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Virus Research 167 (2012) 43– 47

Contents lists available at SciVerse ScienceDirect

Virus Research

jo u r n al hom epa ge: www.elsev ier .com/ locate /v i rusres

atural infection with peste des petits ruminants virus: A pre and post vaccinalssessment following an outbreak scenario

uhammad Abubakara,b, Muhammad Javed Arshedb, Aamir Bin Zahurc, Qurban Alib, Ashley C. Banyardd,∗

PARC Institute of Advance Studies in Agriculture (PIASA), National Agriculture Research Center, Park Road, Islamabad, PakistanNational Veterinary Laboratory, Park Road, Islamabad, PakistanAnimal Science Institute, National Agriculture Research Centre, Park Road, Islamabad, PakistanAnimal Health and Veterinary Laboratories Agency, Woodham Lane, Weybridge, Surrey KT15 3NB, United Kingdom

r t i c l e i n f o

rticle history:eceived 21 December 2011eceived in revised form 23 March 2012ccepted 28 March 2012vailable online 5 April 2012

eywords:NA viruseseste des petits ruminants virusecal matterheddingaccination

a b s t r a c t

Peste des petits ruminants virus (PPRV) infection was confirmed in a herd of goats (n = 55) at an organ-ised farm in Islamabad, Pakistan. PPRV infection was confirmed using both antigen- and antibody-baseddetection methods, haemagglutination (HA) tests and molecular methods. Animals that survived nat-ural infection developed a typical serological response and virus antigen was detected in fecal matter.Following determination of serological response to infection animals were grouped and either vacci-nated or left unvaccinated: group 1 animals succumbed to infection (n = 5) and samples were analysedfor PPRV antigen; group 2 animals developed clinical disease (n = 10) and were divided into 2 groups,half being vaccinated (group 2a) whilst the remainder were unvaccinated (group 2b); group 3 (n = 15)animals included those that developed only very mild clinical disease or no clinical disease; group 4animals (n = 5) were negative for clinical disease and were housed as a negative control group. A variableantibody response was detected following resolution of the initial outbreak. Excretion of virus antigenwas assessed at different time points following vaccination. Importantly, animals that were vaccinated

(group 2a) excreted antigen in fecal matter for 1 month following vaccination whilst unvaccinated ani-mals (group 2b) continued to shed virus antigen for 2 months. The potential for virus excretion in fecalmatter and effects of vaccination upon virus infection are discussed. We postulate that excretion in fecalmaterial may represent a mechanism of virus transmission following natural infection and that this mech-anism may demonstrate a potential method by which PPRV outbreaks occur spontaneously in areas notpreviously known to have circulating virus.

. Introduction

Peste des petits ruminants (PPR) is an acute and highly con-agious viral disease of small ruminants that is caused by aon-segmented negative strand RNA virus, peste des petits rumi-ants virus (PPRV). This virus is a member of the morbillivirus genusnd as such is closely related to rinderpest virus (RPV). The recentradication of (RPV) has increased the global interest in PPRV andas highlighted its potential for elimination using a similar vacci-ation and surveillance strategy (Baron et al., 2011).

Clinically PPR may vary from acute infection with severe clin-

cal disease and death to mild, with little or no visible clinicaligns. Acute infection may include severe pyrexia (40–41.3 ◦C) withffected animals often becoming restless, having a dull coat, dry

∗ Corresponding author. Tel.: +44 1932 357722.E-mail address: Ashley.banyard@ahvla.gsi.gov.uk (A.C. Banyard).

168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.virusres.2012.03.018

© 2012 Elsevier B.V. All rights reserved.

muzzle, congested mucous membranes, and inappetance. Classi-cal clinical signs include both ocular and nasal discharge; a profusecatarrhal conjunctivitis with matting of the eyelids; oral lesionssometimes resulting in large areas of necrotic tissue sloughingoff during late stage disease; and as disease progresses infectedanimals may develop a profuse, bloody diarrhoea that leads todehydration and emaciation. The lympohcytopoenic effects ofinfection may lead to a profound immunosuppression that in turnmay contribute to the development of secondary bacterial dis-eases that exacerbate disease. Generally, a bronchopneumonia,may develop along with hypothermia and, after 5–10 days, deathof the infected animal (Couacy-Hymann et al., 2005). One inter-esting consequence of infection is increased potential for fetalabortion (Abubakar et al., 2008) although the mechanisms behind

this remain unknown.

Morbidity and mortality rates are often higher in younganimals than in adults. Whilst disease can be severe, differentanimals respond differently following infection in both natural and

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to determine the presence of PPRV specific antibodies both pre-and post-vaccination. This assay is based on a monoclonal anti-body that is specific for the PPRV N protein. ELISA plates were read

4 M. Abubakar et al. / Viru

xperimental settings (Diop et al., 2005; Couacy-Hymann et al.,007a,b). In contrast, manifestation of mild disease within hostopulations can mean that virus may circulate undetected withinerds before being passed to a susceptible group of animals thatay succumb with high morbidity and mortality (Bidjeh et al.,

995). Prerequisites for susceptibility remain unknown but thenfecting strain, immunological competence, nutritional status andre-existing parasitic infections are all thought to contribute to theutcome of infection (Couacy-Hymann et al., 2007b). Furthermore,reed susceptibility is poorly understood and remains an area forurther research. In some reports, morbidity rates as high as 100%nd 90%, respectively, have been observed (Couacy-Hymann et al.,007b; Diop et al., 2005). PPRV is considered to be one of the mainonstraints to improving small ruminant productivity in regionshere it is endemic, often disproportionately affecting the poor

Kitching, 1988; Shaila et al., 1989; Perry et al., 2002).PPRV is mainly transmitted by the aerosol route and as such

ften requires close contact between animals to spread within aerd (Anderson, 1995; Banyard et al., 2010). Although no carriertate for PPRV has been defined, virus may be shed during subclin-cal cases or during incubation periods. Whilst it is clear that PPRVisseminates through the herd through droplet spread betweennimals in close contact, other transmission routes are also thoughtossible. Previous studies have suggested that excretion of virus inecal matter is also important in PPRV transmission (Ezeibe et al.,008). Historically, the report of infectious rinderpest virus in theecal matter of animals showing severe clinical disease is of inter-st. Early experiments by Farrell used the fecal matter of infectednimals to infect other animals by smearing infected fomites onhe muzzles of uninfected animals (Spinage, 2003). Certainly, inreas where PPRV is endemic, fecal matter is known to be used asanure to fertilise farms and pasture where sheep and goats graze.

he fragility of infectious virions is largely assumed from data forinderpest virus where, increase in heat (>70 ◦C), pH (<5.6, >9.6) andltraviolet light are all known to inactivate virus particles. Thesehysiological factors reduce the likelihood for transmission by con-act with infected fecal matter. However, the virus often causesporadic outbreaks and these may be explained by the potentialor live virus in fecal material that may act as source material fornward transmission. Proving the presence of live virus in fecalatter may be problematic as generally isolation of live virus is not

lways successful unless taken from correctly stored and processedost mortem samples.

PPRV is endemic in Pakistan. Isolates of PPRV are routinelyenetically typed, where facilities exist, and all isolates are char-cterised into lineages I–IV (Forsyth and Barrett, 1995). Alongsidehe circulation of virus within Pakistan, the virus is also thoughto be endemic in all bordering countries, namely Iran, Afghanistan,ndia and China with virus being reported and genetically typedrom all countries, with the exception of Afghanistan where no virusas been reported, as belonging to lineage IV. Historically, PPRV iselieved to be of Asiatic origin, spreading later into Africa (Banyardt al., 2006) In Pakistan, initial reports of rinderpest like disease inmall ruminants were described by Pervez et al. (1993) and by Athart al. (1995). These early reports were followed by an upsurge inecognition of PPRV in both Pakistan and neighbouring countries.ost recently within Pakistan there have been reports of PPRV in

he Bahadurnagar region (Ahmad et al., 2005), the Punjab province,zad Jammu, Kashmir, the North West Frontier province (NWFP)nd the Sindh (Khan et al., 2007; Hussain et al., 2008; Mehmoodt al., 2009; Abubakar et al., 2011a). Within Pakistan, the endem-icity of PPRV significantly affects the livestock sector, a growth

rea that contributes almost 11% to the Pakistan GDP. The Pak-stani national small ruminant herd is estimated to contain totalopulations of sheep and goats of 25.5 and 61.9 million animals,espectively (Abubakar et al., 2011b; Anonymous, 2005–2006).

arch 167 (2012) 43– 47

Here we detail a sporadic outbreak of PPRV disease in Pakistanwith a post outbreak vaccination initiative. We highlight the use ofboth serological and molecular confirmatory tests during the out-break and the application of vaccine following resolution of thenatural infection within the herd. We report the detection of bothviral antigen and nucleic acid in the fecal matter of animals thathave survived infection and comment on differences in sheddingof antigen between recovered animals both following and in theabsence of post outbreak vaccination.

2. Materials and methods

2.1. Sample processing

All animals were individually tagged for identification purposes.Initial antigen assessments were made on ocular and nasal swabs.Fecal samples were taken both pre- and post-vaccination andwhere possible, samples were linked to individual animals. Sampleswere processed through the addition of 1 ml of phosphate bufferedsaline (PBS, pH 7.2) per gram of fecal matter. The resulting particu-late suspensions were thoroughly mixed and stored at 4 ◦C for 48 hbefore being cleared by pulse centrifugation. Supernatants wereharvested and assessed for the presence of virus specific antigen inthe haemagglutination test as described below.

2.2. Antigen detection

2.2.1. Haemagglutination (HA) testThe HA test was performed as described previously (Wosu,

1985; Seth and Shaila, 2001) using 0.5% sheep red blood cells (RBCs)diluted in normal saline. The presence of virus H protein was indi-cated by agglutination of RBCs to give a characteristic RBC matt inthe wells of micro-titre plate. Where HA activity was absent, RBCssettled to the bottom of the well forming a distinct RBC ‘button’.

2.2.2. Immunocapture ELISA (ICE)ICE was also used to confirm the presence of virus antigen in tis-

sue and fecal samples. The monoclonal antibody-based diagnosticICE kit manufactured by Biological Diagnostic Supplies Ltd. (BDSL),Flow Laboratories and the Institute for Animal Health, Pirbright,Surrey, England, was used for the detection of PPR viral antigen asdescribed previously (Libeau et al., 1994).

2.3. Molecular detection of viral nucleic acid

Total cellular RNA was extracted from tissue samples to assessthe presence of viral nucleic acid using the Qiagen RNAeasy kitas per the manufacturer’s instructions. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed for the F-geneof PPRV using one step RT-PCR kit (Bio-one) as per the manufac-turer’s instructions. PCR was carried out using PCR primers andconditions as described previously (Forsyth and Barrett, 1995).

2.4. Serological detection

The competitive ELISA (cN-ELISA, Libeau et al., 1995) was used

using an immuno-skan reader (BDSL, Finland) at 492 nm and datawas analysed using ELISA data interchange (EDI) software. Per-centage inhibition (PI) values were generated to indicate levels ofserological response.

M. Abubakar et al. / Virus Rese

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Fig. 1. Serological responses in animals post outbreak resolution. cN-ELISA wasused to determine PI values in animals following the resolution of a PPRV outbreak.Groups are as detailed in the text. Mean values are defined by the central horizontallei

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ines for each group with the upper and lower limits of the standard deviation forach data set being shown. For comparison individual data sets are also shown tollustrate the spread of values around the mean.

. Results

This study details an outbreak of PPRV in a herd of goats inslamabad, Pakistan. In this instance, the outbreak affected 55 6-

onth old male Beetal goats being farmed for meat production.linical signs of disease included pyrexia, cough, ocular and nasalischarges and diarrhoea. Animals were grouped according to theresence and severity of clinical disease. Five animals succumbed to

nfection (n = 5; group 1) defining the mortality rate for the outbreaks 9.1% (5/55 animals) with a further 10 animals exhibiting severelinical disease without succumbing to disease (group 2). In total,linical disease signs that are often seen during PPRV infection wereeen in 15 animals (27.3% morbidity). Of the remaining 40 animalsn the herd, a further 35 showed very mild clinical disease signs that

ere consistent with very mild PPRV infection or no disease (group) and 5 animals showed no signs of disease (group 4). The obser-ation of clinical disease consistent with PPRV infection was notednd graded for each animal as either mild or severe. Infection withPRV was confirmed for animals in groups 1 and 2 where severeisease (group 2) and death (group 1) was seen using both antigennd antibody detection methods: Immunocapture ELISA (ICE) andhe competitive ELISA (cN-ELISA), respectively. Following an initialssessment using both ICE on nasal and ocular swabs and cN-ELISAn serum, the animal groups were split and either vaccinated andoused together (groups 1–3) or not vaccinated and housed away

rom the remaining animals (group 4) (Table 1). Animals in group 2ere divided across two groups: group 2a (n = 5) that were all posi-

ive by ICE and cN-ELISA and group 2b (n = 5) that were ICE positiveut only 2/5 were cN-ELISA positive. To these end animals in groupsa, 2b and 3 were housed together whilst the group 4 animals, thatere ICE and cN-ELISA negative, were housed separately.

Initially, ICE was performed on samples with animals fromroups 1, 2a and 2b all testing positive for virus antigen in ocu-ar and nasal swabs as well as fecal matter. Group 3 animals wereot assessed by ICE. Fecal samples from all group 4 animals, thatad shown no clinical disease signs, were all negative by ICE. Serum

rom each animal was then checked by cN-ELISA to assess the devel-pment of PPRV specific antibodies following resolution of the nat-ral infection. For groups 2a (n = 5), 2b (n = 5), a selection of animalsrom group 3 (n = 15) and group 4 (n = 5) the cN-ELISA was used to

etermine serological responses following the outbreak. A PI valuef 50% inhibition was used as a cut off value for the determination of

PPRV positive serological response (OIE, 2008). The results of thisnalysis are detailed in Fig. 1. Across the data set, following natural

arch 167 (2012) 43– 47 45

exposure a variable antibody response was seen and PI values var-ied from 21% to 67% in animals grouped according to the suspicionof PPRV clinical disease alone. The majority of animals that survivedinfection following the development of moderate to severe clinicaldisease (groups 2a and 2b) developed a PPRV specific serologicalresponse although only two animals from group 2b were serolog-ically positive despite being ICE positive (group 2b, samples 1 and2). However, the remaining group 2b animals that were cN-ELISAnegative did have very low ICE OD values when compared to thebaseline control for positivity (Libeau et al., 1994). Group 2a ani-mals, that exhibited severe clinical disease, were all sero-positive(mean PI = 63.4). For group 3 animals that had mild clinical disease,only 46.7% (n = 7/15) had PI values above the 50% threshold with themean PI for group 3 being 43.8%. In group 4, all animals were bothnegative for virus antigen (ICE) and PPRV specific antibodies (Fig. 1).

Following this initial assessment the different groups withinthe herd were vaccinated (groups 2a and 3) or left unvaccinated(groups 2b and 4) with a licensed live attenuated vaccine (Pes-tivac, Jovac, Jordan). Following vaccination, groups 2a, 2b and 3were housed together whilst group 4 animals were housed sepa-rately. Serological responses were then monitored at monthly timepoints following vaccination. Animals that were serologically posi-tive following natural infection generated an anamnestic responseand maintained a strong serological profile at each samplingpoint following vaccination. Animals in group 4 did not developa PPRV specific serological response throughout the remainder ofthe experiment. Following vaccination, animals in groups 2a and3 developed serological profiles consistent with vaccination andmaintained high levels of PPRV specific antibody for 6 months fol-lowing vaccination. Animals in group 2b that were serologicallynegative prior to the vaccination of groups 2a and 3 (n = 3) werehoused with groups 2a and 3 and developed an antibody responsein the absence of vaccination. Generally, however, the antibodyresponses in group 2b animals were lower than those in vaccinatedanimals during the 6 month period following vaccination (Fig. 2).

Fecal samples were collected pre-vaccination and every fif-teenth day post-vaccination for a period of 3 months from animalsin group 2a (n = 5) and 2b (n = 5) to monitor the excretion of PPRVantigen in fecal matter. From group 2a, viral antigen was detectedin fecal matter for 1 month following vaccination. In contrast theunvaccinated animals in group 2b (n = 5) shed virus antigen in fecalmaterial for up to 2 months following outbreak resolution. Further-more, a viral haemagglutinin (HA) assay was carried out on samplesfrom group 2a (n = 5) and 2b (n = 5). Interestingly, HA titres rangedfrom1:16 to 1:128 but these reduced 30 days post vaccination forgroup 2a animals but remained positive for group 2b animals until60 days. The detection of viral material in fecal matter was alsoconfirmed using ICE. Interestingly group 2a animals remained pos-itive for viral antigen by ICE for 30 days following vaccination andas with the HA results, the unvaccinated animals of group 2b wereICE positive 60 days post infection. Both groups were negative forviral antigen excretion at further sampling points. RT-PCR of fecalmaterial was used to attempt detection of viral nucleic acid in fecalmaterial at different time points and 5 samples from group 2 werepositive by PCR for a region of the F gene and sequence of 2 sam-ples resulting amplification confirmed the excretion of materialassociated with the natural infection rather than the vaccination.

4. Discussion

An outbreak of PPRV caused moderate morbidity and mortality

in Beetal goats on a small meat production farm in Pakistan. Theoutbreak was monitored closely and animals were housed accord-ing to the results of ICE on fecal matter and planned vaccination.Animals were also assessed for serological reaction following the

46 M. Abubakar et al. / Virus Research 167 (2012) 43– 47

Table 1Grouping of animals following resolution of natural outbreak.

Group Number of animals Clinical disease ICE (±) cN-ELISA (+/total tested) Vaccinated (±)

1 5 Died from PPRV + n/a n/a2a 5 Severe clinical disease + 5/5 +2b 5 Severe clinical disease + 2/5 −3 35 Mild/no clinical disease n/a 7/15a +4 5 None seen − 0/5 −

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evelopment of, or in the absence of, clinical disease. The mor-idity and mortality rates observed during the outbreak (27% and.1%, respectively) were low in comparison to previously reportedutbreaks of disease in Pakistan. However, the clinical outcomef disease with PPRV is known to be highly variable, affecting dif-erent species (goats and sheep) and even breeds very differentlyCouacy-Hymann et al., 2007a,b; Diop et al., 2005). The variabilityn serological response was also interesting considering what isnderstood about the clinical pathogenesis of PPRV. The observa-ion of animals in group 2b that were ICE positive but serologicallyegative is noted and may be a factor of the sensitivity of theN-ELISA (Libeau et al., 1995) and also highlights the potential fornimal to animal variation. Ocular and nasal swab samples are thereferred samples for assessing antigen excretion by ICE, with fecalamples generally not being recommended due to the low sensitiv-ty of the test with these samples and the lower likelihood of virusntigen being shed through the fecal route (OIE, 2008). However,n aim of this study was to attempt detection of viral antigen inecal matter to assess the potential for transmission via this route.

The low morbidity seen within the herd gave an interest-ng opportunity to both study pre- and post-vaccinal antibodyesponses following natural infection. The separation of 5 ani-als from the herd (group 4, n = 5) also enabled a truly negative

ontrol to exist for the data. The variability in immune responserom the group 2b animals also gave the opportunity to assessransfer of vaccine from vaccinated animals to serologically naivein contact’ animals. This feature clearly highlighted the spreadf vaccine strain virus from animals in groups 2a and 3 to ani-als in group 2b that developed a strong PPRV specific antibody

esponse in the absence of vaccination. Khan et al. (2009) previ-usly described the responses to PPRV vaccination in sheep andoats and assessed serological status at 10, 30 and 45 days post-accination by cN-ELISA. Mean PI values in sheep at 10, 30 and5 days post-vaccination were recorded as 37%, 65% and 91%,espectively, whereas in goats these values were 43%, 78% and 86%,espectively. The results of the current study are generally in agree-

ent with these findings but have extended the observation period

o 6 months post vaccination. As for RPV vaccination, it is widelyccepted that a strong neutralising antibody response develops fol-owing vaccination and that this response protects animals for life.

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ig. 2. Serological responses in animals post vaccination. cN-ELISA was used to determiach data set with error bars showing standard deviation.

Another interesting feature of the experiment was the observa-tion of excretion of viral antigen and for a small sample of animals,viral nucleic acid, in fecal matter. Fecal samples were analysed fromrecovered goats to monitor excretion of PPRV antigen (H protein) intheir fecal matter. From group 2a, RT-PCR positive results showedthe presence of viral nucleic acid within fecal matter and sequenceresults from PCR amplicons confirmed the isolate to be geneticallysimilar to other natural isolates genetically typed within Pakistanand surrounding countries (data not shown). It is important to notethat where nucleic acid has been detected it is likely that antigen isalso present but is beyond the detection limits of the ICE. Follow-ing vaccination, group 2a animals contained viral antigen in fecalmatter, as evidence by the HA test, for 30 days following vaccina-tion but were negative by HA at further sampling time points. Thisfeature highlights the use of the HA test in assessment of antigenexcretion as a simple assay that can be used cheaply in this form ofstudy. In contrast the unvaccinated animals in group 2b continuedto shed viral antigen for 60 days, presumably following exposure tovaccine-derived live virus from groups 2a and 3 vaccinated animalsor, alternatively following the delayed clearance of necrotic mate-rial following natural infection. The genotype of viral material shedfrom group 2b animals was not confirmed. Shedding of PPRV anti-gen in the fecal matter of the recovered goats following a diseaseincursion put forward the possibility that goats may be sheddingthe PPR virus in their fecal matter although we currently have noexperimental evidence to support this. Certainly, the detection oflive virus in the fecal matter of animals that appear to have recov-ered from clinical disease must be confirmed before any strongconclusions are drawn from this data. These findings are, how-ever, in agreement with Ezeibe et al. (2008) and may reinforce theidea that virus can sub-clinically infect animals and excrete and/ortransmit virus to naïve ‘in contact’ animals. Animals may also becontagious during the prodromal phase of infection although thisremains ill defined. Such a hypothesis may aid explanation of sev-eral phenomena regarding the spontaneity of outbreaks of PPRV. Itmay also help to explain the observation that introduction of new

sheep or goats into healthy flocks often leads to fresh cases of PPRV.Of course, it is also possible that breed differences in susceptibilityand clinical progression of disease are the main factor dictating such

Group 2a

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ne PI values in animals at monthly intervals as shown. Mean values are shown for

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Peste des petits ruminants of sheep in India. Veterinary Record 125, 602.Spinage, C.A., 2003. Cattle Plague: A History. Kluwer Academic/Plenum Publishers,

M. Abubakar et al. / Viru

vents where mixing of animals harbouring clinically mild diseaseith naïve animals occurs.

In conclusion, the sporadic nature of PPR outbreaks remainsoorly understood. As described, differences in breed susceptibilityould suggest one mechanism by which virus can circulate silently

n the absence of detectable disease. The role and susceptibility ofifferent wildlife species remains largely unknown (reviewed inanyard et al., 2010). Certainly, the detection of PPRV antigen in thisnd other studies may highlight this as a potential method by whichirus is introduced into new areas. Via this mechanism, virus maye transmitted between ‘in contact’ groups of animals that differ

n susceptibility. Of course, numerous other factors influence hostesponse to viral infection including infecting dose, immunocom-etence of the host and pre-existing parasitic infections that mayender individual animals less able to defend themselves immuno-ogically against an immunosuppressive virus like PPRV. Indeed,ew animals being brought into contact with healthy flocks areften associated with epizootics.

However, the potential for transmission in fecal material mayxplain the sporadic outbreaks of PPR even in farms with no historyf recent contact with animals from other farms.

cknowledgements

We wish to thank the support staff at the Animal Science Insti-ute, National Agriculture Research Centre, Park Road, Islamabad,akistan, for their help in the sampling process. A.C.B. is part fundedy BBSRC CIDLID Grant HH009485/1.

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