Lack of Detectable Equine Herpesviruses 1 and 2 in Paraffin-Embedded Specimens of Equine Sarcoidosis
Corresponding author: Dr Stephen D. White, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA; e-mail: email@example.com.
Background: Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2).
Objective: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis.
Animals: Eight horses with sarcoidosis, reported previously.
Methods: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with EHV-1 and EHV-2. For both herpesviruses the target was their respective glycoprotein B gene. Positive controls consisted of DNA from viral cultures of culturettes from naturally occurring respiratory infections of EHV-1 and EHV-2.
Results: The PCR analyses for both equine herpesviruses' DNA were negative in all 8 horses.
Conclusion: The failure to detect DNA from EHV-1 and EHV-2 in paraffin-embedded skin of these 8 horses does not discount EHV-1 or EHV-2 as causing some cases of ES, but lends support to the presumably multifactorial etiologic nature of the disease.
equine herpesvirus 1
equine herpesvirus 2
polymerase chain reaction
Equine sarcoidosis (ES), also known as equine granulomatous skin disease, is a rare, multisystemic, noncaseating, primarily granulomatous and lymphoplasmacytic disease of unknown etiology.1–4 There is no evidence of Mycobacteria spp., Coccidioides immitis, Cryptococcus neoformans, Corynebacterium pseudotuberculosis, and Borrelia burgdorferi in paraffin-embedded specimens from horses diagnosed with ES.1
A recent report described a horse with recurrent granulomatous skin disease with intranuclear viral inclusions within macrophages and giant cells.5 Electron microscopic examination showed intranuclear and intracytoplasmic viral particles consistent with herpesvirus. Sequence results of the polymerase chain reaction (PCR) product obtained from paraffin-embedded skin biopsies were consistent with equine herpesvirus 2 (EHV-2). The authors suggested that EHV-2 could be a possible etiology for equine granulomatous skin disease. We elected to investigate the presence of equine herpesvirus 1 (EHV-1) and EHV-2 using PCR in the samples from 8 horses evaluated in our previous report.1
Material and Methods
Eight horses were chosen by a retrospective evaluation of data at the Veterinary Medicine Teaching Hospital of the University of California at Davis and IDEXX Laboratories Incorporated in West Sacramento, California. These horses were diagnosed as having ES based primarily on histopathologic examination of affected organs as well as history, laboratory results, clinical signs, and elimination of other causes of granulomatous disease. Information about these horses has been published.1
PCR was used to detect EHV-1 and EHV-2 nucleic acid. DNA was extracted from 57 tissue samples, including skin samples from all 8 horses and lung, lymph node, and gastrointestinal tract from 4 horses. Two 50 μm sections from paraffin-embedded formalin-fixed tissue samples of the skin and the other available lesional tissues were placed in 1.5 mL tubes, then deparaffinized by treatment for 1 hour in xylenea and washed twice with 100% ethanol. After allowing the ethanol to evaporate, tissue was extracted using a kitb following the manufacturer's instructions.
Controls. To prove the presence of amplifiable DNA, a real-time PCR with a target sequence within the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was performed using a kit.c Results of real-time PCR assays were considered positive if the threshold cycle (Ct) value was <40 and there was a characteristic amplification plot.
Positive controls consisted of DNA from viral cultures of culturettes from previously diagnosed naturally occurring respiratory disease in horses caused by EHV-1 and EHV-2.
PCR was performed using a real-time TaqMan system, as described previously.6 The assay, which targets the EHV-1 glycoprotein B gene, was performed using 400 nM of each primer (designated “forward” and “reverse” in the original publication7), 80 nM of the probe, commercial mastermixd and 1 μL of DNA for a final volume of 12 μL. Samples were amplified in a thermal cycler-fluorometere with a protocol consisting of 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Results were considered positive if the threshold cycle was ≤40 and amplification plots showed logarithmic increases in target DNA. While the original paper describing this assay did not give an absolute sensitivity, as for most TaqMan assays the likely lower detection limit is from 10 to 100 organisms/sample.6
Conventional PCR was performed to detect DNA associated with EHV-2 as described previously.7 The gene target was the EHV-2 glycoprotein B gene. For the first round, 1 μL of DNA was added to a reaction mix consisting of 50 mM KCl, 10 mM Tris-hydrochloride (pH 8.3), 1.5 mM MgCl2, 200 mM each dNTP, 15 μL of primers 2-07/forward and 2-09/reverse, and 2 U of Taq polymerase.f Cycling conditions were 5 minutes at 95°C followed by 35 amplification cycles at 95° for 1 minute, 1.5 minutes at 60°C, and 1 minute at 72°C. For the nested reaction, 1 μL of DNA was used with the same mix and reaction conditions, except that primer 2-08/forward was substituted for 2-07/forward. Following electrophoresis, the PCR products were evaluated via ultraviolet transillumination of ethidium-bromide-stained 1% agarose gels. The routine detection limit after ethidium bromide staining was 0.6 fg (femtograms) equivalent to approximately 100 genome copies.7
For all reactions, extraction, PCR, and electrophoresis were performed in different rooms, plugged tips were utilized to prevent DNA carryover, and a downstream water negative control was included in each room to prevent against cross-contamination.
Nested PCR for the target sequence within the housekeeping gene GAPDH showed amplifiable DNA in 100% of the tissues in the 8 horses with ES. The PCR analyses for both EHV-1 and EHV-2 DNA were positive in the samples from the control horses with these viral infections, but negative in all horses with ES.
The failure to find evidence of viral DNA in the 8 horses in this report does not preclude equine herpesvirus as a potential etiologic agent in some cases of ES. Our histologic samples did not have the findings of intranuclear viral inclusions within macrophages and giant cells that were noted in the report which associated EHV-2 with granulomatous dermatitis in a single horse.5 Sarcoidosis in humans (a similar but not identical disease) has been postulated to be an abnormal immunologic response to either an infectious agent or an allergen.8 It therefore seems reasonable to suspect multiple etiologies in ES. While suppositions of mycobacteria3 and Borrelia9 were not supported in our previous paper1 it is still possible that these or other infectious agents are responsible for some cases of ES.
The exact pathogenic importance of EHV-2 remains poorly characterized, as approximately 90% of horses are seropositive.10 In addition, the incidence of EHV-2 antibody and levels of titers are not different between healthy and diseased horses.10 EHV-2 has been isolated from a number of internal organs as well as nasal and ocular swabs and blood buffy coat samples, in both healthy horses and those with various diseases.10–12 Therefore, the recovery of this virus from the skin in the aforementioned report5 is probably not as important in suggesting EHV-2 as a potential cause of ES as is the histologic finding of intranuclear viral inclusions within macrophages and giant cells. Ideally to support an etiologic association between a microorganism and a disease, the organism should be able to be demonstrated from affected tissues on a consistent basis. However, studies in rodents have demonstrated a potential for a “hit-and-run” herpes virus effect: that is, transformation of rodent cell lines to a neoplastic phenotype in vitro. The transfected DNA does not persist long term in the transformed cells, and viral proteins, although transiently expressed, cannot be detected in the established cell lines.13 Whether there might be a similar effect in vivo is controversial.
While EHV-2 is more widely prevalent compared with EHV-1, the latter is endemic in horse populations worldwide,14 although diseases due to EHV-2 are considered of less economic and veterinary importance.15 The abilities of EHV-1 to cause respiratory, reproductive, and neurologic disease, as well as to persist as a latent infection, suggested a possible role in ES, which can involve multiple organ systems and sometimes be a disease of slow insidious onset.1
PCR can be a highly sensitive and specific technique, which has revolutionized the diagnosis of some infectious diseases when run with appropriate controls and technique. It has been used to detect EHV for more than a decade.7,16 False-negative results can be due to F (a) degraded target DNA, (b) PCR-inhibiting substances present in tissue specimens, or (c) insufficient extraction of target DNA.17 The degradation of the target DNA also is a concern in this study as many of our samples were greater than 5–10 years old and had been fixed in formalin, which can diminish the PCR amplification signal with long fixation times.18 However, for all of the samples used in this study, the good quality GAPDH signals suggested that DNA had been efficiently extracted from the samples.
aXylene, Merck, Rahway, NJ
bQiagen DNeasy tissue kit, Valencia, CA
cTaqMan GAPDH Control Reagents, Applied Biosystems, Foster City, CA
dTaqMan Master Mix, Applied Biosystems
eOne Step Plus, Applied Biosystems
fTaq DNA Polymerase, 5-Prime, Gaithersburg, MD