ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 62 - No. 3-4 2007
ANTIGENIC CHARACTERIZATION OF MICROFILARIAL AND ADULT
ANTIGENS OF ONCHOCERCA CERVICALIS
Marques SMTa and Scroferneker MLb
a. School of Veterinary Medicine of Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio
Grande do Sul, Brazil
b. Institute of Basic Health Sciences of Universidade Federal do Rio Grande do Sul ,Porto Alegre, Rio
Grande do Sul, Brazil.
Correspondence to :Sandra Márcia Tietz Marques Rua Aneron Corrêa de Oliveira, 74/201 Porto
Alegre, RS, Brasil CEP: 91410-070 Fax: +55 51 3316 3155
Email : sandra.tietz@terra.com.br scrofern@ufrgs.br
Keywords :Onchocerca cervicalis; antigen; antibody; immunoelectrophoresis;
immunodiffusion; microfilariae.
ABSTRACT
Antigens of adult parasites and microfilariae of Onchocerca cervicalis were analyzed
by double diffusion (DD) and immunoelectrophoresis (IEP) using the sera of rabbits
immunized with these antigens and the serum of horses infected with O. cervicalis.
Both groups of antigens reacted with the horse serum and the anti-O. cervicalis serum.
In DD, the microfilarial antigen displayed 2 reactive bands with the antiserum. In IEF,
the microfilarial antigen showed four reactive bands, while the antigen from the adult
parasite presented 12 bands.
INTRODUCTION
The filarial parasite Onchocerca is the causative agent of onchocerciasis, affecting cattle,
horses, and humans. O. cervicalis is a filarial nematode of the horse with a worldwide
distribution and high prevalence as reported by Fernandes (1), Collobert et al ,)2( .
Monahan et al. (3), Bradley et al. (4), Lyons et al. (5), and Marques and Scroferneker (6). The
adults reside in the ligamentum nuchae and microfilariae (mf) are found in the dermis,
especially in the ventral midline and thorax, withers and eyelids.
Onchocerciasis is an important disease, especially of humans (7),and is endemic in Africa and
Latin America. In Brazil, cases of human onchocerciasis were reported in several states,
where new foci were detected (8, 9, 10 .)Impressive progress has been made in many areas
toward the elimination of the disease by means of vector control and mass distribution of
ivermectin. Control efforts have recently been expanded with implementation of the African
Program for Onchocerciasis Control (APOC )and Onchocerciasis Elimination Program in the
Americas (OEPA). Improved methods are needed for monitoring the success of such control
programs over time and for assessing the efficacy of drug treatment in patients with
onchocerciasis (11.)
Several diagnostic methods have been used, such as skin snips and palpation of nodules, in
addition to immunologic methods (11, 12, 13, 14). In equine onchocerciasis, diagnosis is
based on the detection of adult parasitic forms and microfilariae. Although these methods
are specific to active infection, they are not sensitive for the detection of early infections or
for monitoring the efficacy of control programs. The adult parasite can only be investigated
in a dead animal .Without microfilaremia, clinical diagnosis can be uncertain. To overcome
this difficulty, an intense search is ongoing for an immunodiagnostic method capable of
reliably revealing prepatent or occult infections in mammals and human hosts.
There are two principal advantages associated with the diagnostic use of filarial
excretory/secretory (E/S) antigens ,particularly those antigens expressed by developing
larvae and adult worms of both sexes. Unlike somatic antigens, E/S products are continually
released by living worms and may thus induce high antibody titers, while antibodies to E/S
antigens expressed by worms to both sexes may be detectable in microfilaremic infections
regardless of whether this condition is due to prepatent, postpatent ,or single-sex infections
(15, 16, 17). The fractionation and purification of Onchocerca sp antigens are important in
that they permit standardization and yield reliable, reproducible, and comparable results in
several immunologic techniques (18.)
The present study is aimed to assess the partial characterization and antigenic
homogeneity of microfilariae and adult worms of Onchocerca cervicalis by double
diffusion (DD) and immunoelectrophoresis (IEP) in horses with O. cervicalis and in
immunized rabbits. It forms the basis for a serological survey of onchocercal
antigens.
MATERIALS AND METHODS
Microfilariae
Microfilariae were recovered from biopsies made from the umbilical region of horses
originating from rural areas of the State of Rio Grande do Sul, southern Brazil. All horses
were examined and sampled at a commercial horse-slaughtering plant. For the isolation of
microfilariae, the umbilical samples were cut into multiple fragments, placed on gauze, and
immersed in phosphate buffer solution (PBS) for 24 hours at room temperature. The
suspension was then removed and centrifuged at 300 g for 20 minutes at 4ºC. The sediment
was examined under a light microscope and positive samples were pooled and kept in PBS.
Microfilariae were placed in a mortar containing 10 gr of glass beads sterilized with 5 ml of
distilled water. The mixture was ground with a glass pestle at 0 to 4 °C until the microfilariae
were totally macerated, which was confirmed microscopically. The homogenate was
ultracentrifuged at 36 000 g for 20 min. The protein concentration was quantified by Lowry
et al. (19), lyophilized and stored at -20°C.
Adult worms
Adult parasites were isolated and removed from 300 cervical ligaments. The worms were
repeatedly washed with saline solution to remove any remaining horse tissue, suspended in
saline and homogenized in ice (with glass beads) for 30 min to yield homogenized O.
cervicalis antigens. For the preparation of the soluble crude worm extract, the homogenate
was ultracentrifuged at 36 000 g for 20 minutes at 4°C using a Beckman 50 Ti rotor (Beckman
Instruments, Fullerton, CA, USA). Protein was estimated (19), lyophilized, and stored at 20°C.
Rabbit immunization.
Three New Zealand rabbits from the same litter were used. Rabbit anti-Onchocerca cervicalis
antibodies were produced by repeated subcutaneous injections of O. cervicalis antigens of
the adult and microfilarial forms. One rabbit was kept as control, the second rabbit was
inoculated with the microfilarial antigen, and the third rabbit was inoculated with the adult
parasite antigen. The inoculum consisted of 1 ml antigen and 1 ml incomplete Freund’s
adjuvant. Ten subcutaneous inoculations were performed at weekly intervals in the axillary
region, 0.5 ml inoculum into each axilla. A test bleeding was performed by cardiac puncture
after the 8th inoculation, and final bleeding in the 10th week. The serum was separated and
stored at - 20ºC (20.)
Horses .A pool of sera from horses that tested positive for microfilariae and for the adult
form of O. cervicalis was used at the concentration of0..0 µl/well.
Antigen detection by DD
Serum samples from the rabbits and horses were tested with different Onchocercal
antigens: Crude adult worm (AdAg) and Mf (MfAg) using 0.50µl per well of each antigen and
0.50µl of antiserum per well. DD was carried out as proposed by Ouchterlony (21). The
antigens were placed in the upper part, and their corresponding antisera were added to the
lower part.
Antigen detection by IEP .This technique was performed by Grabar and Williams (22 ,)and
modified by Scroferneker (23) and Geimba (24), 150 µl of AdAg and MfAg were added to the
wells; or2.0 µl of immune serum and horse sera was added to the wells (Ad antiserum in
the upper well and Mf antiserum in the lower well), and electrophoresis was performed
during 290 minutes.
All horses from which O. cervicalis microfilariae and blood were obtained were slaughtered
at a slaughterhouse inspected by the Brazilian Ministry of Agriculture, and the meat was
processed for human consumption. All procedures on rabbits were compliant with the local
animal research laws .
RESULTS AND DISCUSSION
When examined by DD, the protein concentration of antigen to adult was adjusted to 5.5
mg/ml. The protein concentration of microfilariae was adjusted to 5.1 mg/ml. O. cervicalis
antigens reacted with their antisera.
Immunological studies were carried out to assess whether the processed antigens were able
to produce an antigen-antibody reaction using DD and IEP. DD revealed that the antigens
from O .cervicalis adult worms and /or microfilariae differ in terms of precipitin bands,
showing different epitopes, of which the antiserum obtained as response to the microfilarial
antigen induced a smaller number of precipitin bands, followed by antisera from the adult
parasite .The adult antigen presented a wider variation of immunogens compared to
microfilariae, which indicates possible applications of immunoassays to the screening of
onchocerciasis, although it is still difficult to obtain sufficient quantities of adult parasites for
detailed biochemical, molecular and immunological studies (4, 25). On the other hand, it is
relatively easy to obtain large quantities of O. cervicalis microfilariae from the skin by using
the currently available techniques. This material may be used as a source of protein for
further studies and the microfilariae used as a model system for the study of the pathology
and control of human onchocerciasis (15, 17)
The responses obtained in rabbits with homologous and heterologous immune sera
demonstrated that the microfilarial antigen provided better resolution than did the adult
antigen by DD. The antigenic fractions common to the two antigens indicate the possible
presence of adult epitopes in microfilariae. However, the opposite was not true, since no
microfilarial components were detected in the adult antigen in view of the absence of a
response of all the sera to the adult antigen. The presence of multiple antigenic components
of the parasite was also shown by IEP when each antigen was tested in the presence of
rabbit antimicrofilarial and anti-adult immune sera, and the sera reacted to microfilarial
antigen with five and four precipitin bands, respectively. The adult parasite antigen was
prepared from the whole parasite and definitely represented a large amount of antigen with
which the host would not be challenged in natural infection, in addition to the fact that it
possibly contained nonspecific components .
Given that the results suggest that the antigen from the adult parasite was more reactive
under these experimental conditions, further studies are necessary to determine the
different profiles of immune response of hosts to challenge with these antigens .
The fractionation and purification of Onchocerca cervicalis antigens deserve further study in
order to obtain a pattern of sensitivity and specificity for antigens derived from different
developmental stages of the parasite (13). These data agree with those of Phillip et al. (26).
After characterization, these antigens could be used in the immunodiagnosis of human
onchocerciasis, since they are more easily obtained (Forsyth et al. (27) ,Boatin et al. (28).
The results demonstrate the need for more detailed studies of the response of these
antigens in natural and heterologous hosts. Sakwe et al.(16), Boatin et al. (28) and Graham
et al. (29) tested Onchocerca spp. antigens for the diagnosis of human onchocerciasis. In the
present study, we used crude antigen from the adults, and the material that was not used
for antigen isolation may represent another source of antigen .
The present study shows that experimentally infected rabbits showed a higher antibody
response to these antigens; therefore, assessing the immunogenic response of individual
antigens in the target host species will remain an important initial step in the evaluation of
circulating antigens and vaccine candidates. Further definition of antigenic determinants
recognized by the antibodies in the sera from horses with onchocerciasis must be
established and the evaluation of Onchocerca spp. material should aid in the development
of a sensitive, specific, and practical diagnostic antibody test that will be useful for the
diagnosis of onchocerciasis in countries where this disease is endemic .
The methods described are important for diagnosing onchocerciasis in countries with a high
prevalence and where serologic tests are not performed routinely. Furthermore, these
techniques are inexpensive, easily applied, and do not require advanced equipment or
skilled personnel.
Fig. 1: Double diffusion (slides 1 and 2) and immunoelectrophoresis (slides 3 and 4).
FIGURE 1. Slide 1: adult antigens (upper well) and antiserum from microfilariae, adult
worms, control antiserum and positive pooled horse serum from adult parasite (lower well,
from left to right), with at least two identical bands of identity with antisera from
microfilariae and adult parasite and four bands from adult antiserum. Slide 2: microfilarial
antigens (upper well); the lower well is the same as in slide 1, with only one band from
microfilariae and adult antiserum. Slide 3: IEP of antigens to adult worm (central well) and
antiserum to microfilariae (upper well) and to the adult parasite (lower well), with six
common bands in the upper well and at least 12 precipitin bands in the lower well. Slide 4:
IEP of antigen to microfilariae (central well); the antisera were placed as in the previous
slide, with two common bands (upper well) and four precipitin bands (lower well).
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