A caracterização por testes bioquímicos e sorológicos da espécie A. pleuropneumoniae e de amostras NAD-dependentes isoladas de rebanhos sem sintomas de PPS apresenta muitas dificuldades de interpretação de resultados e de reações cruzadas com outras espécies relacionadas do gênero e da Família Pasteurellaceae, principalmente entre A. pleuropneumoniae e A. suis. Devido a estes problemas, torna-se necessário o desenvolvimento de testes mais específicos e sensíveis, que possam ser utilizados como rotina de laboratório.
O A. pleuropneumoniae apresenta vários fatores de virulência que são pouco conhecidos, dificultando o controle da PPS e o desenvolvimento de testes utilizando alvos específicos. No entanto, a cápsula é sugerida como um destes principais fatores e é sugerido que a superóxido dismutase apresente alguma vantagem na sobrevivência do App no organismo do hospedeiro. Portanto, buscamos estudar e analisar estes fatores de virulência do A. pleuropneumoniae.
Como objetivos específicos foram apresentados:
o desenvolvimento de primers para amplificar o gene sodC e padronização de primers para amplificação dos genes cpx responsáveis pela síntese de proteínas envolvidas no transporte da cápsula de A. pleuropneumoniae;
aplicação dos primers cpx e sodC para caracterização de amostras NAD-dependentes isoladas de campo;
caracterização das superóxidos dismutases (SOD) de A. pleuropneumoniae;
análise da presença de cápsula nas amostras de A. pleuropneumoniae.
CAPÍTULO 2
Detection of Actinobacillus pleuropneumoniae by PCR on field strains isolated from healthy and diseased pigs
Detection of Actinobacillus pleuropneumoniae by PCR on field strains isolated from healthy and diseased pigs.
Running title: Detection of A. pleuropneumoniae by PCR
C. S. Klein1,4, I. A. Piffer4, S. C. Silva1,3, A. Schrank1,2, M. B. B. Fávero4 and I. S. Schrank*1,2
Address of institution where the work was performed:
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Prédio 43421, C.P. 15005, Porto Alegre, RS, Brazil.
Catia Silene Klein1,4, Itamar Antônio Piffer4, Sérgio Ceroni da Silva1,3, Augusto Schrank1,2, Maria Bernardete Burin Fávero4 and Irene Silveira Schrank*1,2
Footnote:
1Centro de Biotecnologia – UFRGS
2Depto. de Biologia Molecular e Biotecnologia - UFRGS
3Depto. de Patologia Clínica Veterinária, Faculdade de Veterinária - UFRGS 4Centro Nacional de Pesquisa de Suínos e Aves, EMBRAPA
Irene Silveira Schrank*: Fax: 55 (51) 319 10 79 Telephone: 55 (51) 316 60 55
Correspondig author: Irene Silveira Schrank
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Prédio 43421, C.P. 15005, CEP 91501-970, Porto Alegre, RS, Brazil.
Abstract
We investigated whether primers that specifically amplify a 0.7 kb DNA fragment from the conserved cpx genes could be applied to characterize A. pleuropneumoniae field isolates. The specific cpx primers were applied on 120 strains of A. pleuropneumoniae and other NAD-dependent field isolates from healthy and diseased animals to characterize A. pleuropneumoniae isolates from herds in Brazil. The PCR and hybridization experiments were applicable to discriminate between isolates of A. pleuropneumoniae and isolates of other microorganisms. The 0.7 kb cpx DNA bands were amplified from all 63 A. pleuropneumoniae isolates from herds with clinical symptoms and isolated from lesions of acute cases of swine pleuropneumonia whether serotypeable or nonserotypeable. The method was also applied to fifty-seven field isolates obtained from animals of apparently healthy herds and the amplified cpx product was present in 4 serotypeable and only in 2 out of 11 A. pleuropneumoniae nonserotypeable isolates. All nonserotypeable A. pleuropneumoniae isolates revealed the apxA amplification pattern compatible to previously known serotypes. Our results suggest that some nonserotypeable isolates might represent a population of isolates that originally were serotypeable but lost the ability to react with serotype-specific antisera or might belong to novel serotypes. The PCR method applied is highly sensitive for serotypeable A.
pleuropneumoniae strains and for nonserotypeable strains isolated from acute cases of swine pleuropneumoniae in Brazil.
Introduction
Porcine pleuropneumonia, distributed worldwide, is a major contagious respiratory disease in pigs and causes high mortality with severe economic losses to the swine industry (31). The disease is characterized by fibrinous pleuritis with hemorrhagic and necrotic lesions in the lungs, and pleural adhesions (22). The etiological agent of porcine pleuropneumonia, Actinobacillus pleuropneumoniae can be differentiated in two biovars; biovar 1, NAD dependent and biovar 2 NAD independent (28). At least 12 serotypes with significant differences in virulence have been identified (4). Serotyping is mainly based on capsular antigens. In addition, the serotypes have different lipopolisaccharide (LPS) composition, except serotypes 1, 9 and 11; serotypes 3, 6 and 8; and serotypes 4 and 7 that show common epitopes. Several putative factors have been proposed to be involved in virulence of this agent: capsule, endotoxin, exotoxins (Apx toxins), adhesins, transferrin binding protein, outer membrane proteins and secreted proteases (12).
Animals that survive an A. pleuropneumoniae infection generally suffer from chronic lesions and became subclinical carriers of the pathogen (3). In diseased animals the clinical symptoms and pathological lesions are very characteristic, allowing the isolation of the agent followed by serotyping and drug susceptibility determination.
In cases of subclinical infection, without the presence of symptoms and lesions, the diagnosis is normally based on serology. Several techniques were developed for serologic diagnosis of A. pleuropneumoniae including tube agglutination test, agglutination in the presence of 2- mercapto-ethanol, complement fixation test and enzyme-linked immunosorbent assay (ELISA). Among these tests, the ELISA shows the best sensitivity and specificity and can been run in a totally automated form. Amongst the ELISA tests, the most utilized are those based on long chain LPS (5, 6, 7).
In spite of the ELISA tests sensitivity there are situations of subclinical infections in which the animals are in excellent clinical state, but with few seropositive animals. In this situation the interpretation is very difficult, considering that any ELISA-test is 100% specific. In these situations it is necessary to make the diagnosis based on the isolation of the agent from tonsil (16, 32). However this method is not sensitive enough. In addition, there are several resident Actinobacillus-like species in swine tonsils that are very difficult to differentiate
biochemically from A. pleuropneumoniae (16). The technique of A. pleuropneumoniae immunomagnetic isolation is promising, but is limited to the serotypes to which the test was standardized. Moreover, several studies have shown the incidence of isolates from clinical cases that could not be serotyped (1, 11, 21). Therefore, the detection of A. pleuropneumoniae from live animals by a specific and sensitive method would be helpful for identification of this pathogen within the normal flora of the upper respiratory tract.
The polymerase chain reaction (PCR) is a method of gene amplification that can be used for highly sensitive and specific detection of microorganisms in a wide range of samples. Problems with non-typeable and cross-reactive A. pleuropneumoniae isolates can be solved by PCR amplification and easily identified (13).Recently, Ward and Inzana (33) have characterized a DNA region involved in the exportation of capsular polysaccharides (cpx) by A. pleuropneumoniae serotype 5a. Based on the conserved nature of cpx genes a PCR assay was developed for detection of A. pleuropneumoniae to improve the sensitivity and specificity of identification of serotypes 5 isolates and for early detection (18).
We have used primers that specifically amplify a 0.7 kb DNA fragment present in serotype 5a from the conserved cpx genes to characterize the other serotypes. We have also applied the specific cpx primers on NAD-dependent field isolates from healthy and diseased animals to further characterize A. pleuropneumoniae isolates from herds in Brazil.
Materials and methods
Bacterial strains and culture conditions
The 12 reference strains for the recognized A. pleuropneumoniae serotypes and other bacteria used in this work are listed on Tables 1 and 2. The two A. pleuropneumoniae field isolates (6796 and 7260), a capsule-deficient mutant of strain K17 (K17-C) and a hemolysis- deficient mutant mIT4-H were also utilized for comparative analysis.
All strains were streaked on blood agar with a nursery strain of Staphylococcus aureus and incubated at 37oC during an 18 hours period in a humid atmosphere with 5-10% CO2. A characteristic NAD-dependent colony was picked up and streaked on Columbia agar (Oxoid, Basingstoke, UK) supplemented with 5% bovine serum, 50 µg of NAD (Sigma Chemical Co., St. Louis, MO) and incubated at same conditions. Microbiological presence of capsule on A pleuropneumoniae strains was determined by visualization of an iridescent colony on Columbia agar supplemented with NAD as previously described (14).
Source of the isolates
The 120 field isolates of A. pleuropneumoniae and other NAD-dependent strains (Table 3) were from Centro Nacional de Pesquisa em Suínos e Aves (CNPSA), EMBRAPA, Brazil and were previously characterized (16, 26, 27). All field isolates were characterized using methods and tables previously described (10, 17, 19). Serotyping was performed by immunodiffusion test (ID) as described by Nielsen and O’Connor (23) but using a phenol – water extracted antigen as described by Gunnarsson et al. (9). Strains which resulted in unknown serotypes according to the ID test were subsequently serotyped by the passive hemaglutination test described by Mittal et al. (20). Sixty three field isolates were originated
from herds with clinical symptoms and isolated from lesions of acute cases of swine
pleuropneumonia (ASPP). Among them, forty-nine were assigned to serotypes 3, 5 or 7 and fourteen were nontypeable (27).
The other fifty seven field isolates were from animals of apparently healthy herds. These herds were classified into three different groups as follows: group A; contains twenty five field isolates from tonsils of asymptomatic 9-15 weeks old piglets provinient from three chronically infected herds with previous history of pleuropneumonia outbreaks (CSPP). Groups B and C herds, containing, respectively, 20 and 12 field strains isolated from tonsils of asymptomatic piglets from three herds where never had been observed symptom nor
lesions characteristic of swine pleuropneumoniae and with no record of A . pleuropneumoniae isolation (NSPP) (Table 3). Additionally, these herds were under constant veterinary
surveillance (16). Antibodies detected by an ELISA based in LPS-LC polyvalent for serotypes 3, 5 and 7 were present in all CSPP (ELISA+) and in Group B NSPP herds (16).
DNA isolation and PCR conditions
Total DNA was purified as previously described (24). Briefly, a loopful of colonies from Columbia agar plates were washed in distilled water, resuspended in water with 1.7% SDS, 50mg/ml of proteinase K and incubated at 65oC for 1 h. Total DNA was purified by repeated phenol-chloroform extractions and precipitation from the aqueous phase by adding 0.1 volumes of 3 M sodium acetate and 0.6 volumes of isopropanol. The precipitated DNA was dried and resuspended in distilled water.
The PCR assays were performed in a DNA thermal cycler (MJ Research PTC 200). PCR mixture contained the reaction buffer (50 mM KCl, 10 mM Tris-Cl pH 8.3, 2 mM MgCl2), 30 pmol of each primer, 1U of Taq polymerase (CenBiot Enzymes, Centro de
Biotecnologia, UFRGS), 200 µM of each dNTP, template DNA and distilled water for a final volume of 25 µl. The reaction mixture was subjected to PCR under conditions described by Lo et al. (18). Primers cpx-up and cpx-do (18) were purchased from Oligo ETC & Oligo Therapeutics Inc. (Wilsonville, USA).
DNA manipulation, sequencing and Southern blotting
DNA manipulations were performed using standard techniques (29) and instructions provided by suppliers of kits, enzymes and reagents. Southern blot analysis was performed using the ECL Direct Nucleic Acid Labeling and Detection Systems (Amersham Pharmacia Biotech). The nucleotide sequence determination was performed by the chain-termination method (30) using 33P-dNTPs and ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech).
Results
Using the cpx specific PCR assay it was possible to amplify a 0.7 kb (715 bp) product from the DNA of all A. pleuropneumoniae reference serotypes but the serotype 4 (Figure 1). DNA from all other Actinobacillus, Haemophilus and Pasteurella species, as well as other bacterial strains resulted in no amplification (Figure 1C). These results are in agreement with previously published work showing that even within a highly conserved region there may be areas of nonhomology among different serotypes (18). To evaluate the specificity of the PCR assay the amplified PCR product from serotype 5a was sequenced and used as a probe against the PCR fragments amplified from the other serotypes. The 0.7 kb fragment from serotype 5a showed total homology with the previously published cpx DNA sequence (33). Southern
blotting revealed a similar hybridization pattern for all PCR positive serotypes (Figure 1B) indicating that the amplified product has identical sequence as published for serotype 5a.
Sixty three bacterial strains isolated from clinical infections in pigs (ASPP) were tested using the cpx primers in PCR reactions in order to establish the specificity of the primers for different field strains. A PCR product of 0.7 kb, specifically hybridizing to serotype 5a cpx probe was obtained for all strains considered to be A. pleuropneumoniae by microbiological and biochemistry methods (27).
The 57 NAD-dependent field isolates obtained from apparently healthy herds (CSPP and NSPP) were also analyzed by PCR for the presence of cpx genes. Based on a previous characterization these field isolates were assigned to three distinct biochemical profiles. The first group with 15 field isolates identified as A. pleuropneumoniae. The second group, with 27 strains corresponded to A. minor and the third group with 15 strains corresponded to other NAD-dependent field isolates.
Among the 15 field isolates of A. pleuropneumoniae only four were serotyped and all of then were isolated from CSPP herds (Group A). All remaining 11 A. pleuropneumoniae strains were nontypeable. Serotypes 7 and 12 were identified amongst the four
A. pleuropneumoniae typeable strains (16). The serotype 7 isolated have been previously isolated from the same herds while the serotype 12 was isolated from herds chronically infected with serotype 3. These results together with the PCR / hybridization analysis are summarized on Table 3. Amplification of field isolates total DNA using cpx primers resulted in a single 0.7 kb DNA band in all A. pleuropneumoniae serotypeable and only in 2 out of 11 A. pleuropneumoniae nontypeable isolates. All A. minor and other NAD-dependent field isolates showed negative results for the PCR/hybridization analysis confirming the specificity of the assigned primers. The 0.7 kb PCR fragment from two field isolates was also sequenced revealing total homology with the previously characterized sequence from serotype 5a DNA.
Discussion
Rapid and accurate identification of A. pleuropneumoniae strains potentially virulent in subclinically infected herds is relevant both for limiting the severity of outbreaks and for determining the source of the bacteria entry into specific herds. A PCR assay with primers specific for the transport region of the capsular polysaccharide of serotype 5 has been recently proposed (18). The present study was conducted to evaluate the feasibility of applying the cpx primers to differentiate A. pleuropneumoniae from the other NAD-dependent strains. The results of the PCR and hybridization experiments for the cpx gene revealed that all A. pleuropneumoniae serotypes, with exception of serotype 4, could be detected by the PCR assay (Figure 1). Moreover, no amplification of DNA under conditions tested was observed from any of the other swine respiratory pathogens analyzed, indicating that the PCR assay is very specific.
The PCR assay was also applied to testing NAD-dependent isolates provinient from different herds. The test was able to discriminate between isolates of A. pleuropneumoniae and isolates of other microorganisms (Table 3). The 0.7 kb cpx DNA bands were amplified from all 63 A. pleuropneumoniae isolates from ASPP herds whether serotypeable or nonserotypeable as well from the 4 serotypeable isolates from CSPP or NSPP herds. However, the amplification of the cpx product was positive only from 2 out of 11 A. pleuropneumoniae nonserotypeable isolates provinient from CSPP or NSPP herds.
All the strains isolated from ASPP were isolated from lesions and thus can be
considered pathogenic, in contrast to strains isolated from tonsil of healthy piglets from NSPP herds never reported to have swine pleuropneumoniae outbreaks, neither characteristic lesions observed at abattoir nor isolation of the A. pleuropneumoniae (16). This fact indicates that these strains might be not pathogenic at all or are less pathogenic than those isolated from
field cases. All the strains belonging to the pathogenic groups of A. pleuropneumoniae gave positive results in the cpx PCR even if they were nonserotypeable, while among the less pathogenic or not pathogenic (NSPP isolates) only 1/8 was positive and all were
nonserotypeable. This fact suggests that the PCR test is more prone to detect pathogenic strains than nonpathogenic ones. Based on this observation and taking in consideration that serotype 4 is not detected by this PCR test, the application of cpxPCR directly to tonsil secretions might be a good alternative for defining the status of a herd in regard to the occurrence of pathogenic strains of A. pleuropneumoniae, specially in Brazil where there are few records of serotype 4 isolation from field cases of swine pleuropneumoniae.
The present study describes the use of primers to amplify conserved capsular gene regions that identify all A. pleuropneumoniae serotypes with exception of serotype 4. Therefore, these 9 A. pleuropneumoniae nonserotypeable isolates could represent the
previously known serotype 4 or possiby the existence of a novel serotype(s). Serotypes of A. pleuropneumoniae are distinguished by their unique capsular polysaccharide that is related with the virulence of the A. pleuropneumoniae strains (15). The 9 isolates discussed above could also have differences on the virulence pattern related with the presence of capsule. However, the presence of capsule was demonstrated in all 9 nonserotypeable isolates by the visualization of characteristic iridescent colonies on Columbia agar (data not shown).
A. pleuropneumoniae isolates that do not react with any of the serotype-specific antisera have been detected and normally are not further characterized. Genotypic characterization of nonserotypeable A. actinomycetemcomitans isolates suggest that the isolates are serotype antigen variants originating from isolates of known serotype (25). Additionally, primers specific to amplify a region of the apxA genes from A.
pleuropneumoniae were constructed (2) and applied to characterize all nonserotypeable isolates. The amplification products related to apxIA, apxIIA and apxIIIA genes could be visualized and are shown on Table 4. All nonserotypeable A. pleuropneumoniae isolates
revealed the apxA pattern compatible to previously known serotypes. However, only 3 out of 9 nonserotypeable isolates could be assigned to serotype 4 based on the apxA pattern (apxII and apxIIIA). Our results suggest that some nonserotypeable isolates might represent a population of isolates that originally were serotypeable but lost the ability to react with serotype-specific antisera or might belong to novel serotypes not yet described.
In conclusion, the PCR for detection of cpx genes was 100% sensitive for serotypeable A. pleuropneumoniae strains and for nonserotypeable strains isolated from acute cases of swine pleuropneumoniae in Brazil.
Acknowledgments
This work was supported by grants from FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul). C.S.K is a receiver of a scholarship from CAPES
(Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior).
References
1. Blackall, P. J., R. Bowles, J. L. Pahoff, and B. N. Smith. 1999. Serological characterization of Actinobacillus pleuropneumoniae isolated from pigs in 1993 to 1996. Aust. Vet. J. 77:39-43.
2. Collares, R. M. 2000. Ms.C. thesis. Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Análise molecular dos genes para toxinas de Actinobacillus pleuropneumoniae em isolados de campo.
3. Fenwick, B., and S. Henry. 1994. Porcine pleuropneumonia. J. Am. Vet. Med. Assoc. 204:1334-1340.
4. Frey, J. and Nicolet, J. 1990. Hemolysin patterns of Actinobacillus pleuropneumoniae. J. Clin. Microbiol. 28:232-236.
5. Gottschalk, M., E. Altman, N. Charland, F. De Lasalle, and J. D. Dubreuil. 1994a. Evaluation of a saline boiled extract, capsular polysaccharides and long-chain
lipopolysaccharides of Actinobacillus pleuropneumoniae serotype 1 as antigens for the serodiagnosis of swine pleuropneumonia. Vet. Microbiol. 42:91-104.
6. Gottschalk, M., F. De Lasalle, S. Radacovici, and J. D. Dubreuil. 1994b. Evaluation of long chain lipopolysaccharides (LC-LPS) of Actinobacillus
pleuropneumoniae serotype 5 for the serodiagnosis of swine pleuropneumonia. Vet. Microbiol. 38:315-327.
7. Gottschalk, M., E. Altman, S. Lacouture, F. De Lasalle, and J. D. Dubreuil. 1997. Serodiagnosis of swine pleuropneumonia due to Actinobacillus pleuropneumoniae serotypes 7 and 4 using long-chain lipopolysaccharides. Can. J. Vet. Res. 61:62-65. 8. Gram, T., P. Ahrens, M. Andreasen, J. P. Nielsen. 2000. An Actinobacillus
pleuropneumoniae PCR typing system based on the apx and omlA genes – evaluation of isolates from lungs and tonsils of pigs. Vet. Microbiol. 75:43-57.
9. Gunnarsson, A. 1979. Evaluation of different antigens in the complement - fixation test for diagnosis of Haemophilus pleuropneumoniae (parahaemolyticus) infections in swine. Am. J. Vet. Res. 40:1564-1567.
10. Gutierrez, C. B., R. I. Tascon, J. I. R. Barbosa, O. R. Gonzalez, J. A. Vasquez, and E. F. R. Ferri. 1993. Characterization of V Factor-dependent organisms of the Family Pasteurellaceae isolated from porcine pneumonic lungs in Spain. Comp. Immun. Microbiol. 6:123-130.
11. Hampson, D. J., P. J. Blackall, J. M. Woodward, and A. J. Lymbery. 1993. Genetic analysis of Actinobacillus pleuropneumoniae, and comparison with Haemophilus spp taxon "Minor group"and taxon C. Zbl. Bakt 279:83-91. 12. Haesebrouck, F., K. Chiers, I. van Overbeke, and R. Ducatelle. 1997.