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African Journal of Biomedical Research
Ibadan Biomedical Communications Group
ISSN: 1119-5096
Vol. 6, Num. 3, 2003, pp. 137-140
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African Journal of Biomedical Research, Vol. 6, No. 3, Sept,
2003, pp. 137-140
ASSOCIATION OF TRYPANOSOME INFECTION WITH CIRCULATING ZONA PELLUCIDA (ZP)
ANTIBODIES IN WEST AFRICAN DWARF (WAD) GOATS
O. FAYEMI
Department of Veterinary Surgery and Reproduction, University of Ibadan ,
Ibadan , Nigeria .
Accepted in final form: March 2003
Code Number: md03055
Sera from 967 adult female West African Dwarf (WAD) goats previously screened
for Trypanosome infection by some diagnostic laboratories around Ibadan metropolis,
in Southwestern part of Nigeria, were assayed for zona pellucida (ZP) antibodies
by the enzyme-linked immunoassay (ELISA) technique. Of the 967 female goats,
534 (55.22%) were positive and 433 (44.78%) negative for Trypanosome infection.
Out of those that were positive for Trypanosome infection, 346 (64.79%) and
188 (35.21%) were positive and negative for ZP antibodies respectively. These
represented 35.78% and 19.44% of the total number of animals screened respectively.
The group that was negative for Trypanosome infections had 149 (34.41%) and
284 (65.59%) positive and negative for ZP antibodies, representing 15.41%
and 29.37% of the total number of animals screened, respectively. Seropositivity
for ZP antibodies was positively correlated with Trypanosome infection (P
Key words: - Trypanosomes, zona pellucida antibodies, and infertility,
Goats.
INTRODUCTION
Goats are raised extensively and semi-intensively in Nigeria as a source of
animal protein for human nutrition. The West African Dwarf (WAD) breed is noted
for its ability to survive the harsh environmental conditions of heat stress
and humidity (Devendra and Mcleroy, 1982, Osuagwuh and Akpokodje, 1981) and
it is the predominant breed in the humid Southwestern part of the country.
The WAD goats are kept in small flocks, typically numbering 2 6 animals,
per household as an adjunct to the main business of cropping, thereby contributing
significantly to the economy of third world countries ( Upton, 1985; Ademosun,
1985). Despite the importance of this species of animal (goat); it has received
very little attention (Okere et. al , 1982) and there is low productivity,
which has been attributed to factors including nutrition, infectious and non-infectious
agents (Laing et al , 1988, Cullen, 1990). Low productivity can be caused
by infertility and immunological reactions had been associated with infertility
especially antibodies produced against the zona pellucida in humans
and animals (Sacco, 1979, Wood et. al , 1981 Sacco, et al , 1981,
Haseguwa, 1995).
Trypanosome infections have been reported to cause infertility due perhaps
to ovarian pathology in goats (Ikede and Akpavie, 1981). The ovary produces
follicles which contain ova surrounded by zonae pellucidae. The zona pellucida
is an acellular glycoprotein which is antigenic (Gupta et.al ., 1997,
Brown et al ., 1997) but ordinarily will not stimulate antibody production
because it is protected by the blood-ovary barrier which when disrupted will
lead to antibody production. The ZP antibody production has been associated
with infertility. It has not been established if damage to the ovary can lead
to damage to the blood-ovary barrier thereby stimulating ZP antibody production.
The ZP antibody has not been correlated with Trypanosome infection.
The objective of this study was therefore to check whether there is any correlation
between trypanosome infection and ZP antibody production
MATERIALS AND METHODS
Serum samples previously collected and screened for Trypanosome infections
by the private veterinary clinics, Government and University Laboratories in
the Southwestern part of Nigeria were used for this investigation. The samples
were randomly selected from the pool without previous reference to the trypanosome
screening results until the results of the ZP antibody assay were ready. The
samples were taken in ice-packs and air-freighted to the laboratories in the
University of Minnesota, St. Paul. Minnesota, U.S.A for ZP antibodies assay.
Preparation of Antigen
Caprine ovaries were collected from slaughterhouses at goat farms around St.
Paul, Minnesota, U.S.A. The follicles were punctured and 1 ml. tuberculin syringes
with 26 gauge needles were used to aspirate the follicular fluid into Petri
dishes. The ova were collected in 0.5mls 0.1M phosphate buffered saline (PBS)
using a stereomicroscope. The ova were transferred into a test tube containing
0.01% w/v sodium citrate in PBS and shaken for 60 seconds to remove the cumulus
cells. The zonae pellucidae were then separated from the eggs using
glass pipettes with bores a little narrower than the diameter of the egg.
The zonae were washed three times in PBS, resuspended at a concentration of
200 zonae/ml and then sonicated 20 strikes with a sonicator model W380 (HeatSystems
Inc.). The sonicates were then centrifuged twice using PBS at 1200g for 20
minutes at 4°C. The optical density OD of the second supernatant was estimated
and adjusted to 0.2. This was equivalent to 0.15mg/ml protein and was used
as the zona pellucida antigen. The antigen was divided into aliquots and stored
in the cold room at 196°C until used for the assay of antibodies.
Assay of Zona Pellucida Antibodies
A total of 967 serum samples were assayed for antizona pellucida antibodies
using the methods of Henderson et al . (1987) with slight modifications.
The zonae antigen was dispensed at 50µl well into 96 well polyvinyl
microplates (Falcon 3912 Micro Test III, Becton Dickinson) and left overnight
at 4°C. On the second day, the fluid was decanted and the antigen fixed
using 50µ 0.1% gluteraldehyde in PBS-Tween 20 (Sigma) for 5 minutes.
The plates were then washed three times in PBS before incubating overnight
with 100 1% Bovine Serum Albumin (BSA) at 4°C. After this the plates were
washed three times again before dispensing 50µ/well of various dilutions
of the test and standard negative samples and incubating for 1 hour at 37°C.
The standard negative samples being sera taken from 2-week old kids. The plates
were then washed in PBS-Tween 20 and incubated for 30 minutes with addition
of 5Oµ/well Biotin-labeled rabbit-anti-goat IgG (KPL, 1:4800) at 37°C.
This was followed by washing and incubation for 30 minutes with 50µ streptavidin
peroxidase (KPL, 1:9600) at 37°C. The plates were then washed three times
before adding 50µ/well substrate for 15 minutes in the dark danser. The
substrate consisted of equal volumes of 2,2, azino-di (3-ethylbenzthiazoline
sulfonate) (ABTS) and hydrogen peroxide (H The optical density was read at
405nm with a micro ELISA reader model MR380 (Dynatech).
The mean OD of the standard negative sera was calculated and used as the benchmark.
Any sample with twice the value of the calculated mean was taken as positive.
Correlation with Trypanosome Infection
The results of the selected samples in term of trypanosome infection status
were collected from the laboratories of origin and compared with the results
of the ZP antibodies assay.
Statistical Analysis of Results
The antibody assay results were correlated with the trypanosome infection
status of the samples using the Panacea statistical package, University of
Minnesota
RESULTS
Table 1 shows the absolute numbers and proportions of animals that were positive
and negative for Trypanosome infection and ZP antibodies. Out of 967 animals
tested 534 (55.22%) were positive compared to 433 (44.78%) that were negative
for Trypanosome infection.
Those positive for both ZP antibody and Trypanosome infection were 346(35.78%
of Total) while those positive for ZP antibodies but negative for trypanosome
infection 149(15.41% of Total). The animals that were negative for ZP antibodies
but positive for trypanosome infection were 188 (19.44% of Total) while those
that were negative for ZP antibodies and negative for trypanosome infection
numbered 284 (29.37% of Total). Of the total number of 967, 495 (51.19% of
Total) were positive for ZP antibodies compared to 472 (48.8 1% of Total) that
were negative for the antibodies.
Table 1: Proportions of animals positive and negative for Trypanosome infection and zona
pellucida antibodies.
|
Positive for Trypanosome |
Negative for Trypanosome |
Total |
No. Positive for ZP antibodies |
346 (35.78%) |
149 (15.41%) |
495 (51.19%) |
No. Negative for ZP antibodies |
188 (19.44%) |
284 (29.37%) |
472 (48.81%) |
Total No Tested |
534 (55.22%) |
433 (44.78%) |
967 (100%) |
Table 2: The proportions of Animals positive and negative for zona pellucida antibodies
in each of the groups that tested positive and negative for Trypanosome infection
|
Positive for Trypanosome |
Negative for Trypanosome |
No. Positive for ZP antibodies |
346(64.79%) |
149(34.41%) |
No. Negative for ZP antibodies |
188(35.21%) |
284(65.59%) |
Total |
534(100%) |
433(100%) |
Table 3 The proportions of Animals positive and negative for Trypanosome infection
in each of the groups that tested positive and negative for zona pellucida
(ZP) antibodies.
|
Positive for ZP Antibodies |
Negative for ZP
Antibodies |
No. Positive for Trypanosome |
346(69.90%) |
188(39.83%) |
No. Negative for Typanosome |
149 (30.10%) |
284 (60.17% |
Total |
495(100%) |
472(100%) |
Table 2 shows the proportions of animals that were positive and negative for zona
pellucida antibodies in each of the groups positive and negative for
Trypanosome infection. Of the 534 that were positive for trypanosome infection,
346(64.79%) and 188 (35.21%) were positive and negative for ZP antibodies
respectively. In the group that were negative for trypanosome infection totaling
433, 149(34.41%) and 284(65.59%) were positive and negative for ZP antibodies
respectively.
Table 3 shows that out of the 495 animals that were positive for ZP antibodies,
346 (69.90%) and 149(30.10%) were positive and negative for trypanosome infection
respectively. Of the 472 that were negative for ZP antibodies, 188 (39.83%)
were positive compared to 284(60.17%) that were negative for trypanosome infection.
The proportion of animals positive for trypanosome infection was significantly
higher than those negative for the infection (P<0.01).
The proportion of animals that were positive for ZP antibodies was significantly
higher than those that were negative for the ZP antibodies (P<0.01). Seropositivity
to ZP antibodies was positively correlated with trypanosome infection (P<0.001).
DISCUSSION
The results show that in the group positive for trypanosome infection, a significantly
higher proportion was seropositive for ZP antibodies (P<0.001). Also in
the group that was negative for trypanosome infection the proportion that was
seropositive for ZP antibodies was significantly lower than those that were
seronegative to the antibody (P<0.001). Seropositively was positively correlated
with trypanosome infection.
The zona pellucida has been shown to be antigenic (Subramanian et
al , 1981; Dunbar, et al . 1989, Skinner, et al , 1999)
but does not ordinarily stimulate production of antibodies because of the
blood-ovary-barrier.
Reproductive disorders have been associated with trypanosome infections in
man and animals (Apted, 1970), Ruminants infected with Trypanosoma brucei ,
T. congolense or T. vivaxe showed that the infection can lead to irregular
oestrus, infertility and intrauterine infection with abortion in females (Ikede
and Akpavie, 1982). The pathogenesis of infertility in such cases may be connected
with damage to the blood-ovary-barrier leading to formation of antibodies to
the zonae pellucidae which had been associated with infertility (Wolgemuth et
al ., 1984, Maresh et al , 1990, Hazeguwal et al , 1995,
Kolle et al ., 1996).
The animals that were negative for trypanosomes when tested but were seropositive
for ZP antibodies might have been previously infected since even after successful
treatment of animals, parasites usually disappear but infertility may not disappear
in Ndama cows 3½ to 16 months after infection (Ige and Amodu, 1975).
The correlation of seropositivity for ZP antibodies with trypanosome infection
in this study allows the speculation that trypanosome infection could have
caused enough damage to the ovaries in the affected animals to the point of
exposure of the ZP proteins, secreted by the oocytes as well as granulose cells
(Wolgemuth et al , 1984, Moresh et al , 1990), to the immune
system The possibility of ovarian structural damage in trypanosome infection
which is enzootic in Nigeria causing infertility should therefore be considered
in infertility investigation in farm and domestic animals.
Further studies on the histopathology and electron microscopy of the ovary
during the course of trypanosome infection will throw further light to this
suspected pathogenesis of infertility.
ACKNOWLEDGEMENT
The author is grateful to Dr. H. S. J of the Department of Clinical and
Population Sciences, College of Veterinary Medicine, University of Minnesota,
St. Paul, MN 55108, U.S.A. in whose laboratory the ZP antibody assay was
done.
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