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Small Ruminant Research 96 (2011) 1–10

Contents lists available at ScienceDirect

Small Ruminant Research

journa l homepage: www.e lsev ier .com/ locate /smal l rumres

eview

este des petits ruminants (PPR): Disease appraisal with globalnd Pakistan perspective

uhammad Abubakara,∗, Haider Ali Khanb, Muhammad Javed Arsheda,anzoor Hussainc, Qurban Ali a

National Veterinary Laboratory, Park Road, Islamabad, PakistanDirectorate of Animal Health, Department of Livestock and Dairy Development, 16 Cooper Road, Lahore, PakistanFaculty of Veterinary and Animal Sciences, PMAR-Arid Agriculture University, Rawalpindi, Pakistan

r t i c l e i n f o

rticle history:eceived 9 June 2010eceived in revised form9 September 2010ccepted 7 October 2010vailable online 1 November 2010

eywords:PRackground

nformationakistan and global perspective

a b s t r a c t

Peste des petits ruminants (PPR), is a highly contagious viral disease affecting domesticand wild small ruminants. It is in the list of animal diseases to be notified to the WorldOrganization for Animal Health (Office International des Epizooties, OIE). Because of thehigh magnitude of importance of sheep and goats for the poor and landless farmers, thecontrol of disease, which has a negative impact on their productions, is aimed at povertyalleviation. A single injection of live attenuated monovalent vaccine (Nig/75/1) can induceprotective immunity for at least the economic life of the animals, however vaccine efficacyonly holds if the vaccination is done before exposure and when the animals at sub-clinicallevel of infection. In Pakistan, though published reports on existence of PPR in the coun-try are few but findings on clinical signs and course of the disease are consistent with theinternationally published reports on PPR elsewhere and the neighboring countries. The lim-

ited reports of incidence of PPR from few places/provinces do not exclude the possibilityof presence of infection in other parts/province of the country. Therefore, the epidemiol-ogy, pathogenicity, host susceptibility/resistance and molecular nature of PPR virus havebecome multifaceted than considered formerly. It is anticipated that patent PPR vaccinesand sophisticated diagnostic tests that differentiate infected and vaccinated animals might

ostic an

improve the diagn

. Global distribution

Peste des petits ruminants (PPR) was first noticedn Ivory cost in West Africa during 2nd World WarGargadennec and Lalanne, 1942) and it was nameds pseudo-rinderpest, pneumoenteritis complex andtomatitis-pneumoenteritis syndrome (Braide, 1981).

ater on, the disease was recorded in Nigeria, Senegalnd Ghana where it was called as Kata (Whitney et al.,967). Afterwards, a disease of goats in Sudan, which wasriginally diagnosed as rinderpest in 1972, confirmed to

∗ Corresponding author. Tel.: +92 519255104.E-mail address: hayee42@yahoo.com (M. Abubakar).

921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.smallrumres.2010.10.006

d epidemiological capabilities.© 2010 Elsevier B.V. All rights reserved.

be PPR (Diallo et al., 1988). It was thought that many of thecases mis-diagnosed as rinderpest among small ruminantsmay, instead, have involved the PPR virus, together withthe emergence of the disease in other parts of Westernand South Asia (Shaila et al., 1996), indicating its growingimportance. It has received a rising attention because of itswidespread, economic impacts (Lefevre and Diallo, 1990)and the role it plays in complication of the ongoing globaleradication of rinderpest (Couacy-Hymann et al., 2002). InIndia, PPR was first reported in 1987 (Dhar et al., 2002).

2. Causative agent

The PPR virus (PPRV) was assumed for a long time tobe a variant of Rinderpest (RP) adopted in small rumi-

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nants causes PPR. However, easy and rapid differentiationbetween RP and PPR has been made credible by the devel-opment of specific and sensitive molecular and serologicaltechniques that can differentiate between these virusesgenetically, antigenically and serologically with differ-ent physicochemical traits. It was also revealed to be animmunologically diverse virus with a separate epizootol-ogy in areas where both viruses were enzootic (Taylor,1979). The development of monoclonal antibodies basedELISA, specific nucleic acid probes for hybridization studiesand nucleic acid sequencing have confirmed that PPR viruswas fairly distinct entity from rinderpest virus (Diallo et al.,1989).

PPRV is in the morbilliviruses genus of Paramyxoviri-dae family (Gibbs et al., 1979) and this genus also includessix other viruses, viz., measles virus (MV), rinderpestvirus (RPV), canine distemper virus (CDV), phocid mor-billiviruses (PMV), porpoise distemper virus (PDV) anddolphin morbilliviruses (DMV). All these viruses share thesame genome organization although their RNA lengths dif-fer slightly (Barrett et al., 1993). The electron microscopyrevealed that morbilliviruses display the typical structureof Paramyxoviridae: a pleomorphic particle with a lipidenvelope, which encloses a helical nucleocapsid (Gibbset al., 1979). The nucleocapsids have a typical herringboneappearance.

Morbilliviruses are linear, non-segmented, singlestranded, negative sense RNA viruses with genomes15–16 kb in length and 200 nm in diameter (Norrby andOxman, 1990). The major site of viral propagation islymphoid tissue, and acute diseases are usually accompa-nied by profound lymphopenia and immunosuppression,leading to secondary and opportunistic infections (Appeland Summers, 1995; Murphy and Parks, 1999). Full-lengthgenome sequences of RPV (Baron and Barrett, 1995), CDV(Barrett, 1987), and PPRV (Bailey et al., 2005) are available.The sequence data show striking similarities and it wasbelieved that the morbilliviruses have an identical genomeorganization (Barrett et al., 1991; Banyard et al., 2005).

3. Sequence analysis

The genome of morbilliviruses are organized into sixcontagious, non-overlapping, transcriptional units encod-ing structural protein, namely the nucleocapsid (N), thephosphoprotein (P), the matrix (M) protein, the fusion(F), the hemagglutinin (H) and the large (L) (Bailey et al.,2005; Barrett, 1999; Baron and Barrett, 1995; Diallo, 1990).In addition, all morbillivirus encode two nonstructural (Vand C) proteins. Between the two non-structural proteins(C and V), the C protein or its putative mRNA has beenidentified in cells infected by CDV (Hall et al., 1980) andRPV (Grubman et al., 1988). The V protein or its putativemRNA has been described in cells infected by MV (Cattaneoet al., 1989; Wardrop and Briedis, 1991) and PDV (Curranand Rima, 1992). The functions of C and V proteins stay

unknown but it was assumed that the C protein increasedmRNA transcription in vitro and is an interferon antagonist,whereas V has a putative regulatory role on transcrip-tion and replication and is also an inhibitor of interferonresponse. These non-structural protein, have been shown

t Research 96 (2011) 1–10

the critical roles in infection. The C and V proteins ofparamyxoviruses also act as interferon antagonists, mod-ifying the cellular immune response to infection (Gotohet al., 2001).

Muthuchelvan et al. (2006) determined the completenucleotide sequence of the nucleocapsid (N) protein ofthe peste des petits ruminants vaccine virus (PPRV Sun-gri/96) belonging to the Asian lineage. The gene was 1692nucleotides in length and encoded a polypeptide of 525amino acids. The phosphorylation prediction reveals eightconserved sites across morbilliviruses, whereas in the C-terminal portion of the protein the sites are not conserved.Phylogenetic analysis of different N proteins of morbil-liviruses revealed five well-defined clusters as observedpreviously.

Apparently, in the reported outbreaks mortality varied,strain variations are the likely explanation. Strains fromoutbreaks at Lahore were sent to World Reference Labo-ratory for Rinderpest, Pirbright, UK (Hussain et al., 1998),virus was designated as ‘Pakistan 94’. Studies publishedlater (Ozkul et al., 2002) established phylogenetic rela-tionship based on partial sequence of fusion (F) proteingene and categorized the Pakistan virus falling in Lineage4, which include viruses whose origin are from neighbor-ing countries of Pakistan (Iran, India) and others including,Saudi Arabia, Turkey, Nepal and Bangladesh. In a similarstudy, Dhar et al. (2002) has clustered ‘Pakistan 94’ moreclose to viruses from Saudi Arabia, Kuwait and Iran. There-fore the likely source of infection postulated by Athar et al.(1995) for Lahore and Faisalabad outbreaks could be thecountries sharing boarders with Pakistan.

4. Protein profile

Six structural and two non-structural proteins of mor-billivirus have been observed. Among all the structuralproteins N protein is the major viral protein of morbil-liviruses. The nucleocapsid formed by the viral genomicRNA, a single-stranded RNA of negative sense, wrappedby the nucleoprotein (N) to which two other associatedviral proteins: the phosphoprotein (P) and the RNA poly-merase (L for large protein) are reported. From the envelopepeplomers protrude formed by the two viral glycoproteinsare essential in the first steps of the host cell infection bythe virus, the hemagglutinin (H) and the fusion (F) pro-teins (Diallo, 1990). The properties and biological functionsof these proteins include three viral structural proteins(N, P and L), which include internal polypeptides associ-ated with the viral genome to form the nucleocapsid, otherthree (M, F, H) form the virus envelope (Norrby and Oxman,1990). The sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE) technique was used for thestructural proteins for measles virus (Cattaneo et al., 1989),RPV and PPRV (Diallo et al., 1987). The variability in elec-trophoretic mobility has been detected in the N (Campbellet al., 1980), P, M, and H proteins (Saito et al., 1992). The

N, M, F and L proteins appeared to be the most conservedproteins. The Matrix (M) protein is the most abundant ofthe six structural proteins (Rima, 1983). The mobility dif-ferences have been detected in the N and M proteins of RPV(Anderson et al., 1990; Diallo et al., 1987) and the N pro-

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ein of PPRV (Taylor et al., 1990). The polymerase associatedP) phosphoprotein is a minor component of virions in alltrains of morbilliviruses (Diallo et al., 1987). Due to thessociation of P protein with the nucleocapsid, it is thoughto be required for formation of an active transcription com-lex (Norrby and Oxman, 1990).

. Geographical distribution

PPR is the most important small ruminant disease, par-icularly in endemic areas located between the Sahara andhe Equator in Africa, the Middle East and the Indian subontinent (Lefevre and Diallo, 1990; Taylor et al., 1990;oeder et al., 1994; Amjad et al., 1996). Peste des petitsuminants virus (PPRV), originally endemic in West Africapread across East Africa, the Middle East and Southern Asias far as Bangladesh (Shaila et al., 1996) and Turkey (Ozkult al., 2002). PPR infection has been recognized in many ofhe African countries that lie between the Atlantic Oceannd the Red Sea. The affected area extends north to Egyptnd south to Kenya, in the east, and Gabon, in the west.arked rise in global incidence of PPR outbreaks during

ecent years (Nanda et al., 1996; Ozkul et al., 2002; Shailat al., 1996) indicates the trend of disease. The virus was iso-ated all over the world, in Nigeria (Taylor and Abegunde,979), Jorden (Lefevre et al., 1991), Sudan (El Hag andaylor, 1988), Saudi Arabia (Abu Elzein et al., 1990), IndiaShaila et al., 1989; Nanda et al., 1996), Turkey (Ozkul et al.,002), Ethiopia (Abraham, 2005) and in Morocco (FAO,009). The presence of circulating virus was confirmed byerological determination in Syria, Niger, India, Turkey, Jor-an and Pakistan whereas the virus presence was detected

n Ethiopia and Eritrea (Abubakar et al., 2007; Roeder et al.,994; Sumption et al., 1998).

Sequence comparisons of F-Protein genes were stud-es in different geographical regions for determination ofenetic relationship between PPR viruses (Shaila et al.,996; Dhar et al., 2002). Four lineages of PPR virusere revealed, lineage 1 and 2 viruses have been found

xclusively in West Africa. However, the virus from anutbreak in Burkina Faso in 1999 fell into the lineage 1roup. The lineage 1 represents viruses isolated in African 1970s (Nigeria/1975/1, Nigeria/1975/2, Nigeria1975/3,igeria/1976/1 and Senegalese strain). Lineage 2 that

ncludes viruses isolated in the late 1980s in West AfricaIvory Coast and Guinea) was the only African lineage thatid not cross the Red Sea to the Asian countries (Abraham,005).

Viruses of lineage 3 have been found in East Africa,here an outbreak in Ethiopia in 1996 was of this type,

nd also in Arabia and in Southern India. However, thereave been no further isolations of lineage 3 viruses from

ndia since the one reported in 1992 from Tamil Nadu. Lin-age 3 is a combination of isolates from Sudan (Diallo et al.,988) and Ethiopia (Roeder et al., 1994). A virus of this lin-age was found circulating in Yemen in 2001. In the past 8

ears virus exclusively of the Lineage 4 has spread acrosshe Middle East and the Asian sub-continent, reaching easts far as Nepal and Bangladesh.

The geographical source of the new lineage 4 virusess unknown although it is most closely related to African

t Research 96 (2011) 1–10 3

lineage 1. Lineage 4 of PPR virus isolates that includesthe Asian isolates from Israel/1994, Iran/1994, Nepal/1995,Bangeldesch/1993, India (Shaila et al., 1996) and Pakistan(Amjad et al., 1996) as described in Table 1. It has also beenreported in Turkey (Ozkul et al., 2002). The presence of thetwo African lineages in Asia alongside a distinct Asian lin-eage gave an indication of the trade route of spread of thedisease (Dhar et al., 2002).

In Pakistan, rinderpest-like-disease was reported ingoats by Pervez et al. (1993) for the first time, based ontypical clinical observations on goats. Athar et al. (1995)observed the cases of rinderpest like disease in small rumi-nants in various districts of Punjab. It was assumed thatthese outbreaks were due to infected smuggled animalsfrom the neighboring country. Based on clinical diagno-sis, outbreaks of PPR disease has been reported in variousdistricts of Punjab province, Pakistan (Ayaz et al., 1997;Hussain et al., 1998; Ahmad et al., 2005; Khan et al.,2006).

Khan et al. (2008a) and Abubakar et al. (2008) confirmedthe PPR outbreaks in various places (Punjab province,Northern Areas and Islamabad) of Pakistan by using com-petitive and immuno-capture ELISAs, respectively. The OIEWorld Animal Health in 2000 also confirmed the outbreakof PPR with IcELISA at a wildlife-breeding center of Faisal-abad, Punjab, Pakistan.

6. Epidemiology

6.1. Host range

Goats and sheep are considered as the only natural hostsfor PPR. Goats appear to be more susceptible and suffer amore severe clinical disease than sheep (Lefevre and Diallo,1990).

Although infected sheep rarely suffer clinical disease(El Hag and Taylor, 1988; Roeder et al., 1994), however,high morbidity and mortality in sheep has been observed,assumed that sheep hold an innate resistance to the clinicaleffects of disease (Shaila et al., 1989), but occasional fieldstrains could overcome this resistance and produce highmortality. Different breed might affect the outcome of PPRvirus infection; the Guinean breeds (West African dwarf,Iogoon, kindi and Djallonke) are recognized as highly sus-ceptible (Lefevre and Diallo, 1990).

The field outbreaks are also reported from a zoolog-ical collection in Alain (Furley et al., 1987) and in wildlife breeding center of Faisalabad, Pakistan (OIE, 2000).The disease was reported in Gazelle and deer (Abu Elzeinet al., 1990), Antelope and other small wild ruminantspecies (Abu Elzein et al., 2004), Dorcas Gazelles (Gazelladorcas), Nubian Ibex (Capra ibex nubiana), Laristan sheep(Ovis orientalis laristani), gemsbok (Oryx gazella) and Nigale(Tragelaphinae) has been confirmed. In a recent investi-gation, PPR has been confirmed in Sindh Ibex in Sindh

province, Pakistan; using both antigen and antibodiesdetection methods (Abubakar et al., 2010, unpublished).The antibodies to PPR have also been detected in cattleand buffaloes, Pakistan (Khan et al., 2008a) and in camels(Ismail et al., 1995).

4 M. Abubakar et al. / Small Ruminant Research 96 (2011) 1–10

Table 1Situational review of peste des petits ruminants (PPR) in Pakistan.

Scientist name Year of publication Disease aspect studied Host affected Journal

Athar et al. 1995 Outbreak Goats Pakistan Veterinary JournalPervez et al. 1993 Disease Goats/sheep Pakistan Journal of Livestock ResearchAmjad et al. 1996 Outbreak Goats Veterinary RecordAyaz et al. 1997 Outbreak Goats Pakistan Veterinary JournalTahir et al. 1998 Diagnosis – Pakistan Veterinary JournalHussain et al. 1998 Outbreak Goats/sheep Pakistan Veterinary JournalAhmad et al. 2005 Outbreak Goats/sheep Pakistan Veterinary JournalKhan et al. 2006 Sero-surveillance Goats/sheep Pakistan Veterinary JournalZahur et al. 2006 Disease Goats/sheep Journal of Veterinary Medicine BAbubakar et al. 2007 Prevalence Goats/sheep Tropical Animal Health & ProductionAsim et al. 2008 Vaccine – Pakistan Veterinary JournalAslam et al. 2009 Diagnostic test – Veterinary WorldAbubakar et al. 2008 Antigen detection Goats/sheep Small Ruminants ResearchKhan et al. 2008a Epidemiology Goats/sheep Tropical Animal Health & ProductionKhan et al. 2008b Epidemiology Goats/sheep Small Ruminants Research

Khan et al. 2009 VaccineMunir et al. 2009 Diagnostic testsAbubakar et al. 2009 Sero-prevalenceAsim et al. 2009 Vaccine

6.2. Transmission

PPRV is transmitted through direct contact betweeninfected and susceptible animals and nomadic animals mayoften come into contact with local sheep and goat popula-tions from whom they contract the virus (Shankar et al.,1998). Likewise, infected migratory animals may trans-mit the virus to susceptible sheep and goat population.The movement of animals, therefore, plays an impor-tant role in the transmission and maintenance of PPRV innature, as does the purchase of potentially infected animalsand their subsequent introduction into naïve flocks/herds.Furthermore, limited fodder availability often, leads tonutritional deficiency, resulting in increased susceptibil-ity to infection. Consequently, large numbers of animalsbecome infected during this period and these animals thenhelp to maintain the circulation of the virus throughout theyear by frequent animal-to-animal transmission. These fac-tors may play a key role in limiting the transmission of PPRinfection.

The affected animals during the febrile stage of dis-ease are important source of transmission (Braide, 1981)because of the limited livability of the virus outside the liv-ing host. The discharges from eyes, nose and mouth, andthe loose faeces, contain large amount of the virus. Fineinfective droplets release into the air from these secretionsand excretions, particularly when affected animals coughand sneeze (Bundza et al., 1988; Taylor, 1984) and ani-mals in close contact to inhale the droplets likely to becomeinfected. Although, close contact is mainly important wayof transmitting the disease, however, infectious materialsmay also contaminate water and feed troughs and bedding,turning them into additional sources of infection.

6.3. Disease pattern

The epidemiological pattern of the disease in the diverseecological systems differs in various geographical regions.Sheep, goats and wild animals are reared on free-rangepastureland, shrubs and forest in Indian subcontinent. Due

Goats/sheep Tropical Animal Health & Production– Tropical Animal Health & ProductionGoats/sheep Tropical Animal Health & ProductionGoats Pakistan Veterinary Journal

to an ongoing decrease in available pastureland and forestarea, these animals are often travel long distances duringthe dry season in search of fodder and water (Nanda et al.,1996). The movement of animals, therefore, determines thepattern of PPRV. The PPR in humid areas always occurredin an epizootic form, it may have remarkable consequenceswith morbidity of 80–90% and mortality 50–80%, while inarid and semi-arid regions, PPR is often fatal and usuallyoccur as a subclinical or in-apparent infection opening thedoor for other infections such as Pasteurellosis (Lefevre andDiallo, 1990).

Newborn animals become susceptible to PPRV infec-tion at three to four months of age (Srinivas and Gopal,1996), corresponding with the natural decline in mater-nal antibodies (Saliki et al., 1993) and this large numberof young susceptible stock may also account for theseverity of PPRV infection in goat population. Serologi-cal evidences revealed that antibodies occur in all agegroups from 4 to 24 months indicating a constant circu-lation of the virus (Taylor, 1979). High morbidity (90%)and mortality (70%) has been reported in all age groups(Abu Elzein et al., 1990).

Based on the data of disease outbreaks (Abubakar et al.,2009), the prevalence of PPR in small ruminants in Pak-istan is 40.98%. Findings of active disease situation alsosuggested that the disease is severe in goats than sheep.A greater number of positive cases have been observed inthe southern and northern parts of the country (30–60%)as compared to west and south-west (10–30%).

6.4. Seasonal occurrence

The encouraging climatic factors for the survival andspread of the virus may also contribute to the seasonaloccurrence of PPR outbreaks. During the rainy season

(between June/July and August/September) in Pakistan,the migratory activity of animals is reduced due to theincreased availability of local fodder. The nutritional sta-tus of the animals also improves, resulting in an increasedresistance to infection. These factors may play a key role

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n limiting the transmission of disease. Although, the out-reaks also occurred in West Africa coincide with the wetainy season (Opasina and Putt, 1985). It appears thaturing dry, cold and dusty weather (December–February)ccompanied with poor nutrition by this time combineo spread PPR. The incidence seems to rise rapidly fromecember and get a peak in April (Obi et al., 1983; Durojaiyet al., 1983).

In Pakistan, Khan et al. (2008b) reported high PPRero-prevalence in the months of December, January andebruary and in the months of September and October.ased on the serology, Abubakar et al. (2009) reported thathe frequency of disease outbreaks is greater between the

onths of January to April as compared to other periodsf the year and it is maximum in the month of March33%).

. Diagnosis

Typical epidemiological features considered togetherith clinical signs help to diagnose PPR. Diagnosis of PPRay be performed by virus isolation, detection of viral anti-

ens, antibodies and nucleic acid sequencing.

.1. Virus isolation

PPR virus may be isolated during the acute stage ofhe disease when clinical signs are still apparent. Viruss present for approximately 10 days after the onset ofever. Swabs of the eye (conjunctival sac), nasal secre-ions, mouth and rectal linings, as well as clotted andhole blood (with EDTA anticoagulant), may be used for

solation. Lymph node or spleen biopsies may also be con-idered. Best time of sampling for virus isolation in livenimals is during high temperature and before diarrhoeaas started (Lefevre, 1987). At postmortem fresh samplesf spleen, lymph nodes and affected sections of alimen-ary tract mucosa may be collected for virus isolation. The

ost widely used cell culture systems are primary lambidney (Taylor, 1984), ovine skin (Gilbert and Monnier,962; Laurent, 1968; Taylor and Abegunde, 1979) and Veroells (Hamby and Dardiri, 1976). Vero cells are howeveridely used for their continuity and low liability of con-

amination. PPRV has also been adapted to grow in otherontinuous cell lines, including MDBK and BHK-21 (Lefevre,987). The cytopathic effects (CPE) produced in vero cellsy PPRV consist of giant cells, cell rounding, formation ofypical grape-like clusters, formation of small syncytia andrranged like of spindle cells (Hamby and Dardiri, 1976).ike other morbilliviruses, the inclusion bodies producedy PPRV are eosinophilic intracytoplasmic and intranuclear

n primary cells (Laurent, 1968) and in continuous cell linesHamby and Dardiri, 1976).

The isolated PPRV in cell culture may be identified bynimal inoculation (Gibbs et al., 1979). The cell culture

echnique is time-consuming, labour intensive, insensitive,r expensive to perform, so with the advent of hybridomand molecular biological techniques, new reagents to assistiagnosis have become available and have led to the devel-pment of specific and rapid tests for the diagnosis of PPR.

t Research 96 (2011) 1–10 5

8. Antigen detecting methods

8.1. Agar gel immunodiffusion test

Agar gel immunodiffusion test (AGID) is widely used forthe confirmation of the PPR antigen (Obi, 1984; Abrahamand Berhan, 2001). This test is being used both for the deter-mination of antigen and antibodies and results obtainedwithin 2–4 h when RP hyper-immune serum is used and itrequire 4–6 h with PPR hyper-immune serum (Obi, 1984).The specificity of this test is 92%, determined by Diallo et al.(1995).

8.2. Counter immuno-electrophoresis

Counter immunoelectrophoresis (CIEP) demonstratedby Obi and Patrick (1984) on a glass slide connected withthe tank of buffer and electric current. The test have beenmodified by Tahir et al. (1998) by using ‘U’ shape tube as thesame principle of AGID except that the gel was electricallycharged to improve the sensitivity of the test. Durojaiyeand Taylor (1984) also determined the serology of PPR.

8.3. Immunocapture enzyme linked immunosorbentassay (IcELISA)

The immunocapture (sandwich) ELISA is suitable forroutine diagnosis of RPV and PPRV in field samples such asocular and nasal swabs (Diallo et al., 1995). The monoclonalantibody-based (MAb) sandwich ELISA is highly sensitivein detection of antigen in tissues and secretions of infectedgoats (Saliki et al., 1994). The immunocapture (IcELISA),which is more widely used, utilizes MAb directed againstthe nucleocapsid protein (Libeau et al., 1994). The resultsmay be observed within two hours in precoated plates andfrom samples maintained at room temperature for a periodof seven days with no more than 50% reduction in response(Libeau et al., 1995). The IcELISA permits a quick differentialdiagnosis of PPR or RP viruses. The detecting MAbs used inIcELISA are directed against two non overlapping domainof the N-protein of PPR and RP, but the capture antibodydetects an epitope common to both RP and PPR (Libeauet al., 1994). The test remained very specific and sensitive;it may detect 100.6 TCID50/well for the PPR virus and 102.2TCID50 for the rinderpest virus. The difference betweenthe two viruses in the assay may be due to dissimilarityin the affinity of the detection antibody for the different Nproteins.

Although there are many tests available for detection ofPPR but immuno-capture ELISA was used for the detectionof PPR viral antigen in a major study by Abubakar et al.(2008) to know the PPRV antigen confirmation from majoroutbreaks in Pakistan.

8.4. cDNA probes

In late 1980s, molecular technique based on nucleicacid hybridization using RP and PPR specific cDNA probeswas developed (Shaila et al., 1989; Pandey et al., 1992).Although sensitive, the technique has not been used forroutine diagnosis of PPR. The PPR and RP may be differ-

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entiated by the use of radiolabelled cDNA probes derivedfrom the N-protein gene of the two viruses (Diallo et al.,1989). cDNA probes directed against the matrix protein,fusion protein and phosphoprotein gene could be crosshybridize to a much greater extent and are not suitablefor use as discriminating probes (Diallo et al., 1989). Thishybridization cannot be used widely because it requiresfresh samples, short half-life of radiolabelled substance,and constraints with the handling of isotopes. Therefore,probes using non-radioactive labels such as biotin (Pandeyet al., 1992) or dioxin (Diallo et al., 1995) are developed.The biotin labelled cDNA is as specific as the radioactivelabel and more rapid in differentiation between PPR andRP (Pandey et al., 1992). However, expected sensitivity hadnever been obtained using non-radioactive labels (Dialloet al., 1995). The specific cDNA probes have also been usedto confirm PPR, for the first time in Ethiopia (Roeder et al.,1994).

8.5. Reverse transcription polymerase chain reaction(RT-PCR)

Among the various techniques developed for the detec-tion of PPRV, PCR technique has been the most popular andhighly sensitive tool so far for diagnosis of PPR. The rou-tine serological techniques and virus isolation are normallyused to diagnose morbillivirus infection in samples sub-mitted for laboratory diagnosis. However, such techniquesare not suitable for use on decomposed tissue samples,the polymerase chain reaction (PCR), has proved invalu-able for analysis of such poorly preserved field samples.The PCR test consists of repetitive cycles of DNA denatura-tion, primer annealing and extension by a DNA polymeraseeffectively doubling the target with each cycle leading,theoretically, to an exponential rise in DNA product. Thereplacement of the polymerase now fragment by thermo-stable polymerase derived from Thermus aquaticus (Taq)has greatly improved the usefulness of PCR. These quali-ties have made the PCR one of the essential techniques inmolecular biology today and it is starting to have a wideuse in laboratory disease diagnosis. Since the genome of allmorbilliviruses consists of a single strand of RNA, it must befirst copied into DNA, using reverse transcriptase, in a two-step reaction known as reverse transcription polymerasechain reaction (RT-PCR).

Among the various techniques developed for the detec-tion of PPRV, however, polymerase chain reaction (PCR)technique developed using F-gene primers has been themost popular tool so far, for diagnosis as well as molecularepidemiological studies (Forsyth and Barrett, 1995; Shailaet al., 1996; Brindha et al., 2001). RT-PCR using phospho-protein (P) universal primer and fusion (F) protein genespecific primer sets to detect and differentiate between PPRand RP are described by Barrett et al. (1993) and Couacy-Hymann et al. (2002). Forsyth and Barrett (1995) developeda RT-PCR test, using phosphoprotein (P) gene and fusion

protein (F) gene specific primer sets to detect and differen-tiate RPV and PPRV. They observed that RT-PCR was ableto detect virus secretion in ocular swabs at four days postinfection (PI) in experimentally infected goats, as comparedto eight days PI by IcELISA. RT-PCR assay preclude the need

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for virus isolation and, because of the rapidity with whichcompletely specific results could be obtained, the assayappeared to be the test of choice for PPRV detection (Nandaet al., 1996). Relative specificity and sensitivity of F-genebased RT-PCR with sandwich-ELISA was 100 and 12.5 per-cent, respectively (George, 2002).

8.6. Phylogenetic analysis

On the basis of phylogenetic analysis of morbilliviruses,it was thought that when cattle were domesticated, theyconceded a morbillivirus, a progenitor of modern RPV, tohumans, which ultimately evolved into MV. Likewise, car-nivores might have contracted a morbillivirus infectionfrom their ruminant prey, which then evolved into CDV(Barrett and Rossiter, 1999). MV and RPV are described asclosely related, and CDV and phocine distemper virus arethe most distantly related to MV and RPV among morbil-liviruses (Barrett and Rossiter, 1999). Furthermore, amongall viral proteins, the H protein is found the least con-served among CDV, RPV, and MV (37% identity betweenCDV and MV) (Blixenkrone-Moller, 1993). PPRV exhibitedthe typical characteristics of the Morbillivirus genus in theParamyxoviridae family. PPRV is not only a distinct virusbut may be less closely related to RPV than MV to RPV.Other three members of the Morbillivirus genus (MV, CDV,and RPV) indicate that strains of varying pathogenicity mayoccur naturally. Furthermore the strains distinguish them-selves from virulent strains by including a faster migratingN protein (Diallo et al., 1987) or by their MAb reactivityrange (Libeau et al., 1992). The gene encoding the matrixprotein of PPRV has been cloned by Haffar et al. (1999)and its nucleotide sequence determined. The compari-son of the PPRV, M protein with those of other virusesin the group confirmed the previously noted high degreeof conservation for this protein sequence. The percentof identity within the group range from 76.7 to 86.9%,the highest being with the dolphin morbillivirus matrixprotein. To study the genetic relationship between iso-lates of distinct geographical origin, a selected region ofF-protein gene of the viruses amplified using RT/PCR andthe resulting DNA product sequenced for phylogeneticanalysis.

8.7. Virus neutralization test (VNT)

The virus neutralization test (VNT) is a gold slanderedtest for the diagnosis of PPR and RP, although, it is veryreliable, sensitive and specific, but on the other hand it istime-consuming and expensive. Virus neutralization is animportant protection mechanism, since passively admin-istered anti-H neutralizing MAbs induce full protection inmice (Varsanyi et al., 1987). The standard neutralizationtest is carried out in roller-tube cultures of primary lambkidney cells or Vero cells when primary cells are not avail-

able. The serum against either PPR or RP may neutralizeboth viruses, but would neutralize the homologous virusat a higher titer than the heterologous virus. Thereforefor specific differentiation of PPR and RP, neutralization ismore widely used (Taylor and Abegunde, 1979).

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.8. Competitive enzyme linked immunosorbent assaycELISA)

Monoclonal antibodies raised against hemagglutininrotein of PPR virus have been used either in a block-

ng ELISA (Saliki et al., 1993) or in a competitive ELISAAnderson and McKay, 1994) for differentiation of PPRVnd RPV antibodies. Competitive ELISA based on mono-lonal antibodies (Mab) specific for N-protein (Libeau et al.,995) and H-protein (Saliki et al., 1993; Anderson andcKay, 1994; Singh et al., 2004) developed for detection of

ntibodies in animal sera and diagnostic performance haseen evaluated (Choi et al., 2003). In the N-protein cELISA,he serum antibodies and the Mab compete on specificpitope on nucleoprotein obtained from recombinant bac-lovirus. Although no cross-reaction in N-protein cELISA iseported, a high level of competition up to 45% is observedmong the negative (Libeau et al., 1995). Despite the facthat neutralizing antibodies are not directed against the N-rotein, but the H-protein (Diallo et al., 1995), a correlationf 0.94 between VNT and cELISA was observed suggestinghat the former is more sensitive (Libeau et al., 1995).

The relative sensitivity of this cELISA to VNT is 94.5,hile the specificity is 99.4%. Both blocking ELISA and

ELISA detecting anti-H antibodies are based on compe-ition between an anti-H monoclonal antibody (MAb) anderum antibodies, but in case of blocking ELISA the test serare preincubated with antigen and then incubated with theAb (Saliki et al., 1993). The sensitivity and specificity of

he H-blocking ELISA is found to be 90.4% and 98.9% respec-ively (Saliki et al., 1993). PPR cELISA using MAb directedgainst the H-protein cross reacted to some extent withinderpest, while RP cELISA is specific, therefore an ani-al is assumed to have experienced RP if it is positive in

oth PPR and RP ELISA (Anderson and McKay, 1994). Thebsorbance in PPR ELISA is converted to percentage of inhi-ition (PI). Sera showing PI greater than 50% score positive.

The overall specificity of cELISA test is 98.4% with a sen-itivity of 92.2% when compared with VNT. The anti-H RPELISA has been successfully used for serological monitor-ng of post vaccination herd immunity and it facilitated theradication of rinderpest all over the world. The competi-ive enzyme-linked immunosorbent assay for the detectionf antibodies to rinderpest virus in sera from cattle, sheepnd goats have been standardized by Singh et al. (2000).

In Pakistan, Munir et al. (2009) tested the efficacy of var-ous test for the antigen detection of PPR. Kappa statisticscores for cELISA versus AGID and Precipitinogen Inhibi-ion Test (PIT) were 0.6343 (95% confidence interval CI.5231–0.7456) and 0.7134 (95% confidence interval CI.5987–0.8281), respectively, which indicate a “substan-ial” agreement between cELISA and AGID and “significant”greement between cELISA and PIT.

. Immunity

Tissue culture rinderpest vaccine (TCRV) was widelysed in West Africa, Asia including Pakistan for the con-rol of PPR disease. The tissue culture rinderpest vaccineTCRV) at a dose of 102.5 TCID50 protected goats againstPR for 12 months and the animals were not able to

t Research 96 (2011) 1–10 7

transmit the infection following challenge with PPR virus(Taylor, 1979), although the antigen was detected in lachry-mal swabs from vaccinated animals after challenge withvirulent virus (Gibbs et al., 1979). This vaccine was success-fully used to control PPR in some countries of West Africa(Bourdin, 1983) and was widely used in many other Africancountries (Lefevre and Diallo, 1990). PPRV is antigenicallyclosely related to rinderpest virus (RPV) and antibodiesagainst PPRV are both cross neutralizing and cross protec-tive (Taylor, 1979).

The recombinant vaccinia expressing H and F pro-teins of RPV has been shown to protect goats againstPPR disease (Jones et al., 1993) though the animalsdevelop virus-neutralizing antibodies only against RPVand not against PPRV. The goats immunized with arecombinant baculovirus expressing the H glycopro-tein generate both humoral and cell-mediated immuneresponses (Sinnathamby et al., 2001). The responses gen-erated against PPRV-H protein in the experimental goatsare also RPV cross reactive suggesting that the H pro-tein presented by the baculovirus recombinant ‘resembles’the native protein present on PPRV (Sinnathamby et al.,2001). Lymphoproliferative responses are demonstratedin these animals against PPRV-H and RPV-H antigensby Sinnathamby et al. (2001). Lambs or kids receivingcolostrum from previously exposed or vaccinated with RPtissue culture vaccine acquire a high level of maternal anti-bodies that persist for 3–4 months. The maternal antibodiesare detectable up to 4 months using virus neutralizationtest compared to 3 month with competitive ELISA (Libeauet al., 1992).

Until recently, the most practical vaccination againstPPR is based on the use of tissue culture adapted rinder-pest vaccine. However, to date, there are conflicting reportsboth documented and mostly undocumented by field vet-erinarians as to the effectiveness of TCRV in controllingthis disease. Effective vaccination against PPR is a majoradvancement in the control of the disease even the localvaccine in Pakistan has been made and evaluated (Asimet al., 2009).

10. Control and prophylaxis

PPR virus belonged to the group where causative agentshave been found maintaining a single serotype over anappreciable period of history besides once recovered fromthe infection, the host gains immunity for a life time andthere was no evidence for a persistent or carrier state inthe recovered animals. Immunization of small ruminantswith lymph node and spleen materials containing viru-lent virus inactivated with 1.5–5% chloroform was triedand the animals were immune to subsequent challenge18 months latter (Braide, 1981). Due to close antigenicrelationship between RPV and PPRV, the attenuated tis-sue culture rinderpest vaccine (TCRV) had been used for along time to protect small ruminants against PPR until the

PPRV strain was successfully attenuated by serial passagesin Vero cells (Diallo et al., 1989). This vaccine provided veryefficient protection of sheep and goats against a virulentchallenge. It is the fact that both of the above heterolo-gous and homologous vaccines have required effective cold

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chain and high cost would be required to conduct a vac-cination campaign. To cut down the cost of vaccination,it would be advisable to use not only a thermo-resistantvaccine but also a polyvalent vaccine for the control ofanother important disease together with PPR. The thermo-stability of the current PPR homologous vaccine has beendramatically improved by a new freeze-drying process andaddition of stabilizing agents (Worrwall et al., 2001). Con-trol of PPR may be achieved using classical measures suchas movement restriction of sheep and goats from affectedareas, quarantine of affected animals, slaughter and properdisposal of carcasses and elimination of contact fomitesand decontamination of affected premises. Control of PPRoutbreaks may also rely on movement control (quaran-tine) combined with the use of focused (“ring”) vaccinationand prophylactic immunization in high-risk populations.Sera from animals vaccinated with RP vaccine containsubstantial level of RP antibodies with little or no cross neu-tralizing antibodies to PPR but after challenge with PPR,neutralizing antibodies to PPR increase sharply. RP ther-mostable vaccine was developed for protection of goatsagainst PPR (Stem, 1993). Homologous PPR vaccine atten-uated after 63 passages in vero cell (Diallo et al., 1989) wasused and produced a solid immunity for 3 years (Dialloet al., 1995). The PPRV homologous vaccine was foundto be safe under field conditions even for pregnant ani-mals and it induced immunity in the vaccinated animals(Diallo et al., 1995).

11. Economical significance

Peste des petits ruminants was a relatively less knownviral disease of economic importance of sheep and goats.Due to its nature and consequent capacity of rapid spread,it was regarded as List A disease by the Office of Interna-tional Des Epizooties (OIE, 2000). The published reports onPPR were limited; however, after the recognition of havingseparate epizootiological cycle from closely related virusof rinderpest, PPR virus has been assigned status of con-tinuation in its own right (Taylor and Abegunde, 1979)with initiation of major studies in different countries in theworld. The high mortality due to PPR epidemics (Kitching,1988), were considered for future concern. The economicimpact of PPR was probably underestimated, but it wasbelieved that PPR may be one of the major limitationsof small ruminant farming in the tropic (Taylor, 1984).Although PPR remained the principal killing diseases ofsmall ruminants in most African, Asian and Middle Eastcountries as recognized in an international survey reportpublished in 2002 (Perry et al., 2002), few economic stud-ies have been made on this disease. The most recent onewas published in 1992, conducted in Niger (Chip, 1993).It concluded to an anticipated minimum net present value(NPV) return of 14 millions dollars with an investment of10 millions dollars and internal rate of return (IRR > 100%) ifthe cost of the vaccination programme were to be increased

five-fold. Opasina and Putt (1985) estimated an annual sumranged from £2.47 per goat at high loss and £0.36 per goatat lowest. The loss due to PPR in Nigeria was estimated to be1.5 million dollars annually (Hamby and Dardiri, 1976). Theeconomic losses due to PPR alone in India have been esti-

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mated annually to 1800 millions Indian Rupees (39 millionsUS$) (Bandyopadhyay, 2002).

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