Small Ruminant Research 75 (2008) 256–259

Available online at www.sciencedirect.com

Short communication

Incidence of peste des petits ruminants (PPR) virus in sheep andgoat as detected by immuno-capture ELISA (Ic ELISA)

Muhammad Abubakar ∗, Syed Muhammad Jamal,Manzoor Hussain 1, Qurban Ali

National Veterinary Laboratories, Park Road, Islamabad, Pakistan

Received 13 September 2007; received in revised form 27 November 2007; accepted 4 December 2007

Abstract

Peste des petits ruminants (PPR) is a highly contagious disease of domestic and wild small ruminants. Rapid, sensitive andspecific laboratory assay is useful to enable timely implementation of appropriate control to restrict the spread of disease. Thepresent study reports observations from 50 laboratory confirmed outbreaks of PPR and provides details of the presence or otherwiseof PPR virus (PPRV) in 427 tissue/organ samples from small ruminants. Most of the samples used for the detection of PPR viralantigen were derived from all major regions within the country; however, these samples may not be a true representation of thetarget population still it provided a lot more information. Monoclonal antibody-based diagnostic kit manufactured by BiologicalDiagnostic Supplies Ltd., Flow Laboratories and Institute for Animal Health Pirbright, Surrey, England, were used for the detectionof PPR viral antigen (immuno-capture ELISA). Findings suggested that the disease outbreaks were more severe in goats than sheepand the frequency of disease outbreaks was greater between the months of January to April as compared to other periods of the yearand it was maximum in month of March (almost 33%). Based on the data of 50 outbreaks (427 samples), the prevalence of PPRin small ruminants in Pakistan was 40.98%. A greater number of positive cases were observed in the southern and northern partsof the country (30–60%) as compared to west and south-west (10–30%). These findings may be correlated with variations in the

sheep and goat husbandry practices within different geographic regions and the topography of different areas. The study indicatedthe scenario of virus circulation in the population and prevalence in actual outbreaks situation, which may be kept in mind whiledeciding the vaccination strategy for the control of disease.© 2007 Elsevier B.V. All rights reserved.

Keywords: Incidence; PPR; Immuno-capture ELISA; Sheep and goat

1. Introduction

Sheep and goats are reared by farmers mostly as asubsidiary occupation or by poor people in Pakistan. Itis more a way of life rather than a commercial enterprise.

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

1 Regional Epidemiologist, FAO Project (GTFS/INT/ITA/901).

0921-4488/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2007.12.001

However, in hilly areas sheep and goat herds provide sub-stantial part of farmer’s income. Population of sheep andgoats in Pakistan was estimated 28.6 and 58.8 millions,respectively (economic survey, 2004–2005).

Peste des petits ruminants (PPR) is an acute, con-tagious disease of domestic sheep and goats caused

by a Morbillivirus within the family Paramyxoviri-dae. This disease is characterized by high fever,oculo-nasal discharge, pneumonia, necrosis and ulcer-ation of mucous membranes and inflammation of

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Table 3 and Fig. 1 for better understanding of disease pat-terns which depicted that the disease is endemic almostthroughout the year with incidence increasing in pre-and post-winter months. Outbreaks were reported to be

Table 1Province-wise prevalence of PPR outbreaks (2004–2006)

Serial no. Provinces Total samples Positive (no.) %

1 Punjab 203 71 352 Sindh 174 85 49

M. Abubakar et al. / Small Ru

astro-intestinal tract, leading to severe diarrhoea.t causes up to 50–80% mortality and about 90%orbidity. The virus is closely related to rinderpest

irus which makes the PPR an important disease ofmall ruminants and has created tremendous prob-ems due to its apparent similarity to rinderpestLefever and Diallo, 1990).

The transmission of virus requires close contactetween susceptible and infected animals in the febriletage because of the livability of the virus outside theiving host (Braide, 1981). The discharge from eyes,ose, mouth and the loose faeces contain large amountsf the virus. Fine infected droplets are released intohe air from these secretions and excretions, particu-arly when affected animals cough and sneeze (Bundzat al., 1988; Taylor, 1984). Animals in close con-act inhale the droplets and are likely to becomenfected.

The existence of PPR has been recognized in Pakistanince 1991 when it gave rise to an epidemic in Punjabrovince (Athar et al., 1995). Although sporadically rec-gnized in subsequent years (Hussain et al., 1998, 2003),o systematic epidemiological impact assessment studyas been undertaken, also there was no laboratory-basediagnosis was done. The present study was carried outo report the laboratory confirmation of PPR outbreaksor the last 3 years (2004–2006) using immuno-captureLISA (Ic ELISA).

. Materials and methods

.1. Clinical samples for peste des petitis ruminants virusntigen detection

Based on information available in outbreak records, theost frequent clinical findings in diseased animals were a high

ody temperature (up to 107 ◦F), severe mucopurulent, nasalnd ocular discharges, necrotic stomatitis and respiratory dis-ress. Diarrhea was also present but abortions were reported infew cases. A complete history of disease was not provided forll of the outbreaks. The most common post-mortem lesionseported were necrotic enteritis, pneumonia, splenomegaly andnlargement of the lymph nodes. Interestingly, at least nine outf 50 outbreaks were associated with either the entry of newlyurchased animals from a common market or the minglingf migratory/nomadic animals with local animals. A completeistory of the movement or the nomadic nature of the animalsas not available for all of the outbreaks investigated. Among

he 50 confirmed outbreaks of PPR, 27 outbreaks involvedoats alone, 13 of the outbreaks involved sheep alone, fivef the outbreaks involved both sheep and goats and in theemaining five outbreaks details of the species involved wereot reported.

Research 75 (2008) 256–259 257

2.2. Laboratory confirmation

A total of 427 swab and tissue samples were collected fromthe suspected and dead animals for the presence of PPR viralantigen. Immuno-capture ELISA for the detection of PPR viruswas carried out at the National Veterinary Laboratory, Islam-abad as described by Libeau et al. (1994). The kit used toanalyze these samples was imported from World ReferenceLaboratory for Rinderpest and PPR at PirBright, UK alsothe Nunc Maxisorb ELISA plates were used in this analy-sis. Ortho-phenylenediamine (OPD) was used as chromogenand the absorbance was measured at a wavelength of 492 nm.The reader was connected to computer loaded with ELISAData Interchange (EDI) software (FAO/IAEA, Vienna, Aus-tria), which was used to automate the reading and calculation ofpercent positivity (PP) values. The OD (optical density) valueswere converted to percentage positivity by using the followingformula:

PP = 100 − (OD control/test sample)

Median OD of PPR ref. antigen× 100

The samples with PP > 18% were considered as positive.

3. Results

A total of 427 suspected samples were tested from21 districts of Pakistan associated with 50 outbreaks.The share of samples from all four provinces and north-ern areas is given in Table 1, showing that the diseaseis endemic all over the country. The district-wise inci-dence of samples showed 175 samples positive for PPRantigen, a detail of which is given in the Table 2. Ante-mortem materials (e.g. nasal, buccal and eye swabs)from diseased animals were found to be more suitablethan post-mortem tissues for PPR diagnosis using theimmuno-capture ELISA as among the positive samples60% (no. = 105) were of swab as compared to the tissuesamples.

The month-wise prevalence of disease is also given in

3 NWFP 35 14 404 Balochistan 12 3 255 Northern areas 3 2 67

Total 427 175 –

258 M. Abubakar et al. / Small Ruminant Research 75 (2008) 256–259

Table 2District-wise prevalence of PPR outbreaks in small ruminants of Pak-istan (2004–2006)

Serial no. Cities/area No. of samplestested

Positivesamples

1 Islamabad/Rawalpindi 124 372 Gilgit 03 023 Hyderabad 16 024 Quetta 12 035 Lahore 10 026 Okara 01 017 Sukker 30 158 Ghotki 02 019 Khairpur 13 1010 Attock/Abbotabad 17 0611 Shekarpur 11 0612 Peshawar 20 0613 Jacob abad 04 0214 Multan 04 0315 Sahiwal 12 0516 Karachi/Sindh Rural 98 4917 Bahwalnagar 04 0218 Bhakkar 6 019 Kohat/Karak 15 0820 Faisalabad 21 1221 Bhawalpur 04 03

Fig. 1. Month-wise prevalence of PPR outbreaks (2004–2006) in smallruminants of Pakistan.

Table 4Species-wise prevalence of PPR virus

Species Positive Total Percent prevalence (%)

the eastern, southern and northern parts of the coun-

Total 427 175

more severe in goats than sheep. The greatest frequencyof PPR outbreaks reported for the period of January 2006to April 2006 (Table 2).

The species-wise disease outbreaks were more

severe in goats than sheep as percent prevalence was35.26% in sheep as compared to 46.36% in goat(Table 4).

Table 3Month-wise prevalence of PPR positive cases and outbreaks

Serial no. Months Year 2004 (11 out-breaks = 38 samplespositive)

Year 2005breaks = 47positive)

1 January 4 22 February 2 83 March 12 164 April 8 75 May 2 26 June 0 17 July 0 08 August 0 09 September 2 110 October 1 311 November 5 412 December 2 3

Total 38 47

Sheep 73 207 35.26Goat 102 220 46.36

4. Discussion

The study provided valuable data on the infectionstatus of PPR in sheep and goats in Pakistan. PPR infec-tion was demonstrated in 21 regions/districts of Pakistanwhich are known to be the pockets/suspected areas ofPPR. Variation in prevalence was probably related tothe intensity of movements of the small ruminants inrespect of provinces, weather and religious occasion (Eidfestivals).

A greater number of outbreaks were confirmed in

try (30–60%) as compared to west and south-west(10–30%). These findings may be correlated with vari-ations in the sheep and goat husbandry practices within

(15 out-samples

Year 2006 (24 out-breaks = 90 samplespositive)

Total no. of positive(%)

6 12 (6.8)16 26 (14.85)29 57 (32.57)19 34 (19.43)

5 9 (5.14)2 3 (1.71)0 00 02 5 (2.85)7 11 (6.28)1 10 (5.71)3 8 (4.57)

90 175

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ifferent geographic regions such as mixed grazing inigh prevalent areas and also due to the topography ofifferent areas.

Climatic conditions and seasonal forage availabilityictate grazing patterns in the areas. Livestock migrateetween alpine pastures and the Pothwar Plateau inhe foothills of the Himalayas. The livestock spend

arch and April in subtropical and temperate forestrazing areas below 2000 m. They utilize the alpinereas from June to October, when low temperaturesetard plant growth and then herders descend towardshe plains or low valleys. During winter, livestock grazen Pothwar scrub ranges, abandoned cultivated lands, orrowse in valleys along water channels, roads and graz-ng grounds between agricultural fields (Dost, 1998). Sohe nutritional status of the animals improves during theainy season due to increased availability of fodder thatay lead to the increased resistance which may be the

xplanation for the seasonal variation of the disease asore number of cases appeared in pre- and post-winteronths.Although there are many tests available for detec-

ion of PPR but immuno-capture ELISA was used foretection of PPR viral antigen as this ELISA was alsoesigned to differentiate between rinderpest and PPRiruses and so it is more specific and reliable. Accordingo Abraham and Berhan (2001) as they did a compar-son of tests, found that antigen capture ELISA wasore rapid, sensitive and virus specific than the agar gel

mmuno-diffusion (AGID). Even if the cold chain of thepecimen is compromised for a day or two during sam-le collection and submission, the specimen may still beuitable for testing by ELISA. Diop et al. (2005) alsosed the same immuno-capture ELISA for detection ofPR virus and found that between 85 and 100% of nasalecretions obtained from clinically diseased goats duringhe PPR outbreak reacted positively.

Based on the data of 50 outbreaks (427 samples), therevalence of PPR in small ruminants in Pakistan was0.98% as a whole while it was higher in goat (46.36%)s compared to sheep (35.26%) which indicates that theisease was more severe in goat than sheep and there wassignificant difference of prevalence (P < 0.05) in goat

s compared to sheep which is in congruent with Kumart al. (2002) who reported from India and claimed thatortality due to PPR virus infection was higher (64.7%)

n goats than in sheep (44.4%). Same was the case byhand et al. (2002) who reported that morbidity andortality were significantly higher (P < 0.05) in goats

ompared to sheep. It was shown that goats were more

Research 75 (2008) 256–259 259

susceptible to PPR than sheep, and young animals ofboth species were at higher risk than adults. The sourceof infection was traced to migratory flocks of nomadsfrom Rajasthan who migrate their animals to Punjab forgrazing in the field after harvest. Thus migration of ani-mals was the significant reason for the spread of thisdeadly virus.

Although there are many reports about the sero-prevalence of PPR antibodies in different areas of thecountry, the current study provides a comprehensive pic-ture of PPR detection as well as virus circulation in thepopulation and prevalence in actual disease outbreaks,which may be kept in mind while deciding the vaccina-tion strategy for the control of the disease.

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