Journal of Biosciences and Medicines, 2014, 2, 27-33
Published Online August 2014 in SciRes. http://www.scirp.org/journal/jbm
http://dx.doi.org/10.4236/jbm.2014.26005
How to cite this paper: Zahur, A.B., et al. (2014) Peste des Petits Ruminants Vaccine (Nigerian Strain 75/1) Confers Protec-
tion for at Least 3 Years in Sheep and Goats. Journal of Biosciences and Medicines, 2, 27-33.
http://dx.doi.org/10.4236/jbm.2014.26005
Peste des Petits Ruminants Vaccine
(Nigerian Strain 75/1) Confers Protection
for at Least 3 Years in Sheep and Goats
Aamer Bin Zahur1, Hamid Irshad1*, Aman Ullah1, Muhammad Afzal2, Asma Latif1,
Riasat Wasee Ullah1, Umer Farooq1, Muhammad Humayoon Samo2, Muhammad Jahangir1,
Giancarlo Ferrari3, Manzoor Hussain2, M. Munir Ahmad4
1Animal Health Research Laboratories, Animal Sciences Institute, National Agricultural Research Centre,
Islamabad, Pakistan
2FAO-UN Pakistan (GCP/PAK/127/USA) NARC, Park Road, Islamabad, Pakistan
3AGAH, FAO (Hq), Rome, Italy
4Divisional Diagnostic Laboratory, Livestock and Dairy Development Department, Multan, Pakistan
Email: *hamidirshad@hotmail.com
Received Ju ly 2014
Abstract
The present study reports the duration of immunity and protective efficacy of Peste des Petits
Ruminants (PPR) vaccine (Nigerian strain 75/1) in sheep and goats. A total of 105 sheep and goats
were divided into three gr oup s A, B and C. Gr oup A received normal recommended dose (1.0 ml) of
PPR vaccine, grou p B received half dose (0.5 ml) of PPR vaccine and g rou p C was kept as unvacci-
nated control g rou p in contact with vaccinated animals. The post vaccination dynamics of antibo-
dies against PPR virus was studied. It was found that significant antibody titres persisted for 3
years post vaccination in sheep and goats vaccinated with either full dose or half dose of PPR vac-
cine. The challenge protection studies were carried out in experimental animals at 24 and 36
month post vaccination. The vaccinates withstood challenge and were found completely resistant
clinically and virologically to virulent PPR virus for 24 and 36 months post vaccination. The un-
vaccinated control animals developed typical clinical signs of PPR and the challenged virus was
detected in ocular, nasal and oral secretions of these animals. This study demonstrated that a sin-
gle immunization with PPR vaccine conferred solid protection in sheep and goats for 3 years.
Keywords
PPR Vaccine, Small Ruminants
1. Introduction
Peste des petits ruminant (PPR) is an acknowledged acute and highly contagious newly emerging/evolving dis-
ease of small ruminants. The disease is caused by a Morbilli virus of genus paramyxoviridae [1 ] . Similar to oth-
*
Corresponding author.
A. B. Zahur et al.
28
er Morbilli viruses it is capable of destroying the whole immunologically naive host population by provoking
epidemics and pandemics subsequently damaging economy, undermining food security and livelihood of the
poor farmers [2].
PPR was confirmed for the first time in Pakistan in 1994 when samples collected during a suspected outbreak
were sent to the Institute of Animal Health, Pirbright UK [3]. The disease has been a cause of heavy mortality
among small ruminants in Pakistan since last decade [4]. Since then number of PPR outbreaks have been rec-
orded and now PPR appears to be widespread and endemic in Pakistan [5]. Previously tissue culture rinderpest
vaccine (TCRV) was used for control of PPR in Pakistan. However, with the declaration of freedom from rin-
derpest in 2007 its use in Pakistan was discontinued. Now a homologous PPR vaccine (Nigerian strain 75/1)
appears to be the only remedy in this regard. However, very limited information is available about the duration
of immunity and protective efficacy of this vaccine. Therefore, a study was designed to evaluate the duration of
immunity and protective efficacy of the homologous PPR vaccine. The information thus generated by this study
would help in devising appropriate vaccination strategy for control of PPR in Pakistan.
2. Material and Methods
2.1. Experimental Animals
Sheep (n = 45) and goats (n = 60) between 5 months and 7 years of age were selected for the trial at Barani Li-
vestock Production Research Institute (BLPRI) Kherimurat, Attock. All the animals were sero-negative for PPR
and there was no history of vaccination against PPR in selected animals. The duration of the experiment was 38
months. Animals were randomly allocated to three group s A, B and C. Each group comprised of 35 animals
(sheep = 15 and goats = 20). The animals in gr o up A and B were administered subcutaneously in the sternum
region with 1 ml (recommended dose) and 0.5 ml of an attenuated PPR virus vaccine Pestevac respectively
(each dose contained PPR virus strain 75/1 at a concentration of 102.5 TCID50) produced by Jordan Bio-In-du-
stries Centre (JOVAC). The animals in group C served as unvaccinated in contact control.
2.2. Sample Collection and Analysis
The sera of experimental animals were collected at monthly intervals during first year, at 2 months interval dur-
ing second year and at 3 months interval during third year of the experiment. The sera were stored at 20˚C until
tested. The samples were analyzed using anti-hemaggl uti nin (H) monoclonal antibody based competitive ELISA
(cELISA) [6] [7]. The cELISA kit was manufactured jointly by Biological Diagnostic supplies Limited (BDSL)
with the collaboration of Flow Laboratories and Institute for Animal Health, P ir b r ight, Sur re y, United Kingdom.
The standardized reagents, bench protocol and manual were supplied along with the kit.
2.3. Challenge Protection Studies
The challenge protection studies were also carried out in the experimental animals. For this purpose three of the
vaccinates (goats) which were immunised with normal recommended dose (1 ml) of PPR vaccine and two un-
vaccinated in contact control goats were procured from BLPRI, Kherimurat. These animals were challenged
subcutaneously in the neck region with 3 ml of virus suspension (PAK-KP1-06/NARC3) at a concentration of
103 TCI D50/ml at Animal Sciences Institute, National Agricultural Research Centre. The challenged animals
were examined twice a day to monitor the development of clinical signs and the observations were recorded.
The necropsy examination was carried out immediately after the death of each goat and the gross pathological
lesions were recorded. The ocular, nasal and oral swabs were collected in 500 µl of sterile phosphate buffer sa-
line (PBS) from the challenged animals from day 0 to day 9 post challenge to find the evidence of viral shedding
in challenged animals.
RNA was extracted from swab samples using Trizol reagent (Gibco BRL) following the manufacturer’s in-
structions. One step PCR was performed in a 9902 thermal cycler (Applied Biosystems, Courtaboeuf, France).
The extracted RNA was amplified using PPR virus specific primers based on highly conserved sequences within
F gene of PPR virus [8]. Each PCR reaction contained 5 μl of RNA, One step 5X RT-PCR Buffer (QIAGEN)
with 12.5 mM MgCl2, 10 mM of each dNTP mix, 2 μl enzyme mix, 5 units of RNAse inhibitor and 100 pmol of
each of forward and reverse primers. The PCR conditions were as follows: an initial reverse transcription for 30
minutes at 50˚C, a PCR activation for 5 minutes at 95˚C, 30 cycles of amplification (1 minute at 95˚C, 1 minute
A. B. Zahur et al.
29
at 50˚C , and 2 minute at 72˚C), and a final extension step at 72˚C for 10 minutes. The amplified PCR products
were electrophoresed through 1.5% agarose gel in 1X TBE buffer at 90V for 80 minutes and visualized using
ultra violet illumination. The expected product size was 371 bp.
2.4. Data Analy sis
The experimental animals were divided into three age categories: kids (5 months to 1 year old), young stock (1-2
years old) and adults (more than 2 years of age). One way analysis of variance (ANOVA) was used to assess the
association of species, sex, age, treatment (vaccination against PPR) and time/month of sampling. This associa-
tion was confirmed by repeated measures ANOVA. The multiple regression analysis that allowed for the ran-
dom effects was used to validate the results of ANOVA. Intercooled STATA 9.2 was used for the statistical
anal ysi s.
3. Results
The animals with antibody levels sufficient to cause color inhibition (PI = percent inhibition) values more than
50% were considered positive and responsive to vaccination. The pre vaccination PI values (day 0) of all ani-
mals in the three group s were found to be diagnostically negative (PI values below 50%) and ranged between 5
to 46%. The highest mean PI value of more than 90% was observed on day 30 (one month) post vaccination in
the animals in group A which received normal recommended dose of PPR vaccine. Thereafter, a steady decline
was observed in the subsequent samplings at defined intervals. Significant antibody titres were observed even at
the end of observation period i.e. 36 months post vaccination. A similar trend was observed in the animal vacci-
nated with half dose of PPR vaccine (gro up B). The highest antibody titre was observed 30 days post vaccina-
tion followed by a gradual decline in mean PI values and significant antibody titres were observed even at the
end of observation period. The unvaccinated control animals (group C) did not develop any significant serolog-
ical titres against PPR and remained negative throughout the observation period (Figure 1). The analysis also
indicated weak association (F = 4.58, P = 0.033) between sex and PI values of PPR cELISA. However, this may
be attributed to the fact that only a small number of male animals (n = 5) were included in each group .
This study could not find an association between species and cELISA PI values (one way ANOVA F = 0.02,
P = 0.877) and thus protection offered by PPR vaccine. The age of the animals was strongly associated with PI
values (F = 28.44, P =< 0.001). The Scheffe’s multiple comparison test showed that the mean PI values among
adults were significantly different from those of either young stock and kids (Table 1). These results suggest
that immune response against PPR virus vaccination in adults is stronger than young stock and kids.
For univariate analysis, linear regression was used. There was no evidence of any association between the
cELISA PI values and species of animals (P = 0.509) and sex (P = 0.394). Age of the animals appears to affect
the PI values of the PPR cELISA (P = 0.008) in the univariate analysis. However, the treatment gr oup that an
animal belonged to (z = 22.08, P < 0.001) or the month of sampling (z = −8.00, P < 0.001) appeared to be
strongly associated with the cELISA PI values. The final model contained the treatment gro up , month of sam-
Figure 1. Mean percent inhibition (PI) values of PPR c-ELISA in animals
among three treatment groups during 36 months of experiment.
A. B. Zahur et al.
30
Table 1. Comparison of ELISA PI values by age groups (Scheffe’s multiple comparison test for means).
Treatment Mean PI values
Kids Young stock
Young stock 3.92461
P = 0.06
Adult 13.1179 9.19332
P < 0.001 P < 0.001
pling and the age of animal at the start of trial. Multiple regression analysis for panel/longitudinal data was used
for the final statistical analysis. Generalized least square (GLS) random effects estimation was used to allow for
the mean titre to vary among various groups. After controlling for the effect of treatment gro up and month of
sampling, the age appeared no longer associated with the PI values of PPR cELISA (z = 0.79, P = 0.43). The β0
coefficient of regression was 28.46 (95% C.I., 23.599 - 33.327: z = 11.47, P < 0.001). The treatment is quite
strongly associated with the PI value. The beta coefficient of regression for treatment group (β1) was 37.972
(95% C.I. , 31 . 191 - 44.752, z = 11.70, P <0.001). The beta coefficient of regression for month of sampling (β2)
was 0.339 (95%C.I., 0.415 to 0.263, z = 8.73, P < 0.001): there is a negative relation, i.e. the PI values de-
crease as the month of sampling gets farther from the date of vaccination. Assuming a linear relationship and
after controlling for the effect of treatment group , the PI values can be determined as:
value28.4630.339month ofsamplingPI = −×
Similarly, the treatment gro up appeared to strongly affect the cELISA PI values. After controlling for the ef-
fect of month of sampling, the PI values were strongly associated with the treatment gro up .
Pred iction:
The final regression equation takes the following form:
For the control group
value28.463(0)(0.339month of sampling)PI =+− ×
For the half dose vaccine group, it takes the following form;
( )
value28.46337.9721(0.339month of sampling)PI =+×− ×
Whereas for the full dose vaccine Group, it takes the following form:
()
value28.46343.4921(0.339month of sampling)
PI =+×− ×
Statisticall y, the PI values among both treatment gro up s are not different.
This statistical model was very good at fitting the observed PI values by controlling the variations among
three treatment groups (R2 between gro up s = 0.849) and quite good at fitting the overall observed PI values (R2
overall = 0.684).
All the three challenged vaccinates withstood challenge and were found to be completely resistant clinically
and virologically to virulent PPR virus. There was no evidence of virus shedding in ocular, oral and nasal secre-
tions of three vaccinated animals examined using RT-PCR during the observation period (day 0 - day 9 post
challenge). These animals remained protected and free of any clinical signs of PPR throughout the observation
period i.e. 35 days post challenge.
The PPRV specific RNA was detected in the secretions (ocular, nasal and oral secretions) of the unvaccinated
control animals on day 4 post challenge. Both the animals continued to shed virus till the end of observation pe-
riod (day 9 post challenge).
The unvaccinated control animals exhibited fever (up to 41.2˚C) after an incubation period of four days ac-
companied by depression and anorexia. This was followed by ocular and nasal discharges, mouth lesions, d iarr-
hea, and pneumonia culminating in death of animals on day 16 and day 21 post challenge. At necropsy the car-
casses were dehydrated with sunken eyes and soiling of hind quarter with diarrhea material. The nasal and buc-
cal mucosae were congested with red raw lesions on the cheek papillae in one of the animal. Both the lungs were
A. B. Zahur et al.
31
found pneumonic. Hemorrhages were seen on the mucosal surface of the abomasum. There was hemorrhagic
enteritis in large intestine (cecum and colon). The lymph-nodes particularly the mesenteric lymph nodes were
found swollen along with accumulation of fluid in both the animals.
Second challenge protection study was conducted 36 months post vaccination. All the 4 challenged vaccinates
withstood challenge and were completely found resistant clinically and virologically to virulent PPRV. The
shedding of PPR virus was not observed in the ocular, nasal and oral swabs during the observation period. These
animals remained protected and free of any clinical signs of PPR throughout the observation period i.e. 35 days
post challenge.
The PPRV specific RNA was detected in the secretions (ocular, nasal and oral secretions) of the unvaccinated
control animals on days 4 and 5 post challenge. Both the animals continued to shed virus till the end of observa-
tion period (day 9 post challenge).One of the unvaccinated control animal developed fever after an incubation
period of four days. The second animal developed fever on day 5 post challenge. This was followed by ocular
and nasal discharges; mouth lesions, diarrhea, and pneumonia followed by death of one animal on 23rd day post
challenge while the second goat recovered after a disease episode and remained alive till the end of observation
period.
4. Discussion
Until recently the TCRV was used successfully for the induction of cross protection against PPR owing to the
antigenic similarities between the two viruses [9]. It was demonstrated that TCRV was safe, efficacious and
conferred protection in goats for 1 year [10] [11]. However, the use of TCRV was discontinued owing to the
need for carrying out sero-epidemiological studies for the verification of presence of antibodies against rinderp-
est virus in ongoing rinderpest eradication campaign. A homologous PPR vaccine developed by successful at-
tenuation of Nigeria 75/1 strain of PPRV on Vero cells became available in 1989 [12]. Unfortunately limited
reports are available on the duration of immunity and protection conferred by this vaccine. Awa et al. [13]
demonstrated that the titres remained above the protection threshold 12 months after the vaccination with PPR
vaccine.
Although the experimental animals were from the area which is known to harbour PPRV, the pre-immun i za -
tion sera antibody titres of all the experimental animals were found diagnostically negative for the presence of
PPRV antibodies. A probable explanation is that the animals used in this study were stationed at isolated pre-
mises in an organized public sector farm with vast grazing areas. Under such conditions the chances of trans-
mission of PPRV diminished when these animals had no contact with other animals of the area. Furthermore,
there was no history of PPR at this farm. It was found that the vaccine virus was not transmitted to the in contact
unvaccinated control animals. This observation is in agreement with those reported earlier [12] [14].
The highest serum antibody titres were recorded at 30 days post vaccination in goats and sheep vaccinated ei-
ther with normal recommended dose or half dose of PPR vaccine. The antibody titres gradually increased from
day to day and highest mean cELISA PI value of 81.5% were obtained at 30 days post vaccination in goats [15].
Maximum serum neutralization test tires were detected 1 month post vaccination in goats following vaccination
with PPR vaccine [13]. This was followed by a gradual decrease in the mean PI values of the vaccinated animals
during the entire study period (3 years post vaccination). The mean antibody titres remained diagnostically posi-
tive and above the protection threshold (more than 50% cELISA PI values) in the last sampling 36 months post
vaccination both in animals either vaccinated with recommended dose or half dose of PPR vaccine. It was found
that even a reduced dose (half of the recommended dose) of PPR vaccine is capable of eliciting a strong immune
response in the vaccinated animals. These findings are supported by the observations made during field trials
with PPR vaccine where it was reported that the antibodies produced lasted for 3 years in respondent animals [9]
[12]. The antibody levels declined between 12 and 18 months but then remained stable for 3 years or more after
vaccination with TCRV in cattle [16].
A serological response with frequency distribution of PI values ranging between 50% - 90% was observed in
sheep and goats in response to PPR vaccination. These results are in agreement with those of Taylor [1] who
reported that although sheep are less severely affected by PPRV than goats, yet a similar serological profile was
exhibited by both species. It has been demonstrated that the entire small ruminant flocks sero-converted at the
same time during an outbreak in Northern Cameroon [17]. However, a different sero-conversion rate following
vaccination with PPR vaccine has been reported in sheep than goats where maximum titres were detected 4
A. B. Zahur et al.
32
months post vaccination in sheep [13]. Likewise a different peak frequency distribution of PI values was re-
ported for goats and sheep in convalescent sera tested positive for PPRV antibodies [18]. This variation may
perhaps be due to the differences in breed, age , health status of animals and management conditions for the
rearing of small ruminants. The outcome of PPR virus infection and its epidemiology may be affected by the
breed. For example, the Guinean goat breeds are known to be highly susceptible to PPR [19].
A strong association existed between age of animals and antibody response to vaccination. The PI values of
adult animals were significantly higher than those of young stock and kids. PPRV is known to cause severe dis-
ease with invariably fatal results in lambs, kids and in animals up to 2 years of age [11] [20]. This phenomenon
of enhanced antibodies production in adult animals may be attributed to the increased immuno-competence and
ability to launch strong humoral response on exposure to PPRV. These results are in accordance with Abubakar
et al. [21] who reported a higher prevalence of antibodies against PPRV in adult sheep and goats (more than 2
years of age). Also the proportion of sero-positive animals increases with age for PPR [22].
There was no evidence of virus shedding in the ocular, nasal and oral secretions of vaccinated animals during
the challenge protection studies. All the vaccinated animals challenged after 24 and 36 months post vaccination
remained protected. In Morbilliv irus infection, association between the neutralizing antibody titres and resis-
tance to natural or experimental challenge following vaccination is often emphasized [16]. It is evident by the
kinetics of antibody response in challenged animals that a strong anamnestic (secondary) response was not ob-
served in challenged animals 24 months post vaccination. Similar retarded serological responses have been re-
ported in cattle challenged with virulent rinderpest virus 6 to 11 years after vaccination with TCRV [16]. How-
ever, a significant rise in antibodies titres was observed in the animals challenged 36 months post vaccination. It
was found that animals with weakly positive antibody titres and even with diagnostically negative titres resisted
challenge and remained protected 36 months post vaccination. Plowright [16] demonstrated that vaccinates
which were negative or weakly positive for antibodies against rinderpest virus resisted challenge with virulent
rinderpest virus.
Though sheep and goats vaccinated with half dose of PPR vaccine were not challenged, yet looking at hu-
moral response in these animals it can be speculated that they might have withstood challenge and remained
protected. These results however should not be considered conclusive because only a small number of animals
were used for challenge owing to financial constraints and insufficient facilities needed for the management of
challenged animals. However, these results are strongly suggestive of the protection conferred by a single sub-
cutaneous injection of PPR vaccine 3 years after vaccination.
The role of cell mediated immunity in the protection against challenge with virulent PPRV has not been in-
vestigated. It is difficult to distinguish the relative importance of the role of cell mediated and humoral immunity
in either recovery from infection or protection against challenge by vaccination [23]. The fusion protein of
PPRV has been reported to play an important role in cell mediated immunity [9]. Further studies in this regard
may prove helpful in understanding the phenomena of protection in natural PPRV infection and in experimental
challenge.
5. Conclusion
The economic life of small ruminants is about 3 years. This study indicated that PPR vaccine (Nigerian strain
75/1) provides immunity for 3 years. Therefore, the strategic use of this vaccine will be useful in reducing the
impact of infection in the areas where the disease is endemic.
Acknowledgements
Assistance of FAO projects GCP/PAK/127/USA-Progressive control of Peste des Petits Ruminants in Pakistan
and GT FS/INT /907/IT A-Controlling Transboundary Animal Diseases in Central Asian Countries in undertaking
this study is highly acknowledged.
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