M. Sweeney, J. Watts, E. Portis, M. Lucas, R. Nutsch, D. Meeuwse, D. Bade, V. Oliver, D. W. Morck, D. Shinabarger, S. Poppe, M. Peterson, D. Sweeney, M. Knechtel, and G. Zurenko Identification of Porphyromonas levii Isolated from Clinical Cases of Bovine Interdigital Necrobacillosis by 16S rRNA Sequencing * M. Sweeney, MS a D. W. Morck, DVM, PhD c J. Watts, PhD, RM(NRCM), M(ASCP) a D. Shinabarger, PhD d E. Portis, BS a S. Poppe, MS d M. Lucas, DVM, MS, DACT a M. Peterson, BS d R. Nutsch, DVM a D. Sweeney, BS d D. Meeuwse, BS a M. Knechtel, BS d D. Bade, BS b G. Zurenko, MS d,† V. Oliver, PhD c aPfizer Animal Health cDepartment of Biological Sciences 333 Portage Street Department of Comparative Biology and Kalamazoo, MI 49007 Experimental Medicine University of Calgary bMicrobial Research Inc. 2500 University Drive NW 2649 East Mulberry Street, Suite 15 Calgary, T2N 1N4 Fort Collins, CO 80524 Canada dMicromyx 4717 Campus Drive Kalamazoo, MI 49008 CLINICAL RELEVANCE Laboratories use pigmentation, antibiotic susceptibility, and biochemical tests to identify anaerobic organisms that play a role in bovine interdigital necrobacillosis (bovine foot rot). In this study, 16S rRNA gene sequencing was used to identify strains to the species level that were originally classified as Prevotella or Por - phyromonas spp by conventional phenotype assessment methods. Of 264 qual - ified strains from ceftiofur clinical trials, 241 isolates were definitively identified by 16S rRNA sequencing as Porphyromonas levii . Similarly, of 275 qualified strains from tulathromycin clinical trials, 156 isolates were definitively identified by 16S rRNA sequencing as P. levii . The predominance of P. levii in this study supports the role of this organism as an associative agent of bovine foot rot and may have implications for routine laboratory diagnosis. *Research sponsored by Pfizer Animal Health, Inc. †Correspondence should be sent to Mr. Zurenko: phone, 269-372-3683; fax, 269-353-5567; e-mail, gezurenko@ micromyx.com. E ©Copyright 2009 MediMedia Animal Health. This document is for internal purposes only. Reprinting or posting on an external website without written permission from MMAH is a violation of copyright laws. Veterinary Therapeutics • Vol. 0, No. 4, Winter 2009 I INTRODUCTION sequencing to become the definitive method Bovine interdigital necrobacillosis, also for identifying many types of bacteria. 0, This known as bovine foot rot or interdigital phleg - method offers much greater discrimination be - mon , is a common cause of lameness in beef tween strains, particularly for poorly described, and dairy cattle. –5 This disease is caused by the undefined, or aberrant species, 2,3 and is thus synergistic effects of several species of anaero - preferred as the most accurate and discriminat - bic bacteria, including Fusobacterium necropho - ing technique for identification of Prevotella rum , Porphyromonas asaccharolytica , Porphy - and Porphyromonas spp derived from bovine romonas levii , Prevotella melaninogenica , and foot rot specimens. Prevotella intermedia .2–4,6 The objective of this study was to compare Conventional methods for the identification 6S rRNA sequencing for identification of of anaerobic bacteria involve culture and bio - strains to the species level ( Prevotella or Por - chemical testing under strict anaerobic condi - phyromonas spp) with identification by con - Identification of bovine foot rot pathogens to the species level can be challenging , and conventional methods employed for the identification of anaerobic veterinary pathogens are very time consuming . tions or the use of gas –liquid chromatography ventional methods from clinical cases of acute for fatty acid profiling 7; diagnostic laboratories bovine interdigital necrobacillosis. may use pigmentation, antibiotic susceptibility, and biochemical tests to help in the presump - I MATERIALS AND METHODS tive identification of anaerobic organisms. 8 P. Test Strains for Reference Sequences intermedia and most Porphyromonas spp are The following American Type Culture Col - pigmented, whereas Fusobacterium and Bac - lection (ATCC; Manassas, VA) strains were teroides spp are not. Determination of suscepti - employed as the sources for the reference se - bility to kanamycin, vancomycin, and colistin quences for 6S rRNA identification: P. inter - aids in the separation of these genera. Most media ATCC 256, P. melaninogenica ATCC Bacteroides , Prevotella , and Porphyromonas spp 43982, P. levii ATCC 2947, and P. asaccha - are resistant to kanamycin. Most Bacteroides rolytica ATCC 25260. Table lists the Gen - and Prevotella spp are also resistant to van - Bank accession numbers assigned to 6S comycin with variable susceptibility to colistin, rRNA sequences for each bacterial strain iden - whereas Porphyromonas spp are usually suscep - tified during the study . tible to vancomycin and resistant to colistin. Most Fusobacterium and Porphyromonas spp , as Clinical Trial Isolates well as P. intermedia , are indole variable or pos - Test strains consisted of F. necrophorum and itive; other Prevotella spp are indole negative. 9 Prevotella and/or Porphyromonas spp isolated In recent years, the availability of public ge - from clinical trials using ceftiofur or tu - nomic databases has enabled 6S rRNA gene lathromycin for the treatment of bovine foot E2 M. Sweeney, J. Watts, E. Portis, M. Lucas, R. Nutsch, D. Meeuwse, D. Bade, V. Oliver, D. W. Morck, D. Shinabarger, S. Poppe, M. Peterson, D. Sweeney, M. Knechtel, and G. Zurenko TABLE 1. GenBank Accession Numbers Assigned to 16S rRNA Sequences Strain Accession No. Table 3* Porphyromonas levii FJ82245 through FJ822385 Porphyromonas levii (RDP isolate) FJ848562, FJ848566, FJ848567, FJ848569, FJ848570 Porphyromonas somerae (RDP isolate) FJ848556, FJ848557, FJ848558, FJ848559, FJ848563, FJ848568 Porphyromonas uenonis (RDP isolate) FJ848555, FJ848560, FJ84856 Porphyromonas cansulci (RDP isolate) FJ848564, FJ848565 Table 4* Porphyromonas levii FJ822386 through FJ82254 Porphyromonas levii (RDP isolate) FJ84855, FJ848552, FJ848554 Prevotella buccalis (RDP isolate) FJ848545 Prevotella heparinolytica (RDP isolate) FJ848548 Bacteroides finegoldii (RDP isolate) FJ848546, FJ848549, FJ848550, FJ848553 Bacteroides salyersiae (RDP isolate) FJ848547 Table 6* Prevotella intermedia ATCC 256 FJ792535 Porphyromonas levii ATCC 2947 FJ792536 Porphyromonas asaccharolytica ATCC 25260 FJ792537 Prevotella melaninogenica ATCC 43982 FJ792538 *Table numbers refer to tables in this article. ATCC = American Type Culture Collection, RDP = Ribosomal Database Project II. rot. The organisms were originally isolated and MI) , where they were logged in, maintained identified to the genus level using convention - frozen at –80˚C, then shipped frozen to Mi - al methods at Microbial Research, Inc. (Fort cromyx (Kalamazoo, MI). Collins, CO) or the University of Calgary. Both laboratories followed isolation and genus Clinical Trial Enrollment Criteria level identification procedures based on the Study candidates with clinical signs of foot Wadsworth Anaerobic Bacteriology Manual .8 rot were identified in commercial feedlots in The methodologies followed were standard for Alberta, Canada (70 calves) , and Oakland, NE each laboratory and were not necessarily stan - (00 calves) , in the tulathromycin field study dardized between the laboratories because this and in commercial feedlots in Clinton, IA; was not a good laboratory practices study. Dayton, ID; Sparta, MI; and Perry, NY (07 Frozen cell suspensions of the isolates were dairy cattle) , or Pender, New Brunswick , and shipped to Pfizer Animal Health (Kalamazoo, Nanton, Alberta, Canada (70 beef cattle) in E3 Veterinary Therapeutics • Vol. 0, No. 4, Winter 2009 TABLE 2. Guidelines Used to Group Organisms Recovered From Animals Exhibiting Signs of Foot Rot Group Description By animal, the biopsy results if available, or swab results if no biopsy available; one each for either Prevotella/Porphyromonas or Fusobacterium necrophorum 2 By animal, additional swab or biopsy results; Prevotella/Porphyromonas or F. necrophorum 3 Animals excluded because they either did not meet the inclusion criteria or received treatments prohibited by the protocol 4 Low percentage 6S rRNA–based identification based upon comparison to RDP database, no 6S rRNA–based identification (no PCR product or no sequencing result), contaminated cul - ture, no growth, or not tested 5 Non-necrophorum Fusobacterium RDP = Ribosomal Database Project II. the ceftiofur field study. Candidates were en - through the infected region and by swabbing tered into either study after they met the foot the necrotic lesion. The test animals were beef rot enrollment criteria on 2 consecutive days or dairy cattle that were not vaccinated for F. in a single foot. Specifically, lameness and necrophorum and did not receive antimicrobial swelling scores of 0 to 3 were used to enroll an - or antiinflammatory agents within 4 days of imals in these studies. A lameness score of 0 enrollment. Beef cattle were approximately 6 was interpreted as no lameness, a score of was to 4 months of age. Lactating Holstein cows interpreted as an animal that favored a foot but exhibiting any parity were 2 to 9 years of age. moved readily, a score of 2 was interpreted as Studies were approved by the sponsor’s animal an animal that put minimal weight on a foot care and use committee. and moved slowly, and a score of 3 was inter - Organisms recovered from animals exhibit - preted as an animal that held a foot