Laboratory Animal Science Vol 48, No 2 Copyright 1998 April 1998 by the American Association for Laboratory Animal Science

Further Evaluation of a Diagnostic Polymerase Chain Reaction Assay for Pasteurella pneumotropica

Benjamin J. Weigler,1* Lisa A. Wiltron,1 Susan I. Hancock,1 Julius E. Thigpen,2 Mary F. Goelz,2† and Diane B. Forsythe2

Pasteurella pneumotropica is a common gram-negative Table 1. tested by PCR, using primers PPN-1 and coccobacillus harbored by laboratory rats, mice, hamsters, PPN-2 for P. pneumotropica guinea pigs, and other rodents. This agent typically resides Bacterial species (biotype) Source (animal of origin) PCR result in tissues of the oropharynx, upper respiratory tract, ocu- Pasteurella pneumotropica (Jawetz) ATCC 35149 (mouse) + P. pneumotropica (Heyl) ATCC 12555 (mouse) + lar tissues and adnexa, urinary bladder, skin, mammary P. pneumotropica (Heyl) ATCC 13669 (mouse) + tissues, and reproductive tract without outward signs of P. pneumotropica 9 field isolates (rodent)a + disease (1). Although usually considered an opportunistic P. dagmatis ATCC 43325 (human) – P. multocida ATCC 43137 (pig) – of minimal importance to laboratory animals, muris ATCC 49577 (mouse) + respiratory and genital tract infections, otitis, and cutane- A. ureae ATCC 25976 (human) – A. equuli ATCC 19392 (horse) – ous and retro-orbital have been associated with A. lignieresii ATCC 49236 (ox) – P. pneumotropica (1–3). The importance of P. pneumotropica Escherichia coli ATCC 11775 (human) – and related agents in rodent colonies probably varies with Bordetella bronchiseptica ATCC 31437 (pig) – B. bronchiseptica ATCC 785 (rat) – a variety of host and bacterial factors that are incompletely B. bronchiseptica ATCC 14065 (rat) – understood at present (3–5). Commercial vendors have con- B. bronchiseptica ATCC 19395 (dog) – sidered this agent worthy of elimination from their rodent B. bronchiseptica ATCC 35086 (turkey) – colonies (6). aOropharyngeal swab specimens from six mice, one hamster, and one rat. One isolate was recovered from a uterine in a mouse described pre- Diagnostic methods for P. pneumotropica have included viously (3). standard microbiological culture from tissue swab speci- mens (7), serologic assays (8), and recently, a polymerase method was done, using a heating block thermal cycler chain reaction (PCR) test for the agent in tissue swab speci- (Progene; Techne Inc., Princeton, N.J.) and the forward mens (9). Use of PCR assays for diagnosis of infectious dis- PPN-1 (5'-TTGCATTTCAGACTGGGAATC-3') and reverse eases has the potential to improve on the speed, sensitiv- PPN-2 (5'-GCACAAAACTATCTCTAGTCTC-3') primers as ity, and cost efficiency of disease detection, and these as- reported (9). The template DNA for these experiments was says have come into widespread usage in human and vet- derived from 14 bacterial reference strains obtained from erinary medicine alike (10). By targeting genomic nucle- the American Type Culture Collection (ATCC) and nine field otide sequences unique to the pathogen, PCR assays can isolates of P. pneumotropica recovered from rodents in our have higher specificity than do traditional diagnostic meth- animal facilities (seven mice, one hamster, one rat), as well ods. This approach could especially benefit recognition of as negative (template-free) and positive (P. pneumotropica P. pneumotropica in laboratory animal facilities because of biotype Jawetz, ATCC no. 35149) control specimens (Table the large investment in technician time and biochemical 1). The rodents providing field isolates were on protocols ap- reagents required to distinguish it from related bacteria in proved by the animal care and use committees at the Na- the Haemophilus-Pasteurella-Actinobacillus complex, tional Institute of Environmental Health Sciences (NIEHS) which also are known to occur in rodents (11, 12). and North Carolina State University (NCSU). After aerobic In our efforts to develop resource-optimized testing al- culture and phenotypic identification, as reported previously gorithms for epidemiologic studies of P. pneumotropica (3), each bacterial isolate was incubated overnight at 378C in in laboratory rodent colonies, we evaluated the PCR 6 ml of tripticase soy broth (BBL Inc., Cockeysville, Md.), as- method reported by Wang et al. (9) for its ability to iden- signed a code number, and then submitted single-blind to a tify P. pneumotropica with high specificity. The PCR assay separate laboratory for PCR testing. The PCR evaluations were done on multiple replicates of Department of Companion Animal and Special Species Medicine, College of the test DNA that had been extracted from bacterial sus- Veterinary Medicine, North Carolina State University, Raleigh, North Caro- lina,1 and Comparative Medicine Branch, National Institute of Environmen- pensions via boiling (1008C), via precipitation of lysozyme tal Health Sciences, Research Triangle Park, North Carolina2 (Sigma Chemical Co., St. Louis, Mo.) and proteinase K *Present address: Regional Primate Research Center, University of Wash- (Promega, Madison, Wis.) digests as described (3), and via ington, Box 357330, Seattle, WA 98185. †Address reprint requests to Dr. Mary F. Goelz, Deputy Chief, Comparative standard cetyltrimethylammonium bromide (CTAB)/NaCl Medicine Branch, MD C1-06, NIEHS, Research Triangle Park, NC 27709. purification methods (13) followed by RNAse I (10 mg/ml; 193 Vol 48, No 2 Laboratory Animal Science April 1998

Figure 2. Agarose gel of RAPD-PCR marker profiles from arbitrary primer OPL-7 (Operon Technologies, Inc., Alameda, Calif.) used as de- scribed (3). Lane M is molecular size standard fX174 RF DNA/Hae III digests (Stratagene, La Jolla, Calif.). Lanes 1 and 2, A. muris (ATCC 49577); lane 3, field isolate of P. pneumotropica from a mouse uterine abscess at NIEHS (3); lanes 4–8, field isolates of P. pneumotropica from oropharyngeal swab specimens of mice at NIEHS; lanes 9 and 10, field isolates of P. pneumotropica from oropharyngeal swab speci- mens of mice at NCSU.

tors constant, we found that the observed cross-reactivity with Figure 1. (A) Agarose gel of amplification product of PPN-1 and A. muris disappeared when the annealing step was in- PPN-2 primers. Lane M is molecular size standard 100-bp ladder. creased from 558C to 578C; all amplicons (including those Lanes 1 and 2, (ATCC 49577); lane 3, field isolate of A. muris from P. pneumotropica) disappeared at 608C. Results of RAPD- P. pneumotropica from uterine abscess at NIEHS (3); lanes 4–8, field isolates of P. pneumotropica from oropharyngeal swab specimens at PCR evaluations (Figure 2) indicated that genomic differences NIEHS; lanes 9 and 10, field isolates of P. pneumotropica from oropha- existed among confirmed field strains of P. pneumotropica ryngeal swab specimens at NCSU. (B) Amplification products from recovered from our animal facilities (3) and that the RAPD (A) after enzymatic cleavage with HindIII, as described (9). profile for A. muris was clearly different from that of other isolates via this method. Promega) digestion and reprecipitation. The CTAB/NaCl The type strain of A. muris used in this investigation method of extraction improved the volume and quality of DNA was documented to be a member of Bisgaard’s taxon 12 yield from some and isolates. Purified Pasteurella Bordetella and forms part of the Haemophilus-Pasteurella-Actinoba- DNA was quantified via spectrophotometry (A nm) and 260/280 cillus complex that occurs in laboratory rodents (12). In assayed at 1 mg per reaction. The PCR amplification prod- earlier reports, this agent had mistakenly been ascribed to ucts were stained with ethidium bromide and visualized belong in the P. ureae (now A. ureae) taxon (14), which ap- on 1.5% agarose gels alongside a 100-bp ladder (Promega). pears instead to be limited to humans (15). Actinobacillus Specificity of the amplicons for was evalu- P. pneumotropica muris and related agents not yet fully characterized have ated through HindIII (Promega) restriction endonuclease been isolated from the conjunctiva, the oropharynx, and digestion, as reported (9). The identity of each coded iso- the lower respiratory tract of mice, with or without evi- late was revealed only after all PCR testing was completed. dence of inflammation (13, 16). Other members of Actino- Randomly amplified polymorphic DNA polymerase chain bacillus have also been recognized in laboratory rodents reaction (RAPD-PCR) assays (3) were then done for select (17, 18), and diagnostic laboratories have been cautioned isolates to further differentiate and characterize them by not to mistake these organisms for pasteurellae despite source and host species. their large array of phenotypic and cultural similarities The PCR primers PPN-1 and PPN-2 performed well (18). Both organisms are typically positive, oxi- for identification of all P. pneumotropica reference strains dase positive, and urease positive; reduce nitrate to nitrite; and field isolates recovered from rodents in our facili- acidify the slants and butts of triple sugar iron agar; and ties (Table 1, Figure 1A). However, A. muris (ATCC are nonmotile (11, 12). However, at least 13 phenotypic dif- 49577) also had positive results by use of this procedure, ferences have been identified between A. muris and bio- and the resulting 395-bp amplicon could not be distin- types of P. pneumotropica (12), and these differences were guished from specimens, even after en- P. pneumotropica used to confirm the identity of the A. muris isolate used in zymatic cleavage with HindIII (Figure 1B). By varying this investigation. components of the PCR assay and keeping all other fac-

194 Note

Chromosomal DNA hybridization experiments (12, 15) the annealing temperature to 578C was sufficient to elimi- and comparisons of 16S rRNA nucleotide sequences (5, 19) nate this problem for our application. The development, have greatly helped to clarify the relatedness between evaluation, and interpretation of all tests for laboratory agents taxonomically placed within this complex, and animal diseases should consider the known interrelation- RAPD-PCR could also be feasibly used for this purpose (3). ships between test sensitivity, specificity, and disease preva- At 46.9 mol %, the G+C content of A. muris is higher than lence, especially as they relate to their practical applica- that of either the Jawetz (40.7%) or Heyl (41.7%) biotypes tion in diagnostic use. of P. pneumotropica, whereas its genomic size (1.49 x 109 daltons) is somewhat smaller (12). Using 16S rRNA se- quence data and a Neighbor-Joining analysis, Dewhirst et Acknowledgements al. (19) observed that A. muris and P. pneumotropica fall We thank Gordon Caviness, Jacqueline Locklear, and Carol within a single phylogenetic cluster distinct from other Lemons for technical assistance. L. Wiltron was supported by a members of the . More recent work has es- Howard Hughes undergraduate research internship in biomedi- cal sciences. tablished that Haemophilus influenzae-murium also falls within this group (20). Although highlighting the value of PCR technology for References P. pneumotropica detection in rodent colonies, our work has 1. National Research Council. 1991. Infectious diseases of mice indicated a potential pitfall that can occur in the develop- and rats, p. 187–190. National Academy Press, Washington, D.C. ment of screening tests intended for practical diagnostic 2. Percy, D. H., and S. W. Barthold. 1993. Pathology of labo- use. In this case, false-positive reactions would have oc- ratory rodents and rabbits, p. 34–35, 91. Iowa State Univer- sity Press, Ames, Iowa. curred in the PCR evaluation had been the infect- A. muris 3. Weigler, B. J., J. E. Thigpen, M. F. Goelz, et al. 1996. Ran- ing agent in our mice, thereby resulting in misleading con- domly amplified polymorphic DNA polymerase chain reaction clusions. Extensive biochemical testing of all putative iso- assay for molecular epidemiologic investigation of Pasteurella lates would have provided for their differentiation but ob- pneumotropica in laboratory rodent colonies. Lab. Anim. Sci. viated the value of PCR as a screening assay. Considering 46:386–392. 4. Boot, R., and M. Bisgaard. 1995. Reclassification of 30 the knowledge that both organisms can reside in respira- Pasteurellaceae strains isolated from rodents. Lab. Animals tory and ocular adnexal tissues, this scenario is not incon- 29:314–319. ceivable. Diagnostic test specificity is the proportion of 5. Nicklas, W., and S. Kirschner. 1996. Phenotypic and geno- nondiseased animals that test negative for the agent, and typic relationship of Pasteurella pneumotropic biotypes and additional selected rodent Pasteurellaceae, PS-02. Abstr. should be assessed by examining groups of individuals free AALAS 47th Annu. Meet. 1996. from the disease in question but presenting with the full 6. Bedigian, H. G. 1995. Animal health update. Jackson Labo- clinical and pathologic spectrum of individuals that might ratory, Bar Harbor, Maine (Letter). usually be studied. In particular, one goal should be to in- 7. Pickett, M. J., D. G. Hollis, and E. J. Bottone. 1991. Mis- clude individuals in the comparison that could be associ- cellaneous gram-negative bacteria, p. 410–428. In A. Ballows (ed.), Manual of clinical microbiology. American Society for ated with false-positive results under real-life conditions Microbiology, Washington, D.C. (21, 22). 8. Manning, P. J., D. DeLong, R. Gunther, et al. 1991. An Previous efforts to validate the diagnostic test performance enzyme-linked immunosorbent assay for detection of of PCR primers PPN-1 and PPN-2 for P. pneumotropica de- chronic subclinical Pasteurella pneumotropica infection in mice. Lab. Anim. Sci. 162–165. tection (9) included one reference strain (ATCC 35149, bio- 41: 9. Wang, R-F., W. Campbell, W-W. Cao, et al. 1996. Detection type Jawetz) and 12 field isolates of the agent along with of Pasteurella pneumotropica in laboratory mice and rats by 42 other species of bacteria, most of which do not usually polymerase chain reaction. Lab. Anim. Sci. 46:81–85. occur in rodents. In that work, the authors used the 10. Persing, D. H., T. F. Smith, F. C. Tenover, et al. 1993. GenBank computerized library of genomic sequences to aid Diagnostic molecular microbiology: principals and applica- tions. American Society for Microbiology, Washington, D.C. in their selection of primers specific for the target organ- 11. Adlam, C., and J. M. Rutter (ed.). 1989. Pasteurella and ism, including comparisons with 33 sequences from Acti- pasteurellosis. Academic Press, London. nobacillus spp. These publicly available databases have 12. Bisgaard, M. 1986. Actinobacillus muris sp. nov. isolated from been tremendously valuable for molecular genetic and di- mice. Acta Path. Microbiol. Immunol. Scand. Sect. B 94:1–8. 13. Ausubel, F. M., R. Brent, R. E. Kingston, et al. 1989. Short agnostic work, but it is important to recognize that their protocols in molecular biology, p. 61. John Wiley & Sons, contents are only as complete as the scientific community’s New York. efforts to sequence and submit the results from all agents 14. Ackerman, J. I., and J. G. Fox. 1981. Isolation of Pasteurella examined in this manner. Although more than 1.1 million ureae from reproductive tracts of congenic mice. J. Clin. Microbiol. sequence records were available in the GenBank database 13:1049–1053. 15. Mutters, R., W. Frederiksen, and W. Mannheim. 1984. as of December 1996, citations specific for A. muris were Lack of evidence for the occurrence of Pasteurella ureae in not available at that time. Recent rRNA sequencing stud- rodents. Vet. Microbiol. 9:83–93. ies (20) indicate that P. pneumotropica and A. muris align 16. Kunstyr, I., and D. Hartmann. 1983. Pasteurella at all but three nucleotide positions in the region of PPN-1 pneumotropica and the prevalence of the AHP (Actinobacil- , , )-group in laboratory animals. and all but one position for PPN-2, probably accounting for lus Haemophilus Pasteurella Lab. Animals 17:156–160. the false-positive reactions we observed. Increasing the stringency of the established PCR protocol by increasing

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17. Simpson, W., and D. J. C. Simmons. 1980. Two Actinoba- 20. Dewhirst, F. E. 1997. Personal communication. cillus species isolated from laboratory rodents. Lab. Animals 21. Ransohoff, D. F., and A. R. Feinstein. 1978. Problems of 14:15–16. spectrum and bias in evaluating the efficacy of diagnostic tests. 18. Lentsch, R. H., and J. E. Wagner. 1980. Isolation of Acti- N. Engl. J. Med. 299:926–930. nobacillus lignieresii and Actinobacillus equuli from labora- 22. Martin, S. W. 1988. The interpretation of laboratory results. tory rodents. J. Clin. Microbiol. 12:351–354. Vet. Clin. North Am.: Food Anim. Pract. 4:61–78. 19. Dewhirst, F. E., B. J. Paster, I. Olsen, et al. 1993. Phylog- eny of the Pasteurellaceae as determined by comparison of 16S ribosomal ribonucleic acid sequences. Zentralb. Bakteriol. 279:35–44.

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