ELISA Antibody Test, PCR and a DNA Vaccine for Use with Avian Malaria in African Penguins
Abstract
Avian malaria is the highest cause of mortality in captive penguins housed outdoors.1
There are three aspects of the epidemiology of this disease which can be manipulated to reduce its effects:
Reduce or eliminate the
reservoir of Plasmodium relictum and elongatum,
Reduce or eliminate the
vector, and
Manipulate the host to produce
a more favorable outcome.
Since it is impossible and undesirable in an outdoor setting to eliminate either the reservoir
(native birds) or the vector (Culex and Aedes sp. mosquitoes), one must concentrate on manipulating the
host. To increase the advantages of the host in the equilibrium between host and parasite one can investigate better
diagnostics, treatments, and preventive measures.
The weekly examination of thin blood smears from susceptible birds and treatment with primaquine and
chloroquine when parasitemias are detected has reduced the mortality from 50% to 10-15%. This improvement comes with a
great labor intensive effort and 15% mortality is still undesirable.
An ELISA test was developed to measure circulating antibodies to P. relictum and P.
elongatum utilizing the antigen R32 tet32 from human malaria P. falciparum. It was found that antibody levels
did not correlate with parasitemias or with a safely "protected" bird. The test was not a good diagnostic tool to
indicate when to treat for malaria or when malaria monitoring could safely be stopped for an individual. Since malaria
infections are a life-long event in penguins, the ELISA test is effective for, a) surveys for detecting exposure of the
host to malaria, and b) measuring the effects of vaccination.2,3
A polymerase chain reaction test for Plasmodium was developed to detect parasitemias. It was
found by PCR that all penguins exposed to malaria will turn parasitemic. Approximately 50% of these birds will have
parasitemias at a level detectable by thin blood smears. If parasitemias as detected by PCR were the indication for
treatment, then all birds would be treated very early. This would mean treating 50% of the birds unnecessarily, and
perhaps this early treatment would lessen the degree of immunity obtained. The PCR test is useful to determine whether a
vaccine produces sterile immunity and to detect the prevalence of malaria in mosquitoes. It has been shown that the
prevalence of malaria in the vector is much higher than the 1% previously reported, and it is in a dynamic state with
P. relictum and P. elongatum, waxing and waning in prevalence independent of each other.4
A DNA vaccine was produced utilizing sequences of the circumsporozoite gene of P. relictum and P.
elongatum. The penguins were vaccinated by injecting the vaccine intradermally above the eyes, and intramuscularly in
each gastrocnemius muscle. They were housed in a mosquito-free environment until given booster injections 3-4 wk later.
The unvaccinated birds were injected identically with equal amount of 0.9% NaCl.
In the first 2 yr parasitemias were seen in both vaccinated and unvaccinated birds by PCR, but for
the first time since the colony was established, there were no parasitemias seen on thin blood smear (Table 1). The
vaccine produced antibody levels in vaccinated birds that were boostered by natural exposure. It is known that the birds
were challenged as in other years by the development of antibodies in the unvaccinated birds when released.
The curious element in this study is the lack of parasitemias in the unvaccinated birds. A
fluorescent antibody test was developed to specifically detect sporozoite antigen reacted positively not only to the
sporozoite, but to a subset of erythrocytic stages. Since it is known that mosquitoes with P. relictum and P.
elongatum are in the area with penguins as early as April, but penguin deaths due to malaria are clustered in July
and August, it is felt the transmission from penguin to penguin is important for pathogenicity. The production of
antibodies against the sporozoite vaccine would also attach to similar antigen sequences in the gametocyte, possibly
creating a transmission blocking mechanism and a herd immunity effect, although not every individual is immunized.
Table 1. Comparison of 3 yr of non-manipulated birds to 3 yr of vaccinated birds.
|
Year |
1 |
2 |
3 |
4 |
5 |
6 |
|
Total malaria deaths |
4 |
3 |
1 |
0 |
0 |
1 |
|
No. of naïve birds (plus manipulated) |
28 |
29 |
14 |
2(6) |
6(6) |
17(8) |
|
No. of parasitemic birds |
16 |
21 |
7 |
0 |
0 |
1 |
|
No. of parasitemias |
29 |
36 |
11 |
0 |
0 |
2 |
|
No. deaths in naïve group
(plus manipulated) |
3 |
3 |
0 |
0 |
0 |
0(1)a |
|
Manipulation |
None |
None |
None |
DNA
vaccine |
DNA
vaccine |
DNA
vaccine |
a. Vaccinated bird died of malaria and aspergillosis, the bird was probably immunosuppressed.
References
1. Cranfield MR, MR Shaw, F Beall, M Skjoldager, D Ialeggio. 1990. A review and update of
avian malaria in the African penguin (Spheniscus demersus). Proc. AAZV South Padre Island. 243-248.
2. Graczyk TK, MR Cranfield, ML Skjoldager, ML Shaw. 1994. An ELISA for detecting anti
Plasmodium spp. antibodies in African black-footed penguins(Spheniscus demersus). J. Parasitol.
80(1): 60-66.
3. Graczyk TK, MR Cranfield, JJ Brassy, J Cockrem, JK Jouventin, PJ Geddon. 1995. Detection
of avian malaria infections in wild and captive penguins. J. Helminthol. 62(2): 135-141.
4. McConkey GA, J Li, MJ Rogers, DC Seeley, TK Graczyk, MR Cranfield, TF McCutchan. 1996.
Surveillance of mosquitoes for the malaria parasite responsible for mortality in captive penguins. J. Eucariotic
Microbiol. 43: 393-399.