Journal of Virological Methods 134 (2006) 119–124

Laboratory diagnosis of contagious ecthyma: Comparison of different

PCR protocols with virus isolation in cell culture
Christine Kottaridi a,b , Kiki Nomikou a , Rossella Lelli c ,
Panayotis Markoulatos b , Olga Mangana a,∗
a Centre of Athens Veterinary Institutions, Institute of Infectious and Parasitic Diseases, 25 Neapoleos Street, 15310 Agia Paraskevi, Attiki, Greece
b Department of Biochemistry and Biotechnology, School of Health Sciences, University of Thessaly, 26 Ploutonos and Aeolou Str., Larissa, Greece
c Istituto Zooprofilattico Sperimentale dell’ Abruzzo e del Molise “G. Caporale”, Terremo, Italy

Received 4 July 2005; received in revised form 1 December 2005; accepted 5 December 2005
Available online 18 January 2006

Abstract
A new polymerase chain reaction (PCR) assay for rapid diagnosis of contagious ecthyma was designed and applied to 21 clinical samples from
Greece. This assay, which detects a highly conserved gene from the parapox genome, was evaluated for its sensitivity and specificity in order to be
considered as a useful diagnostic tool. A comparative study with two published PCR protocols one using primers PPP1–PPP3, PPP1–PPP4 which
targets putative virion envelope gene B2L and the other using VIR1–VIR2 primers which amplifies ORF virus interferon resistant (VIR) gene, as
well as cell culture virus neutralization assay was carried out. All samples tested were amplified successfully with the PCR protocol established
in the laboratory. The combination of primers PPP1–PPP3 and PPP1–PPP4 in a semi-nested PCR gave a positive result in 20 of 21 samples while
primers VIR1–VIR2 failed to amplify successfully 7 of 21 samples. The diagnostic value of parapox viral DNA amplification was also compared
with the results of virus isolation by cell culture and was positive in three samples that the virus isolation was obtained.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Contagious ecthyma; Parapox virus; Polymerase chain reaction; Cell culture; Diagnosis

1. Introduction cific geographical distribution of the disease since sheep and

Parapoxvirus genus of the family Poxviridae includes orf
virus (ORFV), which is the prototype member of the genus,
bovine papular stomatitis virus (BPSV), pseudocowpox virus
(PCPV) and parapox virus of deer and seal pox virus (de la
Concha-Bermejilo, 1995). Infected animals develop contagious
ecthyma also known as soremouth, contagious pustular dermati-
tis, infectious labial dermatitis, orf or scabby mouth. Lesions
may also occur on the udder and between the toes (Esposito
et al., 1995; Fenner, 1996; Mayr and Büttner, 1990a, 1990b,
1990c; Robinson and Lyttle, 1992; Mercer et al., 1997; Moss,
1996; Haig and Mercer, 1998). The disease is characterized by
proliferative lesions in the skin of the lips, around the nostrils
and in the oral mucosa, which usually resolve in 1–2 months
(McKeever et al., 1988). There is no information about a spe-
∗ Corresponding author. Tel.: +30 210 6011499; fax: +30 210 6011499.
E-mail address: viruslab@ath.forthnet.gr (O. Mangana).
goats worldwide can be infected. The orf virus can also infect
humans with lesions characterized by large, painful nodules on
the hands and less frequently on the face, after close contact with
skin lesions of infected animals or handling virus-contaminated
materials (Fenner, 1996; Mayr and Büttner, 1990b; Memar and
Tyring, 1995; Robinson and Lyttle, 1992).
Parapoxviruses are distinguished from other poxvirus genera
by their ovoid shape, the crisscross pattern on the particle surface
the relatively small size and the high G + C content (approx-
imately 64%) of the genome (Mercer and Haig, 1999; Moss,
2001; Delhon et al., 2004). Virions are about 260 nm in length
and 160 nm in width, and have an outer membrane that consists
of a single, long spiral tubule wrapped around a homogenous
core. The viral genome is composed of ∼135 kb linear, ds DNA
with closed hairpin loop ends and genes located on both strands
with a bi-directional orientation. Conserved genes are found in
the central region of the genome, while variability is observed
in the terminal ends (Gassmann et al., 1985; Fraser et al., 1990;
Mercer et al., 2002).

0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2005.12.005
120 C. Kottaridi et al. / Journal of Virological Methods 134 (2006) 119–124

The cell culture systems that are used for virus isolation Table 1
include primary lamb testis cells, bovine kidney cells and others. Studied specimens
Typical cytopathic effects (CPE) include cell rounding, clump- Clinical sample Geographic regions Host
ing and detachment. 4945/86a Keffallinia Sheep
Laboratory diagnosis of orf disease is achieved by negative- 202/94 Kavala Sheep
stain electron microscopy from scabs of affected animals where 155/95 Kavala Sheep
the characteristic ovoid shape of the virion is demonstrated 176/95 Lesvos Sheep
(Wilson and Sweeny, 1970; Hiramatsu et al., 1999; Guo et al., 38/96 Evia Sheep
144/96 Rodopi Sheep
2004). However, the lack of an electron microscope by many 1179/00 Argos Sheep
veterinary diagnostic laboratories and the high cost of the test 757/01 Rodopi Sheep
precludes the submission of orf cases for laboratory testing. The 794/01 Farsala Sheep
serological tests used for orf diagnosis include virus neutraliza- 928/02 Velestino Sheep
tion, agar gel immunodiffusion (AGID), complement fixation, 1010/03 Arkadia Sheep
30/96 Chios Goat
or agglutination for the detection of anti-contagious ecthyma 994/00 Kavala Goat
antibodies. The development of PCR methods for the molecular 759/01 Rodopi Goat
detection of parapox DNA has met the demands for specific and 814/01 Farsala Goat
sensitive laboratory diagnosis of orf disease (Mazur et al., 2000; 848/01 Evros Goat
Inoshima et al., 2000, 2001, 2002; de la Concha-Bermejilo et al., 849/01 Evros Goat
1710/03 Thessaloniki Goat
2003; Guo et al., 2003, 2004; Torfason and Gunadottir, 2002; 513/04 Thessaloniki Goat
Tryland et al., 2005). 1635/03 Rodopi Bovine
The objective of the present study was to establish a simple 1090/04 Evros Bovine
and convenient molecular method for identification of parapox a The second number represents the sampling year.
virus directly from skin biopsies. A new parapox specific and
sensitive PCR assay was applied, designed upon a conserved
gene of ORF virus, in order to detect and differentiate parapox ples were moreover inoculated in BHK-21 (baby hamster kidney
infections from those induced by pox virus. A comparison of cells) and GBK (Georgian bovine kidney). The inoculated flasks
the results with those obtained from the amplification of the were incubated at 37 ◦ C for a period of up to 14 days. A second
genes for ORF virus interferon resistance (VIR) and B2L using passage to cells carried out for samples without an appearance of
published primers (Guo et al., 2004; Inoshima et al., 2000) was CPE and sometimes a third passage was necessary for accuracy.
also carried out. When CPE was obvious, two aliquots, 1 ml each, were taken for
toxicity check. After inactivation of one of them (heat at 60 ◦ C,
2. Materials and methods for 1 h) they both were inoculated in two different flasks. When
CPE was observed in the flask with the inactivated inoculum as
2.1. Clinical samples well as at the flask with the non-inactivated,the sample was con-
sidered as toxic. The specificity of CPE was confirmed further
Crusted scab lesions obtained from 11 sheep, 8 goats and by identification (virus neutralization, Manual of Standards for
2 bovines at different geographical locations in Greece, with Diagnostic Tests and Vaccines, 2004) of orf virus. Uninfected
suspected contagious ecthyma infection or bovine papular stom- LT cells were used as negative controls.
atitis, were tested in the present study for the presence of parapox
virus (Table 1). Skin biopsies from naive sheep and from sheep 2.3. DNA extraction
infected with pox virus were included as negative controls.
DNA was extracted from infected LT cells with complete
2.2. Cell cultures and virus Isolation CPE, as well as directly from the resulted supernatant once the
centrifugation of the grinded biopsy was performed. The extrac-
Primary lamb testis (LT) cells were grown in plastic flasks tion procedure was carried out using guanidine thiocynate lysis
of 25 cm3 (Corning, NY, USA) in Eagle’s medium supple- buffer (Casas et al., 1995). DNA was also extracted from healthy
mented with 10% fetal calf serum (FCS) and the usual antibi- tissue and from uninfected cell cultures.
otics (penicillin 100 U/ml, streptomycin 100 ␮g/ml, kanamycin
50 ␮g/ml, amphotericin B 2.5 ␮g/ml). Skin and crusted scab 2.4. Molecular characterization of the viruses
lesions were grinded and Eagle’s medium with antibiotics (peni-
cillin 100 U/ml, streptomycin 100 ␮g/ml, kanamycin 50 ␮g/ml The primers 045F (5 cct act tct cgg agt tca gc 3 ) and 045R
and amphotericin B 2.5 ␮g/ml) making up a 50% (w/v) solution (5 gca gca ctt ctc ctc gta g 3 ) were designed based on the pub-
was added. The suspension was frozen at −70 ◦ C and thawed lished sequence of gene 045 of ORF virus, isolate OV-SA00
three times. After centrifugation at 2000 × g for 30 min, 1 ml (accession no. AY186732) (Delhon et al., 2004) that encodes
of the supernatant was inoculated in LT cells. Sheep and goats for the late transcription factor VLTF-1. All clinical specimens
samples were propagated in LT cells as described previously were screened initially with the primers for ␣-tubulin which
(Mangana-Vougiouka et al., 1999, 2000), while bovine sam- were used as a control of DNA extraction procedure, sample
 C. Kottaridi et al. / Journal of Virological Methods 134 (2006) 119–124 121

Table 2
Oligonucleotides used in the present study
Primers Sequence (5 → 3 ) Polarity Accession number Position References

045 F cct act tct cgg agt tca gc Sense AY186732 46558–78 Present study
045R gca gca ctt ctc ctc gta g Antisense 46968–49
␣-Tubulin L cac ccg tct tca ggg ctt ctt ggt tt Sense X01703 454–480 Markoulatos et al. (2000)
␣-Tubulin R cat ttc acc atc tgg ttg gct ggc tc Antisense 955–981
PPP1 gtc gtc cac gat gag cag ct Sense UO6671 162–182 Inoshima et al. (2000)
PPP4 tac gtg gga agc gcc tcg ct Antisense 757–737
PPP3 gcg agt ccg aga aga ata cg Sense 522–541
VIR1 aca atg gcc tgc gag tg Sense AJ222702 114–131 Guo et al. (2004)
VIR2 tta gaa ctg atg ccg cag Antisense 731–713

integrity, presence of inhibitors and finally PCR reaction effi-
ciency (Markoulatos et al., 2000). Also, the already published
primers shown on Table 2 were used for the molecular detection
of parapox DNA of the samples included in the present study. All
primer sets were synthesized by Invitrogen, Life Technologies
(Paisley, UK). The PCR assay carried out in a reaction mix-
ture of 50 ␮l containing 10 ␮l of extracted DNA, 5 ␮l dNTP’s
10 mM, 5 ␮l 10× PCR buffer, 2 ␮l MgCl2 (50 mM), 2 units/tube
Taq Polymerase (Invitrogen, Life Technologies Paisley, UK),
50 pmol of each of the primer pair and 33 ␮l Rnase-free dis-
tilled water. Forty cycles of denaturation (95 ◦ C, 10 s), annealing
(47 ◦ C, 10 s) and extension (74 ◦ C, 10 s) were carried out, fol-
lowed by a 15 min incubation at 78 ◦ C to final extension of
the primers. Amplification products (10 ␮l) were run on 2%
agarose gel (Gibco, Life Technologies Paisley, UK) in Tris–boric
acid–EDTA (TBE) buffer and strained with ethidium bromide
(1 ␮g/ml). Amplicons were visualized under UV transillumi-
nator FOTO/PHORESIS I, FOTODYNE (Hartland, WI, USA).
Separate rooms and different sets of pipettes were used in order
to minimize the risk of contamination.
2.5. Sensitivity and specificity of the PCR assay
The clinical isolate 176/95 was titrated on LT cells and
TCID50 was determined to be 104 /ml. Serial 10-fold dilutions
of the virus stock up to 10−7 (corresponding from 103 up
to 0.01 TCID50 /ml) in Eagle’s medium, were performed and
each one of the above virus dilution was subjected to DNA
extraction and PCR amplification with primers 045F–045R and
PPP1–PPP4.
The specificity of the PCR assay was determined by apply-
ing the PCR assay with all primer pairs included in the present
study (Table 2). Samples infected with sheep poxvirus as well
as foot-and-mouth disease (FMD), bovine herpesvirus type-2
(BHV-2) and bluetongue virus (BTV) were used as negative
controls.
3. Results
The clinical specimens consisted of 21 parapox samples col-
lected from different geographic regions of Greece. The isolation

Table 3
Results of PCR amplification with all primer pairs
Clinical isolate Primer pairs

␣-Tubulin PPP1–PPP4 PPP3–PPP4 VIR1–VIR2 045F–045R

4945/86 + + + +
202/94 + + + +
155/95 + + + +
176/95 + + + +
38/96 + − + − +
144/96 + + + +
1179/00 + − + − +
757/01 + − + − +
794/01 + − + − +
928/02 + − + + +
1010/03 + + + +
30/96 + + + +
994/00 + − − − +
759/01 + + + +
814/01 + + + +
848/01 + + + +
849/01 + + + +
1710/03 + − + + +
513/04 + + + +
1635/03 + + − +
1090/04 + + − +
122 C. Kottaridi et al. / Journal of Virological Methods 134 (2006) 119–124

year for each of the studied specimens was different and varied Table 4
from 1986 to 2004 (Table 1). Comparison of PCR amplification with primers 045F–045R, with virus isolation
by cell culture
The sequence of the highly conserved gene 045 coding for
the late transcription factor VLTF-1 of ORF virus, isolate OV- Clinical isolate Cell culture PCR
SA00 (accession no. AY186732, Delhon et al., 2004) was taken 4945/86 + +
into consideration for designing a new primer pair. All primer 202/94 + +
pairs that we used in the present study are listed in Table 2. 155/95 + +
Viral DNA was extracted from LT cell cultures inoculated 176/95 + +
38/96 + +
with clinical specimens with suspected contagious ecthyma 144/96 + +
infection. All samples were screened by PCR with ␣-tubulin 1179/00 + +
primers, providing an additional control for false negative 757/01 + +
results. The results of PCR amplification with all primer pairs 794/01 + +
used in the present study are shown in Table 3. 928/02 − +
1010/03 + +
045F–045R amplified successfully a fragment of 392 bp in 21 30/96 + +
out of 21 clinical specimens that were investigated in the present 994/00 + +
study. PCR amplification with the primer pair PPP1–PPP4 gave 759/01 + +
positive result in 14 of 21 samples. The seven samples resulted 814/01 + +
negative were analysed further using a semi-nested PCR analysis 848/01 + +
849/01 + +
with PPP3–PPP4 primers. The results indicated that in six out 1710/03 + +
of the seven negative samples the low copy number of viral 513/04 − +
DNA was successfully detected. Amplification with published 1635/03 − +
primers VIR1–VIR2 did not give a parapox specific band in 1090/04 + +
seven samples. Five of these samples were the same negative
samples, when amplified with PPP1–PPP4 while the remaining
two were isolated from the two bovine isolates. 4. Discussion
In order to minimize the whole diagnostic procedure of con-
tagious ecthyma in laboratory routine, emphasis was given to the Contagious ecthyma, caused by parapox virus is one of the
molecular detection of the virus. Processed biopsy supernatants most common skin diseases of sheep and goats. The lesions
were used for viral DNA extraction and PCR amplification with induced by the virus, prevent infected animals from suckling
oligonucleotides 045F–045R. All the 21 samples were positive. and grazing. Clinical symptoms similar to parapox infection are
The efficiency of orf virus detection by estimation of virus caused in animals by other viruses such as sheep pox, foot-and-
cytopathic effect and subsequent virus neutralization on cultured mouth disease (FMD), bovine herpesvirus type-2, bluetongue
LT cells was comparable to the PCR methods, allowing the iden- virus, etc. Also, as far as bovine hosts are concerned, in our
tification of 85.7% of samples (18/21) (Table 4). In 2 out of the country we have to face the problem of differential diagnosis
21 clinical samples studied, virus was undetectable in cell cul- between bovine papular stomatitis virus and FMD which have,
tures of both BHK-21 (baby hamster kidney) and primary LK in some cases, similar clinical symptoms.
(lamb kidney). Furthermore, one sample showed cytotoxicity A number of procedures have been developed to detect para-
therefore precluding virus isolation. pox virus. However most of these methods are time-consuming,
laborious and sometimes show lack of specificity and sensitiv-
3.1. Sensitivity and specificity of the PCR assay ity with cross-reactions observed (Lard et al., 1991; Rosenbusch
and Reed, 1983; Wittek et al., 1980). PCR analysis, as proved by
To evaluate the sensitivity of our PCR assay, viral DNA many reports, is a rapid, sensitive and specific tool in identifying
extracted from each virus dilution of the titrated isolate 176/95, several infectious diseases of veterinary importance (Mangana-
was amplified with primer pair 045F–045R as well as with Vougiouka et al., 1999, 2000; Inoshima et al., 2001, 2002;
primers PPP1–PPP4. Gel electrophoresis analysis of the PCR Billinis et al., 2001). Several PCR protocols have been described
products with primers 045F–045R, showed a successful ampli- for parapox DNA detection (Mazur et al., 2000; Inoshima et
fication up to 0.1 TCID50 /ml of the virus stock. It is also notewor- al., 2000, 2001, 2002; Torfason and Gunadottir, 2002; de la
thy, that the sensitivity of the reaction was not equivalent with Concha-Bermejilo et al., 2003; Guo et al., 2003, 2004; Tryland
primer pair PPP1–PPP4, when the same dilutions of 176/95 were et al., 2005) emphasizing on the prevention of serological cross-
subjected to DNA extraction and PCR amplification, a parapox reactivity and on diagnosing ecthyma without using cell culture
specific band was obvious up to 1 TCID50 /ml. systems or electron microscopy.
The specificity control of the PCR assay with negative con- Parapox genomics relied initially upon sequencing studies
trols from samples infected with sheep poxvirus as well as of the NZ2 strain of the orf virus. A recent report (Delhon
foot-and-mouth disease (FMD) virus, bovine herpesvirus type-2 et al., 2004) presenting the complete DNA sequences of two
virus, bluetongue virus and uninfected LT cells was successful ORFV and one BPSV isolate showed a detailed overview of their
as no specific band for parapox genome in any of these negative genome organization, gene content and similarities between
controls was detected. isolates. We selected gene 045 as a suitable target for a diag-
 C. Kottaridi et al. / Journal of Virological Methods 134 (2006) 119–124
nostic assay, as this gene is well conserved. This gene encodes
for VLTF-1, a late transcription factor and the vaccinia coun-
terpart to it is designated as G8R. Primers 045F–045R were
designed taking into consideration the specific amplification
of orf virus genome and the absence of homology with mam-
malian genes as false positive results from adjacent healthy tis-
sue may occur. As negative control, adjacent healthy tissue were
used.
According to the results of the present study, the whole pro-
cedure can be simplified further successfully by extracting and
amplifying viral DNA after a quick processing of infected tissue,
without cell culture inoculation. The probability that the PCR-
assay yields false negative results due to low sample quality or
technical errors was excluded by including the amplification of
␣-tubulin as an internal control.
In the cases reported in the present study, isolation of the
virus through lamb testis (LT) cell cultures was unsuccessful for
three samples. However, PCR amplified successfully viral DNA
extracted directly from biopsy of all three samples.
The results of previous studies (de la Concha-Bermejilo et
al., 2003; Guo et al., 2004) suggested that although the virus
interferon-resistance (VIR) gene is exposed to immunological
pressure from the host and tends to be variable, this region
could serve as a genetic marker for parapox virus. In the present
study, we did not succeed in amplifying the region flanked by
the primers VIR1-VIR2, which were designed for the virus
interferon-resistance (VIR) gene, in the two BPSV strains avail-
able for our study and in five more isolates with sheep and goat
species origin, revealing a possible variability resulting from
host specific substitutions. The possibility that samples were not
infected by parapox virus was rejected as there was a clear pos-
itive PCR result for at least one more genomic region included
in the study.
Although primers PPP1–PPP4 followed by the semi-nested
PCR with PPP3–PPP4, amplified 20 out of 21 suspected orf
virus-infected samples, the amplification of a second genomic
region flanked by primers 045F–045R gave a positive result in
all cases tested, indicating once more the diagnostic importance
of using more than one primer pairs targeting different regions
of the viral genome in order to achieve maximal sensitivity and
specificity (Mangana-Vougiouka et al., 1999; Guo et al., 2003;
Kottaridi et al., 2004).
The data described in this study demonstrate that the designed
PCR-assay is a powerful tool for the diagnosis of orf infec-
tions which shows increased sensitivity over existing PCR pro-
tocols, is more rapid when compared with virus isolation by
cell culture and can differentiate successfully poxvirus infec-
tions.
Acknowledgments
The present work was supported by research grants of
the Italian Ministry of Health under the project code number
MSRCTE0602, Diagnosis of infectious exotic diseases of rumi-
nants: evaluation of available diagnostic systems, study on the
sensitivity and specificity of innovative assays.
123
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