The document summarizes an experiment using an ELISA test with recombinant NcSAG1 to detect antibodies for Neospora caninum in canine serum samples. Key points: - The indirect ELISA method was used to test 12 canine serum samples for N. caninum antibodies. This method has advantages like increased sensitivity but requires an extra incubation step. - OD values indicated samples 1, 4, and 3 had the highest antibody levels while samples 9, 6, and 2 had the lowest. - Based on the OD values, samples 1, 3, 4, 5, 7, 8, 10, 11, and 12 tested positive for N. caninum antibodies while 2,

1. ｱﾊﾞｵﾗﾘｰﾈﾙﾋﾞﾙﾊﾞｵ May 18, 2010
Animal Production Hygiene Prof. Xuan
Toxoplasma Gondii Infection
According to Wikipedia, Toxoplasma gondii (T. gondii) is a species of parasitic protozoa of the
genus Toxoplasma. The definitive host of T. gondii is the cat, but the parasite can be carried
by many warm-blooded animals. Toxoplasmosis, the disease caused by T. gondii, is usually
minor and self-limiting but, can have serious or even fatal effects on a fetus whose mother
first contracts the disease during pregnancy or on an immuno-compromised human or cat.
The life cycle of T. gondii has two (2) phases. The sexual part of the life cycle (coccidia like)
takes place only in members of the Felidae family (domestic and wild cats). This makes
these animals the parasite's primary host. The asexual part of the life cycle can take place in
any warm-blooded animal (e.g. other mammals and birds).
In the intermediate hosts (as well the definitive host felines), the parasite invades the cells.
This forms intracellular parasitophorous vacuoles containing bradyzoites, the slowly
replicating form of the parasite (Dubey, et. al., 1998). Vacuoles form tissue cysts mainly
within the muscles and brain. Since they are within cells, the host's immune system does not
detect these cysts. Resistance to antibiotics varies, but the cysts are very difficult to eradicate
entirely. Within these vacuoles, T. gondii propagates by endodyogeny until the infected cell
eventually bursts and tachyzoites are released. Tachyzoites are the motile, asexually
reproducing form of the parasite. Unlike the bradyzoites, the free tachyzoites are usually
efficiently cleared by the host's immune response. However, some manage to infect cells and
form bradyzoites thus, maintaining the infection.
Tissue cysts are ingested by a cat (e.g. by feeding on an infected mouse). The cysts survive
passage through the stomach of the cat and the parasites infect epithelial cells of the small
intestine (where they undergo sexual reproduction and oocyst formation). Oocysts are shed
with the feces. Animals and humans that ingest oocysts (e.g., by eating unwashed
vegetables etc.) or tissue cysts in improperly cooked meat become infected. The parasite
enters macrophages in the intestinal lining and is distributed via the blood stream throughout
the body.
According to the lecture conducted, there are five (5) types of T. Gondii infection. The first
type is primary infection. It refers to the first time an individual is infected with the disease.
The second type is congenital toxoplasmosis. It occurs in newborn babies born to mothers
infected with T. Gondii before or during pregnancy (Vogel, et. al., 1996). The third type is
infection of the eye (Glasner, et. al., 1992). Eye disease from toxoplasmosis usually occurs in
children, either from a congenital infection or from infection in childhood. The fourth type is
latent infection. In this stage, the person is infected with T. Gondii (usually in a cyst form) but
has no signs or symptoms of the disease. The last type is the reactivation of latent infection.
This typically occurs in people who have a weakened immune system, such as those with
HIV/AIDS (Holliman, et. al., 1988) and those who have undergone an organ transplant.
In the Philippines, two (2) studies confirmed the presence of T. Gondii in the country. One (1)
study serologically ascertained T. gondii infection in Rattus spp. inhabiting agricultural,
commercial, and residential sites in Dasmariñas, Cavite, and confirmed the presence of T.
gondii parasites through its bioassay in mice (Salibay, et. al., 2006). It represented the first
confirmed report of T. gondii infection of rats in the Philippines. The second study found that
T. gondii was present among cats in Kabacan, Cotabato as detected by an antibody test kit
(Molina and Dash, 2008). This poses a health hazard as the infection could pass to humans.
In conclusion, it is really very important to observe general sanitation and food safety steps to
reduce the chances of becoming infected with T. Gondii. Also, it is also important to
strengthen one’s immune system so as to prevent illness from the disease if ever it hits an
individual.
1

2. ｱﾊﾞｵﾗﾘｰﾈﾙﾋﾞﾙﾊﾞｵ May 18, 2010
Animal and Food Hygiene Practice II Prof. Xuan
ELISA Test with Recombinant NcSAG1 for Detection of N. Caninum
According to Wikipedia, Enzyme-linked immunosorbent assay (or ELISA) is a
biochemical technique used mainly in immunology to detect the presence of an
antigen or antibody in a sample. Other names, such as enzyme immunoassay (or
EIA), are also used to describe the same technology. The ELISA has been used as
a diagnostic tool in medicine and plant pathology, as well as a quality control check in
various industries. It is a common serological test for the presence of particular
antigens or antibodies. There are two (2) forms of this assay: (1) the direct ELISA
employs monoclonal antibodies to detect the presence of a particular antigen in a
sample; and (2) the indirect ELISA is used to determine the presence of a specific
antibody in a specimen or serum (Perry, et. al., 2002).
For some brief definitions (from www.answers.com website), an antigen is defined as
a substance that when introduced into the body stimulates the production of an
antibody. Antigens include toxins, bacteria, foreign blood cells, and the cells of
transplanted organs. Meanwhile, the antibody is a Y-shaped protein on the surface
of B cells that is secreted into the blood or lymph in response to an antigenic stimulus
(such as a bacterium, virus, parasite, or transplanted organ). It neutralizes the
antigen by binding specifically to it (an immunoglobulin).
1. Principle of ELISA with recombinant NcSAG 1
In the class activity, we did the serodiagnosis of Neospora Caninum (N. caninum)
infection by enzyme-linked immunosorbent assay with recombinant NcSAG3, which
is considered a very usefool tool (Xuan, 2005). As mentioned in the handouts that
were given, NcSAG 1 is a major surface protein of tachyzoites and an
immunodominant antigen. For the experiment, we got twelve (12) samples of canine
sera for testing for the antibody of N. caninum. The protocol used in the testing was
also provided and the students just had to follow it. Other materials necessary (such
as antigen coating buffer, substrate buffer, GST, ELISA plate, etc.) to conduct the
experiment were also provided.
For the class activity, the indirect method of ELISA was used. Using the indirect
method has advantages and disadvantages. The advantages are the following:
• A wide variety of labeled secondary antibodies are available commercially;
• Versatile because many primary antibodies can be made in one species and
the same labeled secondary antibody can be used for detection;
• Maximum immunoreactivity of the primary antibody is retained because it is
not labeled;
• Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing for
signal amplification; and
• Different visualization markers can be used with the same primary antibody.
2

3. Meanwhile, the disadvantages of using the indirect method are:
• Cross-reactivity might occur with the secondary antibody, resulting in
nonspecific signal; and
• An extra incubation step is required in the procedure.
2. OD Values for each sample
Results of the Group 3 OD values (as read by the machine) are presented in Table 1.
It can be seen that samples 1, 4, and 3 have the highest amount of antibodies
present with OD values of 1.5855, 1.4575, and 1.4455, respectively. Meanwhile, the
samples with the lowest OD values are the following: samples 9 (-0.056), 6 (0.016),
and 2 (0.0335).
Table 1. Group 3 Experiment’s OD Values of N. Caninum Antibodies
Dog Sera Calculation Method of OD Values OD Values
Sample 1 - [{(1.639 + 1.645) / 2} – {(0.067 + 0.046) / 2}] = 1.5855
Sample 2 - [{(0.053 + 0.012) / 2} – {(0.011 - 0.013) / 2}] = 0.0335
Sample 3 - [{(1.480 + 1.456) / 2} – {0.040 + 0.005) / 2}] = 1.4455
Sample 4 - [{(1.559 + 1.398) / 2} – {(0.015 + 0.027) / 2}] = 1.4575
Sample 5 - [{(1.356 + 1.510) / 2} – {(0.011 + 0.010) / 2}] = 1.4225
Sample 6 - [{(0.009 + 0.057) / 2} – {(0.017 + 0.017) / 2}] = 0.016
Sample 7 - [{(1.527 + 1.411) / 2} – {(0.064 + 0.040) / 2}] = 1.417
Sample 8 - [{(0.701 + 1.142) / 2} – {(0.038 + 0.014) / 2}] = 0.8955
Sample 9 - [{(0.073 + 0.060) / 2} – {(0.065 + 0.180) / 2}] = -0.056
Sample 10 - [{(1.101 + 1.092) / 2} – {(-0.002 + 0.049) / 2}] = 1.073
Sample 11 - [{(1.458 + 1.324) / 2} – {(0.020 + 0.010) / 2}] = 1.376
Sample 12 - [{(1.418 + 1.362) / 2} – {(-0.020 + 0.010) / 2}] = 1.395
3. Judgment of each sample
Based on the OD values, samples 1, 3, 4, 5, 7, 8, 10, 11, and 12 are positive for
antibodies of N. caninum. Meanwhile, samples 2, 6, and 9 are negative of antibodies
for N. caninum.
In conclusion, ELISA with recombinant NcSAG1 is a very useful serodiagnostic tool
in the detection of N. caninum antibodies from canine sera. In general, ELISA is
really a useful tool in determining antibodies (or antigen) in a sample. Also, it allows
for testing of more samples at low costs.
3