. All horses hospitalized at the Veterinary Teaching Hospital have fecal samples collected biweekly for surveillance of Salmonella spp. None of the febrile horses in this study was associated with fecal Salmonella shedding. Other respiratory tract pathogens contagious to horses were certainly of concern and need to
be investigated; however, the surveillance study of respiratory tract pathogen testing by using PCR for the presence of influenzavirus, EHV-1 or -4, and Streptococcus equi spp. equi genomes by Pusterla et al.[7] was unable to identify a causative agent in more than 60% of the cases investigated. Previous studies have shown similarly low or absent shedding of EHV-1 or -4 in equine populations subjected to various stressors. Following long-distance transportation, the number of horses detected with EHV-1 nasal shedding was low in adult horses (1% [3 of 302 horses]) [10], and nasal swabs from 17 of 451 horses were either positive for EHV-1 or -4 in juvenile horses upon arrival at show grounds or sales [11]. Horses hospitalized with acute gastrointestinal disease requiring an emergency celiotomy or horses with acute colitis all tested negative for cell-associated viremia or for the presence of EHV-1 in nasal secretions on one sample collected on admission and two more swabs between days 2 and 12 [12]. These findings indicate that there is probably a low frequency of EHV recrudescence from its latent location, and stressors like transportation and physical stress resulting from concurrent disease may not be the correct stressors that cause recrudescence with detectable virus replication in the upper respiratory tract. Foals have been shown to seroconvert to EHV-1 or -4 within the first few months of life, and shedding of both EHV-1 and -4 has been detected in populations of broodmares and their foals [13,14]. While nasal shedding appears to be part of a “silent” cycle of herpesvirus infection, it is possible that the event of parturition with endocrinological changes is linked to activation of latent herpesvirus in the mare followed with nasal shedding of virus. For this reason, postpartum mares should be considered an at-risk group for EHV-1 or -4 shedding. Because of this and because our single EHV-4-positive sample was from a febrile postpartum mare, special barrier precautions and EHV-1- or -4specific diagnostic sampling methods are warranted in febrile and maybe also in nonfebrile postpartum mares. Nasal swabs might have been found positive for EHV-1 or -4 more often if we had selected horses with a fever and concurrent respiratory tract disease or febrile horses with concurrent neurological disease, febrile horses coming from a horse show or event, mares that aborted a late-term fetus, peripartum mares with partial or complete placental separation, and febrile postpartum mares [6,7,15]. Hence, solid clinical judgment is necessary to recognize these “red flags” of potential EHV-1 or -4 disease in equine patients that need to be admitted into a referral center. 5. Conclusions Finally, if a case of EHV-1 or EHV-4 infection has been identified, any further fevers in hospitalized horses need to be investigated, and rectal temperatures during a time like this should be collected at least twice daily in all hospitalized horses [4]. Acknowledgments The work was completed at the James L. Voss Veterinary Teaching Hospital, Colorado State University, Fort Collins,
J.M. Sonis and L.S. Goehring / Journal of Equine Veterinary Science 33 (2013) 756-759
CO. The study was supported by a grant from the Animal Population Health Institute at Colorado State University. An abstract was presented at the Forum of the American College of Veterinary Internal Medicine held in Anaheim, CA, June 9-12, 2010. Our special thanks to Dr. Brandy Burgess for carefully editing the script.
[7]
[8] [9]
References [10] [1] van Maanen C. Equine herpesvirus 1 and 4 infections: An update. Vet Q 2002;24:58-78. [2] Slater J. Equine herpesviruses. In: Sellon DC, Long MT, editors. Equine infectious diseases. St. Louis, MO: WB Saunders Elsevier; 2007. p. 134-53. [3] Henninger RW, Reed SM, Saville WJ, et al. Outbreak of neurologic disease caused by equine herpesvirus-1 at a university equestrian center. J Vet Intern Med 2007;21:157-65. [4] Goehring L, Landolt G, Morley P. Detection and management of an outbreak of equine herpesvirus type 1 infection and associated neurological disease in a veterinary teaching hospital. J Vet Intern Med 2010;24:1176-83. [5] Burgess BA, Tokateloff N, Manning S, et al. Nasal shedding of equine herpesvirus-1 from horses in an outbreak of equine herpes myeloencephalopathy in Western Canada. J Vet Intern Med 26:384-392. [6] Goehring LS, van Winden SC, van Maanen C, et al. Equine herpesvirus type 1-associated myeloencephalopathy in The Netherlands: A
[11]
[12]
[13]
[14]
[15]
759
four-year retrospective study (1999-2003). J Vet Intern Med 2006;20:601-7. Pusterla N, Kass P, Mapes S, et al. Surveillance programme for important equine infectious respiratory pathogens in the USA. Veterinary Record 2011;169:12. Goehring L, Wagner B, Bigbie R, et al. Control of EHV-1 viremia and nasal shedding by commercial vaccines. Vaccine 2010;28:5203-11. Hussey SB, Clark R, Lunn KF, et al. Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction. J Vet Diagn Invest 2006;18:335-42. Pusterla N, Mapes S, Madigan JE, et al. Prevalence of EHV-1 in adult horses transported over long distances. Vet Rec 2009;165:473-5. Yactor J, Lunn KF, Traub-Dargatz J, et al. Detection of nasal shedding of EHV-1 & 4 at equine show events and sales by multiplex real-time PCR. Paper presented at: 52nd Annual Convention of American Association of Equine Practitioners. December 2-6, 2006; San Antonio, TX. Carr E, Schott H, Pusterla N. Absence of equid herpesvirus-1 reactivation and viremia in hospitalized critically ill horses. J Vet Intern Med 2011;25:1190-3. Foote CE, Gilkerson JR, Whalley JM, et al. Seroprevalence of equine herpesvirus 1 in mares and foals on a large Hunter Valley stud farm in years pre- and postvaccination. Aust Vet J 2003;81:283-8. Foote CE, Love DN, Gilkerson JR, et al. EHV-1 and EHV-4 infection in vaccinated mares and their foals. Vet Immunol Immunopathol 2006;111:41-6. van Maanen C, Willink DL, Smeenk LA, et al. An equine herpesvirus 1 (EHV1) abortion storm at a riding school. Vet Q 2000;22:83-7.
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Nasal Shedding of Equid Herpesvirus Type 1 and Type 4 in Hospitalized, Febrile Horses Jennifer M. Sonis DVM, MS, Lutz S. Goehring DVM, MS, PhD Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2012 Received in revised form 5 November 2012 Accepted 20 November 2012 Available online 15 March 2013
The objective of this study was to determine the prevalence of shedding of equid herpesvirus 1 (EHV-1) or EHV-4 in nasal swab samples from any febrile, hospitalized horses during a 1-year period. It was hypothesized that some fevers in horses are associated with viral replication following recrudescence of latent virus or following a horizontal viral infection prior to or during admission to a referral hospital. During the observational period, nasal swab samples were collected from 64 febrile and 10 nonfebrile hospitalized horses. Routine DNA extraction was performed, and a validated quantitative polymerase chain reaction (qPCR) assay was used to detect and quantify genomic EHV-1 and -4 DNA. Genomic DNA of EHV-4 was detected in the nasal swab specimen of 1 of 64 febrile horses. EHV-1 DNA was not detected in any of the febrile horses. Samples from all nonfebrile horses were negative for both viruses. Considering the known association between fever and shedding of EHV-1 and EHV-4, we anticipated finding a higher percentage of PCR-positive samples from febrile patients. Fevers detected were likely a result of active disease processes for which the horses were hospitalized; concurrent other diseases appeared not to affect viral recrudescence. Further studies are warranted to examine frequency and factors of EHV latency and reactivation. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Detection Equid herpesvirus Fever Latency recrudescence Nasal swab
1. Introduction Equid herpesvirus 1 (EHV-1) and EHV-4 are ubiquitous equine pathogens that typically lead to primary respiratory disease and, less frequently, to secondary complications such as abortion and myeloencephalopathy, specifically with EHV-1 infection and infrequently with EHV-4 infection [1]. EHV-1 and -4 maintain a presence in equine populations via two mechanisms. One mechanism is latency, which is a nonreplicative, nonimmunogenic stage in a horse. Latency is established after initial replicative
Corresponding author at: Lutz S. Goehring, DVM, MS, PhD, Department of Clinical Sciences, Colorado State University, 300 W. Drake Rd., Fort Collins, CO 80523. E-mail address: [email protected] (L.S. Goehring). 0737-0806/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2012.11.002
infection. Recrudescence from latency will cause viral respiratory tract replication, which may then start further horizontal spread to other in-contact horses. This horizontal spread through respiratory droplet infection or fomite transmission, but also contact with an aborted fetus or fetal fluids or membranes, is considered the second mechanism by which EHV-1 or -4 maintains presence in equine populations. Mechanisms that lead to recrudescence from latency are poorly understood, and longdistance transportation, stress, and participation at a (horse) show event are often cited [2]. Over the last decade, there have been several reports on nosocomial spread of EHV-1 infection in hospitalized horses, including development of equid herpesvirusassociated myeloencephalopathy (EHM) [3-5]. These infections have led to fatal losses of patients and voluntary quarantine of equine hospitals, with substantial financial losses and were associated with significant loss
J.M. Sonis and L.S. Goehring / Journal of Equine Veterinary Science 33 (2013) 756-759
of image for the affected institutions, as perceived by the public. Arguably the most commonly observed clinical signs during outbreaks of EHV-1 or -4 infection are pyrexia and (mandibular) lymphadenopathy [5,6]. Fever was also the main clinical sign in EHV-1-positive horses in a surveillance study that determined infectious agents in respiratory conditions in horses [7]. However, infections with EHV-1 or -4 are just one of numerous causes for development of a fever in hospitalized horses. Fever is a systemic response to inflammation whether it is caused by an infectious agent, an immune-mediated disease, neoplasia, or the result of tissue destruction and necrosis. It is not uncommon for horses in a hospitalized setting to be febrile. However, from a biosecurity standpoint, a horse shedding EHV-1 or -4 in a shared environment must be detected as quickly as possible to limit the risk of nosocomial spread. Fevers during experimental EHV-1 infections are detected during two distinct events following the infection. A fever can be expected during early upper respiratory tract replication of virus, which typically lasts from 36-48 hours following nasopharyngeal instillation of a viral inoculum. A secondary fever is then noticed between days 5 and 10 following the infection and is due to a cell-associated viremia in peripheral blood mononuclear cells. EHM may develop shortly after cessation of viremia. A number of experimental infection studies have shown that viral replication in the nasopharynx is continuous and spans the viremic phase in most cases, which has been shown by using polymerase chain reaction (PCR) assays or viral culture techniques [8]. During a recent EHM outbreak, significant nasal shedding, detected by quantitative PCR (qPCR) methods, continued in horses beyond a secondary fever and beyond the onset of EHM [5]. The objective of this study was to determine the prevalence of nasal shedding of EHV-1 or EHV-4 in nasal swab samples collected from horses with a fever while hospitalized at a university referral center. Because approximately 14% of febrile cases in a field prevalence study were attributed to either EHV-1 or EHV-4 infection, it was hypothesized that >10% of fevers in hospitalized patients would result from EHV-1 or EHV-4 replication in the respiratory tract following either recrudescence of virus from latency or following horizontal virus transmission prior to or during admission into an equine hospital [7]. 2. Materials and Methods Seventy-four horses were enrolled in the study between February 1, 2009, and March 1, 2010. During that time span, horses that developed a rectal temperature higher than 101.3 F (38.5 C) during hospitalization at the Colorado State University Veterinary Teaching Hospital, Fort Collins, CO, were sampled after client consent was obtained. Sixtyfour febrile horses were enrolled. An additional 10 afebrile horses from within the hospital or an out-patient population were enrolled (approximately 1 horse per month) after client consent was obtained. The study was approved by the Colorado State University Animal Care and Use Committee.
757
For the febrile population, swabs were collected within 6 hours after a fever was detected. Two rayon-tipped swabs (synthetic-tipped applicators; Fischer Scientific, Pittsburgh, PA) were inserted into the left or right ventral nasal meatus of each horse. Swabs were gently moved in and out, rubbing over the nasal mucosa for 3-5 seconds. Halter and lead rope were sufficient methods of restraint, and the investigator was wearing disposable gloves during sample collection. All nasal swabs were immediately immersed in 1 mL of a virus transportation medium and stored frozen at 80 C. Following collection of all samples, no more than 10 swab samples at a time were allowed to thaw. The laboratory technician was blinded to the clinical presentation of the horse associated with each sample. Manual routine DNA extraction was performed using a commercially available kit (QIAamp DNA blood mini-kit; Qiagen, Inc., Valencia, CA), and a previously validated qPCR assay using different primer and probe sets was used to detect and quantify a section of viral genomic DNA (glycoprotein B [gB]) of either EHV-1 or -4, as previously described [9]. 3. Results Of the 74 horses enrolled in the study, there were 32 geldings, 11 stallions, and 30 mares. Horses ranged in age from 1-29 years old, with a median age of 6 years old. The horses belonged to several breeds, including Quarter horse (42), Thoroughbred (6), foreign warmblood (7), Tennessee Walker (5), draft (4), Arabian (2), Paint (2), pony (2), Andalusian (1), Appaloosa (1), Missouri Fox Trotter (1), and Spanish Mustang (1). Various organ systems were associated with the primary reason for hospitalization of the horses, including gastrointestinal (36), respiratory (12), musculoskeletal (11), reproductive (3), cardiovascular (1), hepatic (1), and ocular (1). Nine horses were hospitalized as companions to other horses. In total, 1 of 64 of the febrile horses was PCR-positive for EHV-4 infection. None of the febrile horses was PCRpositive for EHV-1 in nasal secretions. None of the afebrile horses was PCR-positive for EHV-1 or -4 in nasal secretions. The PCR-positive horse was a 5-year-old, postpartum Quarter horse mare approximately 10 months in foal. The mare was presented to the hospital for a uterine torsion, which was confirmed and corrected surgically and without noticeable complications. The mare was discharged within 24 hours after the procedure; however, the mare was presented again 10 days later for signs of abdominal pain of approximately 4 hours’ duration. She was treated medically for suspected gas colic. Following resolution of colic signs, the mare remained hospitalized for monitoring and care during the last stages of gestation and during parturition. Normal parturition occurred on the sixth day of hospitalization. On the eighth day of hospitalization, she developed a temperature of 102.5 F in addition to vaginal discharge. The EHV-4-positive swab was collected at that point. The fever and vaginal discharge resolved with medical therapy, which included uterine lavages, antimicrobial drugs, and nonsteroidal antiinflammatory drugs. A specimen of vaginal discharge was not examined for viral presence.
758
J.M. Sonis and L.S. Goehring / Journal of Equine Veterinary Science 33 (2013) 756-759
4. Discussion This study was designed to gain understanding of the frequency of EHV-1 or -4 nasal shedding in the febrile equine population of a veterinary teaching hospital, with the potential risk of spreading into other hospitalized horses. Our results indicate that nasal shedding of EHV-1 or EHV-4 in febrile, hospitalized horses may be a rare event. Based on a previous prevalence study, where investigators found approximately 14% of febrile horses with nasal swabs PCR-positive for either EHV-1 or -4, we anticipated finding a percentage of PCR-positive nasal swab samples collected from hospitalized horses with concurrent fevers that was at least similar [7]. However, the main difference from the above-cited study is that it focused on horses with acute febrile, respiratory tract disease or a fever that was associated with neurological disease. Although we elected to collect nasal swab samples from any horse with a fever, not only from horses with respiratory tract disease, we still assumed we would find a similarly high PCR-positive nasal swab percentage because of two principles that are intricately linked to EHV-1 or -4 pathogenesis. These principles are recrudescence of virus infection from latency because of “stress,” and, potentially, a heightened susceptibility of hospitalized horses to infectious agents. Both the transportation of a horse and the entrance into a hospital environment are generally associated with stress, and admission to an equine hospital is often because of “other” disease. Both factors were thought to facilitate recrudescence of latently present virus, resulting in active respiratory tract replication which results in a concurrent fever. Also, an equine hospital environment presents an increased risk of direct or indirect pathogen transmission among horses. Our study shows the rare event of EHV detection in nasal swabs among febrile and hospitalized horses during a 1-year observational period. However, caution is warranted in interpreting these results. Year-to-year variation of specific pathogens that circulate in horse populations may have been the reason for a low detection frequency of EHV-1 or -4 in this study. Also, despite the fact that high shedding during EHV-1 or -4 infection is associated with especially febrile periods, the single nasal swab sampling technique, which we used, may have been associated with an occasional false-negative result. Detection frequency and sensitivity could have been overcome by sampling more often, for example, on 2-3 consecutive days during or following a fever. However, the PCR-assay used for the analysis of the collected nasal swabs has an excellent track record in sensitivity and specificity [9]. Furthermore, as rapid horizontal transmission is a feature of EHV-1 or -4 infection, none of the febrile horses sampled in this study was associated with clusters of newly febrile cases. This may indicate that either fevers in hospitalized horses are more likely to be of a noncontagious cause or febrile horses were sufficiently contained to avoid transmission of a contagious agent. All horses hospitalized at the Veterinary Teaching Hospital have fecal samples collected biweekly for surveillance of Salmonella spp. None of the febrile horses in this study was associated with fecal Salmonella shedding. Other respiratory tract pathogens contagious to horses were certainly of concern and need to
be investigated; however, the surveillance study of respiratory tract pathogen testing by using PCR for the presence of influenzavirus, EHV-1 or -4, and Streptococcus equi spp. equi genomes by Pusterla et al.[7] was unable to identify a causative agent in more than 60% of the cases investigated. Previous studies have shown similarly low or absent shedding of EHV-1 or -4 in equine populations subjected to various stressors. Following long-distance transportation, the number of horses detected with EHV-1 nasal shedding was low in adult horses (1% [3 of 302 horses]) [10], and nasal swabs from 17 of 451 horses were either positive for EHV-1 or -4 in juvenile horses upon arrival at show grounds or sales [11]. Horses hospitalized with acute gastrointestinal disease requiring an emergency celiotomy or horses with acute colitis all tested negative for cell-associated viremia or for the presence of EHV-1 in nasal secretions on one sample collected on admission and two more swabs between days 2 and 12 [12]. These findings indicate that there is probably a low frequency of EHV recrudescence from its latent location, and stressors like transportation and physical stress resulting from concurrent disease may not be the correct stressors that cause recrudescence with detectable virus replication in the upper respiratory tract. Foals have been shown to seroconvert to EHV-1 or -4 within the first few months of life, and shedding of both EHV-1 and -4 has been detected in populations of broodmares and their foals [13,14]. While nasal shedding appears to be part of a “silent” cycle of herpesvirus infection, it is possible that the event of parturition with endocrinological changes is linked to activation of latent herpesvirus in the mare followed with nasal shedding of virus. For this reason, postpartum mares should be considered an at-risk group for EHV-1 or -4 shedding. Because of this and because our single EHV-4-positive sample was from a febrile postpartum mare, special barrier precautions and EHV-1- or -4specific diagnostic sampling methods are warranted in febrile and maybe also in nonfebrile postpartum mares. Nasal swabs might have been found positive for EHV-1 or -4 more often if we had selected horses with a fever and concurrent respiratory tract disease or febrile horses with concurrent neurological disease, febrile horses coming from a horse show or event, mares that aborted a late-term fetus, peripartum mares with partial or complete placental separation, and febrile postpartum mares [6,7,15]. Hence, solid clinical judgment is necessary to recognize these “red flags” of potential EHV-1 or -4 disease in equine patients that need to be admitted into a referral center. 5. Conclusions Finally, if a case of EHV-1 or EHV-4 infection has been identified, any further fevers in hospitalized horses need to be investigated, and rectal temperatures during a time like this should be collected at least twice daily in all hospitalized horses [4]. Acknowledgments The work was completed at the James L. Voss Veterinary Teaching Hospital, Colorado State University, Fort Collins,
J.M. Sonis and L.S. Goehring / Journal of Equine Veterinary Science 33 (2013) 756-759
CO. The study was supported by a grant from the Animal Population Health Institute at Colorado State University. An abstract was presented at the Forum of the American College of Veterinary Internal Medicine held in Anaheim, CA, June 9-12, 2010. Our special thanks to Dr. Brandy Burgess for carefully editing the script.
[7]
[8] [9]
References [10] [1] van Maanen C. Equine herpesvirus 1 and 4 infections: An update. Vet Q 2002;24:58-78. [2] Slater J. Equine herpesviruses. In: Sellon DC, Long MT, editors. Equine infectious diseases. St. Louis, MO: WB Saunders Elsevier; 2007. p. 134-53. [3] Henninger RW, Reed SM, Saville WJ, et al. Outbreak of neurologic disease caused by equine herpesvirus-1 at a university equestrian center. J Vet Intern Med 2007;21:157-65. [4] Goehring L, Landolt G, Morley P. Detection and management of an outbreak of equine herpesvirus type 1 infection and associated neurological disease in a veterinary teaching hospital. J Vet Intern Med 2010;24:1176-83. [5] Burgess BA, Tokateloff N, Manning S, et al. Nasal shedding of equine herpesvirus-1 from horses in an outbreak of equine herpes myeloencephalopathy in Western Canada. J Vet Intern Med 26:384-392. [6] Goehring LS, van Winden SC, van Maanen C, et al. Equine herpesvirus type 1-associated myeloencephalopathy in The Netherlands: A
[11]
[12]
[13]
[14]
[15]
759
four-year retrospective study (1999-2003). J Vet Intern Med 2006;20:601-7. Pusterla N, Kass P, Mapes S, et al. Surveillance programme for important equine infectious respiratory pathogens in the USA. Veterinary Record 2011;169:12. Goehring L, Wagner B, Bigbie R, et al. Control of EHV-1 viremia and nasal shedding by commercial vaccines. Vaccine 2010;28:5203-11. Hussey SB, Clark R, Lunn KF, et al. Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction. J Vet Diagn Invest 2006;18:335-42. Pusterla N, Mapes S, Madigan JE, et al. Prevalence of EHV-1 in adult horses transported over long distances. Vet Rec 2009;165:473-5. Yactor J, Lunn KF, Traub-Dargatz J, et al. Detection of nasal shedding of EHV-1 & 4 at equine show events and sales by multiplex real-time PCR. Paper presented at: 52nd Annual Convention of American Association of Equine Practitioners. December 2-6, 2006; San Antonio, TX. Carr E, Schott H, Pusterla N. Absence of equid herpesvirus-1 reactivation and viremia in hospitalized critically ill horses. J Vet Intern Med 2011;25:1190-3. Foote CE, Gilkerson JR, Whalley JM, et al. Seroprevalence of equine herpesvirus 1 in mares and foals on a large Hunter Valley stud farm in years pre- and postvaccination. Aust Vet J 2003;81:283-8. Foote CE, Love DN, Gilkerson JR, et al. EHV-1 and EHV-4 infection in vaccinated mares and their foals. Vet Immunol Immunopathol 2006;111:41-6. van Maanen C, Willink DL, Smeenk LA, et al. An equine herpesvirus 1 (EHV1) abortion storm at a riding school. Vet Q 2000;22:83-7.