INTRODUCTIONPeste 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 Côte d’Ivoire 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 Côte d’Ivoire (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.