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J Vet Diagn Invest 21:108111 (2009)

Evaluation of a SRS2 sandwich commercial enzyme-linked immunosorbent assay for the


detection of antiNeospora caninum antibodies in bovine and canine sera
Farida Ghalmi, Bernard China, Rachid Kaidi, Bertrand Losson1
Abstract. Neospora caninum is a parasite responsible for abortion in cows and neuromuscular disease in
dogs. Serology is the most widely used technique to evaluate the prevalence of N. caninum in different host
populations. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the use of an anti-SRS2
monoclonal antibody was evaluated against the indirect fluorescent antibody test for 100 canine sera and
against a well-characterized ELISA for 102 bovine sera. In cattle sera, the relative sensitivity and relative
specificity were 100%. In dog sera, the relative specificity and relative sensitivity were 94% and 86%,
respectively. The kappa value was 1 for bovine sera and 0.77 for canine sera. The seroprevalence was 3.9% in
bovine sera and 2123% in canine sera. The SRS2 sandwich ELISA was considered a valuable tool in both
species.
Key words: Cattle; dogs; enzyme-linked immunosorbent assay; Neospora caninum; serology; SRS2
monoclonal antibody.

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Neospora caninum (order Eucoccidiorida, suborder Eimeriorina, family Sarcocystidae) is an apicomplexan parasite responsible for abortion in cows and neuromuscular
disease in dogs. This heteroxenous parasite uses the dog
and the American coyote (Canis latrans) as definitive hosts.
Cattle are considered as the major intermediate host.9
Serology is commonly used to study the prevalence of N.
caninum in animals. Different methods were developed,
including indirect fluorescent antibody test (IFAT),7 direct
agglutination, Western blot, or enzyme-linked immunosorbent assays (ELISAs).10 Whole tachyzoites18 or purified
antigens15 also can be used for the detection of specific
antibodies. Because intact tachyzoites are used as antigen in
the IFAT, this test mainly detects antibodies directed to
antigens present on the cell surface of the parasite.2
Additionally, in different experimentally infected animal
species, the N. caninum IFAT shows little cross reactivity
with other coccidian parasites, including Toxoplasma
gondii.6 Consequently, IFAT is considered as a reference
technique in N. caninum serology.10 The aim of the present
study was to compare the performance of a new
commercially available SRS2 (also referred to as p38)19
sandwich ELISA kit to the standard IFAT for canine sera
(n 5 100) and to a previously validated and commercially
available ELISA for bovine sera (n 5 102).
Canine sera (n 5 100) were obtained from an animal
control facility in Algiers, Algeria. Blood samples (5 ml)
were collected from the cephalic vein into a dry glass tube.a
After clotting, the tubes were centrifuged at 3,000 3 g for
From the University of Liege, Faculty of Veterinary Medicine,
Infectious and Parasitic Diseases Department, Laboratory of
Parasitology, Lie`ge, Belgium (Ghalmi, Losson), the National
Veterinary School of Algiers, Algiers, Algeria (Ghalmi), the
Scientific Institute of Public Health, Clinical Biology, Brussels,
Belgium (China), and the University Saad Dahlab of Blida,
Veterinary Sciences Department, Blida, Algeria (Kaidi).
1
Corresponding Author: Bertrand Losson, University of Liege,
Faculty of Veterinary Medicine, Laboratory of Parasitology, Sart
Tilman B43, B-4000 Liege, Belgium. blosson@ulg.ac.be

5 min, and the sera were separated and kept at 220uC until
further use.
The IFAT was performed as previously described.7,12
Each slideb comprised a positive and a negative control.
For the canine sera, the positive control was the serum of a
dog experimentally infected with N. caninum, whereas the
negative control was obtained from a noninfected specific
pathogenfree (SPF) dog.14 Slides were observed under
epifluorescence at a magnification of 4003. A sample was
considered as positive when the majority (.50%) of the
tachyzoites fluoresced uniformly. The cut-off used for
canine sera was 1/100 as recommended8 for an optimal
specificity to avoid cross reactions with closely related T.
gondii without affecting the sensitivity. The sensitivity and
specificity for this cut-off were 82% and 100%, respectively.7 The specificity of the method is difficult to address
because neosporosis in dog is mostly asymptomatic; thus,
sera from SPF dogs are necessary to guarantee their
seronegativity.
The SRS2 sandwich ELISA kitc was used following the
manufacturers recommendations. The odd columns were
coated with a monoclonal antibody specific to SRS2
(p38),17 and a crude extract of tachyzoites was then
deposited to specifically capture the SRS2 antigen. The
even columns were left uncoated (negative controls). One
hundred microliters of each diluted serum sample was
added to twin wells (a coated and an uncoated well for each
sample). The plates were incubated for 1 hr at room
temperature and then washed 3 times with the washing
solution provided with the kit. For the canine sera,
horseradish peroxidase conjugate (antidog immunoglobulin G)d was diluted 1:10,000 in the appropriate dilution
solution, whereas for the bovine sera, the kit conjugate was
diluted according to the manufacturers recommendations.
One hundred microliters of the conjugate was added to
each well and was incubated for 1 hr at room temperature.
The plates were then washed 3 times, and 100 ml of the
substrate solution (3,39,5,59-tetramethylbenzidine, H2O2)
was added to each well and incubated for 15 min in the

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Figure 1. Serological status of the sera tested using the SRS2 sandwich enzyme-linked immunosorbent assay. A, after the first
reading, sera were classified into negative I (S/P ratio ,5), negative II (S/P ratio 510), borderline (S/P ratio 1015), slightly positive (S/P
ratio 1520), and strong positive (S/P .20). B, repeatability was assessed by repeating some sera twice (on separated plates) after the first
reading. Seven negative II sera (N1N7), 4 borderline sera (BL1BL4), and 4 slightly positive sera (P1P4) were tested. Black diamonds
represent the average of the 3 measurements. Vertical lines represent the 95% confidence interval (average 61.96 SD). Region I 5
positive region; region II 5 borderline region; region III 5 negative region.

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110

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dark at room temperature. The reaction was stopped by the


addition of 50 ml of stop solution (H3PO4, 1 mol). The
optical density at 450 nm (OD450) was read using a plate
reader.e For each serum sample, the difference between the
ODs recorded on the coated and uncoated wells was
calculated (DOD450). The ratio between each DOD450, and
the DOD450 of the positive control, was calculated and
multiplied by 100 (S/P). A ratio value .15 or ,10 was
considered as positive or negative, respectively, whereas a
ratio value between 10 and 15 was considered as borderline.
Among the 100 canine sera, using the SRS2 sandwich
ELISA, 23 were positive (S/P .15), 73 were negative (S/P
,10), and 4 sera were classified as doubtful (S/P 5 10.2,
10.5, 10.9, and 11.5, respectively; Fig. 1A). The borderline
sera were repeated twice, resulting in negative results
(serum BL1 average S/P 5 8.83, coefficient of variation
(CV): 16.5%; serum BL2 average S/P 5 8.97, CV:13.9%;
serum BL3 average S/P 5 9.17, CV: 16.7%; serum BL4
average S/P 5 9.7, CV: 16.2%; Fig. 1B).
The seroprevalence was 21% (95% confidence interval [CI]:
1329%) using IFAT and 23% (95% CI: 14.737.7%) using
SRS2 sandwich ELISA. The relative specificity and sensitivity
were 94% and 86%, respectively. The relative accuracy was
97%, indicating a good general agreement between the 2
methods, confirmed by a kappa value of 0.77 (calculated with
the Win Episcope 2.0 softwaref ). McNemars test was
nonsignificant (P 5 0.72). Therefore, the results of canine
sera were similar for IFAT and SRS2 sandwich ELISA.
Cattle (n 5 102) blood samples (10 ml) were collected
from the tail vein of adult cows belonging to 7 different
herds located around Algiers and processed as described
above. A previously validated ELISAg,23 was used to
evaluate the performance of the SRS2 sandwich ELISA in
bovine sera. The ELISA kit was based on whole antigens
(WA ELISA) and was used following the manufacturers
recommendations, including the dilution of bovine sera (1/
100). Using reference sera, the sensitivity and specificity of
the WA ELISA were determined as 100% and 93.3%,
respectively, in one study23 and 100% and 99.7% in another
study.20 When compared to IFAT, the sensitivity and
specificity were 93% and 96%, respectively.21 Using sera
from aborting and nonaborting cows, the sensitivity and
specificity were 98% and 92%, respectively.22 For bovine
sera, the SRS2 sandwich ELISA resulted in 98 negative
results (S/P , 10), 4 positive sera (S/P . 15), and no
borderline serum. The results were identical for both
ELISA kits with specificity, sensitivity, and agreement of
100%, respectively. The seroprevalence for WA ELISA and
SR2 sandwich ELISA was 3.9% (95% CI: 0.17.7%).
Finally, the repeatability of the SRS2 sandwich ELISA
kit was addressed as 4 borderline sera were retested and
considered as negative. Therefore, 7 randomly selected
(among the 17) negative sera with an S/P ratio ranging from
5 to 10 and the 4 positive sera with an S/P ratio ranging
from 15 to 20 were repeated twice. The average S/P, CV,
and CI at 95% were calculated and plotted (Fig. 1).
Coefficient of variation values ranged from 6% to 24%.
The negative samples stayed negative, and the positive
samples stayed positive. However, for negative serum N7,
the upper border of the CI was located in the borderline

region (zone II on Fig. 1B). For positive sera P7, the lower
border of the CI was located in the borderline zone. The
presence of the borderline zone avoids the transformation
of a positive into a negative and vice versa. In neosporosis,
only high serological titers are of clinical relevance for
borderline sera; thus, it is better to consider these
borderline sera as negative, but a followup of the suspect
animal is recommended. In a screening procedure, the
borderline sera can be considered as positive since all
positive samples will be confirmed by a confirmation test
such as Western blot.
The present study was conducted to evaluate a new
commercial sandwich ELISA for the detection of antibodies against N. caninum in both the dog and cattle. This kit
uses a monoclonal antibody to capture SRS2 antigen from
a crude lysate of tachyzoites. The performance of this kit
was evaluated in comparison with the IFAT as the
standard method for canine sera and in comparison with
WA ELISA for bovine sera. Others studies have compared
different ELISAs, based on the use of recombinant N.
caninumpurified antigens, with the IFAT.10 In cattle,
previous studies4,13 showed perfect agreement between
ELISA and IFAT when the recombinant Ncp29 (NcsSRS1)
antigen was used. ELISAs based on the use of rNcGra6
and NcGra7 also showed good correlation with the
IFAT.15 The RNcSRS2 antigen also gave similar results
when compared to the IFAT.11,17,19 In dogs, a competitive
ELISA was validated against the IFAT for N. caninum
circulating antibodies with a specificity and sensitivity of
89.3% and 72%, respectively.3 ELISAs were also validated
against previously validated ELISA or Western blot.23
ELISA validation can also be performed without reference
method by using true positive and true negative sera
previously confirmed by several methods.1
Although it was not the main purpose of the current
study, preliminary information about the potential impact
of N. caninum in Algeria was collected for the first time.
The seroprevalence in cattle (3.9%) was low compared to
the authors previous study conducted in Belgium (29%).5
Interestingly, the 4 positive bovine sera originated from 3
herds with abortion problems. Nevertheless, a larger
epidemiological control study should be performed to
evaluate the seroprevalence in this region. The seroprevalence in Algerian dogs (2123%) was similar to the
prevalence obtained in other studies in rural or farm
dogs.16 This could be related to the fact that the dogs were
stray dogs coming mainly from peri-urban areas. Once
again, a larger epidemiological study must be performed in
different canine populations (rural, stray, and urban dogs).
Acknowledgements. F. Ghalmi was financially aided by
both the Belgian Technical Cooperation (Brussels, Belgium) and the Algerian Ministry of Higher Teaching via the
National Veterinary School of Algiers.

Sources and manufacturers


a. BD VacutainerH Dry Tubes (368434), BD Diagnostics, Plymouth, Devon, UK.
b. Black Teflon Slide (W0149D), Fisher Scientific Bioblock,
Tournai, Belgium.

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Brief Research Reports


c. Neospora caninum Antibody Detection Kit (BIO K 192), Bio-X
Diagnostics SPRL, Jemelle, Belgium.
d. Anti-Dog IgG (whole molecule)Peroxidase Antibody (A6792),
Sigma-Aldrich NV/SA, Bornem, Belgium.
e. Multiskan AscentH, Thermo Fisher Scientific BV, Breda, The
Netherlands.
f. Win Episcope 2.0, CLIVE: Computer-aided Learning In
Veterinary Education, Edinburgh, UK (http://www.clive.ed.
ac.uk/winepiscope/).
g. HerdChek, anti-Neospora caninum, IDEXX Laboratories Inc.,
Westbrook, ME.

12.

13.

14.

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