TOXIN DETECTION IN Pasteurella multocida

Arquivos da Faculdade de Veterinária. UFRGS. 29(2):79-85, 2001. ISSN 0101-5230

Recebido/Received: dez 2000

TOXIN DETECTION IN Pasteurella multocida STRAINS ISOLATED FROM SWINE LUNGS IN THE STATE OF RIO GRANDE DO SUL, BRAZIL

DETECÇÃO DA PRODUÇÃO DE TOXINA EM AMOSTRAS DE Pasteurella multocida ISOLADAS DE PULMÕES DE SUÍNOS NO RIO GRANDE DO SUL

S.M. BOROWSKI1 , S.C. SILVA2,3, I. SCHRANK2 & M. CARDOSO3

RESUMO

ABSTRACT

Foi determinada a produção de toxina e a presença de gene toxA que a codifica, utilizando 24 amostras de Pasteurella multocida isoladas de suínos com pneumonia e pleurite. Para determinar a capacidade toxigênica foi utilizado o teste de letalidade em camundongo. Para a detecção do gene toxA foi utilizada a técnica do PCR, utilizando 2 primers que possibilitaram a amplificação de um produto de PCR de 300 pb. Com a finalidade de comprovar que o produto do PCR amplificado correspondia ao segmento do gene toxA foi feita a clivagem do fragmento da P. multocida tipo capsular D padrão toxigênica com a endonuclease de restrição Taq I, sendo que os fragmentos de clivagem corresponderam aos tamanhos esperados. Apenas uma dentre as 24 amostras de P. multocida foi toxigênica pelo teste biológico em camundongo. Entretanto, com o uso da técnica do PCR, nove amostras foram positivas. São discutidas hipóteses para explicar essa diferença, como possibilidade da ocorrência da supressão da expressão gênica “in vivo” ou a existência de um gene defectivo, não funcional.

Toxin production and the presence of the toxA gene was assayed in 24 Pasteurella multocida strains isolated from pigs with pneumonia and pleuritis in the State of Rio Grande do Sul, Brazil. Toxigenicity was assessed through a mouse lethality test and a PCR amplification assay was used to obtain a 300 bp product from the toxA gene. In order to demonstrate that the amplified product corresponded to the toxA gene, the same segment of a reference P. multocida capsular type D toxigenic strain was amplified and treated with the restriction enzyme Taq1, and the restriction fragments corresponded to the expected sizes. Only one out of 24 strains of P. multocida was toxigenic in the mouse lethality test. However, with the use of PCR, nine strains carried the toxA gene. Hypothesis to explain this difference is the suppression of genic expression in vivo or the presence of a defective, non-functional gene.

Descritores: Pasteurella multocida , toxina, pulmão, suínos, PCR.

Key words: Pasteurella multocida, toxin, lung, pigs, PCR.

1Centro

de Pesquisa Veterinária “Desidério Finamor”, Fundação Estadual de Pesquisa Agropecuária, Caixa Postal 47, CEP 92990-000, Eldorado do Sul, RS, Brazil, e-mail: [email protected] 2Centro de Biotecnologia/Universidade Federal do RS, Brazil 3Faculdade de Veterinária/Universidade Federal do RS, Brazil

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S.M. BOROWSKI , S.C. SILVA, I. SCHRANK & M. CARDOSO. 2001. Toxin detection in Pasteurella multocida strains isolated from swine lungs in the state of Rio Grande do Sul, Brazil. Arq. Fac. Vet. UFRGS. 29:79-85.

INTRODUCTION

strains which cause pneumonia and pleuritis are not well defined. Some authors reported isolation of toxigenic strains from pig lungs (Pijoan et al., 1984; Iwamatsu and Sawada, 1988). However, toxigenicity does not seems important for the experimental reproduction of pneumonia by P. multocida (Baekbo and Nielsen, 1988). There are several laboratory tests available for discrimination between toxigenic and nontoxigenic strains of P. multocida. They include biological assays for toxin activity, such as mouse lethality test (Ahn and Kim, 1994) and dermal necrosis test in guinea pigs (Avella et al., 1993). More recently, molecular tests such as polymerase chain reaction (PCR) (Nagai et al., 1994; Lichtensteiger et al., 1996) have been used. The present study evaluated toxin production in P. multocida strains isolated from pigs with pneumonia and pleuritis using a mouse lethality test and investigated the presence of the toxA gene by PCR.

Pasteurella multocida (P. multocida) can be present as a commensal in the nasopharynx of apparently healthy animals and as a primary or secondary pathogen in several animal species (Rimler and Rhoades, 1989). Toxigenic strains of the bacteria have been isolated from humans with tonsillitis, rhinitis, sinusitis, pleuritis, appendicitis, and septicemia, suggesting a potential zoonosis (Nilsen and Frederiksen, 1990, Donnio et al., 1991). Most human isolates belong to capsular types A and D (Carter and Bain, 1960), similar to types isolated from swine (Pijoan, 1992). In pigs, P. multocida is a normal inhabitant of the nasal cavity and is one of the agents of progressive atrophic rhinitis (De Jong et al., 1980). Toxigenic strains of P. multocida alone or in association with factors such as previous infection by Bordetella bronchiseptica is considered the main etiological agent of the condition (Pijoan, 1992). Toxigenic strains of P. multocida synthesize a 145 kDa toxin codified by the chromosomal gene toxA (Lichtensteiger et al., 1996). The importance of P. multocida as a primary agent of pneumonia in swine has been thoroughly investigated. According to Pijoan and Fuentes (1987), the microorganism is unable to perform as a primary pathogen, depending on the interaction with other organisms to produce pneumonia (such as adenovirus, Classical Swine Fever virus, Reproductive and Respiratory Syndrome virus, Mycoplasma hyopneumoniae and Aujeszky Disease virus). However, the same authors state that the isolation of P. multocida from swine lungs in slaughterhouses demonstrates the importance of the agent in pneumonic processes, causing great economical losses to the swine industry. In Brazil, Stepan (1995) examined 5987 pigs and collected 230 lungs with pleuritis and pneumonia in 4 slaughterhouses in the State of Rio Grande do Sul. Bacteriological examination yielded 99 isolates of P. multocida (43%). The virulence factors of P. multocida

MATERIALS AND METHODS

1.Strains A total of 24 strains of P. multocida were used. Eleven were obtained from pig lungs from four slaughterhouses in the State of Rio Grande do Sul, Brasil, isolated by Stepan (1995) at Laboratório de Patologia Suína do Centro de Pesquisa Veterinária “Desidério Finamor” (CPVDF) (Swine Pathology Laboratory of the Center of Veterinary Research), Eldorado do Sul, identified as PI5, PI23, PI21, PI7, FII14, PIII47, PII8, PIII19, FII9, PIII43, CII2, and 13 strains isolated in the same laboratory from piglets with pneumonia and pleuritis, identified as 76, 805, Ca, Ta, 710, Fr, 1/95, 1265, 1133, 903, 1628, 1363, 1621. The strains were initially preserved in brain and heart infusion (BHI) media with the addition of 5% horse serum, preserved at –70o C, and later lyophilized. A reference strain of toxigenic P. multocida, capsular type D (obtained

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multocida by PCR, a reference strain of capsular type D was used. All strains were cultivated in BHI for 24 hours at 37o C.

from American Type Culture Collection – ATCC N0 12948) and a strain isolated from a pig with atrophic rhinitis (fimbriated, capsular type D, obtained from Centro Nacional de Pesquisa de Suínos e Aves – National Poultry and Swine Research Center – EMBRAPA, Concórdia, Santa Catarina, Brasil) were used as controls.

4.2 PCR primers Two specific primers for P. multocida toxA gene were designed, based on Nagai et al. (1994).

2. Classification All strains were classified into capsular types A and D. Neutral acriflavine test 1:1000 (Carter and Subronto, 1973) was used to identify D-type strains. Hyaluronidase test was employed to identify A-type strains, using a hyaluronidase producing strain of Staphylococcus aureus (Carter and Rundell, 1975).

4.3 DNA amplification by PCR The technique used was essentially as described by Sambrook et al. (1989). DNA extracted from reference strains and field isolates was obtained by heating at 100o C of 100 ml of the culture for 5 minutes, followed by cooling at 4o C and centrifugation at 14000 rpm for 2 minutes. PCR reactions was performed in a 25 ml volume, containing 30 pmol of each primers, 200 mM of each tri-phosphate de-oxy-ribo-nucleotide, 1 unit of Taq DNA polymerase (CENBIOT Enzimas) in 1x reaction buffer (10 mM Tris-HCl, pH 8,3; 50 mM KCl, 2,5 mM MgCl2) and 5 ml of the sample. The following protocol was used in the thermocycler (MJ Research): a denaturation stage at 94o C (5 minutes), followed by a annealing at 55o C (45 seconds) and extension at 72o C (45 seconds) and denaturation at 94o C (45 seconds). The three last cycles were repeated 35 times and the strains were incubated at annealing temperature for 2 minutes, followed by extension at 72o C (5 min).

3. Dermonecrotic toxin detection in mouse In order to assess the toxigenicity of P. multocida strains, the technique described by Ahn and Kim (1994) was modified as follows:

3.1 Strain cultivation Strains were propagated in TSB (trypticase soy broth) for 36 hours at 37o C. Each bacterial growth was frozen and thawed repeatedly 10 times in liquid nitrogen and centrifuged for 30 minutes at 4000 g. The supernatant was then filtered in a 0.22 mm- membrane.

3.2 Mouse inoculation 21-day-old Webster mice were inoculated intraperitonially with 0.5 ml of the filtrate described above and observed for 10 days. The strains causing death were considered toxigenic.

4.4 Restriction of PCR products with enzymes To demonstrate that the amplified PCR products corresponded to the toxA gene segment, the PCR amplicons of reference P. multocida type D strains were treated with TaqI restriction endonuclease (Fig. 2B, lane 4). An aliquot of 10 ml of the amplicon was treated with the enzyme according to the manufacturer’s specification (Pharmacia).

4. Detection of the Gene toxA by PCR 4.1 Strains To determine optimal conditions for the amplification of specific DNA fragments of P.

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4.5 Analysis of PCR products by electrophoresis in agarose gel Aliquots corresponding to 1/5 of PCR reactions or ½ of the restriction reaction were submitted to electrophoresis in 2% agarose gel, stained with ethidium bromide and the DNA bands were visualized under UV light, essentially as described by Sambrook et al. (1989).

RESULTS

1. Strain classification All strains were classified as capsular type A, except for sample 1/95, which belonged to type D. 2. Toxin assay in mouse Table 1 shows the results obtained in mouse lethality test. Two strains, the standard P. multocida D and 1/95 were considered toxigenic. Table 1. Results of PCR and mouse lethality assays STRAINS GROUP I CLINICAL CASES Ca, Ta, 805, 903, 1133,1265, 1363,1621, SLAUGHTERHOUSES FII9, FII14, PI7, PI21, PI23, PIII19, PIII47 GROUP II CLINICAL CASES 1/95 REFERENCE P. multocida D GROUP III CLINICAL CASES 76, 710, 1628, Fr, SLAUGHTERHOUSES CII2, PI5, PII8, PIII43 REFERENCE FimbD

PCR

Mouse lethality

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3. ToxA gene detection Using PM1 and PM2 primers, a 300 bp PCR product was obtained with the D-type P. multocida strain (Fig. 2A, lane 12). The restriction fragments corresponded to the expected sizes, according to the analysis of the restriction map presented in Fig. 1. In the present study, 9 out of the 24 analyzed strains presented bands with sizes corresponding to the segment of the toxA gene observed in the reference strain (lane 1, 2, 7, 8, 15, 17, 19, 22 and 26), besides the fimbriated sample (fimbD), isolated from the nasal turbinate of an animal with atrophic rhinitis (lane 25), (Fig. 2A).

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Fig. 1. Restriction map of P. multocida toxA gene:The main characteristics are indicated in the map: RBS, Ribosome Linking Site; CDS1, codifying site of toxA gene. The number in brackets indicate the position in the sequence where restriction sites are found.

A

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Fig. 2. Electrophoresis in agarose gel of PCR products amplified from toxA gene. In A are presented the fragments of 300 bp amplified from P. multocida strains: lane M, 100 bp ladder; 1, 1628; 2, PII8; 3, 1133; 4, 903; 5, 805; 6, 1265; 7, 1/95; 8, CII2; 9, 1363; 10, PIII19; 11, PIII47; 12, tipo D; 13, PI23; 14, 1621; 15, 76; 16, CA; 17, FR; 18, FII9; 19, PIII43; 20, PI7; 21, PI21; 22, PI5; 23, FII14; 24, TA; 25, FIMBD; 26, 710; 27, negative control. In B are presented the fragments of PCR from strains 1/95 (lane 1) and type D (lane 3) and the respective restriction products with Taq1 (lane 2 and 4). M, 100 bp ladder.

DISCUSSION

Only one out of 24 strains isolated from pneumonia and pleuritis in our work was classified as capsular type D. All other strains were identified as capsular type A. These results are consistent with other authors (Pijoan et al., 1983, Iwamatsu and Sawada, 1988, Lopez et al., 1988, Awad-Masalmeh et al., 1994), which support the assertion of Rutter (1983) and Pijoan (1992) that P. multocida type A strains are more prevalent in pneumonic processes than type D strains. Type A strains are more frequently found in the lung, probably due to the presence of a capsule of hyaluronic acid, which difficults phagocytosis by alveolar macrophages (Pijoan, 1992).

Pneumonia and pleuritis associated to P. multocida are very important to pig production and most strains isolated from the lesions belong to capsular type A (Pijoan, 1992). The ability of P. multocida strains to produce toxin is the main factor in the pathogenesis of progressive atrophic rhinitis, frequently associated with capsular type D (Pijoan et al., 1984). However, some toxigenic capsular type A strains have been isolated from pigs with progressive atrophic rhinitis and pneumonia (Pijoan et al., 1984; Ahn e Kim, 1994).

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Only one out of twenty four strains was toxigenic in the mouse lethality test, sample 1/ 95, classified as capsular type D. However, using PCR, 9 strains amplified the segment of the toxA gene. The relationship between P. multocida capsular type, toxin production and pneumonia in pigs is not well defined. Results obtained by diverse authors present differences. Iwamatsu and Sawada (1988), examining 116 strains of P. multocida isolated from pneumonic pig lungs by dermal necrosis assay in guinea pigs, found that only 14.3% of the toxigenic strains belonged to capsular type A. Most (85.7%) of the toxin-producing strains belonged to capsular type D. However, Ahn and Kim (1994), studying 77 strains of type A P. multocida isolated from pig lungs by mouse lethality test, determined that 77% of the strains were toxigenic, and, out of 3 type D strains, 66.3% produced the toxin. We observed that some strains which yielded PCR products for the toxA gene were negative in the mouse lethality test. However, Lichtensteiger et al. (1996), comparing PCR with other tests, including mouse lethality assay, concluded that PCR was 100% sensitive and specific in the detection of toxigenic P. multocida strains. Amigot et al. (1998), evaluated three techniques for detection of toxigenic P. multocida strains isolated from swine: ELISA test, cytotoxyc effect in cat fetal lung cell culture and detection of toxA gene by PCR. He did not observe complete consistency among the 3 tests, as some strains that were negative by ELISA showed weak bands in PCR reaction when in cell culture. The primers used in our experiment had been previously used in the differentiation of toxigenic and non-toxigenic strains of P. multocida (Nagai et al., 1994). 187 isolates of P. multocida and related bacteria were analyzed in this work, and the PCR method was shown to be sensitive and specific in the detection of toxigenic strains. Some hypothesis can be raised to ex-

plain the fact that some non-toxigenic strains in the mouse test amplified a specific PCR product of the toxA gene. It includes the suppression of the expression of the gene in vitro after subculture. Another possibility would be the presence of a non-functional, defective gene. In this case, the PCR test could be used to detect the mutant or non-functional gene. Lichtensteiger et al. (1996) stresses that a genetic test, such as PCR, could avoid potential problems such as the suppression of the expression of a gene. Some authors suggest PCR as a fast and safe alternative for toxin detection in P. multocida strains isolated from diseased swine (Nagai et al., 1994, Lichtensteiger et al., 1996). However, this may not always be true, as PCR assay can present false-positive results. Additional studies, including analysis of new isolates of P. multocida isolated from pneumonia lesions in pigs are suggested, in order to confirm the present results. They may indicate that toxigenicity can be an important factor in strains isolated from this condition in swine.

ACKNOWLEDGEMENTS This work was supported by funds from Fundação Estadual de Pesquisa Agropecuária (FEPAGRO), Centro de Pesquisa Veterinária “Desidério Finamor” and Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Brazil. This report represents a portion of a thesis submitted by first author to the Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Brazil, in partial fulfillment of the requirements for the PhD degree.

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S.M. BOROWSKI , S.C. SILVA, I. SCHRANK & M. CARDOSO. 2001. Toxin detection in Pasteurella multocida strains isolated from swine lungs in the state of Rio Grande do Sul, Brazil. Arq. Fac. Vet. UFRGS. 29:79-85. teurella multocida de origen porcino. Revista de la Facultad de Ciencias Veterinarias U.C.V., v. 35, n. 1-4, p. 21-28. NAGAI, S.; SOMENO, S.; YAGIHASHI,T. 1994. Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by PCR. J. Clin. Microbiol.,v. 32, n. 4, p. 1004-1010. NIELSEN, J.P.; FREDERIKSEN, W.1990. Atrophic rhinitis in pigs caused by a human isolate of toxigenic Pasteurella multocida. In: INTERNATIONAL PIG VETERINARY SOCIETY CONGRESS, 11, Proceedings, p.75. PIJOAN, C.; MORRISON, R.B.; HILLEY, H.D.1983. Serotyping of Pasteurella multocida isolated from swine lungs collected at slaughter. J. Clin. Microbiol., v.17, n. 6, p. 1074-1076. PIJOAN, C.; LASTRA, A.; RAMIREZ, C.; LEMAN, A.D.1984. Isolation of toxigenic strains of Pasteurella multocida from lungs of pneumonic swine. J. Am. Med. An., v.185, n. 5, p. 522- 523. PIJOAN, C. 1992. Pneumonic Pasteurellosis. In: Leman, A. (Eds) Diseases of Swine. 7 ed. Iowa; Wolfe Publshing Ltd., p. 552-559. PIJOAN, C; FUENTES, M. 1987. Severe pleuritis associated with certain strains of Pasteurella multocida in swine. J. Am. Vet. Med. Assoc., v. 191, n. 7, p. 823-826. RHIMLER, R.B.; RHOADES, K .R.1989. Pasteurella multocida. In: Adlam,C.; Rutter, J.M. ed. Pasteurella and Pasteurellosis Academic Press Limited , London, p. 37-73, RUTTER, J.M.1983. Virulence of Pasteurella multocida in athrophic rhinitis of gnotobiotic pigs infected with Bordetella bronchiseptica . Res. Vet. Sci., p. 34, p. 285-287. SAMBROOK, J., FRITSCH, E.F., MANIATIS, T.1989. Molecular Cloning: A Laboratory Manual, 2ed., Cold Sprig Harbor, Cold Spring Harbor Laboratory, STEPAN, A.L.1995. Tipificação e sensibilidade de amostras de Pasteurella multocida isoladas a partir de lesões de pleurite em suínos terminados. Tese de mestrado, UFRGS.

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Recebido/Received: dezembro 2000 Aceito/Accepted: junho 2001

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