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Abstract
Background: The orf virus of sheep and goats is one of several zoonotic parapoxviruses. In the ovine/caprine host
it causes contagious ecthyma (contagious pustular dermatitis, scabby mouth), but in humans it normally causes
solitary or clustered orf lesions, typically on hands, arms or face. In addition to disease in the animals, the virus can
be quite a nuisance as an occupational hazard in farmers and butchers. Clinical diagnosis is often possible, but
laboratory diagnosis is sometimes necessary. For virus isolation, primary ovine or bovine cells, not routinely present,
are needed. Serological methods exist, but electron microscopy is the most commonly used method. Objecti6es: To
develop a reliable method for the laboratory diagnosis of orf zoonoses, without virus culture and without access to
an electron microscope. Study design: A suitable primer pair was designed for orf polymerase chain reaction (PCR),
using the Oligo software and sequence information from GenBank. Orf positive controls and specimens were kindly
provided by several public health centers. Molluscum contagiosum specimens were provided by a dermatologist.
HSV-1, HSV-2 and VZV positive swab specimens came from our routine diagnostic service. Asymptomatic skin
specimens were obtained from sheep heads from the abattoir, and swab specimens from the heads of asymptomatic
sheep. Selected amplified orf PCR positive specimens were sequenced to ensure the authenticity of the PCR products.
Orf positive specimens were sent to another laboratory for electron microscopy. Results and conclusions: A robust
PCR was developed, with very small inter-run variation. All specificity demands were met, and the sensitivity seems
to be good or excellent. All negative specificity controls from cell cultures and non-orf viruses were negative.
Twenty-two (95.7%) of 23 scab or swab specimens with suspected orf etiology were orf PCR positive. Five of eight
skin specimens from sheep heads from the abattoir were positive, and all 11 swab specimens from asymptomatic sheep
were negative. Electron microscopy demonstrated orf-like particles in orf-PCR positive specimens. This PCR seems
to be suitable as a diagnostic test for orf in humans, but asymptomatic virus shedding in sheep or goats may
complicate veterinary applications of the assay. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Contagious ecthyma; Contagious pustular dermatitis; Parapoxvirus; Scabby mouth; Zoonosis
1. Introduction
* Corresponding author. Tel.: + 354-560-2400; fax: + 354-
560-2406. The orf virus is the type species of the para-
E-mail address: einarg@landspitali.is (E.G. Torfason). pox6irus genus. It is the causal agent of conta-
1386-6532/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 6 - 6 5 3 2 ( 0 1 ) 0 0 2 3 2 - 3
80 E.G. Torfason, S. Guðnadóttir / Journal of Clinical Virology 24 (2002) 79–84
gious ecthyma (contagious pustular dermatitis, tion period of 2–4 days (Murphy et al., 1999).
scabby mouth) in sheep and goats worldwide The lesions are most often localized on hands,
(Fenner, 1996; Murphy et al., 1999). The orf virus arms or face. Solitary lesions are more frequent
can also infect other ruminants, as well as hu- than multiple lesions. Duration is from 4 to 9
mans. Other zoonotic parapoxviruses are pseudo- weeks (Murphy et al., 1999), usually 6– 7 weeks
cowpox virus (paravaccinia, milker’s nodule) and (Buchan, 1996). Healing is complete without
bovine papular stomatitis virus in cattle, ausdyk scars, but secondary infections may retard heal-
virus in camels, and seal parapox virus in grey ing. Fever, swelling of the draining lymph nodes
seals (Fenner, 1996; Murphy et al., 1999). or blindness following an eye infection are seen
Zoonotic parapoxviruses in Finnish and Norwe- only rarely. Erythema multiforme, toxic erythema
gian reindeer and Norwegian musk ox have been or allergic reaction, as well as blisters on arms,
reported (Büttner et al., 1995; Falk, 1978). Para- body, face or mouth in association with orf have
poxviruses have also been found in chamois an- been reported in at least one study (Buchan,
telopes, red deer and squirrels (Fenner, 1996; 1996). Reinfection in humans is frequent (Buchan,
Murphy et al., 1999; Haig et al., in press). 1996; Murphy et al., 1999), but usually less severe
The virus particles are ovoid, 260 by 160 nm, (Haig et al., in press).
containing : 135 kbp linear dsDNA with closed The purpose of the present study was to de-
hairpin loop ends (Moss, 1996). Genes are located velop a reliable method for the laboratory diagno-
on both strands with left and right orientation. sis of orf zoonoses. Orf in humans can resemble
Variation is greatest near the termini, but essential or coincide with herpes or papilloma and possibly
genes are located in the conserved central part of
other infections. Several specimens are sent to our
the genome. The viral DNA and RNA poly-
laboratory each year with requests for orf diag-
merases are eucaryotic-like. At least eight genes
nostics. Electron microscopy is widely used for
encode the viral RNA polymerase complex. Infec-
diagnosis of ‘orf/paravaccinia’ (Baxby, 1998;
tivity is resistant to organic solvents and desicca-
Murphy et al., 1999), but we do not have access
tion but is destroyed by heating to 58– 60 °C for
to an electron microscope. Until now, virus isola-
30 min (Fenner, 1996; Moss, 1996).
tion under suboptimal conditions has been the
In sheep and goats the lesions commonly ap-
pear on the muzzle and lips (scabby mouth), sole available method in our laboratory. Appro-
sometimes also affecting the gums and tongue, priate primary cell cultures for orf isolation are
especially in young lambs. Eyelids, feet and teats not routinely available, and virus isolation at-
can also be affected (Murphy et al., 1999). Severe tempts have therefore aimed at excluding other
facial and oral lesions in lambs may interfere with viruses, rather than isolating the orf agent itself.
suckling. Ewes suckling infected lambs may de- Therefore, we decided to develop polymerase
velop lesions on their udders (Fenner, 1996). Re- chain reaction (PCR) for the laboratory diagnosis
infection, chronic infections, and virus remaining of orf infections. We decided to design a new
viable in scabs shed by infected animals make the assay from orf sequence information and aim at
virus difficult to eradicate from flocks, but vacci- sufficient sensitivity for original specimens, with-
nation of ewes with commercial non-attenuated out any need for tissue culture enhancement.
virus several weeks before lambing can minimize One of the obstacles we had to encounter was
the risk of orf in young lambs (Baxby, 1998; the lack of positive reference material, because we
Murphy et al., 1999). had never isolated the virus, and attempts to
Zoonosis occurs most frequently during lamb- acquire orf strains from abroad have not met with
ing, shearing, docking, drenching or slaughtering success. Therefore, we sent a circular to rural
(Murphy et al., 1999). Infection occurs through public health centers in Iceland, requesting viral
abrasions in the skin. Lesions in humans proceed culture specimens from lesions with the clinical
from macular through papular to large nodules, diagnosis of orf. This elicited a good response and
sometimes papillomatous, following an incuba- we got some orf positive specimens to start with.
E.G. Torfason, S. Guðnadóttir / Journal of Clinical Virology 24 (2002) 79–84 81
2. Materials and methods from non-infected human, simian and ovine cell
cultures, as well as from an untyped human aden-
2.1. Primer design ovirus, herpes simplex type 1 and 2 viruses, vari-
cella-zoster virus, human cytomegalovirus, human
Orf sequence information was downloaded papilloma type 18 virus and Coxsackie B4 virus.
from GenBank. A 5261 bp sequence denominated Non-orf parapoxviruses were not available in our
‘ORF-RPA’ (Accession number U33419), which laboratory.
is similar to the A24R gene of vaccinia (U30342,
Mercer et al., 1995) was selected as a suitable 2.3. Preparation of specimens for PCR
target. This gene encodes RPO132, a major com-
ponent of the viral RNA polymerase (Moss, The specimens were extracted with phe-
1996). The following primers flanking a 140 bp nol:chloroform:isoamyl alcohol (25:24:1), precipi-
sequence (including the primers) at position 985– tated with sodium acetate and isopropanol,
1125 were selected with the Oligo software. washed with 70% ethanol, and resuspended in 100
ml of lysis buffer (10 mM Tris pH 8.3, 50 mM
Orf1: cgcagacgtggctgagtacgt
KCl, 2.5 mM MgCl2, 1 mg/ml gelatin, 0.45%
Orf2: tgagctggttggcgctgtcct NP40, 0.45% Tween 20).
The present study included the following PCR Buffer II 50 mM KCl, 10 mM Tris–HCl,
specimens.)\ pH 8.3
Twenty-three scab or swab specimens from 21 MgCl2 2.5 mM
humans and one sheep with suspected orf. dNTPs 200 mM each
Two human serum specimens where orf was Primers 0.5 mM each
suspected. AmpliTaq 2.5 units in each 100 ml
Three biopsies and one swab from three indi- Gold reaction
viduals with molluscum contagiosum.
Three HSV-1 positive, two HSV-2 positive and
two VZV positive swab specimens from seven Of each specimen, 25 ml dissolved in lysis buffer
individuals with lesions caused by these or tenfold dilutions of this in water were added to
herpesviruses. 75 ml of a master mix. Reaction volume was 100
Eight :0.5 cm2 healthy skin specimens from ml in thin-walled 0.2 ml tubes.
the area around the horn base of five sheep
heads that another research group had ob- 2.5. Cycling profile
tained from the abattoir.
Five of these came from farms in the mid-south A hot-start, two temperature cycling profile was
part of the country, and three from the south- designed as follows in a PE9600 cycler.
west area. One cycle of 94 °C for 12 min (‘Gold’ enzyme
Eleven swab specimens from the horn area of activation).
the skin of ten asymptomatic sheep at a small Forty cycles of 94 °C for 30 s and 68 °C for 45 s.
hobby farm in the southwest part of Iceland. One cycle of 65 °C for 3 min.
Final soaking at 4 °C.
Positive controls were prepared from specimens
with solid clinical evidence of orf and positive orf 2.6. Electrophoresis
PCR where the PCR product had been sequenced
to demonstrate orf specificity. Polyacrylamide gel electrophoresis was per-
Negative specificity controls were prepared formed in 4–20% gradient minigels (Mighty Small
82 E.G. Torfason, S. Guðnadóttir / Journal of Clinical Virology 24 (2002) 79–84
Fig. 1. Polyacrylamide gel electrophoresis of orf PCR positive and negative specimens and controls. Lane 1: marker (pBR322 Hae
III digest), lanes 2 –6: orf positive human specimens, lane 7: herpes simplex type 1 virus, lane 8: varicella-zoster virus, lane 9:
Coxsackie B4 virus, lane 10: human papilloma virus type 18, lane 11: herpes simplex type 2 virus, lanes 12 – 13: orf positive sheep
skin specimens, lane 14: negative control (lysis buffer, no template), and lane 15: positive control.
E.G. Torfason, S. Guðnadóttir / Journal of Clinical Virology 24 (2002) 79–84 83
Fig. 2. Sequence comparison of GenBank sequence U33419 and eight amplified orf positive specimens. Primer sequences are shown
in boldface.
amplified positive specimens (Fig. 2) demonstrated was collected, there was one lesion, described as
that the amplicons were almost identical to the ‘abscess like’, that had been unsuccessfully culti-
original sequence in GenBank, validating these vated and treated for bacteria. Differential diagno-
specimens as positive controls. Geographical isola- sis for herpes vs. orf was requested. When the
tion of flocks of sheep for decades did not produce second specimen was collected, a second lesion had
variation in this particular sequence. Electron mi- developed on the same finger after surgical inter-
croscopy (Fig. 3) demonstrated the presence of vention. It is unclear whether the surgical interven-
‘brick shaped’ particles, :140 ×88 nm, pre- tion may have complicated the course and the
sumably naked orf virus particles. duration of the clinical infection, which according
to textbooks does normally not exceed 9 weeks
(Murphy et al., 1999).
4. Discussion One of the orf-PCR positive specimens was from
a garbage disposal worker with orf-like lesions on
Our orf PCR seems to be quite sensitive, and we fingers and the scalp. He had not been in any
have seen only minimal inter-run variation. Read- contact with sheep, but he had been working with
ing of results is easy, because the background is garbage containers from an abattoir. One week
negligible around the size (140 bp) of the orf later his fiancée developed an orf PCR positive
amplicon. Molluscum contagiosum, a common lesion on one of her thumbs. She had not either
poxviral infection in humans, does not react in this
been in any contact with sheep.
PCR. Therefore, this PCR seems suitable for labo-
ratory diagnosis of orf in humans.
The negative results with two human serum
specimens is consistent with the fact that orf lesions
are normally singular or as a localized cluster. This
may indicate that viremia is rare. The five positive
skin specimens from sheep heads indicate that orf
may be present in considerable amounts where
sheep are around. There were absolutely no visible
lesions on any of the sheep heads. Therefore, the
origin of the virus particles is uncertain, but virus
contamination from other individuals in the herd
seems likely. This should be kept in mind if this
PCR is used for laboratory diagnosis of orf in
animals, and also in humans shortly after exposure
to environment where sheep or goats are present.
In one human an infection of a little finger
seemed to persist for at least 14 weeks, and swab
specimens collected at 3 weeks and 14 weeks after
the onset of symptoms were both orf PCR positive, Fig. 3. Composite picture of four electron micrographs of
see lanes 4 and 5 in Fig. 1. When the first specimen orf-like particles seen in orf-PCR positive specimens.
84 E.G. Torfason, S. Guðnadóttir / Journal of Clinical Virology 24 (2002) 79–84