Prevalence, Distribution, and Host Range of Peste des petits ruminants virus, Turkey

Abstract

Peste des petits ruminants virus (PPRV, genus Morbillivirus), which causes a severe disease in sheep and goats, has only recently been officially declared to be present in Turkey. We carried out a study to determine the prevalence, distribution, and host range of PPRV in Turkey. A total of 1,607 animals, reared in 18 different locations, were monitored for the presence of antibodies to PPRV and the related virus of large ruminants, Rinderpest virus (RPV). Only two farms had animals that were free of antibody responses to either disease. Prevalence for PPRV infection varied (range 0.87%-82.6%) and was higher in sheep (29.2%) than in goats (20%). The overall antibody responses to PPRV and RPV were 22.4% and 6.28%, respectively. Two PPRVs of lineage 4, which comprises many other PPRVs whose origins are in the Middle East, the Arabian Peninsula, and southern Asia, were isolated from Turkish sheep.

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708 Emerging Infectious Diseases • Vol. 8, No. 7, July 2002
Prevalence, Distribution, and
Host Range of Peste des petits
ruminants virus, Turkey
Aykut Özkul,* Yilmaz Akca,* Feray Alkan,* Thomas Barrett,*† Taner Karaoglu,*
Seval Bilge Dagalp,* John Anderson,*† Kadir Yesilbag,* Can Cokcaliskan,*
Ayse Gencay,* and Ibrahim Burgu*
Peste des petits ruminants virus (PPRV, genus Morbillivirus), which causes a severe disease in sheep
and goats, has only recently been officially declared to be present in Turkey. We carried out a study to
determine the prevalence, distribution, and host range of PPRV in Turkey. A total of 1,607 animals, reared
in 18 different locations, were monitored for the presence of antibodies to PPRV and the related virus of
large ruminants, Rinderpest virus (RPV). Only two farms had animals that were free of antibody responses
to either disease. Prevalence for PPRV infection varied (range 0.87%–82.6%) and was higher in sheep
(29.2%) than in goats (20%). The overall antibody responses to PPRV and RPV were 22.4% and 6.28%,
respectively. Two PPRVs of lineage 4, which comprises many other PPRVs whose origins are in the Mid-
dle East, the Arabian Peninsula, and southern Asia, were isolated from Turkish sheep.
este des petits ruminants virus (PPRV) is a morbillivirus
that primarily infects sheep and goats. The virus is
present in Africa (1–3), the Middle East (4), the Arabian Pen-
insula (5), and southern Asia (6,7) and is closely related to
Rinderpest virus (RPV), Canine distemper virus, and human
measles virus (8). Infection with PPRV results in an acute,
highly contagious disease characterized by fever, anorexia,
necrotic stomatitis, diarrhea, purulent ocular and nasal dis-
charges, and respiratory distress (9,10). Infection rates in
sheep and goats rise with age, and the disease, which varies in
severity, is rapidly fatal in young animals (10,11). As with
other morbillivirus infections, PPRV needs close contact
between infected and susceptible animals to spread (10). The
two ruminant morbilliviruses, PPRV and RPV, have common
antigens demonstrable in a variety of serologic test systems,
and they also show a degree of cross-neutralization (12,13).
Originally PPRV was considered a variant of RPV adapted to
small ruminants; however, the two viruses have separate epi-
zootiologic cycles in nature, and each exists in its own right
(14,15).
PPRV infection has only recently been officially reported
in Turkey, in September 1999 (16,17), but some reports indi-
cate it was present before then (18,19). The objectives of our
research were to determine the seroprevalence of PPRV infec-
tion in cattle, sheep, and goats; determine the regional distribu-
tion of PPRV in Turkey; isolate and characterize the Turkish
virus; and compare its genome sequence with those of other
PPRV sequences in the sequence database maintained at the
World Reference Laboratory, Pirbright, United Kingdom.
Materials and Methods
Animals Used in the Study
Domestic ruminant species (cattle, sheep, and goats) from
throughout Turkey were examined for virus-specific antibod-
ies. The sampling procedure depended on the presence of sus-
pected infection and focused on two groups of animals. The
first included 193 sheep that local authorities reported as hav-
ing clinical signs of PPRV infection. These animals were
examined, blood samples were collected, and any animals with
signs of disease were sampled by swabbing for virus isolation.
Cattle grazing with sheep or goats were also sampled to moni-
tor for antibodies to the two viruses. The second group con-
sisted of 1,414 animals randomly selected for serologic
screening for PPRV and RPV antibodies from herds near the
flocks of sheep and goats in which PPRV-like infection was
reported. The numbers of serum samples collected from sheep,
goats, and cattle were 884, 209, and 321, respectively.
Tests for PPRV- and RPV-Specific Antibodies
Competitive enzyme-linked immunosorbent assays were
performed as described in the manual of Peste des Petits
Ruminants enzyme-linked immunosorbent assay (ELISA) kit
(20) and the Office International des Epizooties Manual of
Standards (9). Each serum sample, regardless of the species
from which it was obtained, was tested for the presence of
antibodies to RPV and PPRV.
Virus Isolation Material and Infection of Cell Cultures
A total of 328 field samples, including heparinized blood,
organ (lung), and swab specimens, were cultured to obtain
virus isolates. The processed samples were spread onto Vero
cells seeded in rolling culture tubes. The cells were grown in
Dulbecco’s modified Eagle medium enriched with 5% fetal
*Ankara University, Ankara, Turkey; and †Institute for Animal Health,
Pirbright Laboratory, Surrey, United Kingdom
P

Emerging Infectious Diseases • Vol. 8, No. 7, July 2002 709
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bovine serum as a regular culture medium. The cell culture
media were changed every 2 days and the inoculated cells
observed for 12–14 days. The positive culture tubes were fro-
zen at -80°C when the cytopathic effect (CPE) was 90%, and
virus stocks were prepared from the positive samples.
Detection of PPRV RNA
Detection of PPRV RNA by reverse transcription-poly-
merase chain reaction (RT-PCR) was performed as described
(21). PCR amplification was carried out by with a PPRV-spe-
cific primer set (PPRVF1b: 5´AGTACAAAAGATTGCTGA
TCACAGT and PPRVF2d: 5´GGGTCTCGAAGGCTAGGCC
CGAATA) selected from the F protein gene sequence, which is
expected to amplify a 448-bp DNA product. RT-PCR products
were digested by using EcoRI at 37°C for 1 hour. Samples
were then analyzed on 1.7% agarose gels to determine the
cleavage patterns of the amplicons. DNA products obtained
with PPR F1b and F2d primers were sequenced by using a T7
polymerase-based commercial kit (Pharmacia Diagnostics AB,
Uppsala, Sweden) with
35
SdATP as the radiolabel.
Phylogenetic Analysis
Sequence data were analyzed with the GCG (Genetics
Computer Group Inc., Madison, WI) package. The nucleic
acid sequences obtained from PCR products were aligned with
known sequences from representatives of the Morbillivirus
genus, and the phylogenetic tree was generated with the DNA-
DIST and FITCH programs of the PHYLIP 3.73 software (22).
Results
Clinical Findings
Animals with clinical signs of PPRV were detected in 11
provinces (Table 1). In many cases, inspections of flocks con-
firmed PPRV-suspect cases reported by local veterinarians or
identified symptoms indicative of PPRV infection. Most clini-
cal cases were characterized by excessive oculonasal dis-
charge, mild ulcerative stomatitis, dyspnea, and coughing.
Severe mucosal eruptions and intestinal signs were not
detected.
Serologic Status of Sampled Animals
A total of 1,607 animals from 18 farms were sampled for
antibodies to PPRV and RPV (Figure 1). Only two farms
(Cihanbeyli and Amasya) had no animals with antibodies spe-
cific to either virus. The overall percentages of antibody
response to PPRV and RPV were 22.4% and 6.28%, respec-
tively (Table 2). Prevalences of PPRV infection varied
between flocks, ranging from 0.87% to 82.60%; however,
these figures may not be accurate because of the small sample
sizes. In general, the level of PPRV infection was higher in
sheep; however, the highest seroprevalence (82.6%) was found
in goats in Sakarya Province, where two PPRV isolates were
identified from sheep during this project. Of 1,077 sheep
examined, 315 (29.2%) were seropositive for PPRV and 1.2%
for RPV. The 10 RPV-seropositive sheep in Bursa Province (in
a flock with no clinical PPRV) were reported to have been vac-
cinated against RPV, while only 1 sheep in Konya was found
to have seroconverted, probably following natural infection
with RPV before 1999. The overall occurrence of PPRV infec-
tion in cattle was 15.57% (a total of 50 animals), and approxi-
mately 27% of cattle were antibody positive for RPV,
indicating previous exposure to the virus either by natural
infection or, most probably, by vaccination, since all cattle in
the study were >6 months of age.
The study showed no substantial relationship between the
occurrence of PPRV infection and geographic location.
Although the main portal of entry of the disease is thought to
be in the southeastern part of Anatolia, distribution of the
prevalence values did not show a clear pattern across the coun-
try, and the disease was detected in varying percentages in
almost every region studied (Figure 1).
Table 1. Peste des petits ruminants virus (PPRV)–specific antibody
prevalence in animals with clinical symptoms indicative of PPRV,
Turkey
Location Animal
Animals with PPRV-suspected symptoms
No. PPRV positive %
Batman Sheep 8 7 87.5
Denizli Goat 10 6 60.0
Cihanbeyli Sheep 8
Amasya Sheep 20
Sakarya Sheep 19 3 15.8
Eskisehir Sheep 5 4 80.0
Malatya Sheep 3 2 66.6
Sivas Sheep 23 6 26.0
Isparta Sheep 32 32 100.0
Aydin Sheep 42 10 23.8
Van Sheep 43 40 93.0
Total 213 110 51.6
Figure 1. Areas of Turkey sampled to detect the presence of infection
with Peste des petits ruminants virus and Rinderpest virus. Numbers in
parentheses indicate the number of serologic test materials collected
from each location. Rectangles indicate a single outbreak; shaded prov-
inces had multiple outbreaks. Key: 1, Aydin (100); 2, Denizli (164); 3,
Balikesir (40); 4, Bursa (40); 5, Kocaeli (100); 6, Sakarya (100); 7,
Eskisehir (5); 8, Bolu (160); 9, Isparta (100); 10, Ankara (20); 11, Cihan-
beyli (75); 12, Konya (50); 13, Amasya (20); 14, Sivas (109); 15,
Malatya (3); 16, Elazig (272); 17, Batman (50); 16, Van (199).

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710 Emerging Infectious Diseases • Vol. 8, No. 7, July 2002
Virus Isolation
A total of 328 samples were spread onto Vero (African
Green Monkey Kidney) cells. Two nasal swab samples
(Sakarya 1 and Sakarya 2), from sheep in Sakarya Province,
showed CPE on Vero cells. The CPE was observed on day 3
after inoculation and was initially characterized by the forma-
tion of rounded cells; later, syncytia developed. RT-PCR was
performed on cell culture supernatants after the first passage
on the Vero cells. The expected amplification product of 448
bp was observed by using RNA prepared from culture super-
natants from only the two samples (data not shown). Restric-
tion fragment length polymorphism analysis of the RT-PCR
products indicated nucleotide substitutions in the EcoRI recog-
nition sequence site in the amplified genome region of the iso-
lates. While the PPR vaccine strain (Nigeria 75/1) produced,
as expected, two fragments of 202 bp and 246 bp on cleavage
with EcoRI, isolates Sakarya 1 and Sakarya 2 were not
digested by this restriction enzyme (data not shown). Partial
sequencing of the F protein–coding region of the two PPRV
isolates showed them to be identical (GenBank accession
number AF384687). The Turkey 2000 sequence was then
aligned with the sequences of other PPRV isolates from
around the world. Figure 2 shows the inferred phylogenetic
relationship between the isolates recovered in this research and
other PPRVs. The Turkish isolates belonged to PPRV lineage 4
(7), which originates in the Middle East, Arabia, and southern
Asia.
Discussion
We investigated the prevalence, host range, and distribu-
tion of PPRV in small private farms in Turkey. We also dem-
onstrated the presence of the disease by observing animals in
the field and by isolating virus from clinical specimens. This
wide-ranging survey is the first to be carried out on this dis-
ease in Turkey. PPRV infection has only recently been offi-
cially declared to be present in Turkey in the Elazig Province
in eastern Anatolia (16,17). Our research provided valuable
data on the serologic status of the three domestic ruminant spe-
cies (cattle, sheep, and goats) with respect to PPRV. Infection
with PPRV was demonstrated in 16 of 18 farms we sampled
(except for Cihanbeyli and Amasya). On a flock basis, the
highest virus prevalence (82.6%) was in goats in Sakarya,
where two isolates were identified from sheep. The second
highest prevalence (80%) was in sheep in Eskisehir, followed
by 72% in sheep in Van Province and 66.6% in sheep in
Malatya Province. Van and Malatya Provinces are in south-
eastern Turkey near the Iranian border; the remaining prov-
inces are mainly in central Anatolia (Figure 1). Variation in
prevalence is probably related to the intensity of trade of ille-
gally imported small ruminants (23).
The prevalence of the disease was as high as 28.5% in
sheep and goats reared in small private flocks, and the disease
was found in almost every region across Turkey. Occurrence
of infection did not vary substantially by geographic locations
of the livestock tested. Although the presence of PPRV infec-
tion in Turkey has been reported before (18,19), the impact of
the disease on production of livestock animals has not previ-
ously been investigated. The overall prevalence of PPRV was
22.4% of the ruminant population. These results indicate much
lower prevalence than the 88.3% reported by Tatar (19). How-
ever, if the overall percentage of PPRV infection takes into
account animals reported as having clinical signs, the level
increases to 51.6% (Table 1). Another ruminant morbillivirus
infection, RPV in cattle, caused great economic losses from
the deaths or slaughter of affected (or suspected infected) ani-
mals in Turkey in recent years (24). Because PPRV and RPV
are antigenically related, the attenuated RPV vaccine has been
used to protect small ruminants against PPRV. According to
anecdotal reports from the field, veterinarians and animal
Table 2. Analysis of antibody response against Rinderpest virus (RPV)
and Peste des petits ruminants virus (PPRV), by species, Turkey
Species Yr of sampling No. of sera
No. (%) of animals with
antibodies to
PPR RPV
Sheep 1999–2000 1,077 315 (29.20) 13 (1.20)
Goats 1999 209 42 (20.00) 1 (0.47)
Cattle 1999–2000 321 3 (0.90) 87 (27.10)
Total 1,607 360 (22.40) 101 (6.28)
Figure 2. Phylogenetic relationship of the Peste des petits ruminants
viruses isolated in Turkey in 2000 to other virus isolates. The tree is
based on partial sequence data from the fusion (F) protein gene (7) and
was derived by using the PHYLIP DNADIST and FITCH programs (22).
Branch lengths are proportional to the genetic distances between
viruses and the hypothetical common ancestor at the nodes in the tree.
The bar represents nucleotide substitutions per position.

Emerging Infectious Diseases • Vol. 8, No. 7, July 2002 711
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owners widely used the RPV vaccine to protect small rumi-
nants against PPRV infection in some parts of Turkey before
RPV vaccination was stopped in the year 2000. This might be
one reason for the lower percentage of PPRV-positive animals
found in this study.
Cattle act as dead-end hosts for PPRV and show no clinical
signs of infection. Nevertheless, they develop a humoral
immune response to PPRV that protects them against natural
or experimental challenge with virulent RPV (12). In our
study, the percentage of natural PPRV infection in cattle was
low (0.9%), and all these were in cattle that had contact with
infected sheep flocks. Cattle seropositive for PPRV could
cause confusion in monitoring for antibodies to RPV after a
vaccination campaign to eradicate RPV. Natural infection of
cattle with PPRV might prevent the immune response to the
RPV vaccine because the PPRV-specific antibodies could neu-
tralize the live attenuated vaccine virus. The cattle would still
be protected from subsequent RPV challenge by heterologous
PPRV antibody but would register as seronegative when tested
in the RPV competitive ELISA. This false value could lead to
a low estimation of herd immunity to RPV or suggest that the
vaccination coverage was inadequate (12). This risk is high in
mixed breeding systems, such as the small-scale production
units common in rural regions of Turkey. On the other hand,
14.6% of the cattle had antibodies to both viruses (PPRV and
RPV). This finding may indicate that in some cases no inter-
ference occurs. Another possible reason is the cross-reactivity
of the PPRV assay for antibodies to RPV, as noted by Ander-
son et al. (25).
Only two PPRVs were isolated—from nasal swab samples
of two sheep from Sakarya Province. The reason for the poor
success in isolating virus could be the nature of the samples. In
previous studies, virus isolations were made from spleen,
mesenteric lymph nodes (7,26), or intestinal epithelial smears
(15) collected during necropsy of affected animals by inocula-
tion onto Fetal Lamb Kidney (FLK) (19,26) or Vero (15,19)
cells. In our study, however, most samples were taken from
surviving animals that may have been past the clinical phase of
the disease, when the virus is secreted, and so were not likely
to yield virus isolates from swabs. Moreover, owners of many
sick animals did not grant permission to euthanize them, so
internal organs were not available for gross pathologic analy-
sis in most cases. According to a previous study (19), FLK and
Vero cells are equally susceptible to PPRV; thus, the use of
Vero cells was probably not a factor in our poor success with
virus isolation.
Our use of molecular epidemiologic techniques provided
data that suggest cross-border transmission into Turkey of
PPRV infection that is actively circulating in neighboring
countries. The viruses we isolated are PPRV lineage 4, which
includes viruses whose origins are in the Middle East, Arabia,
and south Asia (7). Because of its geographic location, Turkey
has borders with countries where many economically impor-
tant infectious diseases are endemic. Thus, one of the neigh-
boring countries in the Middle East region is most likely the
source of infection. Since the terrain of eastern and southeast-
ern Anatolia permits uncontrolled animal movement, restrict-
ing the spread of infectious diseases into the country has been
difficult. Therefore, the importance of PPRV as a threat to live-
stock should be considered, together with other economically
important diseases, and measures taken to prevent the import
and subsequent spread of such diseases.
Acknowledgments
Competitive enzyme-linked immunosorbent assay test kits were
provided by the Institute for Animal Health, Pirbright Laboratory.
This research was supported by grants of the British Council
(Link Project ANK/992/102) and Research Fund of Ankara Univer-
sity (98.10.00.08).
Dr. Özkul is a faculty member in the Virology Department of the
Veterinary School at Ankara University. His main research interests
are the molecular epidemiology of morbilliviruses and the neuropa-
thology of herpes and morbillivirus infections.
References
1. Taylor WP. The distribution and epidemiology of peste des petits rumi-
nants. Prev Vet Med 1984;2:157–66.
2. Awa DN, Njoya A, Ngo Tama AC. Economics of prophylaxis against
peste des petits ruminants and gastrointestinal helminthosis in small rumi-
nants in north Cameroon. Trop Anim Health Prod 2000;32:391–403.
3. Roeder PL, Abraham G, Kenfe G, Barrett T. Peste des petits ruminants in
Ethiopian goats. Trop Anim Health Prod 1994;26:69–73.
4. Lefevre PC, Daillo A, Schenkel S, Hussein S, Staak G. Serological evi-
dence of peste des petits ruminants in Jordan. Vet Rec 1991;128:110.
5. Abu-Elzein EME, Hassanien MM, Al-Afaleq AI, Abdelhadi MA, Hon-
sawai FMJ. Isolation of peste des petits ruminants from goats in Saudi
Arabia. Vet Rec 1990;27:309.
6. Shaila MS, Purushothaman V, Bhavasar D, Venugopal K, Venkatesan RA.
Peste des petits ruminants of sheep in India. Vet Rec 1989;125:602.
7. Shaila MS, Shamaki D, Forsyth M, Diallo A, Goatley L, Kitching P, et al.
Geographic distribution and epidemiology of peste des petits ruminants
viruses. Virus Res 1996;43:149–53.
8. Barrett T. Morbilliviruses: dangers old and new. In: Smith GL, McCauley
JW, Rowlands DJ. New challenges to health: the threat of virus infection.
Society for General Microbiology, Symposium 60. Cambridge: Cam-
bridge University Press; 2001. p. 155–78.
9. Office International des Epizooties (OIE). OIE manual of standards for
diagnostic tests and vaccines. List A and B diseases of mammals, birds
and bees. Paris: The Office; 2000.
10. Lefevre PJ, Diallo A. Peste des petits ruminants. Rev Sci Tech (OIE)
1990;9:951–65.
11. Wosu LO. Current status of peste des petits ruminants (PPR) disease in
small ruminants—a review article. Stud Res Vet Med 1994;2:83–90.
12. Anderson J, McKay JA. The detection of antibodies against peste des petits
ruminants virus in cattle, sheep and goats and the possible implications for
rinderpest control programmes. Epidemiol Infect 1994;112:225–31.
13. Taylor WP. Protection of goats against peste des petits ruminants with
attenuated rinderpest virus. Res Vet Sci 1979;27:321–4.
14. Dardiri AH, De Boer CJ, Hamdy FM. Response of American goats and
cattle to peste des petit ruminants.Vet Lab Diagn 1976;337–44.
15. Taylor WP, Abegunde A. The isolation of peste des petits ruminants virus
from Nigerian sheep and goats. Res Vet Sci 1979; 26:94–6.
16. Emergency Prevention System (EMPRES) for Transboundary Plant and
Animal Pests and Diseases 2000; no. 13. Available from: URL: http://
www.fao.org/empres

RESEARCH
712 Emerging Infectious Diseases • Vol. 8, No. 7, July 2002
17. Office International des Epizooties (OIE). OIE disease information
1999;12:137.
18. Alcigir G, Vural SA, Toplu N. Türkiye'de kuzularda peste des petits rumi-
nants virus enfeksiyonunun patomorfolojik ve immunohistolojik ilk tan-
imi. Ankara Universitesi Veteriner Fakültesi Dergisi 1996;43:181–9.
19. Tatar N. Koyun ve keçilerde küçük ruminantlarin vebasi ve sigir vebasi
enfeksiyonlarinin serolojik ve virolojik olarak arastirilmasi [dissertation].
Ankara: Ankara Üniversitesi, Saglik Bilimleri Enstitusu; 1998.
20. Peste des Petits Ruminants ELISA kit, Competitive Enzyme Immunoas-
say for Detection of Antibody to PPR Virus. Bench protocol, version PPR
1.0, January 1993. Joint FAO/IEAE Programme, Animal Production and
Health. Pirbright, United Kingdom: World Reference Laboratory for
Rinderpest; 1993.
21. Forsyth M, Barrett T. Evaluation of polymerase chain reaction for the
detection of rinderpest and peste des petits ruminants viruses for epiemio-
logical studies. Virus Res 1995;39:151–63.
22. Felsenstein J. PHYLIP – Phylogeny inference package. Cladistics
1989;5:164–6.
23. Al-Naeem A, Abu-Elzein EME, Al-Afaleq AI. Epizootiological aspects
of peste des petits ruminants and rinderpest in sheep and goats in Saudi
Arabia. Rev Sci Tech (International Office of Epizooties) 2000;19:855–8.
24. Burgu I, Akca Y, Ozkul A. Rapid detection of rinderpest virus antigen
using pig-anti-CDV-PO conjugate in cell culture. In: Proceedings of
International Symposium on Morbillivirus infections. 12–13 June 1994,
Hannover Veterinary School, Germany. Hannover: European Society for
Veterinary Virology; 1995.
25. Anderson J, McKay JA, Butcher RN. The use of monoclonal antibodies
in competitive ELISA for the detection of antibodies to rinderpest and
peste des ruminants viruses. In: Jeggo MH, editor. The sero-monitoring of
rinderpest throughout Africa phase one. The proceedings of a final
research co-ordination meeting of the FAO/IAEA/SIDA/OAU/IBAR/
PARC Co-ordinated Research Programme. Ivory Coast. IAEA-Techdoc
1990;623:43–53.
26. Furley CW, Taylor WP, Obi TU. An outbreak of peste des petits rumi-
nants in a zoological collection. Vet Rec 1987;121:443–7.
Address for correspondence: Aykut Ozkul, Ankara University, IrfanBastug
Cd. Diskapi, 06110 Ankara, Turkey; fax: 90 312 3164472; e-mail:
Aykut.Ozkul@veterinary.ankara.edu.tr
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