INFECrION AND IMMUNITY, Apr. 1992, p. 1401-1405 0019-9567/92/041401-05$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 60, No. 4
Use of Restriction Endonuclease Analysis and Ribotyping To Study Epidemiology of Pasteurella multocida in Closed Swine Herds GUOSONG ZHAO,1 CARLOS PIJOAN,1* MICHAEL P. MURTAUGH,2
AND
THOMAS W.
MOLITOR'
Departments of Clinical and Population Sciences' and Veterinary PathoBiology, 2 College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 Received 29 October 1991/Accepted 10 January 1992
One hundred and sixty-four clinical isolates of PasteureUla multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTPlabeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.
Pasteurella multocida induces adhesive pleuritis and pneumonia in swine, playing a central role in pneumonic pasteurellosis and/or mycoplasma-induced respiratory disease syndrome (9, 10). The microorganism is widespread in swine herds, but epidemiological studies have been hampered by a lack of appropriate discriminatory methods which can identify separate clones and differentiate virulent strains. Since the epidemiology of this disease in a closed population of pigs is still not well understood, disease control remains difficult. It is generally accepted that conventional phenotyping techniques, including serotyping, biotyping, antibiogram determination, and phage typing, are not sensitive enough to identify individual bacterial strains involved in natural outbreaks. However, restriction endonuclease analysis (REA) and ribotyping have been successfully used as epidemiological tools for the differentiation of strains in a variety of bacterial infections, including some of those caused by P. multocida (1, 6-8, 15, 18). The objective of this study was to utilize REA and ribotyping techniques to differentiate isolates of P. multocida recovered from two closed swine herds during a natural outbreak of pneumonic pasteurellosis. These techniques were evaluated in comparison with serotyping and biochemical analysis in order to study the epidemiology of this disease. The results presented here suggest that genomic fingerprinting is an important and useful method for epidemiological studies of P. multocida in swine infections.
barns
MATERIALS AND METHODS Herd descriptions. The herds used in this study were from feeder pig contracting facilities in Minnesota. These closed herds had complete all-in-all-out management. The originating nucleus herd comprised 1,000 crossbred sows, and the facility consisted of crated gestation, farrowing, and nursery *
well
replacement gilt barn. The farrowing and had eight rooms each, with each room holding 1 week's production. After animals were moved out from the farrowing or nursery facilities, the rooms were completely cleaned and disinfected before any pigs were introduced. Piglets were weaned at 3 weeks of age and placed in a nursery room. Feeder pigs were contracted out to a finishing farm at 9 weeks of age. This sow herd was the only source of feeder pigs for the two herds described in the study. Herd A was housed in two naturally ventilated growerfinisher barns with 20 pens each, with 26 pigs per pen. An entire barn was filled with feeder pigs in 1 day, and the pigs were marketed 14 to 16 weeks later. According to previous health inspections of the herd, the animals were apparently free of Actinobacillus pleuropneumoniae lesions but had Mycoplasma hyopneumoniae lesions. Nose-to-nose contact occurred between pigs. Herd B was very similar, holding 587 finish pigs in a mechanically ventilated barn with 20 pens. Pigs were raised on a totally slatted concrete floor, like the pigs in herd A, for 4 months and were marketed at about 245 lb (1 lb = 453.59237 g) of body weight. Bacteriological sampling. In the nucleus herd, the following animals were randomly sampled from both nose and tonsils by using sterile cotton swabs (Culturette; Becton Dickinson Microbiology Systems, Cockeysville, Md.): 15 farrowing sows of various parities, 3 piglets from the litter of each sow, and 32 nursery pigs 7 to 9 weeks of age. In addition, nasal swabs were also taken from 6 boars and 10 replacement gilts. In both herd A and herd B, 30 apparently healthy pigs of the same group were screened at random by swabbing their noses and tonsils 4 weeks before marketing. Lung samples (290 and 74) were also collected at slaughter from these pigs in herds A and B, respectively, for pasteurella isolation. Assessment of pneumonia was made at slaughter. The pneumonic lesions of lungs were scored as previously described (17). Representative lung samples were fixed in phosphate-buffered 10% Formalin, embedded in paraffin, as
nursery barns
Corresponding author. 1401
as a
1402
ZHAO ET AL.
sectioned, and stained with hematoxylin and eosin by routine procedures. Isolation of P. multocida. Isolation of P. multocida was achieved by dilution in brain heart infusion broth (13). Briefly, swabs were inoculated in a tube containing 10 ml of broth and 5 ,ug of lincomycin per ml. This was labeled as 10-1 and then diluted to 10-5. All broth cultures were incubated overnight at 37°C. The highest dilution showing turbidity was plated onto 5% sheep blood agar and incubated at 37°C overnight. Organisms were obtained from lung tissue by scorching the lung surface with a hot spatula, collecting a sample of approximately 1 cm3, and directly smearing the cut surface onto sheep blood agar plates. Plates were then incubated at 37°C overnight. Characterization of P. multocida isolates. Suspect colonies were identified by standard bacteriological techniques, including Gram stain, oxidase, indole, hydrogen sulfide, urease, catalase, growth in MacConkey agar, motility, and fermentation of glucose, sucrose, mannitol, lactose, maltose, dulcitol, and arabinose. Serotyping of strains. Capsular serotype A and D strains of P. multocida were identified by hyaluronidase sensitivity and acriflavine autoagglutination tests, respectively, as previously described (2, 3). DNA extraction and analysis. Bacterial DNA was extracted as described by Mogollon et al. (8). Cells were grown overnight at 37°C in brain heart infusion broth, recovered by centrifugation at 10,000 x g, and washed twice with phosphate-buffered saline. Cells were suspended in TE sucrose buffer (25% sucrose in 50 mM Tris hydrochloride-50 mM EDTA-10 mM NaCl [pH 8.0]) containing 10 mg of lysozyme per ml and incubated at room temperature for 60 min with frequent mixing. Proteinase K and Sarkosyl were added to final concentrations of 100 ,ug/ml and 1%, respectively. The resulting suspension was thoroughly mixed and incubated at 55°C for 3 h. Ammonium acetate was added to a final concentration of 2.5 M, and the tubes were kept on ice for 20 min. One hundred microliters of Phenylmethylsulfonyl fluoride was added (2-mg/ml stock in ethanol) to inactivate the proteinase K, and the preparation was centrifuged at 10,000 x g for 20 min. DNA was precipitated from the supernatant with 2 volumes of cold absolute ethanol, washed with 70% ethanol, dried, dissolved in 500 ,u of TE buffer, and stored at 4°C. The final DNA concentration was measured spectrophotometrically at 260 nm (14). Restriction endonuclease digestion of DNA and electrophoresis. Pasteurella DNA samples were digested for 2.5 h at 37°C in 30-,u volumes containing the restriction endonuclease in the appropriate buffer, according to the instructions of the manufacturer (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Selected digests with HpaII were repeated at least three times to determine the reproducibility of the method. Digested DNA was electrophoresed in a horizontal gel containing 0.55% agarose at 30 V for 17 h in Tris borate buffer (89 mM Tris Base, 89 mM boric acid, 2.4 mM sodium EDTA [pH 8.0]). DNA was visualized with UV light after staining with ethidium bromide and photographed with type 55 Polaroid film. Southern blotting. DNA fragments were transferred to nylon membranes (Nytran; Schleicher & Schuell, Inc., Keene, N.H.) by the method of Southern (16). Membranes were fixed by UV irradiation for 4 min and stored at room temperature until use. Ribotyping. Escherichia coli rRNA (Sigma Chemical Co., St. Louis, Mo.) was suspended in sterile distilled water to a concentration of 1 ,ug/ul. The rRNA was reverse transcribed
INFECT. IMMUN.
into cDNA with avian myeloblastosis virus reverse transcriptase. The cDNA was then labeled with digoxigenindUTP according to the manufacturer's directions (Boehringer Mannheim Corp., Indianapolis, Ind.). Blots were prehybridized at 68°C for 2 h with a solution of Sx SSC (lx SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.5% blocking reagent, 0.1% N-lauroylsarcosine, and 0.02% sodium dodecyl sulfate (SDS). They were then hybridized with the labeled cDNA probe (25 ,ul of probe in 25 ml of fresh hybridization solution). Hybridization proceeded for 6 h at 68°C with occasional remixing of the solution. After hybridization, membranes were washed twice for 5 min each time at room temperature with 2x SSC-0.1% SDS and twice for 15 min each time at 68°C with 0.lx SSC-0.1% SDS. Antibody binding and Lumi-Phos visualization of hybrids by using Lumi-Phos 530 was performed according to the manufacturer's instructions (Boehringer Mannheim). Results were visualized by autoradiography using X-Omat AR film (Eastman Kodak Co., Rochester, N.Y.) and exposure at room temperature for 2 to 5 min.
RESULTS Clinical signs, gross lesions, and histopathologic examinatiQns. No obvious respiratory signs were detected in the nucleus herd or in finishing herd B. In herd A, however, clinical pneumonia characterized by coughing, labored breathing, and atrophic rhinitis with snout distortion or turbinate hypoplasia was detected in finishing pigs, beginning at 5 weeks after pigs were introduced to the facilities. At slaughter, over 70% of the animals examined in herd A had pneumonic lesions. In contrast, only 18% of lungs in herd B had macroscopic lesions of pneumonia. The major pneumonic lesions were found in the anteroventral portions of the lungs. These regions were purple to grey. Affected areas were firm and appeared atelectatic. The common histological lesion was acute purulent pneumonia indicative of pasteurellosis and chronic lymphocytic peribronchiolitis indicative of mycoplasma infections. Bacteriological analysis. No P. multocida was isolated from the 200 and 60 nasal and tonsillar swabs collected from the nucleus herd and herd B, respectively. However, 14 isolates were recovered from nasal and tonsillar swabs from herd A. Ten isolates belonged to serotype A (two from noses and eight from tonsils), and the other four belonged to serotype D (two each from noses and tonsils). Only one pig yielded multiple isolates: a serotype A isolate from the tonsil and a serotype D isolate from the nasal cavity. One hundred forty-three P. multocida isolates were recovered from 290 lung samples collected at slaughter from herd A. Of these 143 isolates, 139 were serotype A. In a subsequent slaughter check of pigs from herd A, 85 lungs were collected and evaluated for pneumonia scores. The association between P. multocida isolation and pneumonic lesions is shown in Table 1. Of the 65 isolates, 63 (97%) (including 1 serotype D isolate) were recovered from lungs with pneumonic lesions. The extent of pneumonic lung tissue involved ranged from 2 to 38%, and the mean pneumonia score was 10.9%. In contrast, the pneumonia score of 17 pneumonic lungs with negative isolation ranged from 2 to 12%. Mean pneumonia score was 4.4% for these negative samples (Table 1). In herd B, seven P. multocida serotype A isolates were recovered from 74 lung samples collected at slaughter. Only 12 lungs had pneumonia, with scores ranging from 1 to 4%, with a mean of 2.7% for lungs with positive isolation and 1.7% for those with negative results.
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EPIDEMIOLOGY OF P. MULTOCIDA IN SWINE
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TABLE 1. Association between pneumonic lesions and
P. multocida isolation from lungs collected at slaughter Herd
Pneumonic
lesionsa
Isolationb
No. of
lungs
pneumonia'
A (n = 85)
+ +
+ +
63 17 2
10.9 4.4 0.0
-
3 3 9 4 58
0.0 2.7 1.7 0.0 0.0
-
B (n = 74)
+ +
+
-
-
+
-
-
Mean %
K h
a Detected macroscopically at slaughter. +, lung with visible lesion; -, lung without visible lesion. b By directly smearing a lung section on blood agar plate. +, P. multocida isolated; -, P. multocida not obtained. c Determined by the method of Straw et al. (17).
Restriction endonuclease patterns of P. mulocida isolates. All P. multocida strains isolated from herds A and B were analyzed for their restriction endonuclease patterns. HpaII was selected to digest DNA in all experiments because, among 10 endonucleases tested, it provided the best resolution of bands and produced a clear cleavage pattern. Four different REA patterns, designated REA types 1 through 4, were observed among the 149 serotype A strains recovered from swabs and lung samples from herd A (Table 2). A representative example of these patterns is presented in Fig. 1. Only patterns 1 and 2 were observed in swab isolates. Types 1 and 2 were also the most common REA types found in lung samples (Fig. 1, lanes 2 to 4 and lanes 5 and 6, respectively). Two other REA types (types 3 and 4) were also observed in lung isolates (Fig. 1, lanes 7 and 8 and lanes 9 and 10, respectively), but they represented only a small number of isolates (8%) and were not observed in the upper respiratory tract (nose and tonsil; Table 2). Two REA patterns which differed from the four patterns found in serotype A strains were observed among the eight serotype D isolates (data not shown). In herd B, restriction analysis showed that six of the seven isolates had identical DNA fingerprints belonging to REA type 2 and probably originated from a single clone. The other isolate belonged to REA type 4 (Fig. 2). Ribotyping. A representative result of ribotyping experiments of swine P. multocida serotype A DNA digested with HpaII is shown in Fig. 3. Autoradiography revealed that four to five HpaII restriction fragments hybridized with E. coli rRNA. Two different ribotypes, numbered I and II, were observed among the 156 serotype A isolates from herd A (Table 2) and herd B. Ribotype I, showing five hybridization bands, was found in all isolates with REA types 1 and 2 (Fig. 3, lanes 1 and 2). The ribotype II pattern had four HpaII
TABLE 2. REA profiles and ribotype patterns of P. multocida serotype A isolates from herd A
No. of isolates from:
REA
Ribotype
profile
pattern
Lung
Nose
Tonsil
1 2 3 4
I I II II
57 71 4 7
1 1 0 0
4 4 0 0
: ofIt*.
:
FIG. 1. Restriction fragment patterns of the four REA types of P. multocida serotype A isolated from pigs in herd A. Samples were digested with HpaII and electrophoresed in 0.55% agarose as described in Materials and Methods. Lanes: 1, lambda DNA digested with HindIll; 2, strain C63; 3, strain 71T; 4, strain 140; 5, strain 60T; 6, strain 37; 7, strain 82; 8, strain 89; 9, strain 173; and 10, strain 179. Profiles 1, 2, 3, and 4 are indicated at the bottom.
fragments and was characteristic of REA types 3 and 4 (Fig. 3, lanes 3 and 4). DISCUSSION The purpose of this study was to use REA and ribotyping for epidemiological studies of P. multocida isolated from swine herds. The results demonstrate that the genomic fingerprinting techniques were reliable tools for the identification of P. multocida strains involved in natural outbreaks of swine pneumonic pasteurellosis in closed populations. Sampling of the upper respiratory tract with nasal and tonsillar swabs and culturing of diluted aliquots with lincomycin led to the isolation of 10 serotype A and 4 serotype D strains from herd A. Attempts to isolate P. multocida from similar swabs for the nucleus herd and finishing herd B by this method were not successful. It is possible that herds of high health status have few infected animals or the numbers of P. multocida in the noses and tonsils were too low for detection, since the dilution technique was originally designed to enhance isolation of A. pleuropneumoniae from pig lungs (13). P. multocida is considered part of the normal flora in the upper respiratory tracts of pigs. It will not usually produce pneumonia unless other microorganisms, such as M. hyopneumoniae or pseudorabies virus, predispose pigs to second-
INFECT. IMMUN.
ZHAO ET AL.
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1
2
3
4
5
6
7
8
1
2
AI0
._
4
3
kb kb
6.6 -
,..
23.1-
444.4 -
*
9.46.64 .4.-
2 .32 .02 32 .0-
Aba.,
Ribotype: M_ A
M&_ M_ _
I
II
___
FIG. 2. Restriction fragment patterns of P. multocida strains isolated from pig lungs in herd B. Lanes: 1, lambda DNA digested with HindIII; 2, strain M27; 3, strain M41; 4, strain M55; 5, strain M62; 6, strain M66; 7, strain M74; and 8, strain M52.
infections by causing an initial insult to the lung (4, 5). In agreement with previous studies (11, 12), the most common isolates belonged to serotype A. The pneumonic lesions presented in animals from herd A correlated well with P. multocida isolation from lungs at slaughter (Table 1). Three isolates were recovered from 3 of 12 lungs with small, macroscopically visible lesions in herd B. The other four isolates were obtained from lungs with no pneumonic lesions. The few pneumonic lungs with negative isolations may be the result of primary uncomplicated mycoplasma infection, since pneumonia scores for these kinds of lungs ary
from both herds were lower than those for lungs with positive pasteurella isolation. It is also possible that the low number of organisms in the lungs may not have been detected by the direct-smearing isolation method. On the other hand, strains isolated from nonpneumonic lungs in both herds may have resulted from early colonization without visible macroscopic lesion or, conversely, from a late, healed pneumonic lesion. The epidemiology of swine pneumonic pasteurellosis is poorly understood. In this study, a single strain characterized by two restriction fragment patterns predominated among affected pigs. The epidemic strain was isolated from both swabs and lungs of pigs in finishing herd A but only from pig lungs in herd B. The organism was apparently transmitted horizontally, since no pasteurella isolates or
FIG. 3. Ribotypes of HpaII-digested P. multocida serotype A DNAs isolated from pigs in herd A. Lanes: 1, strain C7 (REA type 1); 2, strain 37 (REA type 2); 3, strain 89 (REA type 3); 4, strain 173 (REA type 4). Numbers on the left indicate the lengths in kilobases of the relevant HindIII-digested lambda DNA restriction fragments.
clinical signs were evident in the nucleus herd. In herd A, clinical signs were detected at about 5 weeks after the feeder pigs were introduced to the farm and occurred throughout the finishing period. Disease transmission within this herd was probably by direct nose-to-nose contact between infected and healthy animals. The prevalent strains detected in herd A were REA types 1 and 2, ribotype I. This ribotype represented 92% of the isolates and was detected both in swabs and lungs (Table 2). In herd B, predominantly REA type 2 isolates were recovered from the lung samples. However, the number of isolates from herd B was too small to draw meaningful conclusions. Vaccination programs to prevent pneumonic pasteurellosis have been largely ineffective, although several killed vaccines for this disease are available (9). A reason for this ineffectiveness may be lack of epidemiological information, which may contribute to improper strain selection for vaccine production. The demonstration that a single strain predominated in these herds indicates that virulent strains for pigs exist and are responsible for natural outbreaks. In developing REA for P. multocida, DNA digestion with 10 different restriction endonucleases was performed. The most consistent and informative results were obtained with the enzyme HpaII. This enzyme produced clear and reproducible DNA fingerprinting patterns that could easily be assessed for comparing different P. multocida isolates. For-
EPIDEMIOLOGY OF P. MULTOCIDA IN SWINE
VOL. 60, 1992
tunately, HpaII also produced informative results in the ribotyping analysis in terms of number and distribution of restriction fragments observed. The REA and ribotyping results from both herds suggest that the isolates of P. multocida serotype A associated with pneumonia belonged to a single strain within these closed populations, even though two or more different REA types were circulating in the herds. A greater degree of variation was observed in P. multocida isolates obtained from turkeys and wildlife (15). Seven different REA patterns and an equal number of ribotypes were observed among 25 isolates (15). In our study, ribotyping with the E. coli rRNA gene could not distinguish between REA patterns 1 and 2 or patterns 3 and 4. Each of these patterns differed in one band, indicating a close genomic relationship between these REA types. Different ribotypes for these different REA types would indicate variation in the highly conserved rRNA genes, which are not subject to frequent mutation (15). In conclusion, REA and ribotyping with HpaII digests proved to be reproducible and reliable methods of typing and identifying isolates of P. multocida in swine. In addition, the use of nonradioactive digoxigenin-dUTP-labeled probes and chemiluminescence detection allowed for safe and rapid detection of results, which may facilitate the use of this method in clinical and diagnostic settings. ACKNOWLEDGMENTS This work was supported in part by grants 0725-5686 from the Minnesota Pork Producers Association and MIN 63-027 from the University of Minnesota Agricultural Experiment Station. We thank Peter Bahnson, Bill Christianson, and Julie Grass for their generous assistance in the collection of samples.
REFERENCES 1. Carpenter, T. E., K. P. Snipes, R. W. Kasten, D. W. Hird, and D. C. Hirsh. 1991. Molecular epidemiology of Pasteurella multocida in turkeys. Am. J. Vet. Res. 52:1345-1349. 2. Carter, G. R., and S. N. Rundell. 1975. Identification of type A strains of P. multocida using a staphylococcal hyaluronidase. Vet. Rec. 93:343. 3. Carter, G. R., and P. Subronto. 1973. Identification of type D strains of Pasteurella multocida with acriflavine. Am. J. Vet. Res. 34:293-294. 4. Ciprian, A., C. Pijoan, T. Cruz, J. Camacho, J. T6rtora, G. Colmenares, R. L6pez-Revilla, and M. de la Garza. 1988. Myco-
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plasma hyopneumoniae increases the susceptibility of pigs to experimental Pasteurella multocida pneumonia. Can. J. Vet. Res. 52:434-438. 5. Fuentes, M., and C. PiJoan. 1987. Pneumonia in pigs induced by intranasal challenge exposure with pseudorabies virus and Pasteurella multocida. Am. J. Vet. Res. 48:1446-1448. 6. Harel, J., S. Cote, and M. Jacques. 1990. Restriction endonuclease analysis of porcine Pasteurella multocida isolates from Quebec. Can. J. Vet. Res. 54:422-426. 7. Irino, K., F. Grimont, I. Casin, P. A. D. Grimont, and the Brazilian Purpuric Fever Study Group. 1988. rRNA gene restriction patterns of Haemophilus influenzae biogroup aegyptius strains associated with Brazilian Purpuric Fever. J. Clin. Microbiol. 26:1535-1538. 8. Mogollon, J. D., C. Pijoan, M. P. Murtaugh, E. L. Kaplan, and P. P. Cleary. 1990. Characterization of prototype and clinically defined strains of Streptococcus suis by genomic fingerprinting. J. Clin. Microbiol. 28:2462-2466. 9. Pijoan, C. Pneumonic pasteurellosis. In A. D. Leman, B. Straw, R. D. Glock, W. L. Mengeling, R. H. C. Penny, and E. Scholl (ed.), Diseases of swine, 7th ed., in press. Iowa State University Press, Ames. 10. Pijoan, C., and M. Fuentes. 1987. Severe pleuritis associated with certain strains of Pasteurella multocida in swine. J. Am. Vet. Res. 191:823-826. 11. Pjoan, C., A. Lastra, C. Ramirez, and A. D. Leman. 1984. Isolation of toxigenic strains of Pasteurella multocida from lungs of pneumonic swine. J. Am. Vet. Med. Assoc. 185:522523. 12. Pijoan, C., R. B. Morrison, and H. D. Hilley. 1983. Serotyping of Pasteurella multocida isolated from swine lungs collected at slaughter. J. Clin. Microbiol. 17:1074-1076. 13. Pioan, C., R. B. Morrison, and H. D. Hilley. 1983. Dilution technique for isolation of Haemophilus from swine lung collected at slaughter. J. Clin. Microbiol. 18:143-145. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Snipes, K. P., D. C. Hirsh, R. W. Kasten, L. M. Hansen, D. W. Hird, T. E. Carpenter, and R. H. McCapes. 1989. Use of an rRNA probe and restriction endonuclease analysis to fingerprint Pasteurella multocida isolated from turkeys and wildlife. J. Clin. Microbiol. 27:1847-1853. 16. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 17. Straw, B. E., E. J. Burge, H. D. Hilley, and A. D. Leman. 1983. Pneumonia and atrophic rhinitis in pigs from a test station. J. Am. Vet. Med. Assoc. 185:607-611. 18. Stull, T. L., J. J. LiPuma, and T. D. Edlind. 1988. A broadspectrum probe for molecular epidemiology of bacteria: ribosomal RNA. J. Infect. Dis. 157:280-286.
Vol. 60, No. 4
Use of Restriction Endonuclease Analysis and Ribotyping To Study Epidemiology of Pasteurella multocida in Closed Swine Herds GUOSONG ZHAO,1 CARLOS PIJOAN,1* MICHAEL P. MURTAUGH,2
AND
THOMAS W.
MOLITOR'
Departments of Clinical and Population Sciences' and Veterinary PathoBiology, 2 College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 Received 29 October 1991/Accepted 10 January 1992
One hundred and sixty-four clinical isolates of PasteureUla multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTPlabeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.
Pasteurella multocida induces adhesive pleuritis and pneumonia in swine, playing a central role in pneumonic pasteurellosis and/or mycoplasma-induced respiratory disease syndrome (9, 10). The microorganism is widespread in swine herds, but epidemiological studies have been hampered by a lack of appropriate discriminatory methods which can identify separate clones and differentiate virulent strains. Since the epidemiology of this disease in a closed population of pigs is still not well understood, disease control remains difficult. It is generally accepted that conventional phenotyping techniques, including serotyping, biotyping, antibiogram determination, and phage typing, are not sensitive enough to identify individual bacterial strains involved in natural outbreaks. However, restriction endonuclease analysis (REA) and ribotyping have been successfully used as epidemiological tools for the differentiation of strains in a variety of bacterial infections, including some of those caused by P. multocida (1, 6-8, 15, 18). The objective of this study was to utilize REA and ribotyping techniques to differentiate isolates of P. multocida recovered from two closed swine herds during a natural outbreak of pneumonic pasteurellosis. These techniques were evaluated in comparison with serotyping and biochemical analysis in order to study the epidemiology of this disease. The results presented here suggest that genomic fingerprinting is an important and useful method for epidemiological studies of P. multocida in swine infections.
barns
MATERIALS AND METHODS Herd descriptions. The herds used in this study were from feeder pig contracting facilities in Minnesota. These closed herds had complete all-in-all-out management. The originating nucleus herd comprised 1,000 crossbred sows, and the facility consisted of crated gestation, farrowing, and nursery *
well
replacement gilt barn. The farrowing and had eight rooms each, with each room holding 1 week's production. After animals were moved out from the farrowing or nursery facilities, the rooms were completely cleaned and disinfected before any pigs were introduced. Piglets were weaned at 3 weeks of age and placed in a nursery room. Feeder pigs were contracted out to a finishing farm at 9 weeks of age. This sow herd was the only source of feeder pigs for the two herds described in the study. Herd A was housed in two naturally ventilated growerfinisher barns with 20 pens each, with 26 pigs per pen. An entire barn was filled with feeder pigs in 1 day, and the pigs were marketed 14 to 16 weeks later. According to previous health inspections of the herd, the animals were apparently free of Actinobacillus pleuropneumoniae lesions but had Mycoplasma hyopneumoniae lesions. Nose-to-nose contact occurred between pigs. Herd B was very similar, holding 587 finish pigs in a mechanically ventilated barn with 20 pens. Pigs were raised on a totally slatted concrete floor, like the pigs in herd A, for 4 months and were marketed at about 245 lb (1 lb = 453.59237 g) of body weight. Bacteriological sampling. In the nucleus herd, the following animals were randomly sampled from both nose and tonsils by using sterile cotton swabs (Culturette; Becton Dickinson Microbiology Systems, Cockeysville, Md.): 15 farrowing sows of various parities, 3 piglets from the litter of each sow, and 32 nursery pigs 7 to 9 weeks of age. In addition, nasal swabs were also taken from 6 boars and 10 replacement gilts. In both herd A and herd B, 30 apparently healthy pigs of the same group were screened at random by swabbing their noses and tonsils 4 weeks before marketing. Lung samples (290 and 74) were also collected at slaughter from these pigs in herds A and B, respectively, for pasteurella isolation. Assessment of pneumonia was made at slaughter. The pneumonic lesions of lungs were scored as previously described (17). Representative lung samples were fixed in phosphate-buffered 10% Formalin, embedded in paraffin, as
nursery barns
Corresponding author. 1401
as a
1402
ZHAO ET AL.
sectioned, and stained with hematoxylin and eosin by routine procedures. Isolation of P. multocida. Isolation of P. multocida was achieved by dilution in brain heart infusion broth (13). Briefly, swabs were inoculated in a tube containing 10 ml of broth and 5 ,ug of lincomycin per ml. This was labeled as 10-1 and then diluted to 10-5. All broth cultures were incubated overnight at 37°C. The highest dilution showing turbidity was plated onto 5% sheep blood agar and incubated at 37°C overnight. Organisms were obtained from lung tissue by scorching the lung surface with a hot spatula, collecting a sample of approximately 1 cm3, and directly smearing the cut surface onto sheep blood agar plates. Plates were then incubated at 37°C overnight. Characterization of P. multocida isolates. Suspect colonies were identified by standard bacteriological techniques, including Gram stain, oxidase, indole, hydrogen sulfide, urease, catalase, growth in MacConkey agar, motility, and fermentation of glucose, sucrose, mannitol, lactose, maltose, dulcitol, and arabinose. Serotyping of strains. Capsular serotype A and D strains of P. multocida were identified by hyaluronidase sensitivity and acriflavine autoagglutination tests, respectively, as previously described (2, 3). DNA extraction and analysis. Bacterial DNA was extracted as described by Mogollon et al. (8). Cells were grown overnight at 37°C in brain heart infusion broth, recovered by centrifugation at 10,000 x g, and washed twice with phosphate-buffered saline. Cells were suspended in TE sucrose buffer (25% sucrose in 50 mM Tris hydrochloride-50 mM EDTA-10 mM NaCl [pH 8.0]) containing 10 mg of lysozyme per ml and incubated at room temperature for 60 min with frequent mixing. Proteinase K and Sarkosyl were added to final concentrations of 100 ,ug/ml and 1%, respectively. The resulting suspension was thoroughly mixed and incubated at 55°C for 3 h. Ammonium acetate was added to a final concentration of 2.5 M, and the tubes were kept on ice for 20 min. One hundred microliters of Phenylmethylsulfonyl fluoride was added (2-mg/ml stock in ethanol) to inactivate the proteinase K, and the preparation was centrifuged at 10,000 x g for 20 min. DNA was precipitated from the supernatant with 2 volumes of cold absolute ethanol, washed with 70% ethanol, dried, dissolved in 500 ,u of TE buffer, and stored at 4°C. The final DNA concentration was measured spectrophotometrically at 260 nm (14). Restriction endonuclease digestion of DNA and electrophoresis. Pasteurella DNA samples were digested for 2.5 h at 37°C in 30-,u volumes containing the restriction endonuclease in the appropriate buffer, according to the instructions of the manufacturer (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Selected digests with HpaII were repeated at least three times to determine the reproducibility of the method. Digested DNA was electrophoresed in a horizontal gel containing 0.55% agarose at 30 V for 17 h in Tris borate buffer (89 mM Tris Base, 89 mM boric acid, 2.4 mM sodium EDTA [pH 8.0]). DNA was visualized with UV light after staining with ethidium bromide and photographed with type 55 Polaroid film. Southern blotting. DNA fragments were transferred to nylon membranes (Nytran; Schleicher & Schuell, Inc., Keene, N.H.) by the method of Southern (16). Membranes were fixed by UV irradiation for 4 min and stored at room temperature until use. Ribotyping. Escherichia coli rRNA (Sigma Chemical Co., St. Louis, Mo.) was suspended in sterile distilled water to a concentration of 1 ,ug/ul. The rRNA was reverse transcribed
INFECT. IMMUN.
into cDNA with avian myeloblastosis virus reverse transcriptase. The cDNA was then labeled with digoxigenindUTP according to the manufacturer's directions (Boehringer Mannheim Corp., Indianapolis, Ind.). Blots were prehybridized at 68°C for 2 h with a solution of Sx SSC (lx SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.5% blocking reagent, 0.1% N-lauroylsarcosine, and 0.02% sodium dodecyl sulfate (SDS). They were then hybridized with the labeled cDNA probe (25 ,ul of probe in 25 ml of fresh hybridization solution). Hybridization proceeded for 6 h at 68°C with occasional remixing of the solution. After hybridization, membranes were washed twice for 5 min each time at room temperature with 2x SSC-0.1% SDS and twice for 15 min each time at 68°C with 0.lx SSC-0.1% SDS. Antibody binding and Lumi-Phos visualization of hybrids by using Lumi-Phos 530 was performed according to the manufacturer's instructions (Boehringer Mannheim). Results were visualized by autoradiography using X-Omat AR film (Eastman Kodak Co., Rochester, N.Y.) and exposure at room temperature for 2 to 5 min.
RESULTS Clinical signs, gross lesions, and histopathologic examinatiQns. No obvious respiratory signs were detected in the nucleus herd or in finishing herd B. In herd A, however, clinical pneumonia characterized by coughing, labored breathing, and atrophic rhinitis with snout distortion or turbinate hypoplasia was detected in finishing pigs, beginning at 5 weeks after pigs were introduced to the facilities. At slaughter, over 70% of the animals examined in herd A had pneumonic lesions. In contrast, only 18% of lungs in herd B had macroscopic lesions of pneumonia. The major pneumonic lesions were found in the anteroventral portions of the lungs. These regions were purple to grey. Affected areas were firm and appeared atelectatic. The common histological lesion was acute purulent pneumonia indicative of pasteurellosis and chronic lymphocytic peribronchiolitis indicative of mycoplasma infections. Bacteriological analysis. No P. multocida was isolated from the 200 and 60 nasal and tonsillar swabs collected from the nucleus herd and herd B, respectively. However, 14 isolates were recovered from nasal and tonsillar swabs from herd A. Ten isolates belonged to serotype A (two from noses and eight from tonsils), and the other four belonged to serotype D (two each from noses and tonsils). Only one pig yielded multiple isolates: a serotype A isolate from the tonsil and a serotype D isolate from the nasal cavity. One hundred forty-three P. multocida isolates were recovered from 290 lung samples collected at slaughter from herd A. Of these 143 isolates, 139 were serotype A. In a subsequent slaughter check of pigs from herd A, 85 lungs were collected and evaluated for pneumonia scores. The association between P. multocida isolation and pneumonic lesions is shown in Table 1. Of the 65 isolates, 63 (97%) (including 1 serotype D isolate) were recovered from lungs with pneumonic lesions. The extent of pneumonic lung tissue involved ranged from 2 to 38%, and the mean pneumonia score was 10.9%. In contrast, the pneumonia score of 17 pneumonic lungs with negative isolation ranged from 2 to 12%. Mean pneumonia score was 4.4% for these negative samples (Table 1). In herd B, seven P. multocida serotype A isolates were recovered from 74 lung samples collected at slaughter. Only 12 lungs had pneumonia, with scores ranging from 1 to 4%, with a mean of 2.7% for lungs with positive isolation and 1.7% for those with negative results.
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TABLE 1. Association between pneumonic lesions and
P. multocida isolation from lungs collected at slaughter Herd
Pneumonic
lesionsa
Isolationb
No. of
lungs
pneumonia'
A (n = 85)
+ +
+ +
63 17 2
10.9 4.4 0.0
-
3 3 9 4 58
0.0 2.7 1.7 0.0 0.0
-
B (n = 74)
+ +
+
-
-
+
-
-
Mean %
K h
a Detected macroscopically at slaughter. +, lung with visible lesion; -, lung without visible lesion. b By directly smearing a lung section on blood agar plate. +, P. multocida isolated; -, P. multocida not obtained. c Determined by the method of Straw et al. (17).
Restriction endonuclease patterns of P. mulocida isolates. All P. multocida strains isolated from herds A and B were analyzed for their restriction endonuclease patterns. HpaII was selected to digest DNA in all experiments because, among 10 endonucleases tested, it provided the best resolution of bands and produced a clear cleavage pattern. Four different REA patterns, designated REA types 1 through 4, were observed among the 149 serotype A strains recovered from swabs and lung samples from herd A (Table 2). A representative example of these patterns is presented in Fig. 1. Only patterns 1 and 2 were observed in swab isolates. Types 1 and 2 were also the most common REA types found in lung samples (Fig. 1, lanes 2 to 4 and lanes 5 and 6, respectively). Two other REA types (types 3 and 4) were also observed in lung isolates (Fig. 1, lanes 7 and 8 and lanes 9 and 10, respectively), but they represented only a small number of isolates (8%) and were not observed in the upper respiratory tract (nose and tonsil; Table 2). Two REA patterns which differed from the four patterns found in serotype A strains were observed among the eight serotype D isolates (data not shown). In herd B, restriction analysis showed that six of the seven isolates had identical DNA fingerprints belonging to REA type 2 and probably originated from a single clone. The other isolate belonged to REA type 4 (Fig. 2). Ribotyping. A representative result of ribotyping experiments of swine P. multocida serotype A DNA digested with HpaII is shown in Fig. 3. Autoradiography revealed that four to five HpaII restriction fragments hybridized with E. coli rRNA. Two different ribotypes, numbered I and II, were observed among the 156 serotype A isolates from herd A (Table 2) and herd B. Ribotype I, showing five hybridization bands, was found in all isolates with REA types 1 and 2 (Fig. 3, lanes 1 and 2). The ribotype II pattern had four HpaII
TABLE 2. REA profiles and ribotype patterns of P. multocida serotype A isolates from herd A
No. of isolates from:
REA
Ribotype
profile
pattern
Lung
Nose
Tonsil
1 2 3 4
I I II II
57 71 4 7
1 1 0 0
4 4 0 0
: ofIt*.
:
FIG. 1. Restriction fragment patterns of the four REA types of P. multocida serotype A isolated from pigs in herd A. Samples were digested with HpaII and electrophoresed in 0.55% agarose as described in Materials and Methods. Lanes: 1, lambda DNA digested with HindIll; 2, strain C63; 3, strain 71T; 4, strain 140; 5, strain 60T; 6, strain 37; 7, strain 82; 8, strain 89; 9, strain 173; and 10, strain 179. Profiles 1, 2, 3, and 4 are indicated at the bottom.
fragments and was characteristic of REA types 3 and 4 (Fig. 3, lanes 3 and 4). DISCUSSION The purpose of this study was to use REA and ribotyping for epidemiological studies of P. multocida isolated from swine herds. The results demonstrate that the genomic fingerprinting techniques were reliable tools for the identification of P. multocida strains involved in natural outbreaks of swine pneumonic pasteurellosis in closed populations. Sampling of the upper respiratory tract with nasal and tonsillar swabs and culturing of diluted aliquots with lincomycin led to the isolation of 10 serotype A and 4 serotype D strains from herd A. Attempts to isolate P. multocida from similar swabs for the nucleus herd and finishing herd B by this method were not successful. It is possible that herds of high health status have few infected animals or the numbers of P. multocida in the noses and tonsils were too low for detection, since the dilution technique was originally designed to enhance isolation of A. pleuropneumoniae from pig lungs (13). P. multocida is considered part of the normal flora in the upper respiratory tracts of pigs. It will not usually produce pneumonia unless other microorganisms, such as M. hyopneumoniae or pseudorabies virus, predispose pigs to second-
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ZHAO ET AL.
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1
2
3
4
5
6
7
8
1
2
AI0
._
4
3
kb kb
6.6 -
,..
23.1-
444.4 -
*
9.46.64 .4.-
2 .32 .02 32 .0-
Aba.,
Ribotype: M_ A
M&_ M_ _
I
II
___
FIG. 2. Restriction fragment patterns of P. multocida strains isolated from pig lungs in herd B. Lanes: 1, lambda DNA digested with HindIII; 2, strain M27; 3, strain M41; 4, strain M55; 5, strain M62; 6, strain M66; 7, strain M74; and 8, strain M52.
infections by causing an initial insult to the lung (4, 5). In agreement with previous studies (11, 12), the most common isolates belonged to serotype A. The pneumonic lesions presented in animals from herd A correlated well with P. multocida isolation from lungs at slaughter (Table 1). Three isolates were recovered from 3 of 12 lungs with small, macroscopically visible lesions in herd B. The other four isolates were obtained from lungs with no pneumonic lesions. The few pneumonic lungs with negative isolations may be the result of primary uncomplicated mycoplasma infection, since pneumonia scores for these kinds of lungs ary
from both herds were lower than those for lungs with positive pasteurella isolation. It is also possible that the low number of organisms in the lungs may not have been detected by the direct-smearing isolation method. On the other hand, strains isolated from nonpneumonic lungs in both herds may have resulted from early colonization without visible macroscopic lesion or, conversely, from a late, healed pneumonic lesion. The epidemiology of swine pneumonic pasteurellosis is poorly understood. In this study, a single strain characterized by two restriction fragment patterns predominated among affected pigs. The epidemic strain was isolated from both swabs and lungs of pigs in finishing herd A but only from pig lungs in herd B. The organism was apparently transmitted horizontally, since no pasteurella isolates or
FIG. 3. Ribotypes of HpaII-digested P. multocida serotype A DNAs isolated from pigs in herd A. Lanes: 1, strain C7 (REA type 1); 2, strain 37 (REA type 2); 3, strain 89 (REA type 3); 4, strain 173 (REA type 4). Numbers on the left indicate the lengths in kilobases of the relevant HindIII-digested lambda DNA restriction fragments.
clinical signs were evident in the nucleus herd. In herd A, clinical signs were detected at about 5 weeks after the feeder pigs were introduced to the farm and occurred throughout the finishing period. Disease transmission within this herd was probably by direct nose-to-nose contact between infected and healthy animals. The prevalent strains detected in herd A were REA types 1 and 2, ribotype I. This ribotype represented 92% of the isolates and was detected both in swabs and lungs (Table 2). In herd B, predominantly REA type 2 isolates were recovered from the lung samples. However, the number of isolates from herd B was too small to draw meaningful conclusions. Vaccination programs to prevent pneumonic pasteurellosis have been largely ineffective, although several killed vaccines for this disease are available (9). A reason for this ineffectiveness may be lack of epidemiological information, which may contribute to improper strain selection for vaccine production. The demonstration that a single strain predominated in these herds indicates that virulent strains for pigs exist and are responsible for natural outbreaks. In developing REA for P. multocida, DNA digestion with 10 different restriction endonucleases was performed. The most consistent and informative results were obtained with the enzyme HpaII. This enzyme produced clear and reproducible DNA fingerprinting patterns that could easily be assessed for comparing different P. multocida isolates. For-
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VOL. 60, 1992
tunately, HpaII also produced informative results in the ribotyping analysis in terms of number and distribution of restriction fragments observed. The REA and ribotyping results from both herds suggest that the isolates of P. multocida serotype A associated with pneumonia belonged to a single strain within these closed populations, even though two or more different REA types were circulating in the herds. A greater degree of variation was observed in P. multocida isolates obtained from turkeys and wildlife (15). Seven different REA patterns and an equal number of ribotypes were observed among 25 isolates (15). In our study, ribotyping with the E. coli rRNA gene could not distinguish between REA patterns 1 and 2 or patterns 3 and 4. Each of these patterns differed in one band, indicating a close genomic relationship between these REA types. Different ribotypes for these different REA types would indicate variation in the highly conserved rRNA genes, which are not subject to frequent mutation (15). In conclusion, REA and ribotyping with HpaII digests proved to be reproducible and reliable methods of typing and identifying isolates of P. multocida in swine. In addition, the use of nonradioactive digoxigenin-dUTP-labeled probes and chemiluminescence detection allowed for safe and rapid detection of results, which may facilitate the use of this method in clinical and diagnostic settings. ACKNOWLEDGMENTS This work was supported in part by grants 0725-5686 from the Minnesota Pork Producers Association and MIN 63-027 from the University of Minnesota Agricultural Experiment Station. We thank Peter Bahnson, Bill Christianson, and Julie Grass for their generous assistance in the collection of samples.
REFERENCES 1. Carpenter, T. E., K. P. Snipes, R. W. Kasten, D. W. Hird, and D. C. Hirsh. 1991. Molecular epidemiology of Pasteurella multocida in turkeys. Am. J. Vet. Res. 52:1345-1349. 2. Carter, G. R., and S. N. Rundell. 1975. Identification of type A strains of P. multocida using a staphylococcal hyaluronidase. Vet. Rec. 93:343. 3. Carter, G. R., and P. Subronto. 1973. Identification of type D strains of Pasteurella multocida with acriflavine. Am. J. Vet. Res. 34:293-294. 4. Ciprian, A., C. Pijoan, T. Cruz, J. Camacho, J. T6rtora, G. Colmenares, R. L6pez-Revilla, and M. de la Garza. 1988. Myco-
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plasma hyopneumoniae increases the susceptibility of pigs to experimental Pasteurella multocida pneumonia. Can. J. Vet. Res. 52:434-438. 5. Fuentes, M., and C. PiJoan. 1987. Pneumonia in pigs induced by intranasal challenge exposure with pseudorabies virus and Pasteurella multocida. Am. J. Vet. Res. 48:1446-1448. 6. Harel, J., S. Cote, and M. Jacques. 1990. Restriction endonuclease analysis of porcine Pasteurella multocida isolates from Quebec. Can. J. Vet. Res. 54:422-426. 7. Irino, K., F. Grimont, I. Casin, P. A. D. Grimont, and the Brazilian Purpuric Fever Study Group. 1988. rRNA gene restriction patterns of Haemophilus influenzae biogroup aegyptius strains associated with Brazilian Purpuric Fever. J. Clin. Microbiol. 26:1535-1538. 8. Mogollon, J. D., C. Pijoan, M. P. Murtaugh, E. L. Kaplan, and P. P. Cleary. 1990. Characterization of prototype and clinically defined strains of Streptococcus suis by genomic fingerprinting. J. Clin. Microbiol. 28:2462-2466. 9. Pijoan, C. Pneumonic pasteurellosis. In A. D. Leman, B. Straw, R. D. Glock, W. L. Mengeling, R. H. C. Penny, and E. Scholl (ed.), Diseases of swine, 7th ed., in press. Iowa State University Press, Ames. 10. Pijoan, C., and M. Fuentes. 1987. Severe pleuritis associated with certain strains of Pasteurella multocida in swine. J. Am. Vet. Res. 191:823-826. 11. Pjoan, C., A. Lastra, C. Ramirez, and A. D. Leman. 1984. Isolation of toxigenic strains of Pasteurella multocida from lungs of pneumonic swine. J. Am. Vet. Med. Assoc. 185:522523. 12. Pijoan, C., R. B. Morrison, and H. D. Hilley. 1983. Serotyping of Pasteurella multocida isolated from swine lungs collected at slaughter. J. Clin. Microbiol. 17:1074-1076. 13. Pioan, C., R. B. Morrison, and H. D. Hilley. 1983. Dilution technique for isolation of Haemophilus from swine lung collected at slaughter. J. Clin. Microbiol. 18:143-145. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Snipes, K. P., D. C. Hirsh, R. W. Kasten, L. M. Hansen, D. W. Hird, T. E. Carpenter, and R. H. McCapes. 1989. Use of an rRNA probe and restriction endonuclease analysis to fingerprint Pasteurella multocida isolated from turkeys and wildlife. J. Clin. Microbiol. 27:1847-1853. 16. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 17. Straw, B. E., E. J. Burge, H. D. Hilley, and A. D. Leman. 1983. Pneumonia and atrophic rhinitis in pigs from a test station. J. Am. Vet. Med. Assoc. 185:607-611. 18. Stull, T. L., J. J. LiPuma, and T. D. Edlind. 1988. A broadspectrum probe for molecular epidemiology of bacteria: ribosomal RNA. J. Infect. Dis. 157:280-286.
