Canine distemper antibodies in lions of the Masai Mara

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Canine distemper antibodies in lions of the Masai Mara
R. Kock, W. S. K. Chalmers, J. Mwanzia, C. Chillingworth, J. Wambua, P. G. Coleman, W. Baxendale
Veterinary Record (1998) 142, 662-665
Canine distemper virus (CDV) has been implicated in some
recent deaths of lions, which showed clinical signs of distemper, in the the Serengeti plain. Similar clinical findings have
since been reported in lions of the Masai Mara. Fifty-five per
cent of serum samples obtained from wild lions of the Masai
Mara have been found to contain neutralising antibody to
CDV, indicating that they had been exposed to the virus. Adult
orphan lions kept in captivity, were vaccinated with the live
attenuated Onderstepoort strain of CDV. The results indicated
that the vaccine is both safe and immunogenic, and may be
potentially useful for the prophylactic vaccination of lions at
high risk.
CANINE distemper virus (CDV) is a member of the
Morbilliviridae and a potentially fatal disease of Canidae,
although it has a wider host range under natural and experimental
conditions (Appel and Gillespie 1972). In 1994, CDV infections
were responsible for the deaths of a significant proportion of the
wild lion (Panthero leo) population in the Serengeti plain in
northern Tanzania (Spencer 1995). Seventy-one of 83 (85 per
cent) of the blood samples taken from the lions were found to be
CDV antibody-positive, and viral antigen was obtained from two
lions (Roelke-Parker and others 1996).
Barrett and others (1993) have found that the phosphoprotein
(P) gene sequence varies between moribilliviruses and they have
used this property to distinguish differences or show similarities
between isolates. The differences in the (P) gene sequence are
also useful for determining phylogenetic relationships. The (P)
gene fragment of the morbillivirus isolated from the diseased lions
was amplified by polymerase chain reaction (PCR) and analysed
and it was concluded that the isolate was more similar to CDV than
to the other morbilliviruses (Harder and others 1995). One
hypothesis to account for CDV infection in lions is that the virus
crossed the species barrier from the domestic dog population, of
which approximately 30,000 are reported to live in close proximity to the Serengeti, and in which CDV has previously been recorded (Roelke-Parker and others 1996). The role of hyaenas, jackals,
and other susceptible species in the spread of the disease is however still uncertain, although CDV was isolated from a bat-eared
fox (Otocycon megalotis) and a spotted hyaena (Crocuta crocuta)
during the 1994 Serengeti outbreak (Roelke-Parker and others
1996).
There are conflicting theories about how to prevent or reduce
disease conditions in wildlife. Harder and others (1995) have suggested that the best strategy would be to vaccinate the surrounding
domestic dog population, the suspected reservoir of the infection,
with CDV vaccines which have been shown to be safe. Their main
concern was that some attenuated vaccines might induce CDV disease in susceptible wild carnivores, a concern also voiced by
Morrell (1994). Conversely, Appel and others (1994), reporting
on deaths due to CDV in exotic Felidae in North American zoos
and wildlife parks, proposed that attenuated chick embryo-adapted
vaccines could be used once their safety had been demonstrated in
wildlife.
The Masai Mara in Kenya and the Serengeti plain in Tanzania
form one continuous ecosystem divided only by political boundaries which are recognised by neither animals nor pathogens.
There is a constant exchange of wildlife between the two areas.
This paper presents the results of a small serological survey of
CDV antibodies in serum samples from lions of the Masai Mara
and describes the responses of lions inoculated with a live attenuated CDV vaccine.
Materials and methods
Serum samples
Between October 1994 and February 1995, 55 wild lions (cubs
less than one year old to adults over 10 years old; 27 males and 28
females) from several prides in the Masai Mara National Reserve
and adjacent areas which form part of the Serengeti-Masai Mara
ecosystem were bled by the Kenya Wildlife Services. Whenever
possible, estimates of age based on the size of the lion and signs
of tooth wear were made by the attending veterinarian. The samples were examined for antibodies to CDV because of earlier confirmed cases in Serengeti lions (Roelke-Parker and others 1996)
and reports from the Masai Mara of sick lions showing the characteristic signs of distemper. For comparison, seven serum samples
from six lions and one leopard (Panthera pardus) were obtained
from other regions of Kenya. All the samples were heat inactivated at 56°C for 30 minutes before they were imported into the UK.
Serum neutralisation test
R. Kock, MA, VetMB, MRCVS, J. Wambua, BVM, J. Mwanzia, BVM,
MVPH, Veterinary Unit, Kenya Wildlife Services, Nairobi, Kenya
W. S. K. Chalmers, BSc, PhD, MIBiol, C. Chillingworth, W. Baxendale,
BVM&S, MRCVS, Intervet (UK) Ltd, The Elms, Houghton, Cambridgeshire
PE17 2BQ
P. G. Coleman, BA, London School of Hygiene and Tropical Medicine,
Keppel Street, London WC1E 7HT
All the sera were diluted successively four-fold, from an initial
dilution of 1/4 to 1/256 in tissue culture medium. A double
strength suspension of neutralising antigen (CDV-Bussel strain),
containing 200 to 600 plaque forming units/ml, was also prepared
in tissue culture medium. Equal volumes of the various dilutions
of sera and neutralising antigen were mixed together to give final
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The Veterinary Record, June 13, 1998
663
16
14
12
>
0
1/8
ctofpoint
.0 10
E
6
z 4
<4
4
8
16
32
64
128
256
512
1024
>1024
Antibody titre (reciprocal)
FIG 2: Distribution of plaque reduction-serum neutralising antibody
titres to canine distemper virus in 55 lions. Only titres greater than
1/8 were considered true positives because some sera may show nonspecific neutralisation at low dilutions
Vaccine
The CDV (Onderstepoort strain) component of Nobivac vaccines
(Intervet) was prepared as a monovalent vaccine (Nobivac D)
containing at least 1050 TCID50 per dose and sent to Kenya
Wildlife Services for inoculation into four orphan adult lions. The
same dose of the vaccine was initially tested in domestic cats to
determine its safety before the orphan lions were inoculated.
Legend:
* Positives only
A Negatives only
* Positives anid negatives frozn the samne pride
Masai Mara Nationial Reserve boundary
SSerengeti National Park boundary
International border
....
FIG 1: Distribution of serum samples collected from lions in Masai
Mara National Reserve and adjacent area between October 1994 and
February 1995
Vaccination
Cats. One dose of Nobivac D vaccine was inoculated into each
of eight domestic cats, approximately nine months old, which
were then kept for three weeks before testing for antibody
response (SN5o) against CDV. The cats were bled before and three
weeks after they were vaccinated.
-
Lions. One dose of Nobivac D vaccine was inoculated into each
of four orphan adult lions which were kept in separate compounds
together with in-contact lions which-were monitored to detect any
potential spread of the vaccinal virus. The four vaccinated lions
and in-contacts (IC) were as follows: Sheru (IC with Irene);
Malaika (IC with George); Joy (IC with Singh); Pascal did not
have an in-contact lion in the same compound but he was separated from Joy and Singh only by a wire fence through which contact
could be made; Singh therefore doubled as the in-contact for both
lions. Blood samples were taken before and three weeks after the
vaccination to measure the antibody response (SN50) to CDV.
-
dilutions of 1/8 to 1/512, and the mixture was incubated at 37°C
for one hour for neutralisation to occur. Vero cell monolayers in
5 cm petri dishes (three plates per dilution) were each inoculated
with 200 VI aliquots of serum/antigen suspension. The plates were
then incubated at 37°C for one hour to allow absorption to occur,
then overlaid with a 1 per cent agar tissue culture medium and reincubated for five days for viral plaque development. After five
days, the agar was removed, the cell monolayer stained with
Naphthalene Black and the plaques were counted. Serum samples
with antibody titres (SN50) .1/8 were scored as CDV antibody-positive.
Results
Serological survey of wild lions, October 1994 to February 1995
Statistical analysis
The proportion of seropositive lions was compared with the
seroprevalence reported from the Serengeti (Roelke-Parker and
others 1996) by using a X2 test. The same method was used to
compare the difference in seroprevalence between the male and
female lions.
To investigate the variation with age in the proportion of
seropositive lions it was necessary to control for any sex difference. A multivariate analysis of variance was therefore carried out
by using the statistical software package GLIM (Crawley 1993).
The appropriate link function (logistic) and error structure (binomial) for the analysis of proportional data were specified. Any
non-significant variables (P>0-05) were removed from the statistical model. Consecutive yearly age classes were then grouped
together, provided there was no significant impact on the fit of the
model, to produce a minimal adequate model. The absence of bias
in the seroprevalence of the lions whose ages had been estimated
compared with those for which no estimates of age were available
was confirmed by using a x2 test.
The spatial distribution of the serological results is shown in
Fig 1. A total of 30 (55 per cent) of the 55 samples contained neutralising antibodies against CDV, with the titres ranging from >1/8
to >1/1024 (Fig 2). This seroprevalence was significantly less than
the 85 per cent recorded in Serengeti lions (P<0-001).
Although the sample population was relatively small, the
results indicated a significant difference in seroprevalence
between the sexes (P<0.001) with considerably more positive
males (21 of 27) than females (nine of 28).
The age to the nearest year was estimated for 38 (69 per cent)
of the 55 lions sampled. The seroprevalence in these lions was 53
per cent (20 of 38) and was not significantly different from the 59
per cent (10 of 17) recorded in the lions whose ages were not
known. Seropositive lions were detected in all age groups ranging
from a cub (aged between three and 12 months) to 10-year-old
adult lions. Both sex (P<0-001) and age (P<0.05) were significant
explanatory variables for the seroprevalence. The most parsimonious grouping of age was into two classes, less than five years of
age and five years old or over, corresponding to animals bom after
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The Veterinary Record, June 13, 1998
664
TABLE 1: Antibody responses of lions after vaccination with attenuated
canine distemper virus
11/12
5/7
08
0
c
>
14/18
T
-
06 +
!
O0 4j
6/20
Status
Sheru
Irene
Malaika
George (IC)
Pascal
Joy
Singh (IC)
Vaccinated
Unvaccinated
Vaccinated
Unvaccinated
Vaccinated
Vaccinated
Unvaccinated
Prevaccination titre*
Postvaccination titre
(SN50)
(SN50)
<1/4
<1/4
<1/4
<1/4
<1/4
<1/4
<1/4
.1/1024
<1/4
.1/1024
Not available
>1/1024
.1/1024
<1/4
*Plaque reduction - serum neutralisation antibody responses as reciprocal
titres
IC In-contact animals
Pascal was in an adjacent pen to Joy and Singh, with contact through the
wire. George died of unrelated causes before the second blood sampling
02
__,
Age group (years)
Lion
>5
FIG 3: Seroprevalence of antibodies against canine distemper virus in
38 lions sampled between October 1994 and February 1993. For the
two age groups, < 4 years of age and 5 years of age, the seroprevalence and standard error bars are shown for all the lions (empty
bars), and the male (shaded bars) and female (filled bars) lions
1990 and those born in 1990 or before. The aggregation of age
classes was achieved without any significant change to the fit of
the model. The seroprevalence in the older lions was greater than
in the younger lions. In both age groups, the seroprevalence in
male lions exceeded that in females. The level of difference
between the sexes did not differ significantly with age. The data
are summarised in Fig 3.
The only leopard tested came from the Naivasha area of central
Kenya and was found to have a high CDV neutralising antibody
titre of >1/1024. The six lion sera obtained from outside the Masai
Mara were seronegative (<1/8).
Serological response of cats to CDV vaccination
A moderate, but protective, neutralising antibody response
ranging in titre from 1/16 to 1/256 (mean titre 1/58) was detected
in six of the eight vaccinated cats; the other two cats had titres
below 1/2. All the cats remained clinically normal throughout the
study.
Serological response of wild lions (orphaned and in captivity)
CDV vaccination
to
All the vaccinated lions responded within three weeks with
high titres of antibody (.1/1024) without any adverse clinical
effects (Table 1). The in-contact lions remained seronegative
throughout the study and showed no clinical effects, indicating
that no distemper virus had been transmitted from lion to lion. The
in-contact lion kept in the same compound as Malaika, died from
non-CDV related causes. Singh, an in-contact lion, remained seronegative when bled two years later.
Discussion
Serological survey
The positive CDV antibody titre in a lion cub aged between three
and 12 months, indicated that the disease had been transmitted
recently in the Masai Mara lion population, because the cub was
too old to have significant levels of maternally derived antibodies.
This finding, together with other more anecdotal evidence, is consistent with the theory that the CDV epidemic reported in the lions
of the Serengeti moved northwards and infected the lion population in the Masai Mara (Roelke-Parker and others 1996). The
absence of any reports of CDV outbreaks in the domestic dog pop-
ulation close to the Masai Mara since 1990 (Alexander and Appel
1994) also provides support for this theory. The first lions in the
Masai Mara to show distemper-like signs were recorded close to
the border with Tanzania, and when there was active disease in
the Serengeti lions. The Serengeti and Masai Mara are part of the
same ecosystem with no natural barriers which could effectively
block the progression of infectious disease. Lions cross between
the Masai Mara and Serengeti as part of their natural home range.
The timing of the first anecdotal reports of CDV infection in the
Masai Mara, around August 1994, also suggests that the movement of nomadic male lions, following the wildebeest migration
from Tanzania to Kenya, may have been important in the spread
of the epidemic.
Seropositive lions were recorded throughout the Masai Mara
and adjacent area, reaching as far as Aitong, the northern limit of
the Serengeti-Masai Mara ecosystem. However, as the Aitong
lions were the only prides sampled immediately outside the Masai
Mara, the full range of the exposure of lions to CDV is not known.
The six other lions sampled from other regions of Kenya were all
CDV-negative. The range of other wildlife species in the Masai
Mara which may have been infected during the CDV epidemic has
not been investigated. The one seropositive leopard was unlikely
to have been exposed to CDV as part of the Serengeti-Masai Mara
epidemic because it came from Naivasha, approximately 150 km
north east of Aitong. Leopards are known to prey on domestic
dogs, and around Naivasha there are domestic dog populations in
which CDV may be present.
Unfortunately, unlike the Serengeti, no previous serological
data are available from the Masai Mara from which the CDV status
of its lion population before August 1994 can be assessed. The
results suggest a 'step-like' age seroprevalence, with lions alive in
1990 having a significantly higher seroprevalence than lions born
after 1-990. As the antibody response to CDV lasts many years, the
pattern of seroprevalence with age may be due either to previous
exposure to infection, or to the variation in infection with age during a recent outbreak. More specifically, the age seroprevalence
may be explained by the differential transmission of CDV with age
during the 1994 epidemic, or by the differential age mortality
rates due to CDV infection in 1994, or by the age-constant CDV
infection during two epidemics, one starting in 1990 and the other
starting in 1994.
The results from the Serengeti, however, are not consistent with
the first two explanations. In the Serengeti lion population, CDV
infection and increased mortality were reported among all ageclasses equally (Roelke-Parker and others 1996). In the Serengeti,
there have been long-term research studies of the lion population,
but there have been no comparable studies with the Masai Mara.
As a result, it was not possible to estimate increased disease-related mortality rates. However, there is no reason to believe that the
progression of disease in the Masai Mara lions would have been
different from that observed in the Serengeti.
The possibility of previous exposure to distemper infection is,
however, consistent with the last- reported CDV epidwaeiic in the
area which started in late 1990 in the domestic dog population
surrounding the Masai Mara (Alexander and Appel 1994). An ear-
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The Veterinary Record, June 13, 1998
665
lier serological survey by Alexander and Appel (1994) suggested
that during this 1990 epidemic, CDV was transmitted from the
domestic dog population to other wild carnivores in the region,
including wild dogs (Lycaon pictus) and hyaenas. In Tanzania, a
retrospective analysis of serum samples collected in the 1980s,
indicates that lions in the Serengeti ecosystem had previously
been exposed to CDV without an increase in disease-related mortality (Roelke-Parker and others 1996). It therefore seems plausible that during the 1990 CDV epidemic in the Masai Mara region,
the lion population may have been exposed to CDV either directly
from domestic dogs or from other wild carnivores, such as hyaenas. Assuming age-constant infection and disease-related mortality rates, the observed cross-sectional seroprevalence data may
therefore be explained by a CDV outbreak in 1990, followed by a
more recent epidemic starting in 1994.
The significant male bias in seroprevalence in the Masai Mara
is different from the Serengeti, where there were no differences in
the proportions of the sexes which were seropositive. There is no
clear explanation for this sex difference in seroprevalence.
The results of the serological survey and the findings from
Tanzania, support the theory that the recent CDV epidemic spread
through the lion populations of the Serengeti-Masai Mara ecosystems. The higher seroprevalence in animals alive at the time of the
last known CDV epidemic in the Masai Mara region is consistent
with the previous exposure of Masai Mara lions to CDV.
Vaccination
The experimental inoculation of domestic cats with canine distemper virus has produced no evidence of typical clinical signs of
CDV, the only evidence of infection being found by the histopathological examination of infected tissue (Appel and others 1974).
The Onderstepoort strain of CDV (Nobivac D; Intervet) passaged
in tissue culture cells, has been shown to produce good antibody
responses in fox, mink and ferret without spreading or being disseminated or causing adverse clinical reactions (W. S. K.
Chalmers, unpublished observations). The results of this small
study have shown that vaccinal virus will replicate and stimulate
an antibody response in the majority of vaccinated cats. However,
the vaccinal strain was more efficacious in wild lions in that the
take rate was 100 per cent, it did not spread to in-contact lions, it
produced much higher protective neutralising antibody titres, and
no adverse clinical signs were recorded.
It has been proposed that inactivated vaccines should be used to
prevent infection because it is believed that attenuated vaccines
may produce distemper in susceptible carnivores. However, the
neutralising antibodies produced by inactivated vaccines are often
poor and short-lived compared with those stimulated by live attenuated vaccines. It is not suggested that the lion population in the
Masai Mara-Serengeti should be vaccinated, first because it would
be impractical, and secondly because the population is large
enough to recover without intervention. Ring vaccination of the
domestic dogs which are the main reservoir of infection may be
feasible, because these dogs can be collected in the surrounding
Masai villages for sampling, vaccination and monitoring over a
defined period. The results of this study have shown that lions
respond well to vaccination without adverse clinical reactions;
vaccination with a safe, live, attenuated CDV vaccine strain may
therefore still be an option in small isolated populations of wild or
captive lions when there is need to protect individual lions against
CDV disease.
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Suspected bacterial meningoencephalitis in two adult horses
S. A. Newton
Veterinary Record (1998) 142, 665-669
Bacterial infections (such as meningitis or meningoencephalitis) of the central nervous system are rare in horses. They are
most prevalent in neonates as a result of septicaemia. A few
cases have been reported in the adult and most have been
fatal. Streptococcal species appear to be the organism most
commonly identified in these cases. Thus, this disease may be
a secondary complication of upper respiratory tract infections. Clinical signs are extremely variable making diagnosis
difficult. In most cases, postmortem has been the definite diagnostic procedure. This paper describes the clinical course of
disease, diagnosis and successful treatment of two presumptive cases of meningoencephalitis in adult horses.
S. A. Newton, BVSc, CertEM (IntMed), MRCVS, Division of Equine Studies,
University of Liverpool, Leahurst, Neston, South Wirral L64 7TE
INFECTIOUS agents incriminated in equine neurological disease
include viruses, bacteria, protozoa, helminths and fungi (Seiler
and others 1981, Steckel and others 1982, Emmons and others
1983, Burgess and Mattison 1987). Bacterial infections of the central nervous system (CNS) of adult horses have rarely been reported and are generally fatal (Mayhew and Mackay 1982, Foreman
and Santschi 1989). They can occur in all ages and breeds but are
more prevalent in neonates when they are most often secondary to
septicaemia (Mackay and Mayhew 1991). Beta-haemolytic streptococci are probably the most commonly isolated bacteria, and
meningoencephalitis can be a complication of strangles or respiratory disease (Ford and Lokai 1980, Smith and others 1987,
Sweeney and others 1987, Johnston 1994).
The clinical signs are diverse because the nervous system, like
most other body systems, responds to insult in limited ways
(Smith and others 1987). Diagnosis can therefore be difficult but
if treatment is delayed until overt clinical signs are evident, the
prognosis is poor (Foreman and Santschi 1989).
Downloaded from veterinaryrecord.bmj.com on September 23, 2014 - Published by group.bmj.com
Canine distemper antibodies in lions of the
Masai Mara
R. Kock, W. S. K. Chalmers, J. Mwanzia, et al.
Veterinary Record 1998 142: 662-665
doi: 10.1136/vr.142.24.662
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