J Vet Diagn Invest 4:270-278 (1992)
Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 in growing and finishing pigs Rodney K. Frank, M. M. Chengappa, Richard D. Oberst, Kristina J. Hennessy, Steven C. Henry, Brad Fenwick Abstract. Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow far-row-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.
Actinobacillus pleuropneumoniae, sometimes in conjunction with Pasteurella multocida, is 8the most common cause of porcine pleuropneumonia. Actinobacillus pleuropneumoniae pleuropneumonia (APP) is most common in 2-5-month-old pigs (especially 3050-kg pigs), with occasional septicemia in pigs 30 days 13,17 Typical cases of APP are assoof age or younger. ciated with pulmonary necrosis, hemorrhage, and characteristic involvement of the caudodorsal aspect 12-14,21 of the caudal lung lobe. Characteristically, necrotic areas are surrounded by darkly stained bands of streaming, swirling alveolar leukocytes; similar cells 18 often line subpleural lymphatics. Fibrin is prominent 12 in alveoli from 2 to 4 days postinfection. Other Actinobacillus species causing disease in swine include A. suis and A. equuli. Actinobacillus suis, A. equuli, and unspeciated actinobacilli are most commonly isolated from the organs of septicemic nursing and weanling pigs. 6,9,12-14,19,24,29,30 Actinobacillus suis was isolated from finishing pigs that died suddenly with pleuropneumonia characterized by pulmonary lesions 3,4 similar to those caused by A. pleuropneumoniae. This report documents a persistent problem with From the Departments of Veterinary Diagnosis (Frank), Laboratory Medicine (Chengappa), and Pathology (Oberst, Hennessy, Fenwick), College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502, and the Abilene Animal Hospital, Abilene, KS (Henry). Received for publication September 6, 1991.
pleuropneumonia caused by A. pleuropneumoniae biotype 2 in a swine herd in the United States. This isolate was used to experimentally reproduce the disease. Materials and methods Case history. A 1,200-sow farrow-to-finish swine herd had a persistent problem with respiratory disease in growing and finishing pigs. Clinical signs varied from sudden death to chronic pneumonia with weight loss and slow growth. The overall mortality rate for growing and finishing pigs on the farm varied from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease varied from 0.17% to 0.52% per month for the same period. Pigs with clinical signs of respiratory disease had variable responses to intramuscular procaine penicillin G. The herd also had a recurrent problem with swine influenza in nursing and recently weaned pigs, but no associated death loss or significant effect on rate of gain was noted. Specimens. Nine 45-70-kg pigs with acute to chronic stages of respiratory disease as evidenced by coughing and expiratory dyspnea (“thumping”) were euthanized by intravenous injection.a Necropsyprocedure. Necropsy was performed on all 9 pigs immediately after death. Sections of lung and hilar lymph node were fixed in 10% buffered neutral formalin and processed routinely for histologic examination. Bacteriologic examination. Lung specimens were submitted for bacterial culture and processed according to standard procedures.1 Actinobacillus pleuropneumoniae biotype 2-like colonies were cross-reacted with antisera to biotype 1 serotypes 1-12 using a slide agglutination test.15
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Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 Table 1. Bacterial strains used in DNA comparisons with Actinobacillus pleuropneumoniae biotype 2-like organisms.
Virology. The lungs of 6 pigs were examined for the presence of swine influenza virus and the tonsils of 4 pigs were examined for pseudorabies virus by direct fluorescent antibody tests. Experimental infection. The A. pleuropneumoniae biotype 2 (Table 1) was grown in brain-heart infusion broth for 6 hr at 37 C. Two 4-wk-old pigs were inoculated intratracheally with either 1 ml or 5 ml of broth culture (109 colony forming units [CFU]/ml) via an 18-gauge needle. Necropsy was performed immediately following death or euthanasia at 7 hr postinfection, and specimens were collected as above. Sections of brain, heart, spleen, kidney, stomach, ileum, colon, and mesenteric lymph node were fixed and processed routinely for histological examination. Extraction of total DNA. DNA was extracted from the A. pleuropneumoniae biotype 2 field strain and 6 other Actinobacillus strains (see Table 1 for sources). Actinobacillus strains were cultivated overnight in 100 ml of Luria-Bertani brothb and pelleted by centrifugation at 1,100 x g for 20 min. Cells were resuspended in lysis buffer (0.15 M NaCl, 0.1 M ethylenediaminetetraacetic acid [EDTA], 20 mM TrisHC1 [pH 8.0], 1% sodium dodecyl sulfate [SDS]) and incubated at 37 C for 1 hr. Total DNA was extracted by adding an equal volume of 10 mM Tris-HC1 (pH 8.0) 1 mM EDTA (TE)-saturated phenol : chloroform (1:1, v/v) and freezing at -70 C for 15 min. The solutions were then thawed at 37 C and centrifuged at 1,700 x g for 20 min. The top aqueous layers were collected and subjected to 2 more phenol : chloroform extractions but without freezing. Total DNA from each strain was precipitated by adding 0.1 x volume of 4M ammonium acetate and 2 x volume of 100% ethanol, and samples were stored at -70 C. Purification and labeling of chromosomal enriched DNA. The total DNA of Actinobacillus isolated from the experimentally infected pigs was resuspended in TE buffer, and aliquots of the solution were loaded on 1% agarose gels in TAE (40 mM Tris-acetate, 1 mM EDTA)c containing ethidium bromide and electrophoresed at 70 volts for 2 hr. Gels were visualized by UV illumination, and the largest band or chromosomal enriched fraction was excised from the gel. The fragment was warmed to 65 C, and an equal volume of TE phenol was added. Samples were centrifuged at 1,100 x g for 20 min, and the top aqueous layer was collected and
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Table 2. Differentiation of Actinobacillus pleuropneumoniae biotype 2 field strain (AP2) from other important Actinobacillus species.*
mixed with an equal volume of chloroform : isoamyl alcohol (24: 1). The solutions were gently mixed and centrifuged at 1,100 x g for 20 min. The top aqueous layer was collected, and the chromosomal enriched DNA was precipitated in
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Figure 1. Lungs of pig with pleuropneumonia from which Actinobacillus pleuropneumoniae biotype 2 was isolated. Note small amount of normal lung and large raised coalescing foci in the remaining lung and a roughened pleural surface.
100% ethanol as described above. Aliquots of this chromosomal enriched DNA were labeled with random primer ex-
tensiond using [α-32P] dCTP (3,000 Ci/mmol). Restriction enzyme digests and hybridizations. The total DNA of Actinobacillus was digested with EcoRIe as described by the manufacturer, except that 15-20 U of enzyme were used per microgram of DNA and samples were incubated at 37 C overnight. The DNA was electrophoresed on 20- x 25cm 1% agarose gels in 45 mM Tris-borate and 1 mM EDTA at 50 volts for 16-24 hr. Gels were stained in ethidium bromide, visualized by UV illumination, and photographed. The gels were then sequentially placed in denaturing solution (0.5 M NaOH, 1.5 M NaCl) and neutralizing solution (1.5 M
NaCl, 1.0 M Tris-HCl, pH 8.0) for 30 min each. DNA was transferred to nylon membranes by overnight capillary blotting in buffer containing 1.5 M NaCl with 0.15 M sodium citrate (10 x SSC).20 The membranes were dried, and the DNA was cross-linked on the membrane by UV illumination. Membranes were prehybridized in 50% deionized formamide, 0.1% SDS, 10 mg/ml glycine, 0.5 mg/ml denatured salmon sperm DNA, 5 x SSC, and 5 x Denhardt’s solution for 1 hr at 42 C.5 The [32P]-labeled DNA of the A. pleuropneumoniae biotype 2 was denatured at 95 C for 5 min, quenched on ice, and mixed with hybridization buffer (50% deionized formamide,
Figure 2. Photomicrograph of necrotizing pneumonia in a pig infected with Actinobacillus pleuropneumoniae biotype 2 field strain. Note large zone of coagulative necrosis surrounding a partially collapsed bronchiole and delineated by a dark basophilic band. HE stain.
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Figure 3.
10% sodium dextran sulfate, 5 x SSC, 1 x Denhardt’s,f 0.1% SDS, 0.1 mg/ml denatured salmon sperm DNA).2
at 55 C for 5 hr. Primers were then lyophilized under vacuum and resuspended in 500 µl of double distilled water. Gel
(2 x SSC, 0.1% SDS), then SDS) at 42 C for 30 min, dried, and autoradiographed at -70 C with an intensifying screen. APLPrimer synthesis. Oligonucleotide primers XP-1 126 and sequencing primers28 were synthesized using standard phosphoramidite chemistry on a DNA synthesizer.f Primers were removed from columns with 100% ammonium hydroxide, and protective groups were removed by heating
oligonucleotides. Primers were quantitated on a spectrophotometerh at 260 nm.
g
DNA preparation for polymerase chain reaction. Actinobacillus suis, A. pleuropneumoniae biotype 1 serotypes 1-12, A. pleuropneumoniae biotype 2 field strain were grown overnight on enriched chocolate agar in a humidified 7% CO2 incubator at 37 C. DNA preparation, single colonies were boiled in 25 µl of water for 5 min before pelleting in a mi-
Figure 4. Lungs from a pig experimentally infected with Actinobacillus pleuropneumoniae dorsal caudal lobes.
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Figure 5. Ethidium bromide-stained restriction enzyme profiles of the EcoRI-digested DNA of Actinobacillus pleuropneumoniae biotype 2 field strain (lanes A, B), A. equuli strain 0102 (lane C), A. suis strain 60408 (lane D), A. pleuropneumoniae biotype 1 serotype 1 (lane E), and A. lignieresii strain 0201 (lane F). Gel was 1% agarose in TBE buffer and was electrophoresed at 50 volts for 16 hours. crocentrifuge.i Supernatant from these samples (5 µl) was used as the DNA source for amplification by polymerase
chain reaction (PCR). Polymerase chain reaction and amplification analysis. Reactions were performed in 100 µl volumes containing 4 mM MgCl2, 10 mM Tris-HC1 (pH 8.3), 50 mM KCl, 0.001% j gelatin, 1 mM each of dATP, dCTP, dGTP, and TTP, 10 µM primers, 5 µl DNA preparation (described above), and 2.5 U of Taq DNA polymerase. j Amplification was performed in a thermocycler programmed for 2 cycles of 5 min at 94 C, 5 min at 40 C, and 5 min at 72 C followed by 35 cycles of 1 min at 94 C, 1 min at 60 C, and 2 min at 72 C,
using the fastest available ramp times between temperatures.
Reaction amplification products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide gels using 75 µl sample volume and ethidium bromide staining.
Results Bacteriology. Beta-hemolytic colonies were isolated in pure culture on blood agar plates from the lungs of 3 pigs and as the predominant isolate from the other 6 pigs. On the basis of biochemical and phenotypic characteristics, the colonies were classified as Actinobacillus pleuropneumoniae biotype 2 (Table 2). Additional isolates from the lungs of the 9 pigs included
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Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2
P. multocida (6 pigs), Haemophilus parasuis (2 pigs), and an alpha-hemolytic Streptococcus species that was not identified to species (2 pigs). The A. pleuropneumoniae biotype 2-like organism was the only isolate from the lungs of 3 pigs. This isolate agglutinated with hyperimmune rabbit antisera to A. pleuropneumoniae biotype 1, serotype 8, but not to serotypes 1-7 or 912. Pathology. Pulmonary gross and histologic lesions resembled those caused by A. pleuropneumoniae. Gross lesions varied from multifocal mottled tan to dark red raised areas in both cranial and caudal lung lobes (Fig. 1) to extensive necrosis of caudal lobes with many fibrinous and fibrous adhesions to the thoracic wall. Histologic lesions included necrosis, hemorrhage, streaming degenerate alveolar leukocytes (“oat cells”), fibrin, and lymphatic thrombosis. Eight pigs had various-sized foci of coagulative parenchymal necrosis with alveoli variously filled with fibrin, neutrophils, and macrophages. These foci were often oriented around bronchioles (Fig. 2) and were bordered by a thick band of degenerate, often streaming leukocytes and necrotic neutrophils (Fig. 3). In the more chronic cases, the necrotic foci were surrounded by bands of fibrous tissue; moderate numbers of lymphocytes and macrophages were diffusely interspersed in the fibrous tissue. Fibrin thrombi were occasionally present in blood vessels and often in interlobular and subpleural lymphatits of pigs with the most extensive lesions. The pleura often had moderate numbers of neutrophils and macrophages and abundant fibrin on the surface, with fibroplasia in chronic cases. Six of 9 pigs had mild to severe suppurative cranioventral bronchopneumonia characterized by bronchi, bronchioles, and surrounding alveoli partially or totally filled with neutrophils and fewer macrophages. Five of 9 pigs also had mild to severe lymphofollicular interstitial pneumonia; bronchi, bronchioles, and blood vessels were surrounded by lymphoid aggregates with mild to moderate thickening of adjacent alveolar septa by lymphocytes and macrophages. Experimental infection. The pig that received 109 CFU A. pleuropneumoniae biotype 2-like organism in 5 ml of inoculum intratracheally died 6 hr later; the pig receiving 1 ml exhibited severe dyspnea at that time and was euthanized at 7 hr postinoculation. Both pigs had severe necrohemorrhagic pneumonia involving the caudodorsal lung lobes. Large dark red foci resembled those present with A. pleuropneumoniae (Fig. 4). A few fine fibrin strands were in the pleural cavities. No other gross or microscopic lesions were detected in either of the 2 pigs. Microscopically, alveolar hemorrhage, degenerate alveolar leukocytes, fibrin exudation, and necrosis were present. DNA studies. Restriction enzyme analysis of the A.
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Figure 6. Hybridization profiles of EcoRI-digested chromosomal DNA of various Actinobacillus species that were probed with [32P]labeled Actinobacillus pleuropneumoniae biotype 2 field strain chromosomal DNA. Gel was electrophoresed for 24 hours (as described in Fig. 5). Actinobacillus pleuropneumoniae biotype 2 (lane A), A. pleuropneumoniae biotype 1 serotype 8 (lane B), A. suis strain 60408 (lane C), A. suis strain 3764 (lane D), A. equuli strain 0201 (lane E). Figure is a composite of lanes from the same gel.
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Figure 7. Polymerase chain reaction amplification DNA fragments of ActinobacilIus species using the universal M13 sequencing primers. Samples (50 µl) were electrophoresed overnight at 50 volts on a 6% nondenaturing polyacrylamide gel and stained with ethidium bromide for visualization. Lane 1:1-kb DNA ladder. Lanes 2-13: A. pleuropneumoniae biotype 1, serotypes 1-12, respectively. Lane 14: A. pleuropneumoniae biotype 2 field strain. Lane 15: A. suis.
pleuropneumoniae biotype 2-like organism with other Oligonucleotides APL-1 and XP-1 served as PCR Actinobacillus species resulted in a complex restriction primers for examining. A. pleuropneumoniae serotypes pattern that was difficult to interpret (Fig. 5). However, 1 and 5 reference strains, A. suis, and the A. pleuropneua distinctive pattern of hybridization was evident when moniae biotype 2 field strain. Using the thermocycling A. pleuropneumoniae biotype 2-like DNA was used as protocol established for arbitrarily primed PCR, a 1.8a probe in DNA hybridizations with other Actinoba- kb DNA fragment was demonstrated for A. pleurocillus DNA (Fig. 6). Restriction fragment length poly- pneumoniae reference strains as well as the biotype 2 morphism (RFLP) was evident among the different field strain (Fig. 8). Actinobacillus suis displayed a organisms and could be demonstrated most effectively unique DNA pattern following PCR amplification, and when electrophoresis was extended to 24 hr. Overall no 1.8-kb DNA fragment was identified. radiographic signals were most intense in hybridizaVirology. Lungs tested negative for swine influenza tions of the probe to itself and less evident in hybrid- virus and tonsils tested negative for pseudorabies virus izations with A. suis strains 60408 and 3764, A. equuli, by direct fluorescent antibody technique. A. pleuropneumoniae biotype 1 serotype 1 (not shown), and A. lignieresii. The A. pleuropneumoniae biotype Discussion 2-like DNA did hybridize intensely with A. pleuropneumoniae biotype 1 serotype 8, but the RFLP patMorphological features of pleuropneumonia protern was slightly different. duced by this A. pleuropneumoniae biotype 2 strain in Polymerase chain reaction amplification of bacterial both field and experimental cases are identical to those DNA was used to compare known PCR-generated DNA of A. pleuropneumoniae biotype 2 cases reported from fragment patterns of A. pleuropneumoniae serotypes Europe.1,10 The experimentally infected pigs had gross 1-12 and A. suis with the biotype 2 field strain. The lesions consistent with acute APP, including firm, M13 universal sequencing primers were used in an raised, sharply delineated dark red areas in the caudal 16,23 arbitrarily primed PCR reaction28 to amplify serotype- lung lobes with some fibrin in the pleural cavity. specific patterns of DNA from the organisms listed Similar light microscopic lesions included alveolar above (Fig. 7). Actinobacillus pleuropneumoniae bio- hemorrhage, degenerate alveolar leukocytes, fibrin extype 2 was identified as having a novel DNA ampli- udation, and necrosis. The 2 acutely affected pigs from fication pattern when compared with A. pleuropneu- field cases had gross and histologic pulmonary lesions resembling those of APP 2-4 days postinfection, inmoniae reference serotypes and an A. suis strain. Downloaded from vdi.sagepub.com at UNIVERSITE DE SHERBROOKE on May 2, 2015
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polyclonal antibody to A. pleuropneumoniae biotype 1 serotype 8, it was classified as biotype 2 because V factor was not required for growth. Polymerase chain reaction data demonstrate the A. pleuropneumoniae biotype 2 organism isolated in this study is unique at the genomic level when compared with all reference serotypes of A. pleuropneumoniae biotype 1 and A. suis. The arbitrarily primed PCR method with the universal M13 sequencing primers indicated that this isolate has a DNA fragment size pattern distinct from all biotype 1 serotypes of A. pleuropneumoniae and A. suis. Although DNA hybridization studies revealed some cross-hybridization with A. pleuropneumoniae serotype 8, PCR-amplified patterns of DNA from these 2 organisms are distinctly different. Amplified DNA fragment sizes produced by PCR using the APL-1 and XP-1 primers were markedly different from those previously described.26 This difference is likely due to the more stringent temperature cycling protocol used in the present study. Also, DNA bands of less intensity are better visualized with polyacrylamide gels than agarose gels. However, these primers produced the 1.8-kb DNA fragment previously identified as specific for A. pleuropneumoniae and, under different reaction conditions, A. lignieresii. 26 This is the first documented case of A. pleuropneumoniae biotype 2 pleuropneumonia in the United States. Figure 8. Polymerase chain reaction amplification DNA fragments of Actinobacillus species using APL-l and XP-1 oligonucleotide primers. Samples were electrophoresed overnight at 50 volts on a 6% nondenaturing polyacrylamide gel and stained with ethidium bromide for visualization. Lane 1:1-kb DNA ladder. Lane 2: A. pleuropneumoniae biotype 1 serotype 1. Lane 3: A. pleuropneumoniae biotype 1 serotype 5. Lane 4: A. pleuropneumoniae biotype 2 field strain. Lane 5: A. suis.
eluding very roughened pleura with extensive adhesions and firm irregularly swollen deep red lung.16 Pasteurella multocida was isolated from the lungs of all 6 of the chronically affected pigs with pleuropneumonia. Pasteurella multocida acts synergistically with A. pleuropneumoniae to produce pleuropneumonia.11 Although lung tissue samples were not tested for the presence of mycoplasmas, the lymphofollicular interstitial pneumonia detected was consistent with mycoplasma infection. The Actinobacillus species isolated in the present study most closely resembles A. pleuropneumoniae biotype 2 based on biochemical and phenotypic features and DNA hybridization studies.8,10,20 Biochemical reactions were also similar to those of A. lignieresii; however, A. lignieresii has never been isolated from swine and is nonhemolytic on 5% sheep blood agar.26 The A. pleuropneumoniae biotype 2 differed markedly from A. suis, A. lignieresii, and A. equuli by DNA hybridization. Although the organism cross-reacted with
Acknowledgements We acknowledge the technical assistance of Roxanna Maddux and Michael Hays.
Sources and manufacturers a. b. c. d.
Beuthanasia, Schering-Plough Animal Health, Kenilworth, NJ. Difco, Detroit, MI. Seaplaque GTG, FMC Bioproducts, Rockland, ME. Oligolabelling Kit, Pharmacia LKB Biotechnology, Piscataway, NJ. e. GIBCO BRL, Life Technologies, Gaithersburg, MD. f. PCR-MATE™ 391, Applied Biosystems, Foster City, CA. g. NICK Columns, Pharmacia LKB Biotechnology, Piscataway, NJ. h. RESPONSE UV-VIS, Gilford, Oberlin, OH. i. Microspin 24S, Sorvall Instruments, The Du Pont Co., Wilmington, DE. j. Perkin-Elmer Cetus, Norwalk, CT. k. Model 60 Tempcycler, Coy Corp., Ann Arbor, MI.
References 1. Bertschinger HU, Seifert P: 1978, Isolation of a Pasteurella haemolytica-like organism from porcine necrotic pleuropneumonia. Proc Int Pig Vet Soc 5:M19 (Abstr.). 2. Bewsey KE, Johnson ME, Juff JP: 1991, Rapid isolation and purification of DNA from agarose gels: the phenol-freeze-fracture method. Biotechniques 10:725-735. 3. Carter GR: 1990, Isolation and identification of bacteria from clinical specimens. In: Diagnostic procedures in veterinary bacteriology and mycology, ed. Carter GR, Cole JR, pp. 19-39. Academic Press, San Diego, CA.
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4. Christenson C: 1986, Finishing house pneumonia pathogenActinobacillus suis. Proc Annu Meet Am Assoc Swine Pract., pp. 317-319. 5. Christenson C: 1990, Clinical significance of Actinobacillus suis. Proc Annu Meet Am Assoc Swine Pract., pp. 267-270. 6. Cutlip RC, Amtower WC, Zinober MR: 1972, Septic embolic actinobacillosis of swine: a case report and laboratory reproduction of the disease. Am J Vet Res 33:1621-1626. 7. Denhardt D: 1966, A membrane filter technique for detection of complementary DNA. Biochem Res Commun 23:641-652. 8. Fodor L, Varga J, Molnar É, Hajtós I: 1989, Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine. Vet Microbiol 20: 173-180. 9. Jones JET, Simmons JR: 1971, Endocarditis in the pig caused by Actinobacillus equuli: a field and an experimental case. Br Vet J 127:25-29. 10. Kielstein P, Bocklisch H, Zepezauer V: 1981, Hämorrhagischnekrotisierende Pleuropneumonien beim Schwein durch Aktinobazillen. Arch Exp Vet Med 35:879-902. 11. Little TWA, Harding JDJ: 1980, The interaction of Haemophilus parahaemolyticus and Pasteurella multocida in the pig respiratory tract. Br Vet J 136:371-383. 12. Liven E, Larsen HJ: 1978, Infection with Actinobacillus suis in pigs. Acta Vet Scand 19:313-315. 13. MacDonald DW, Hewitt MP, Wilton GS, et al.: 1976, Actinobacillus suis infections in Alberta swine, 1973-75: pathology and bacteriology. Can Vet J 17:251-254. 14. Mair NS, Randall C J, Thomas GW, et al.: 1974, Actinobacillus suis infection in pigs: a report of four outbreaks and two sporadic cases. J Comp Pathol 84:113-119. 15. Mittal KR, Higgins R, Lariviere S: 1982, Evaluation of slide agglutination and ring precipitation tests for capsular serotyping of Haemophilus pleurpneumoniae. J Clin Microbiol 15:10191023. 16. Myrlea PJ, Fraser G, MacQueen P, Lamboum DA: 1974, Pleuropneumonia in pigs caused by Haemophilus parahaemolyticus. Aust Vet J 50:255-259. 17. Nielsen R: 1970, Pleuropneumoni hos svin, fremkaldt af Haemophilus parahaemolyticus. I. Kliniske, patalogisk-anatomiske og epidemiologiske under sogeleser. Nord Vet Med 22:240-245. 18. Olander HJ: 1963, A septicemic disease of swine and its cau-
sative agent, Haemophilus parahaemolyticus. Ph.D. Thesis, University of California, Davis, CA. 19. Pedersen KB: 1977, Actinobacillus infektioner hos svin. Nord Vet Med 29: 137-140. 20. Phillips JE: 1984, Actinobacillus. In: Bergey’s manual of systematic bacteriology, ed. Krieg NR, vol. 1, pp. 560-575. Williams and Wilkins, Baltimore, MD. 21. Phillips JE: 1990, Actinobacillus. In: Diagnostic procedures in veterinary bacteriology and mycology, ed. Carter GR, Cole JR, pp. 143-149. Academic Press, San Diego, CA. 22. Pohl S, Bertschinger HU, Frederiksen W, Mannheim W: 1983, Transfer of Haemophilus pleuropneumoniae and the Pasteurella haemolytica-like organism causing porcine necrotic pleuropneumonia to the genus Actinobacillus (Actinobacillus pleuropneumoniae comb. nov.) on the basis of phenotypic and deoxyribonucleic acid relatedness. Int J Syst Bacteriol 33:510-514. 23. Sanford SE, Josephson GKA: 1981, Porcine Haemophilus pleuropneumonia epizootic in southwestern Ontario: clinical, microbiological, pathological and some epidemiological findings. Can J Comp Med 45:2-7. 24. Sanford SE, Josephson GKA, Rehmtulla AJ, Tilker AME: 1990, Actinobacillus suis outbreaks in pigs in Ontario: pathology and bacteriology of a creeping epidemic. Proc Int Pig Vet Soc 11: 183. 25. Schiefer B, Greenfield J: 1974, Porcine Haemophilus parahaemolyticus pneumonia in Saskatchewan. 1. Bacteriological and experimental studies. Can J Comp Med 38:105-110. 26. Sirois M, Lemire EG, Levesque RC: 1991, Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction. J Clin Microbiol 29: 11831187. 27. Southern EM: 1975, Detection of specific sequences among DNA fragments separated by electrophoresis. J Mol Biol 98: 503-507. 28. Welsh J, McClelland M: 1990, Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18:7213-7218. 29. Windsor RS: 1973, Actinobacillus equuli infection in litter of pigs and a review of previous reports on similar infections. Vet Rec 92:178-180. 30. Zimmerman NT: 1965, Die Actinobacillose des Schweines. [Actinobacillosis in swine.] Tierärzt1 Umsch 20:565-568.
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Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 in growing and finishing pigs Rodney K. Frank, M. M. Chengappa, Richard D. Oberst, Kristina J. Hennessy, Steven C. Henry, Brad Fenwick Abstract. Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow far-row-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.
Actinobacillus pleuropneumoniae, sometimes in conjunction with Pasteurella multocida, is 8the most common cause of porcine pleuropneumonia. Actinobacillus pleuropneumoniae pleuropneumonia (APP) is most common in 2-5-month-old pigs (especially 3050-kg pigs), with occasional septicemia in pigs 30 days 13,17 Typical cases of APP are assoof age or younger. ciated with pulmonary necrosis, hemorrhage, and characteristic involvement of the caudodorsal aspect 12-14,21 of the caudal lung lobe. Characteristically, necrotic areas are surrounded by darkly stained bands of streaming, swirling alveolar leukocytes; similar cells 18 often line subpleural lymphatics. Fibrin is prominent 12 in alveoli from 2 to 4 days postinfection. Other Actinobacillus species causing disease in swine include A. suis and A. equuli. Actinobacillus suis, A. equuli, and unspeciated actinobacilli are most commonly isolated from the organs of septicemic nursing and weanling pigs. 6,9,12-14,19,24,29,30 Actinobacillus suis was isolated from finishing pigs that died suddenly with pleuropneumonia characterized by pulmonary lesions 3,4 similar to those caused by A. pleuropneumoniae. This report documents a persistent problem with From the Departments of Veterinary Diagnosis (Frank), Laboratory Medicine (Chengappa), and Pathology (Oberst, Hennessy, Fenwick), College of Veterinary Medicine, Kansas State University, Manhattan, KS 66502, and the Abilene Animal Hospital, Abilene, KS (Henry). Received for publication September 6, 1991.
pleuropneumonia caused by A. pleuropneumoniae biotype 2 in a swine herd in the United States. This isolate was used to experimentally reproduce the disease. Materials and methods Case history. A 1,200-sow farrow-to-finish swine herd had a persistent problem with respiratory disease in growing and finishing pigs. Clinical signs varied from sudden death to chronic pneumonia with weight loss and slow growth. The overall mortality rate for growing and finishing pigs on the farm varied from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease varied from 0.17% to 0.52% per month for the same period. Pigs with clinical signs of respiratory disease had variable responses to intramuscular procaine penicillin G. The herd also had a recurrent problem with swine influenza in nursing and recently weaned pigs, but no associated death loss or significant effect on rate of gain was noted. Specimens. Nine 45-70-kg pigs with acute to chronic stages of respiratory disease as evidenced by coughing and expiratory dyspnea (“thumping”) were euthanized by intravenous injection.a Necropsyprocedure. Necropsy was performed on all 9 pigs immediately after death. Sections of lung and hilar lymph node were fixed in 10% buffered neutral formalin and processed routinely for histologic examination. Bacteriologic examination. Lung specimens were submitted for bacterial culture and processed according to standard procedures.1 Actinobacillus pleuropneumoniae biotype 2-like colonies were cross-reacted with antisera to biotype 1 serotypes 1-12 using a slide agglutination test.15
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Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 Table 1. Bacterial strains used in DNA comparisons with Actinobacillus pleuropneumoniae biotype 2-like organisms.
Virology. The lungs of 6 pigs were examined for the presence of swine influenza virus and the tonsils of 4 pigs were examined for pseudorabies virus by direct fluorescent antibody tests. Experimental infection. The A. pleuropneumoniae biotype 2 (Table 1) was grown in brain-heart infusion broth for 6 hr at 37 C. Two 4-wk-old pigs were inoculated intratracheally with either 1 ml or 5 ml of broth culture (109 colony forming units [CFU]/ml) via an 18-gauge needle. Necropsy was performed immediately following death or euthanasia at 7 hr postinfection, and specimens were collected as above. Sections of brain, heart, spleen, kidney, stomach, ileum, colon, and mesenteric lymph node were fixed and processed routinely for histological examination. Extraction of total DNA. DNA was extracted from the A. pleuropneumoniae biotype 2 field strain and 6 other Actinobacillus strains (see Table 1 for sources). Actinobacillus strains were cultivated overnight in 100 ml of Luria-Bertani brothb and pelleted by centrifugation at 1,100 x g for 20 min. Cells were resuspended in lysis buffer (0.15 M NaCl, 0.1 M ethylenediaminetetraacetic acid [EDTA], 20 mM TrisHC1 [pH 8.0], 1% sodium dodecyl sulfate [SDS]) and incubated at 37 C for 1 hr. Total DNA was extracted by adding an equal volume of 10 mM Tris-HC1 (pH 8.0) 1 mM EDTA (TE)-saturated phenol : chloroform (1:1, v/v) and freezing at -70 C for 15 min. The solutions were then thawed at 37 C and centrifuged at 1,700 x g for 20 min. The top aqueous layers were collected and subjected to 2 more phenol : chloroform extractions but without freezing. Total DNA from each strain was precipitated by adding 0.1 x volume of 4M ammonium acetate and 2 x volume of 100% ethanol, and samples were stored at -70 C. Purification and labeling of chromosomal enriched DNA. The total DNA of Actinobacillus isolated from the experimentally infected pigs was resuspended in TE buffer, and aliquots of the solution were loaded on 1% agarose gels in TAE (40 mM Tris-acetate, 1 mM EDTA)c containing ethidium bromide and electrophoresed at 70 volts for 2 hr. Gels were visualized by UV illumination, and the largest band or chromosomal enriched fraction was excised from the gel. The fragment was warmed to 65 C, and an equal volume of TE phenol was added. Samples were centrifuged at 1,100 x g for 20 min, and the top aqueous layer was collected and
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Table 2. Differentiation of Actinobacillus pleuropneumoniae biotype 2 field strain (AP2) from other important Actinobacillus species.*
mixed with an equal volume of chloroform : isoamyl alcohol (24: 1). The solutions were gently mixed and centrifuged at 1,100 x g for 20 min. The top aqueous layer was collected, and the chromosomal enriched DNA was precipitated in
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Figure 1. Lungs of pig with pleuropneumonia from which Actinobacillus pleuropneumoniae biotype 2 was isolated. Note small amount of normal lung and large raised coalescing foci in the remaining lung and a roughened pleural surface.
100% ethanol as described above. Aliquots of this chromosomal enriched DNA were labeled with random primer ex-
tensiond using [α-32P] dCTP (3,000 Ci/mmol). Restriction enzyme digests and hybridizations. The total DNA of Actinobacillus was digested with EcoRIe as described by the manufacturer, except that 15-20 U of enzyme were used per microgram of DNA and samples were incubated at 37 C overnight. The DNA was electrophoresed on 20- x 25cm 1% agarose gels in 45 mM Tris-borate and 1 mM EDTA at 50 volts for 16-24 hr. Gels were stained in ethidium bromide, visualized by UV illumination, and photographed. The gels were then sequentially placed in denaturing solution (0.5 M NaOH, 1.5 M NaCl) and neutralizing solution (1.5 M
NaCl, 1.0 M Tris-HCl, pH 8.0) for 30 min each. DNA was transferred to nylon membranes by overnight capillary blotting in buffer containing 1.5 M NaCl with 0.15 M sodium citrate (10 x SSC).20 The membranes were dried, and the DNA was cross-linked on the membrane by UV illumination. Membranes were prehybridized in 50% deionized formamide, 0.1% SDS, 10 mg/ml glycine, 0.5 mg/ml denatured salmon sperm DNA, 5 x SSC, and 5 x Denhardt’s solution for 1 hr at 42 C.5 The [32P]-labeled DNA of the A. pleuropneumoniae biotype 2 was denatured at 95 C for 5 min, quenched on ice, and mixed with hybridization buffer (50% deionized formamide,
Figure 2. Photomicrograph of necrotizing pneumonia in a pig infected with Actinobacillus pleuropneumoniae biotype 2 field strain. Note large zone of coagulative necrosis surrounding a partially collapsed bronchiole and delineated by a dark basophilic band. HE stain.
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Figure 3.
10% sodium dextran sulfate, 5 x SSC, 1 x Denhardt’s,f 0.1% SDS, 0.1 mg/ml denatured salmon sperm DNA).2
at 55 C for 5 hr. Primers were then lyophilized under vacuum and resuspended in 500 µl of double distilled water. Gel
(2 x SSC, 0.1% SDS), then SDS) at 42 C for 30 min, dried, and autoradiographed at -70 C with an intensifying screen. APLPrimer synthesis. Oligonucleotide primers XP-1 126 and sequencing primers28 were synthesized using standard phosphoramidite chemistry on a DNA synthesizer.f Primers were removed from columns with 100% ammonium hydroxide, and protective groups were removed by heating
oligonucleotides. Primers were quantitated on a spectrophotometerh at 260 nm.
g
DNA preparation for polymerase chain reaction. Actinobacillus suis, A. pleuropneumoniae biotype 1 serotypes 1-12, A. pleuropneumoniae biotype 2 field strain were grown overnight on enriched chocolate agar in a humidified 7% CO2 incubator at 37 C. DNA preparation, single colonies were boiled in 25 µl of water for 5 min before pelleting in a mi-
Figure 4. Lungs from a pig experimentally infected with Actinobacillus pleuropneumoniae dorsal caudal lobes.
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Figure 5. Ethidium bromide-stained restriction enzyme profiles of the EcoRI-digested DNA of Actinobacillus pleuropneumoniae biotype 2 field strain (lanes A, B), A. equuli strain 0102 (lane C), A. suis strain 60408 (lane D), A. pleuropneumoniae biotype 1 serotype 1 (lane E), and A. lignieresii strain 0201 (lane F). Gel was 1% agarose in TBE buffer and was electrophoresed at 50 volts for 16 hours. crocentrifuge.i Supernatant from these samples (5 µl) was used as the DNA source for amplification by polymerase
chain reaction (PCR). Polymerase chain reaction and amplification analysis. Reactions were performed in 100 µl volumes containing 4 mM MgCl2, 10 mM Tris-HC1 (pH 8.3), 50 mM KCl, 0.001% j gelatin, 1 mM each of dATP, dCTP, dGTP, and TTP, 10 µM primers, 5 µl DNA preparation (described above), and 2.5 U of Taq DNA polymerase. j Amplification was performed in a thermocycler programmed for 2 cycles of 5 min at 94 C, 5 min at 40 C, and 5 min at 72 C followed by 35 cycles of 1 min at 94 C, 1 min at 60 C, and 2 min at 72 C,
using the fastest available ramp times between temperatures.
Reaction amplification products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide gels using 75 µl sample volume and ethidium bromide staining.
Results Bacteriology. Beta-hemolytic colonies were isolated in pure culture on blood agar plates from the lungs of 3 pigs and as the predominant isolate from the other 6 pigs. On the basis of biochemical and phenotypic characteristics, the colonies were classified as Actinobacillus pleuropneumoniae biotype 2 (Table 2). Additional isolates from the lungs of the 9 pigs included
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Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2
P. multocida (6 pigs), Haemophilus parasuis (2 pigs), and an alpha-hemolytic Streptococcus species that was not identified to species (2 pigs). The A. pleuropneumoniae biotype 2-like organism was the only isolate from the lungs of 3 pigs. This isolate agglutinated with hyperimmune rabbit antisera to A. pleuropneumoniae biotype 1, serotype 8, but not to serotypes 1-7 or 912. Pathology. Pulmonary gross and histologic lesions resembled those caused by A. pleuropneumoniae. Gross lesions varied from multifocal mottled tan to dark red raised areas in both cranial and caudal lung lobes (Fig. 1) to extensive necrosis of caudal lobes with many fibrinous and fibrous adhesions to the thoracic wall. Histologic lesions included necrosis, hemorrhage, streaming degenerate alveolar leukocytes (“oat cells”), fibrin, and lymphatic thrombosis. Eight pigs had various-sized foci of coagulative parenchymal necrosis with alveoli variously filled with fibrin, neutrophils, and macrophages. These foci were often oriented around bronchioles (Fig. 2) and were bordered by a thick band of degenerate, often streaming leukocytes and necrotic neutrophils (Fig. 3). In the more chronic cases, the necrotic foci were surrounded by bands of fibrous tissue; moderate numbers of lymphocytes and macrophages were diffusely interspersed in the fibrous tissue. Fibrin thrombi were occasionally present in blood vessels and often in interlobular and subpleural lymphatits of pigs with the most extensive lesions. The pleura often had moderate numbers of neutrophils and macrophages and abundant fibrin on the surface, with fibroplasia in chronic cases. Six of 9 pigs had mild to severe suppurative cranioventral bronchopneumonia characterized by bronchi, bronchioles, and surrounding alveoli partially or totally filled with neutrophils and fewer macrophages. Five of 9 pigs also had mild to severe lymphofollicular interstitial pneumonia; bronchi, bronchioles, and blood vessels were surrounded by lymphoid aggregates with mild to moderate thickening of adjacent alveolar septa by lymphocytes and macrophages. Experimental infection. The pig that received 109 CFU A. pleuropneumoniae biotype 2-like organism in 5 ml of inoculum intratracheally died 6 hr later; the pig receiving 1 ml exhibited severe dyspnea at that time and was euthanized at 7 hr postinoculation. Both pigs had severe necrohemorrhagic pneumonia involving the caudodorsal lung lobes. Large dark red foci resembled those present with A. pleuropneumoniae (Fig. 4). A few fine fibrin strands were in the pleural cavities. No other gross or microscopic lesions were detected in either of the 2 pigs. Microscopically, alveolar hemorrhage, degenerate alveolar leukocytes, fibrin exudation, and necrosis were present. DNA studies. Restriction enzyme analysis of the A.
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Figure 6. Hybridization profiles of EcoRI-digested chromosomal DNA of various Actinobacillus species that were probed with [32P]labeled Actinobacillus pleuropneumoniae biotype 2 field strain chromosomal DNA. Gel was electrophoresed for 24 hours (as described in Fig. 5). Actinobacillus pleuropneumoniae biotype 2 (lane A), A. pleuropneumoniae biotype 1 serotype 8 (lane B), A. suis strain 60408 (lane C), A. suis strain 3764 (lane D), A. equuli strain 0201 (lane E). Figure is a composite of lanes from the same gel.
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Figure 7. Polymerase chain reaction amplification DNA fragments of ActinobacilIus species using the universal M13 sequencing primers. Samples (50 µl) were electrophoresed overnight at 50 volts on a 6% nondenaturing polyacrylamide gel and stained with ethidium bromide for visualization. Lane 1:1-kb DNA ladder. Lanes 2-13: A. pleuropneumoniae biotype 1, serotypes 1-12, respectively. Lane 14: A. pleuropneumoniae biotype 2 field strain. Lane 15: A. suis.
pleuropneumoniae biotype 2-like organism with other Oligonucleotides APL-1 and XP-1 served as PCR Actinobacillus species resulted in a complex restriction primers for examining. A. pleuropneumoniae serotypes pattern that was difficult to interpret (Fig. 5). However, 1 and 5 reference strains, A. suis, and the A. pleuropneua distinctive pattern of hybridization was evident when moniae biotype 2 field strain. Using the thermocycling A. pleuropneumoniae biotype 2-like DNA was used as protocol established for arbitrarily primed PCR, a 1.8a probe in DNA hybridizations with other Actinoba- kb DNA fragment was demonstrated for A. pleurocillus DNA (Fig. 6). Restriction fragment length poly- pneumoniae reference strains as well as the biotype 2 morphism (RFLP) was evident among the different field strain (Fig. 8). Actinobacillus suis displayed a organisms and could be demonstrated most effectively unique DNA pattern following PCR amplification, and when electrophoresis was extended to 24 hr. Overall no 1.8-kb DNA fragment was identified. radiographic signals were most intense in hybridizaVirology. Lungs tested negative for swine influenza tions of the probe to itself and less evident in hybrid- virus and tonsils tested negative for pseudorabies virus izations with A. suis strains 60408 and 3764, A. equuli, by direct fluorescent antibody technique. A. pleuropneumoniae biotype 1 serotype 1 (not shown), and A. lignieresii. The A. pleuropneumoniae biotype Discussion 2-like DNA did hybridize intensely with A. pleuropneumoniae biotype 1 serotype 8, but the RFLP patMorphological features of pleuropneumonia protern was slightly different. duced by this A. pleuropneumoniae biotype 2 strain in Polymerase chain reaction amplification of bacterial both field and experimental cases are identical to those DNA was used to compare known PCR-generated DNA of A. pleuropneumoniae biotype 2 cases reported from fragment patterns of A. pleuropneumoniae serotypes Europe.1,10 The experimentally infected pigs had gross 1-12 and A. suis with the biotype 2 field strain. The lesions consistent with acute APP, including firm, M13 universal sequencing primers were used in an raised, sharply delineated dark red areas in the caudal 16,23 arbitrarily primed PCR reaction28 to amplify serotype- lung lobes with some fibrin in the pleural cavity. specific patterns of DNA from the organisms listed Similar light microscopic lesions included alveolar above (Fig. 7). Actinobacillus pleuropneumoniae bio- hemorrhage, degenerate alveolar leukocytes, fibrin extype 2 was identified as having a novel DNA ampli- udation, and necrosis. The 2 acutely affected pigs from fication pattern when compared with A. pleuropneu- field cases had gross and histologic pulmonary lesions resembling those of APP 2-4 days postinfection, inmoniae reference serotypes and an A. suis strain. Downloaded from vdi.sagepub.com at UNIVERSITE DE SHERBROOKE on May 2, 2015
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polyclonal antibody to A. pleuropneumoniae biotype 1 serotype 8, it was classified as biotype 2 because V factor was not required for growth. Polymerase chain reaction data demonstrate the A. pleuropneumoniae biotype 2 organism isolated in this study is unique at the genomic level when compared with all reference serotypes of A. pleuropneumoniae biotype 1 and A. suis. The arbitrarily primed PCR method with the universal M13 sequencing primers indicated that this isolate has a DNA fragment size pattern distinct from all biotype 1 serotypes of A. pleuropneumoniae and A. suis. Although DNA hybridization studies revealed some cross-hybridization with A. pleuropneumoniae serotype 8, PCR-amplified patterns of DNA from these 2 organisms are distinctly different. Amplified DNA fragment sizes produced by PCR using the APL-1 and XP-1 primers were markedly different from those previously described.26 This difference is likely due to the more stringent temperature cycling protocol used in the present study. Also, DNA bands of less intensity are better visualized with polyacrylamide gels than agarose gels. However, these primers produced the 1.8-kb DNA fragment previously identified as specific for A. pleuropneumoniae and, under different reaction conditions, A. lignieresii. 26 This is the first documented case of A. pleuropneumoniae biotype 2 pleuropneumonia in the United States. Figure 8. Polymerase chain reaction amplification DNA fragments of Actinobacillus species using APL-l and XP-1 oligonucleotide primers. Samples were electrophoresed overnight at 50 volts on a 6% nondenaturing polyacrylamide gel and stained with ethidium bromide for visualization. Lane 1:1-kb DNA ladder. Lane 2: A. pleuropneumoniae biotype 1 serotype 1. Lane 3: A. pleuropneumoniae biotype 1 serotype 5. Lane 4: A. pleuropneumoniae biotype 2 field strain. Lane 5: A. suis.
eluding very roughened pleura with extensive adhesions and firm irregularly swollen deep red lung.16 Pasteurella multocida was isolated from the lungs of all 6 of the chronically affected pigs with pleuropneumonia. Pasteurella multocida acts synergistically with A. pleuropneumoniae to produce pleuropneumonia.11 Although lung tissue samples were not tested for the presence of mycoplasmas, the lymphofollicular interstitial pneumonia detected was consistent with mycoplasma infection. The Actinobacillus species isolated in the present study most closely resembles A. pleuropneumoniae biotype 2 based on biochemical and phenotypic features and DNA hybridization studies.8,10,20 Biochemical reactions were also similar to those of A. lignieresii; however, A. lignieresii has never been isolated from swine and is nonhemolytic on 5% sheep blood agar.26 The A. pleuropneumoniae biotype 2 differed markedly from A. suis, A. lignieresii, and A. equuli by DNA hybridization. Although the organism cross-reacted with
Acknowledgements We acknowledge the technical assistance of Roxanna Maddux and Michael Hays.
Sources and manufacturers a. b. c. d.
Beuthanasia, Schering-Plough Animal Health, Kenilworth, NJ. Difco, Detroit, MI. Seaplaque GTG, FMC Bioproducts, Rockland, ME. Oligolabelling Kit, Pharmacia LKB Biotechnology, Piscataway, NJ. e. GIBCO BRL, Life Technologies, Gaithersburg, MD. f. PCR-MATE™ 391, Applied Biosystems, Foster City, CA. g. NICK Columns, Pharmacia LKB Biotechnology, Piscataway, NJ. h. RESPONSE UV-VIS, Gilford, Oberlin, OH. i. Microspin 24S, Sorvall Instruments, The Du Pont Co., Wilmington, DE. j. Perkin-Elmer Cetus, Norwalk, CT. k. Model 60 Tempcycler, Coy Corp., Ann Arbor, MI.
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