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





* Contact address:
Mr. Boris Habrun,
Department of Bacteriology, Croatian Veterinary Institute, 10000 Zagreb, Savska c. 143, Croatia.
Phone: 385 1 6190 838; Fax: 385 1 6190 841

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).


Table 1. Antibodies to serovar 2 of A. pleuropneumoniae in gilts

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.


Table 2. Antibodies to serovar 2 of A. pleuropneumoniae in sows

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)


Table 3. Antibodies to serovar 2 of A. pleuropneumoniae in boars

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.


Table 4. Antibodies to serovars 4-7 of A. pleuropneumoniae in gilts

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)


Table 5. Antibodies to serovars 4-7 of A. pleuropneumoniae in sows

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)


Table 6. Antibodies to serovar 4-7 of A. pleuropneumoniae in boars

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.

References

ALTON, G. G., L. M. JONES, D. E. PIETZ (1975): Laboratory techniques in brucellosis. 2nd ed. World Health Organisation. Geneva.

ANDERSON, R. K., R. JENNESS, H. P. BRUMFIELD (1964): Brucella agglutinating antibodies: relation of mercaptoethanol stability to complement fixation. Science 143, 1334-1335.

BILIC, V. (1993): Pleuropneumonija svinja u Hrvatskoj. 1. Nalazi protutijela za bakteriju Actinobacillus pleuropneumoniae u rasplodnih svinja. Praxis vet. 41, 165-174.

FENWICK, B. (1992): Critical comparison of the serologic tests used to diagnose porcine pleuropneumoniae. Proc. 12th IPVS Congress. p. 226. The Hague, Netherland.

FREY, J. (1995): Virulence in Actinobacillus pleuropneumoniae and RTX toxins. Trends in Microb. 3, 257-261.

GOYETTE, G., S LARIVIÉRE, K. R. MITTAL, R. HIGGINS (1986a): Development of enzyme-linked immunosorbent assay for the serodetection of pigs exposed to Haemophilus pleuropneumoniae. p. 256. Proc. 9th IPVS Congress. Barcelona, Spain

GOYETTE, G., S LARIVIÉRE, K. R. MITTAL, R. HIGGINS, G. P. MARTINEAU (1986b): Comparison of CFT, ELISA and tube agglutination test with 2-ME in pigs from herds. Proc. 9th IPVS Congress. p. 258. Barcelona, Spain.

MITTAL, K. R., R. HIGGINS, S. LARIVIÉRE, D. LEBLANC (1984): A 2-mercaptoetanol tube agglutination test for diagnosis of Haemophilus pleuropneumoniae infection in pigs. Am. J. Vet. Res. 45, 715-719.

NICOLET, J. (1988): Taxonomy and serological identification of Actinobacillus pleuropneumoniae. Can. Vet. J. 29, 578-580.

NICOLET, J., P. A. DE MEURON, P. BACHMANN (1971): Sur l'hémophilose du porc. IV. L'épruve de déviation du complément, un test de dépistage des infections Haemophilus pleuropneumoniae (H. parahaemolyticus) infection. Schweis. Arch. Tierheilkd. 113, 191-200.

NICOLET, J., P. PAROZ, M. KRAWINKLER, A. BAUMGARTNER (1981): An Enzyme-linked immunosorbent assay, using an EDTA-extracted antigen for the serology of Haemophilus pleuropneumoniae. Am. J. Vet. Res. 42, 2139-2142

NIELSEN, R. (1988): Seroepidemiology of Actinobacillus pleuropneumoniae. Can. Vet. J. 29, 580-582.

SEBUNYA, T. N. K., J. R. SAUNDERS (1983): Haemophilus pleuropneumoniae infection in swine: a review. J. A. V. M. A. 182, 1331-1337.

VAILLANCOURT, J. P., G. P. MARTINEAU, S. LARIVIÉRE, R. HIGGINS, K. R. MITTAL (1988): Serological follow-up in breeding herds infected with Actinobacillus pleuropneumoniae serotype 1 using the tube agglutination test with 2-mercaptoetanol. Prev. Vet. Med. 6, 263-274.

WILLSON, P. J., C. SCHIPPER, E. D. MORGAN (1988): The use of an enzyme-linked immunosorbent assay for diagnosis of Actinobacillus pleuropneumoniae infection in pigs. Can. Vet. J. 29, 583-585.

ZIMMERMAN, W., M. STAEGER, W. BOMMELI (1990): A sero-epidemiological study on the occurence of serotypes of A. pleuropneumoniae (APP) on Swiss breeding herds surveyed by pig health service. Proc. 11th IPVS Congress. p. 32. Lausanne, Switzerland.

Received: 1 October 1997
Accepted: 12 January 1998



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


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