INTRODUCTION

Peste des petits ruminant (PPR), which literally means Plague of small ruminants is an economically
significant disease of sheep and goats. The disease is caused by a morbillivirus of
the family Paramyxoviridae. Following the first report of the disease in sheep and goats
(Gargadennec and Lalanne, 1942), for many years it was believed to have remained restricted to
western part of the African continent. However, in recent years, the disease has been recorded in
several parts of the world, Southern Asia including India, Pakistan and Nepal; Near East and
the Arabian Peninsula including Islamic Republic of Iran, Iraq, Israel, Jordan, Kuwait, Lebanon,
Oman, Saudi Arabia, the United Arab Emirates, and Yemen, and there is serological evidence
from the Syrian Arab Republic.
PPR is a highly contagious disease, morbidity and mortality can be as high as 100 and 90
per cent respectively (Abu-Elzein et al., 1990). The disease naturally affects mainly sheep and
goats, but it is usually more severe in goats than sheep. A case of clinical disease has been
reported in wildlife resulting in deaths of gazelle, ibex, gemsback and larsian sheep (Abu-Elzein
et al., 1990; Furley et al., 1987). A report of PPR in buffaloe has also been described in India by
Govindarajan et al. (1997). In India, the first outbreak of PPR was described in Tamilnadu by
Shaila et al. (1989). Following major outbreaks in northern India in 1994, the disease became
endemic in the country (Nanda et al., 1996; Mondal et al., 1995).
Peste des petits ruminants virus (PPRV) is grouped under genus Morbillivirus, which
also includes Measles virus (MV), Rinderpest virus (RPV) and Canine distemper virus (CDV) as
well as phocine, porpoise and dolphin distemper viruses that infect marine mammals (Barret,
2001). PPRV has negative sense single strand RNA genome.
The disease is clinically characterized by stomatitis-pneumo-enteritis complex. Following
the infection, there is a three-day incubation period during which, the virus replicates in the
draining lymphnodes of oro-pharynx before spreading via blood and lymph to other tissues and
organs including the lung, causing a primary viral pneumonia in the infected animal. This
pneumonia is exacerbated by secondary bacterial infection. Other overt clinical signs include
ocular and nasal discharges, which usually become mucopurulent, as well as conjunctivitis,
necrotic stomatitis, and severe diarrhea (Gibs, 1979).
Because PPR is clinically indistinguishable from Rinderpest, a laboratory confirmation of
PPR is of utmost importance, especially in regions where both the viruses are prevalent.
Conventional serological tests like Agar gel immuno-diffusion (AGID) and
Counterimmunoelectrophoresis (CIEP) can be used for the diagnosis of PPR, but these techniques
are not sensitive enough so as to be used as reliable diagnostic tools. Specific diagnosis of PPR
can be made by virus neutralization test (VNT), c-ELISA and immunocapture ELISA (Libeau et
al., 1994). More sensitive tests such as polymerase chain reaction (PCR) has been developed and
widely used for detection of PPRV. Forsyth and Barret (1995) developed a PCR using a set of
primers amplifying a conserved region of fusion protein (F) gene of the PPRV. The technique has
become popular for specific diagnosis as well as for molecular epidemiological studies based on
the nucleotide sequence of the amplification product. PPRV has been placed into four groups or
lineages based on nucleic acid sequence of a segment of fusion protein gene (Shaila et al., 1996).
There are four distinct lineages of the PPRV circulating in the world. Lineage 4 is most common
in India, excepting a solitary isolate (India/TN/92) of lineage 3 from Tamilnadu (Shaila et al.,
1996).
A couple of recent studies (Tiwari, 2004; George, 2002) revealed that, the widely used F
gene based PCR technique (Forsyth and Barret, 1995) failed to detect PPRV from few of the
samples that had been tested positive by sandwich ELISA. There fore, a need was felt to assess
the suitability of other PPRV gene targets for the detection of PPRV in field samples. Moreover,
literature appeared scanty with regards to the sequence information on local isolates of PPRV,
which can enable in identifying possible genetic variation and the resulting changed behaviour
pattern of the virus in the field.
Considering the aforementioned points, the present study was planned with following objectives.
i. To study the incidence of PPRV in sheep and goats of Gujarat
ii. To study comparative efficacy of sandwich ELISA and the conventional F gene based
RT-PCR in detecting PPRV
iii. Comparative evaluation of F and N gene based RT-PCR in detection of PPRV from
clinical samples. Additionally, development of a new N gene based PCR primer set for
sensitive and specific diagnosis of PPRV in clinical samples.
iv. To study possible genetic variation among the field PPR viruses and their phylogenetic
comparison with the other reported isolates.
Towards achieving the said objectives, following parameters were undertaken.
a) Detection of PPRV in clinical samples by sandwich ELISA and and deriving
estimates of overall, locationwise and specieswise incidence and samplewise
positivity rates.
b) Detection of PPRV in clinical samples by F gene based PCR using F1/F2 primers and its
comparative efficacy with sandwich ELISA.
c) Development and application of a PCR diagnostic by designing a new N gene based
primer pair for specific detection of PPRV in field samples.
d) Standardization and applying the N gene based RT-PCR technique using primer pairs
NP3/NP4, pprn_fr2/ pprn_rev and N1/N2, and comparing their sensitivity and specificity
with F-gene based RT-PCR.
e) Cloning of F and N gene PCR products into pTZ57R/T vector supplied with the
InsT/Aclone PCR product cloning kit and sequencing of the targeted inserts of the
respective genes for determining possible genetic variation among the field viruses.
f) Assessing the genetic distance among the field viruses and the vaccine virus under the
study as well as with the isolates from different geographical locations by in silico
analysis using Clustal W (1.82), PHYLIP 3.65 and Network 4.111 softwares.

Peste des petits ruminants (PPR) is an acute viral disease of sheep and goats
characterized by fever, catarrhal inflammation of ocular and nasal mucous membrane,
erosive stomatitis, gastroenteritis and pneumonia. The causal agent is PPR virus
(PPRV) an envelope, pleomorphic particle containing single-stranded RNA,
approximately 16kb long with negative polarity genome (Barrett et al., 2005). The
genome of the virus codes for six structural (N, P, M, F, H and L) and two
nonstructural (C and V) proteins in the order of 3-N-P(C/V) - M-F-H-L-5 (Bailey et al.,
2005, Mahapatra et al., 2006). The disease is prevalent in most African and Middle
Eastern countries and the Indian subcontinent (Taylor, 1984). Morbidity and mortality
of the disease can be as high as 100% and 90%, respectively (Abu-Elzein et al., 1990,
Dhar et al., 2002). In Bangladesh, outbreaks of PPR were first reported in 1993 (Sil et
al., 1995; Islam et al., 2001). Since then the disease has caused severe losses and is
presently considered one of the major threats to about 22 million small ruminants in
Bangladesh where mortality may reach 100%. At present, more than 1 billion sheep
and goats in Africa and Asia are at risk of PPR (EMPRES, 2009). Rapid diagnosis is
essential for successful control. Reverse transcriptase polymerase chain reaction (RTPCR),
a molecular diagnostic test based on amplification of the gene target offers a
new strategy for diagnosis of PPRV, and is more sensitive than other tests. RT-PCR
also offers the possibility of analysing the relationship betweendifferent PPRV strains for epidemiological
studies (Shaila et al., 1996; Ozkul et al.,
2002; Kwiatek et al., 2007; Wang et al., 2009; Balamurgan et al., 2010). Although RTPCR
overcomes the limitations of conventional tests, the sensitivity varies depending
on the primer used and the gene targeted. This may be due to high rate of nucleotide
substitution error in RNA viruses (Steinhauer and Holland, 1986). The present study
was undertaken to find an efficient primer set and gene, which would be specific and
sensitive for detecting PPRV in field samples.
Peste des petits ruminants (PPR) is an acute, highly
contagious, notifiable and economically important
transboundary viral disease of goats and sheep, which is
listed by the World Organisation for Animal Health (OIE).
The mortality usually ranges from 50% to 90%, although it
sometimes can be zero, and morbidity varies from 10% to
100%, or sometimes lower than 10%, depending on
circumstances (2). The disease is considered to be one of the main constraints to improving the productivity of small
ruminants in enzootic countries (14). The causative agent,
PPR virus (PPRV), belongs to the Morbillivirus genus of the
Paramyxoviridae family. It affects sheep and goats primarily,
and occasionally infects wildlife. The disease is
characterised clinically by severe pyrexia, oculonasal
discharge, necrotising and erosive stomatitis, enteritis and
pneumonia (10, 18, 31). Although PPRV has a single
serotype, it is grouped genetically into four lineages (I, II,
III and IV) on the basis of partial sequence analysis of the
fusion (F) gene. Lineages I to III circulate in Africa and
lineage IV in Asia (6, 22). Peste des petits ruminants was
first reported in Cte dIvoire in West Africa (10), and later
in other parts of the world, namely sub-Saharan Africa, the
Arabian Peninsula, the Middle East and the Indian
subcontinent (22). In recent years, the disease has also
been reported from China (34) and Morocco (35), which
raises the threat of its introduction into Europe.
In India, PPR was first recorded in 1987 from Arasur
village, in the Villupuram district of Tamil Nadu (21), and
it continued to be present in the southern peninsula until
1994. Later, a number of PPR outbreaks were reported
from the northern states of India (13, 16), with a solitary
report in Indian buffalo in a southern state (11). Peste des
petits ruminants is enzootic in India, and outbreaks occur
in small ruminants, such as sheep and goats, regularly
throughout the country (13, 24). It is a major constraint on
small ruminant production (24), causing great economic
losses because of morbidity, mortality, and losses of
productivity due to trade restrictions. Economic losses due
to PPR have been estimated to be 1,800 million INR
(US$39 million) annually (23, 33).
Small ruminants are important livestock species, both
numerically and economically, in developing countries
such as India. The world population of sheep and goats is
approximately 2.1 billion, of which India has about
78 million sheep and 140 million goats (5). In India, small
ruminants make important contributions to the lives of
small, marginal and landless rural farmers and allow them
to sustain their livelihood by providing meat, fibre, milk,
skin and manure (20). The husbandry of small ruminants
also generates self-employment, raises income, improves
household nutrition and plays an important role in
sustainable agriculture and generation of employment (3,
12). The proportion of sheep to goats and the population
density vary greatly under different agro-climatic
conditions.
Information on the prevalence of antibodies to PPRV in
small ruminants and other species is available from a
number of countries in which the disease is reported,
including the Sultanate of Oman, Jordan, Sudan, Turkey
and various African countries (1, 8, 17, 30). Only a few
reports have described systematic study of the pattern of
PPRV infection and its seroprevalence in small ruminants
in India (19, 24). The prevalence of PPRV antibodies in
sheep and goats indicates subclinical or inapparent or nonlethal
clinical infection, which may be of epidemiological
significance. Efficient and sensitive diagnostic tests are a
great help in rapidly providing evidence that PPRV is not
circulating in a free-ranging population. Data on the
molecular epidemiology and sero-epidemiology of the
disease play an important role in effective disease
management. A monoclonal antibody (MAb)-based
competitive enzyme-linked immunosorbent assay
(c-ELISA) and a sandwich ELISA, for detection of PPRV
antibody and antigen respectively, were developed at the
Indian Veterinary Research Institute (IVRI), Mukteswar
(25, 26). These are the tests currently employed for
serosurveillance and seromonitoring of the clinical
prevalence of PPR throughout India.
In view of the economic importance of the disease and the
dense sheep and goat population in the region, the authors
undertook the present study with the objectives of
generating baseline data on the prevalence of PPR in India
between 2003 and 2009, and of investigating the seroepidemiology
of the disease using serum samples from
sheep and goats suspected of the disease.
PPR was first described in Cte dIvoire
(Gargadennec L. & Lalanne A., 1942) and then after, it
has been recognized in many of the sub-saharian
countries that lie between the Atlantic Ocean and the
Red Sea (Lefevre and Diallo, 1990). The affected area
extends north to Egypt and south to Kenya, in Eastern-
Africa, and to Gabon, in Western-Africa. PPR has not
been recognized in most of Northern and Southern-
Africa. In 1998, serological survey in the United
Republic of Tanzania did not detect any antibodies to
PPR suggesting that infection has not extended that
far to the south. PPR is present in nearly all Middle
Eastern countries up to Turkey (Furley et al.. 1987;
Lefevre et al. 1991; Perl et al. 1994; Taylor et al. 1990,
Ozkul et al. 2002). It is also widespread in India and
southwest Asia (Shaila et al. 1989).
Presently, PPR occurs in most African countries
situated in a wide belt between the Sahara and equator,
the middle east ( Arabian peninsula, Israel, Syria and
Jordon) and the Indian subcontinent. Outbreaks of PPR
are now known to be common in India, Nepal,
Bangladesh, Pakistan and Afghanistan ( Abdollahpour
et al, 2006). It still causes serious economic losses(Diallo, 2003) and remains a major constraint on the
development of small ruminant farms in these countries.
PPR is considered to be one of the main constraints to
improve productivity of small ruminants in the regions
where it is endemic.
Thus its control is a major goal for programmes
aim at poverty alleviation. Major outbreaks in Turkey
and India in recent years have indicated a marked rise
in the global incidence of PPRV (Bailey et al, 2005). It
is of great economic importance on the basis of
mortalities, morbidity, losses through body wastage,
poor food efficiency, loss of meat, milk and milk
products and offspring (Nawathe, 1984). A
consequence of this high mortality was the inclusion
of PPR in the list A of the former animal disease
classification of the OIE , the world organization for
the Animal health. In the new OIE classification it is
included in a group of economically important animal
diseases , which must be notified to the Organization
in all the regions where PPR is endemic.
There are four groups of phylogeny of which, 3
are located in Africa. The fourth group is the only one
present into the Indian sub-continent but it also coexists
in the Middle East with the East African groupIII . It was deduced that outbreaks in North and South
India were caused by PPR viruses of different lineages
and was suggested that either two viruses were
independently introduced in India in the past decade
or the South Indian virus has been enzootic infection
in that region for many decades and was confused with
RP virus, as before 1988 all Morbilli viruses infections
were considered to be rinder pest. (Singh, 2002). The
presence of the two African lineages in Asia beside a
distinct Asian lineage may be taken as indication of
the trade route of spread of the disease.
In India, PPR was first reported in 1987 from Arasur
village in the Villapurum district of Tamilnadu, South
India (Shaila et al, 1987). Since, its first reported
occurrence in 1987, PPR was thought to be restricted
to southern India up to 1993, after which the epidemics
of PPR swept away large number of small ruminants
from Northern India (Nanda et al, 1996). Since, then
the disease has been reported regularly from different
parts of the country and is considered as an endemic
disease causing a great loss to small ruminants of the
country. Now the disease has spread all over India. In
Gujarat, incidence of PPR was reported by various
workers, namely , Hinsu et al, (2001), Tiwari (2005),
Nagraj (2006), Sannat (2006) and Bulbule (2007).
It is still not clear whether the apparent
geographical spread of the disease in the last 50 years
is real or reflects increased awareness, wider
availability of diagnostic tools or even a change in the
virulence of the virus. It seems most likely that a
combination of factors is responsible for the present
knowledge of the disease distribution. It is also known
that confusion of PPR with pneumonic pasteurellosis
and other pneumonic diseases of small ruminants has
precluded and delayed its recognition in some
countries.