Pathological, serological and virological findings in goats experimentally infected with Sudanese Peste des Petits Ruminants (PPR) virus isolates

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Journal of General and Molecular Virology Vol.1 (1), pp. 001-006, June, 2009
Available online at http://www.academicjournals.org/JGMV
© 2009 Academic Journals



Full Length Research Paper

Pathological, serological and virological findings in
goats experimentally infected with Sudanese Peste des
Petits Ruminants (PPR) virus isolates

Nussieba A. Osman1*, A. S. Ali2, M. E. A/Rahman3 and M. A. Fadol

1Department of Pathology, Parasitology and Microbiology, College of Veterinary Medicine and Animal Production, Sudan
/Sudan University of Science and Technology, P.O.Box 204, Khartoum North, Sudan.
2Department of Preventive Medicine and Veterinary Public Health, Faculty of Veterinary Medicine, University of
Khartoum, Post code 13314, Khartoum North, Sudan.
3Department of Virology, Central Veterinary Research Laboratories, Soba, P.O.Box 8067, Khartoum, Sudan.
4Viral Vaccine Production Unit, Central Veterinary Research Laboratories, Soba, P.O.Box 8067, Khartoum, Sudan.

Accepted 9 June, 2009

Four Peste des Petits Ruminants virus (PPRV) Isolates were collected from clinical cases of three goats
and one sheep from Khartoum State; Soba/Khartoum State and Bashaier/River Nile State. These PPR
viruses were isolated in Lamb kidney cells (LKC) and Lamb testis cells (LTC) and identified by Agar Gel
Precipitation Test (AGPT) and Hemagglutinition (HA) tests. Four PPRV isolates were used for
experimental infection in four groups (n = 4) of Sudanese goats. Goats of group A and B were
inoculated with the 4th passage of two Sudanese PPRV cultured in lamb testis cells, 6 × 10 TCID /ml of
Bashaier and 6 × 10 TCID /ml of Soba isolates, isolated from sheep and goat respectively. Whereas,
group C and D were received 6ml of the fifth passaged 20% infected tissue suspensions of Khartoum
and Soba PPR isolates propagated in goats. The inoculated goats showed typical PPR clinical signs,
gross lesions and histopathological changes while control animals (group E) appeared healthy. Two
goats from group A died on the 16th and 19th days post inoculation. PPR viruses were detected by HA
test from lacrymal fluid and nasal swabs on the 6th and 7th dpi. Serum samples were collected and
tested for PPRV antibodies by C-ELISA from the sixteen experimentally infected goats and from control
animals. Traces of PPRV antibodies were shown on day 7 and they were continued to rise till the 28th
day and dropped on the 30th day which is the 9th day post challenge. The observed clinical signs, post
mortem lesions and the detectable antibodies indicated that the tissue culture propagated PPR viruses
and the infected tissue homogenate were effective for initiation of infection.

Key words: PPRV isolates, tissue culture virus, goat adapted virus, experimental infection, immune response.


INTRODUCTION

Peste des petits ruminants virus (PPRV) is a member of
genus Morbillivirus, closely related to rinderpest virus
(RPV), causes an acute febrile disease of small rumi-
nants with morbidity and mortality rates as high as 100
and 90% respectively (Abu-Elzein et al., 1990).
Clinically, the disease is characterized by severe pyre-
xia, ocular and nasal discharges, necrotizing and stoma-
titis, conjunctivitis, gastroenteritis, diarrhoea and pneumo-
nia (Ismail et al., 1995 and Jones et al., 1993). The prin-



*Corresponding author. E-mail: nussieba@yahoo.com. Tel:
00249 912446180.
ciple host of PPR are sheep and goats, with goat being
more susceptible to infection and erosive subsequent dis-
ease (Ezeokoli et al., 1986). In goats and sheep PPR and
RP viruses produce clinical disease and pathology that
are indistinguishable. However, for the diagnosis of rin-
derpest in small ruminants it is essential to differentiate it
clearly from PPR. Infection rates in sheep and goats rise
with age and the disease, which varies in severity, is
rapidly fatal in young animals (Lefevre and Diallo, 1990
and Wosu, 1994). The disease appears with a higher
incidence in the rainy season. The infection is transmitted
by close contact between infected and susceptible ani-
mals (Lefevre and Diallo, 1990).

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002 J. Gen. Mol. Virol.



Experimentally, the virus has been transmitted parante-
rally through different routes: nasal, oral, subcutaneous,
intraocular, intratracheal and intravenous or by contact
(Durtnell, 1972 and Durojaiye, 1980).
The aim of this study is to determine the pathological,
virological and serological findings in goats experiment-
tally infected with tissue culture virus and infected tissue
suspensions of PPRV. On the other hand, is to investi-
gate the possibilities of identification of PPR virus from
the samples of experimentally infected goats.


MATERIALS AND METHODS

Animals

Twenty one healthy goats of local breed aged between 4-5 months
were purchased from the local market. These animals were
grouped in 5, A, B, C, D, and E. First 4 groups contain 4 animals in
each and group E contains 5 animals as control. Goats were qua-
rantined for 7 days and kept under close observations for any signs
of the disease. All animals were free from detectable PPR anti-
bodies as judged by C-ELISA.


Collection of samples

Four Lymph nodes and spleen samples were collected from sheep
and goats suspected to be infected by PPRV. The first sample
originated from goats was collected from Khartoum State; the
second and the third originated from goats were collected from
Soba, Khartoum State and the fourth originated from sheep was
collected from River Nile State.
10 - 20% (w/v) suspensions of lymph nodes and spleen samples
were prepared by grinding with sterile sand using mortar and pestle
in PBS pH 7.4 supplemented with antibiotics. The supernatant was
used for PPRV isolation after identification by AGPT (White, 1958)
and HA test (Nussieba et al., 2008).


Virus isolation and titration

10 - 20% suspension of spleen and lymph nodes from suspected
animals were used for initial PPRV isolation which was carried in
primary lamb kidney (LK) and lamb testis (LT) cells. The 2nd pas-
sage of LK and LT cells were used for propagation of PPR virus.
For experimental inoculation of goats each PPRV isolate was adap-
ted to LT cells with a minimum of 4 passages following the tech-
nique described previously by Plowright and Ferris (1959). The
tissue culture dose end point TCID50 (50% Tissue Culture Infective
Dose) of a virus suspension was determined as described by Plow-
right and Ferris (1962). The titre was calculated by the Spearman-
Karber method (Spearman, 1908 and karber, 1931) and expressed
as log10 TCID50/ ml.


PPRV isolates

The 4th passage of PPRV Bashaier (LTC P4) and Soba (LKC P3,
LTC P4) isolates were used for inoculation of goats as tissue cul-
ture adapted virus. These isolates originated from sheep and goat,
respectively. The 3rd passage of another two PPRV isolates, Kha-
rtoum and Soba (LKC P3) originated from sheep was subjected to
further two passages in goats (Goat P5). Viruses from the 5th goat
passage were used as 20% infected tissue suspensions.




Virus propagation in goats

The procedure of the propagation of PPRV in goats was followed
as described earlier by Durtnell (1972). Two goats were inoculated
with 6 ml subcutaneously (s/c) accompanied by 1 ml intranasally
(i/n) of the 3rd virus passage. On the 12th dpi goats were slaugh-
tered and organs were collected aseptically. Spleen and lymph
nodes were prepared as 20% suspension in PBS for further pas-
sage in goats. The 5th passage of PPRV (LTC. P3 Goat P5) was
used for inoculation of goats.


Experimental inoculation of goats with PPRV isolates

Group A and B were treated with tissue culture isolates (TCV) while
group C and D were treated with infected tissue suspensions (ITS).
Goats of group A and B were inoculated with 6×10
5 . 4
TCID50/ml of
PPRV Bashaier (LTC P4) and 6×10
3 . 4
TCID50/ml PPRV Soba
(LKC P3 LTC P4) isolates, respectively. Goats of group C and D
were inoculated with 20% infected spleen and lymph nodes
suspensions of PPRV Khartoum (LTC P3 Goat P5) and PPRV
Soba (LTC P3 Goat P5) isolates, respectively. Each goat received
5 ml subcutaneously and 1 ml intranasally following a combinations
of the procedures described by Mann et al. (1974) and Bundza et
al. (1988).
Challenge experiment of immunity was carried out on the 21st dpi
4 . 5
10
TCID50/ml of virulent PPRV Sinnar strain (72/1) at a
dose of 5 ml/animal subcutaneously. Rectal temperatures were re-
corded at 8.30 am daily. Blood was collected from all the infected
goats at peak of temperature for virus isolation. Recovered goats
were slaughtered after a week of the challenged experiments and
postmortem findings were recorded. Slices of mesenteric lymph
nodes, spleen, lung, small and large intestine were collected for
histopathological examinations following the procedure of Carleton
(1967).
For determining the humoral immune response of PPRV isolates,
serum samples were collected at 7, 10, 14, 18, 21, 28 and 30 days
post inoculation. PPR C-ELISA (BDSL, 2000) was carried out to
determine the antibody titres induced by PPRV isolates.


Detection of PPRV in samples of inoculated goats

PPR viruses were detected in nasal and lacrymal swabs using
haemagglutination (HA) test as described previously by Nussieba et
al. (2008). Lacrymal and nasal swabs were collected on 6 and 7
dpi, respectively in 150 µl of PBS pH 7.2 for antigen detection. The
swab fluid was centrifuged 1 h after collection, at 3000 rpm for 20 -
30 min and then stored at -40° C.


RESULTS

Clinical response to PPR viral isolates in infected
goats

Inoculated goats developed clinical signs similar to those
of the naturally infected animals while uninoculated (con-
trol) goats remained apparently healthy. Inoculated goats
of all groups remained healthy for 3 - 4 days following
inoculation passaged tissue culture and infected tissue
suspensions PPR viruses. Clinical signs began with an
elevated temperature up to 40 - 40.6° C in infected goats
with

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Nussieba et al. 003



Table 1. Clinical observation of the goats after infection with PPRV.

Type of virus Fever Stomatitis Pneumonia Diarrhoea Ocular
involvement
6 G
75%
6 G
75%
Nasal
involvement
8 G
100%
8 G
100%
Labial
scabs
3 G
37.5%
2 G
25%
Death
TCV1
%
ITS2
%
8 G
100%
8 G
100%
3 G
37.5%
2 G
25%
4 G
50%
3 G
37.5%
4 G
50%
4 G
50%
2 G
25%
0
0%




Table 2. Days of the onset of clinical signs in goats infected with PPRV.

Experiment
No.
virus period
Days of onset of
Nasal
excretion
5th
5th
Type of Incubation
Viraemia Lacrymal
excretion
5th
5th
Mouth
lesions
7th
7th
Diarrhoea
Days killed (k)
or dead (D)
Exp. 1
Exp. 2
TCV1
ITS2
5 - 7th
5 - 7th
5 - 6th
5 - 6th
6 - 13th
9 - 13th
16 and 19th
?

Notes:
TCV1 : Tissue culture virus.
ITS2 : Infected tissue suspension.



while animals in the control group showed no thermal
response. Pyrexia was detected in all experimentally
infected goats on the 5 - 7th dpi. Goats developed a very
slight superficial necrosis of the lips on the 7th day. On the
6th day, the fever was sustained. On the 5th day, serous
nasal and lacrymal dischargesappeared. Serous nasal
and lacrymal discharges involved all the 16 goats (100%)
on the 7th day. During this phase animals in general were
dull, depressed and anorexic with congested mucous
membranes. A cough was usually noticed early in the
disease on the 8th and the 9th day. The respiration was
usually fast and shallow. On the 9th day, conjunctivitis
appeared and involved goats on the 13th day. Mucoid
nasal discharges were observed in goats on the 13th day
and on the 15th day.
Diarrhoea with abdominal pain was a common feature
of the disease and it occurred on the 9th day following the
onset of fever. Goats showed evidence of diarrhoea on
the 13th day. The signs persisted for 6 days and the
animals become progressively weaker.
Between the 11 and 13th days, the fever regressed and
the oral and the encrusted lip lesions began to resolve.
The nasal discharges and crust formation occurred while
the oral lesions extended to cross the muco-cutaneous
portion of the lip with scab formation at mouth com-
missure at the 12 and 15th day respectively. Death was
usually preceded by severe emaciation, dehydration,
subnormal temperature and collapse. Two goats (25%) of
group (A) which were inoculated with PPRV Bashaier
isolate died on day 16 and 19th post inoculation res-
pectively. The incidence of the different clinical signs is
shown in Table 1 and the onset of the disease is
summarized in Table 2.
Immune response to PPR viral isolates in infected
goats

Serum samples were collected from goats experimentally
inoculated with PPRV at 7, 10, 14, 18, 21, 28 and 30th dpi
(Figure 1) and examined using C-ELISA to detect anti-
bodies against PPRV. 4 sera (25%) out of the 16 goats
experimentally inoculated with PPRV showed detectable
antibodies at 7 dpi while all the goats (100%) showed
detectable antibodies at 10 dpi. The titre of antibodies
induced by PPRV started from weak positive (PI 51 -
70%) at 7 dpi to moderate (PI 73 - 82%) and strong posi-
tive (PI 85-90%) at the following days till 21 dpi. Following
challenge of experimental animals at 21 dpi, a rise in
antibodies titre was observed at 28 dpi. In 30 dpi the anti-
body titres were less than in 28 dpi.


Detection of PPRV antigen in samples from inocu-
lated goats

The HA titres of PPRV antigen detected in nasal and
lacrymal swabs of experimentally infected goats ranged
between 4 and 16.


Post-mortem examination of inoculated goats

Gross pathology: The most characteristic post mortem
lesions were found in the gastrointestinal and respiratory
tract. There was evidence of emaciation in all the inocu-
lated goats (100%). In some goats of the two groups the
rumen, reticulum, omasum and abomasum were filled
with foetid watery fluid. The small intestine showed evi-
dence of severe inflammation. The small intestine was

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004 J. Gen. Mol. Virol.











7 10 14 18 21 28 30
50
55
60
65
70
75
80
85
90
95
100
Days post infection
PI values


Figure 1. PI values for PPRV antibodies.





Figure 2a. Lung: note proliferation of bronchiolar epithelium,
intense diffusion of mononuclear cells. H and E X10.




Figure 2b. Lung: note interlobular proliferation of fibrous tissue
(white arrows). H&E X10.


filled with watery fluid as found in the abomasum. The
colon and caecum showed evidence of linear haemor-
rhage and areas of ulcerations. Lymph nodes were
engorged and oedematous. Spleen was enlarged and
oedematous. In the respiratory system, the trachea show-
ed evidence of inflammation and filled with frothy exu-
dates. Pneumonia was usually observed in a few lung
lobes. The most involved was the right apical lobe and


Figure 3a. Lymph node: note oedema (white arrows). H and
E X10.


the less involved were the intermediate and cardiac
lobes. Post mortem examination showed evidence of
pneumonia, congestion of the lung lobes and hydro-
thorax. Fatty change in kidneys was observed.


Histopathology

The lung showed bronchiointerstitial pneumonia charac-
terized by proliferation of bronchiolar lining epithelium,
intense diffusion of mononuclear cells mainly lymphoid,
macrophages and plasma cells in the periductal, the
interstitial tissue and alveoli lumina (Figure 2a). Areas
of scarring were seen in interlobular connective tissue
(Figure 2b). In lymph nodes, there was oedema in the
cortical and medulla (Figure 3a) and infiltration of mono-
nuclear cells and some giant cells in subcapsular areas
and medullary sinuses (Figure 3b). The spleen showed
haemorrhage and haemosidren pigment deposition (Fi-
gure 4a and 4b). The intestine showed atrophic villi with
partial denudation of epithelial lining and intense diffusion
of mononuclear cells in the lamina propria and submu-
cosa (Figure 5).

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Figure 3b. Lymph node: note proliferation of mononuclear
and giant cells (white and yellow arrows). H and E X40.





Figure 4a. Spleen: note accumulation of extravagated
erythrocytes. H and E X10.




Figure 4b. Spleen: note accumulation of extravasated
erythrocytes. H and E X40



DISCUSSION

The examination of nasal and lacrymal swabs from
goats experimentally inoculated with PPRV by HA test
resulted in agglutination of RBCs. Chicken RBCs were
used for detection of PPRV in swabs depending on the
highest sensitivity of the RBCs of this species upon the
others. The HA test of swab samples resulted in slightly
low titres ranging from 4 to 16. This result was in agree-
Nussieba et al. 005





Figure 5. Small Intestine: note atrophic villi, denudation of
lining epithelium and intense infiltration of mononuclear cells.
H and E X10.


ment with Wosu (1991) who documented that the HA titre
was not a reflection of the concentration of the virus in
secretions, but rather a reflection of the degree of dilution
of the virus in the secretion with the diluents. The
obtained HA titres were indication of the shedding of PPR
virus in nasal and lachrymal swabs. This result was simi-
lar to that mentioned earlier by Abegunde and Adu (1977)
whom detected the existence of PPRV in nasal and
conjunctival secretions. Virus was confirmed in ocular
and nasal swabs at the onset of clinical signs which is the
most important epidemiological aspect in spread of the
disease.
Of the 16 infected goats, 8 were inoculated with tissue
culture propagated PPRV and the other 8 were inocu-
lated with infected tissue suspension. The experimental
infection of goats with PPRV revealed observable clinical
signs and postmortem lesions in inoculated animal. This
infection was accompanied with morbidity and mortality
rates reaching 100% and 12.5% respectively. Serological
studies indicated the presence of detectable antibodies
against PPRV. Following challenge of inoculated animals,
with virulent PPRV which was carried out at day 21 p.i.,
goats did not show rise in body temperature or any signs
of the disease. From this result it was obvious that experi-
mental infection with PPRV resulted in high percentage of
morbidity but low percentage of mortality in contrast to
natural PPRV infection. This is in agreement with Elhag
Ali (1973) and Mann et al. (1974) whom found that ani-
mals experimentally infected with PPRV develop mild
form of the disease. Although they demonstrated the
appearance of the disease in experimentally infected ani-
mals, Mann et al. (1974) suggested that the successful
transmission of the acute disease may require more than
one challenge. Moreover some authors suggested that a
more severe disease results from mixed infection of bac-
teria and viruses than a single infection. This substan-
tiated the result of Onoviran et al. (1984) who reported
that a combined infection of Mycoplasma capri and PPR
was found to be much more severe in goats than infec-
tion by a single agent. Nutritional and environmental
factors have an important effect on the appearance of the

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disease in a flock of animals. On the other hand, Saliki
(1998) previously reported that poor nutrition status,
stress of movement and concurrent parasitic and bac-
terial infections enhance the severity of clinical signs.
Examination of serum samples from goats infected with
PPRV showed detectable antibodies at the 7th day and
the 10th day p.i. with increase in the antibody titre at the
following days. The detectable PPR antibodies were
indicative of the humoral immune response due to the
exposure of animals to the virus. This observation was in
concordance with those reported by Taylor (1979).


Conclusion

The observed clinical signs, post mortem lesions and the
detectable antibodies indicated that PPRV in the form of
tissue culture propagated viruses or infected tissue
homogenate were proved effective for initiation of infec-
tion.


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