VETERINARSKI ARHIV 68 (1), 19-26, 1998
Enzyme-linked immunosorbent assay in serological diagnosis of swine pleuropneumonia in Croatia
Boris Habrun1*, Vitomir Bilic1, Tomo Naglic2, and Andrea Humski1
1Department of Bacteriology, Croatian Veterinary Institute, Zagreb, Croatia
2Department of Microbiology and Infectious Diseases, Faculty of Veterinary
Medicine,
University of Zagreb, Zagreb, Croatia
ISSN 0372-5480
Printed in Croatia
HABRUN, B., V. BILIC, T. NAGLIC, A. HUMSKI: Enzyme-linked immunosorbent assay in serological diagnosis of swine pleuropneumonia in Croatia. Vet. arhiv 68, 19-26, 1998.
ABSTRACT
The present study was the first in Croatia which employed ELISA to establish the presence of antibodies against Actinobacillus pleuropneumoniae serovars 2 and 4-7 in the sera of breeding swine. A total of 782 sera were tested. The sera had been taken from 345 gilts (aged 6 months), 334 sows (over 12 months of age) and 103 boars (aged 6-12 months). Sera samples, taken from the animals kept at nine large pig-breeding farms, were examined for the presence of antibodies against A. pleuropneumoniae serovar 2. The results showed that infection with this serovar existed at seven of the nine farms. The percentage of serologically positive gilts for serovar 2 at the seven infected farms ranged between 61% and 91%, of serologically positive sows from 26% to 61% and boars from 9% to 50%. Sera from farms 7, 8 and 9 were also examined for the presence of antibodies against serovar 4-7; findings were positive. The percentage of serologically positive gilts for A. pleuropneumoniae serovar 4-7 at these farms ranged from 78% to 91%, of sows from 74% to 78% and boars from 20% to 50%. ELISA has shown to be reliable method in the serological diagnosis of swine pleuropneumonia in breeding swine of various age, on a farm level.
Key words: swine pleuropneumonia, serological diagnosis, ELISA, Croatia
Introduction
Swine pleuropneumonia is caused by Actinobacillus pleuropneumoniae which has two biovars and twelve serovars (FREY, 1995). The incidence and economic significance of porcine pleuropneumonia increased as a result of the intensification of pig breeding (SEBUNYA and SAUNDERS, 1983; VAILLANCOURT et al., 1988).
Complement fixation test (CFT) was the first method to be applied in serological
diagnosis of porcine pleuropneumonia (NICOLET et al., 1971). Despite the
fact that anticomplementary activity of swine sera also interferes frequently
in the evaluation and interpretation of the CFT, the complement fixation
test had been the basic serological method for detection of antibodies
against A. pleuropneumoniae for quite some time. After which, ELISA was
introduced (NICOLET et al., 1981; GOYETTE et al., 1986a). This method proved
to be reliable for the serological diagnosis of porcine pleuropneumonia
on a farm level (GOYETTE et al., 1986a; WILLSON et al., 1988). Additionally,
ELISA was more sensitive and specific than CFT or 2-mercaptoethanol agglutination
(GOYETTE et al., 1986b; NICOLET, 1988; NIELSEN, 1988; FENWICK, 1992). Serological
diagnostic methods for porcine pleuropneumonia were further supplemented
by 2-mercaptoethanol agglutination (MITTAL et al., 1984), previously employed
in serological diagnosis of brucellosis (ALTON et al., 1975). Agglutination
in saline solution or formalinized saline solution having proved to be
nonspecific (NICOLET et al., 1971; MITTAL et al., 1984), 2-mercaptoethanol
was then used as a diluent which inhibits agglutination reaction of IgM
antibodies (ANDERSON et al., 1964) and being almost always responsible
for cross-reactions (MITTAL et al., 1984).
2-mercaptoethanol agglutination has been employed in serological diagnosis
of swine pleuropneumonia in Croatia since 1985. ELISA was introduced in
Croatia in 1995. The present study was designed to determine, through the
ELISA test, just how widely porcine pleuropneumonia had spread at Croatia's
largest pig-breeding farm at that point in time.
Materials and methods
The study was carried out on 782 sera samples of breeding swine (345 were
gilts, 334 sows and 103 boars). The sera had been taken from nine big pig-breeding
farms accounting for 50% of national pig production. All sera were examined
for the presence of antibodies against serovar 2 of A. pleuropneumoniae.
The sera from farms 7, 8 and 9 were also examined for the presence of antibodies
against serovars 4-7 of A. pleuropneumoniae, because at that time serovars
2 and 4-7 were prevalent in the country.
Blood samples were examined at the Croatian Veterinary Institute where
they were centrifuged (over 15 minutes at 3,000 r/min) to separate the
sera, which were kept at -30 °C until testing.
ELISA was performed with commercial kits ("CHEKIT APP-TEST", Dr. Bommeli
AG, Switzerland). For each tested serovar (2 and 4-7) a corresponding kit
was available comprising all necessary reagents: microtiter plates covered
with corresponding antigen (2 or 4-7), diluent, peroxidase conjugate, positive
control serum, negative control serum, chromogen and stop solution. The
test was performed in accordance with manufacturer's recommendations.
Each serum sample was double tested. Arithmetic mean of optical density
of the two obtained results was calculated initially. Results for each
sample were then calculated as a percentage of optical density of positive
control serum, corrected by the optical density of negative control serum.
Read-out and calculation of the results was automatic ("Anthos ht II" Labtec,
Salzburg, Austria). Values below 30% were considered as negative findings,
from 30 to 50% as suspect and over 50% as positive.
Calculation of t-distribution showed a significant difference between the
results obtained from gilts, sows and boars.
Results
Results of determination of antibodies against serovar 2 of A. pleuropneumoniae
are given in Tables 1 (gilts), 2 (sows) and 3 (boars), while results of
determination of antibodies against serovar 4-7 are given in Tables 4 (gilts),
5 (sows) and 6 (boars).
Discussion
The success of the control and elimination of porcine pleuropneumonia depends
on how efficiently intra- and inter-farm transmission of the infection
is prevented (FENWICK, 1992). Optimal results in this aspect are achieved
by reliable serological methods. Serological diagnostic of A. pleuropneumoniae
infection is an essential tool for identifying a latently infected herd,
recognizing multiple serovars in the herd, control over quarantined animals,
elimination of the infection in the herd and evaluating a vaccine (GOYETTE
et al., 1986b).
Gilts were the youngest age group to be included in this work. At the time
of blood sampling they were 6 months of age. Reliability of serological
control over them was very important, due to the detection of infected
animals before breeding having been a basis for successful control and
elimination of infection. This was because of the fact that after farrowing
the mothers probably transmit the infection to their young, which subsequently
become sick at the beginning of the fattening period (BILIC, 1993).
Farm
No. of samples
No. of positive (%) samples
No. of suspective (%) samples
No. of negative (%) samples
1.
46
36 (78.2)
7 (15.2)
3 (6.5)
2.
46
28 (60.8)
4 (8.6)
14 (30.4)
3.
46
0 (0.0)
0 (0.0)
46 (100.0)
4.
46
39 (84.7)
6 (13.0)
1 (2.1)
5.
46
37 (80.4)
4 (8.7)
5 (10.8)
6.
46
39 (84.7)
6 (13.0)
1 (2.1)
7.
23
21 (91.3)
1 (4.3)
1 (4.3)
8.
23
19 (82.6)
4 (17.3)
0 (0.0)
9.
23
2 (8.7)
0 (0.0)
21 (91.3)
Total
345
221 (64.0)
32 (9.2)
92 (26.6)
Of the 345 samples of sera taken from the gilts and examined for the presence
of antibodies against serovar 2 of A. pleuropneumoniae, 221 (64%) were
positive, 35 (9%) suspect and 92 (27%) negative. At farms 1, 2, 4, 5, 6,
7, and 8 the percentage of serological positive gilts ranged from 61% to
91% (Table 1). Consequently, these gilts were considered to be infected
with serovar 2. Sera of gilts from farm 3 were serologically negative;
therefore, these animals were considered to be free from infection with
serovar 2 of A. pleuropneumoniae. With regard to farm 9, two (9%) sera
were serologically positive. All A.
Farm
No. of samples
No. of positive (%) samples
No. of suspective (%) samples
No. of negative (%) samples
1.
35
10 (28.5)
13 (37.1)
12 (34.2)
2.
46
27 (58.7)
11 (23.9)
8 (17.3)
3.
46
8 (17.3)
4 (8.7)
34 (73.9)
4.
46
21 (45.6)
12 (26.0)
13 (28.2)
5.
46
25 (54.3)
13 (28.2)
8 (17.3)
6.
46
28 (60.8)
12 (26.0)
6 (13.0)
7.
23
6 (26.0)
12 (52.1)
5 (21.7)
8.
23
14 (60.8)
8 (34.8)
1 (4.3)
9.
23
5 (21.7)
7 (30.4)
11 (47.8)
Total
334
144 (43.1)
92 (27.5)
98 (29.3)
Farm
No. of samples
No. of positive (%) samples
No. of suspective (%) samples
No. of negative (%) samples
1.
11
1 (9.0)
0 (0.0)
10 (90.9)
2.
18
6 (33.3)
6 (33.3)
6 (33.3)
4.
10
2 (20.0)
0 (0.0)
8 (80.0)
5.
10
5 (50.0)
5 (50.0)
0 (0.0)
6.
8
5 (62.5)
1 (12.5)
2 (25.0)
7.
11
1 (9.0)
4 (36.6)
6 (54.5)
8.
15
5 (33.3)
5 (33.3)
5 (33.3)
9.
20
2 (10.0)
3 (15.0)
15 (75.0)
Total
103
27 (26.2)
24 (23.3)
51 (50.4)
Although all the above mentioned authors (GOYETTE et al., 1986a; NICOLET,
1988; NIELSEN, 1988; FENWICK, 1992) agree that ELISA is the most sensitive
and reliable method for serological diagnosis of porcine pleuropneumonia,
the risk of false positive results still remains, particularly in the examination
of contaminated sera.
Farm
No. of samples
No. of positive (%) samples
No. of suspective (%) samples
No. of negative (%) samples
7.
23
18 (78.2)
2 (8.7)
3 (13.0)
8.
23
18 (78.2)
5 (21.7)
0 (0.0)
9.
23
21 (91.3)
2 (8.7)
0 (0.0)
Total
69
57 (82.6)
9 (13.0)
3 (4.3)
Farm
No. of samples
No. of positive (%) samples
No. of suspective (%) samples
No. of negative (%) samples
7.
23
18 (78.2)
3 (13.0)
2 (8.7)
8.
23
17 (73.9)
3 (13.0)
3 (13.0)
9.
23
17 (73.9)
5 (21.7)
1 (4.3)
Total
69
52 (75.3)
11 (15.9)
6 (8.7)
Farm
No. of samples
No. of positive (%) samples
No. of suspective (%) samples
No. of negative (%) samples
7.
11
5 (45.4)
2 (18.1)
4 (36.3)
8.
15
3 (20.0)
6 (40.0)
6 (40.0)
9.
20
10 (50.0)
3 (15.0)
7 (35.0)
Total
46
18 (39.1)
11 (23.9)
17 (36.9)
A total of 334 sera of sows were tested for the presence of antibodies
against serovar 2 of A. pleuropneumoniae. The sows (over 12 months of age)
were the oldest group included in the study. Positive results were achieved
in 144 (43%) cases, 92 (28%) were suspect and 98 (29%) were negative (Table
2).
Results achieved for gilts and sows show that there were more serologically
positive gilts (64%) than sows (43%), although both were kept at the same
farms. On the other hand, there were more suspect sows than gilts (28%
as against 27%). The percentage of serologically negative gilts and sows
was almost equal (27% as against 29, respectively) (Tables 1 and 2). This
can be explained by the fact that exposure to the infection in gilts might
have occurred only 2-3 months before the study, and in the sows definitely
more than 9 months before.
Farm 3. All gilts were serologically negative, while 8 sows (17%) were
positive. With respect to the total number of examined sera (92), 9% were
positive. Consequently, the farm could be considered free of infection
from serovar 2 of A. pleuropneumoniae.
Farm 9. Five (22%) sows had a positive titer of antibodies against serovar
2. However, these positive values were slightly above the limits (from
50% to 65%; an average of 58%). Given the relatively small number of sows
(23) examined in this farm, it is considered necessary to include more
animals in order to exclude the possibility of false positive reactions,
or serum contamination.
Farms 1, 2, 4, 5, 6, 7 and 8. Serologically positive sows accounted for
from 26% (farm 7) to 61% (farm 6). Consequently, these sows were considered
to be infected with serovar 2 of A. pleuropneumoniae.
It was not possible to determine the age of boars precisely. Of 103 sera
examined for the presence of antibodies against serovar 2, twenty seven
(26%) were positive, 24 (23%) suspect and 52 (51%) negative (Table 3).
Irrespective of the small number of examined sera, the percentage of positive
findings was significantly smaller compared to gilts and sows (Tables 1,
2 and 3). The difference between positive boars (26%) and gilts (64%) was
statistically significant (P<0.05) This could be explained by the fact
that the boars were kept in individual pens, provided with enclosures,
usually with the best quality facilities and with an optimal microclimate,
to ensure good spermatogenesis. The high zoohygienic conditions under which
the boars were kept, contributed in all respects to the lower incidence
of swine pleuropneumonia.
Sera of studied animals of all age groups at farms 7, 8 and 9 were also
examined for the presence of antibodies against serovar 4-7 of A. pleuropneumoniae.
Of the 69 samples taken from gilts, 57 (83%) were positive, 9 (13%) suspect
and 3 (4%) negative. Expressed in percentage terms for each farm, results
ranged from 78% (farms 7 and 8) to 91% (farm 9) (Table 4). Gilts from all
these three farms were serovar 4-7 positive, which conformed to the fact
that isolates of A. pleuropneumoniae from farms 7 and 8 were serotyped
as serovar 2 and serovar 4-7. On the other hand, all isolates from farm
9 were serotyped as serovar 4-7.
Of 69 sera of the sows examined for the presence of antibodies against
serovar 4-7, 52 (75%) were positive, 11 (16%) suspect and 6 (9%) negative
(Table 5). Sows from all three farms could be considered as being infected
with the serovar in question. Results indicating the presence of serovar
4-7 antibodies in sows were not as low as they were for serovar 2. This
requires cautious interpretation due to the modest number of samples taken
for examination of the presence of antibodies against serovar 4-7.
With regard to examination of boar samples for the presence of antibodies
against serovar 4-7 antibodies, 18 (39%) were positive, 11 (24%) suspect
and 17 (37%) negative (Table 6). Statistically, the percentage of serological
positive boars for serovar 4-7 was significantly lower (P<0.02) at the
same farms.
Conclusions
Based on results of the study it may be concluded that the employed ELISA
test is a reliable method for serological diagnosis of swine pleuropneumonia
in breeding swine of various ages, on a farm level. The test is specific
for the detection of antibodies against each different serovar of A. pleuropneumoniae.
In a small number of serological positive animals on a farm, caution is
required in the interpretation of results, due to possible false positive
reactions and/or serum contamination. In such instances, blood sampling
and testing should be repeated. The number of serological positive boars
was significantly lower compared with the gilts and sows at the same farms,
which was the outcome of the individual keeping of boars and better zoohygienic
management conditions.
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VAILLANCOURT, J. P., G. P. MARTINEAU, S. LARIVIÉRE, R. HIGGINS, K. R. MITTAL
(1988): Serological follow-up in breeding herds infected with Actinobacillus
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Received: 1 October 1997
HABRUN B., V. BILIC, T. NAGLIC, A. HUMSKI: Primjena imunoenzimnog testa
u seroloskoj dijagnostici pleuropneumonije svinja u Hrvatskoj, Vet. arhiv
68, 19-26, 1998.
SAZETAK
Prvi su put u Hrvatskoj serumi rasplodnih svinja pretrazeni imunoenzimnim
testom na prisutnost protutijela za serovarove 2 i 4 - 7 bakterije Actinobacillus
pleuropneumoniae. Pretrazeno je ukupno 782 uzorka krvnih seruma svinja
(od toga 345 seruma nazimica, 334 seruma krmaca i 103 seruma nerastova).
Uzorci seruma koji su poticali s 9 velikih svinjogojskih farmi pretrazeni
su na prisutnost protutijela za serovar 2 bakterije A. pleuropneumoniae.
Dobiveni rezultati pokazuju da je 7 od 9 pretrazenih farmi inficirano serovarom
2 bakterije A. pleuropneumoniae. Postotak seroloski pozitivnih nazimica
na prisutnost protutijela za serovar 2 uzrocnika na 7 inficiranih farmi
kretao se od 61% do 91%, seroloski pozitivnih krmaca od 26% do 61% i seroloski
pozitivnih nerastova od 9% do 50%. Serumi dostavljeni s farmi 7, 8 i 9
pretrazeni su i na prisutnost protutijela za serovar 4 - 7 bakterije A.
pleuropneumoniae. Te tri farme su zarazene serovarom 4 - 7 bakterije A.
pleuropneumoniae. Dobiveni rezultati pokazuju da se na farmama broj 7,
8 i 9 postotak seroloski pozitivnih nazimica na prisutnost protutijela
za serovar 4 - 7 uzrocnika kretao od 78% do 91%, seroloski pozitivnih krmaca
od 74% do 78% i seroloski pozitvnih nerastova od 20% do 50%. Imunoenzimni
test se pokazao pouzdanom metodom u seroloskoj dijagnostici pleuropneumonije
svinja na razini farme u rasplodnih svinja razlicite starosti.
Kljucne rijeci: pleuropneumonija svinja, seroloska dijagnoza, imunoenzimni
test, Hrvatska
Table 1. Antibodies to serovar 2 of A. pleuropneumoniae in gilts
Table 2. Antibodies to serovar 2 of A. pleuropneumoniae in sows
Table 3. Antibodies to serovar 2 of A. pleuropneumoniae in boars
Table 4. Antibodies to serovars 4-7 of A. pleuropneumoniae in gilts
Table 5. Antibodies to serovars 4-7 of A. pleuropneumoniae in sows
Table 6. Antibodies to serovar 4-7 of A. pleuropneumoniae in boars
Accepted: 12 January 1998
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