http://vdi.sagepub.com/Investigation

Journal of Veterinary Diagnostic

http://vdi.sagepub.com/content/22/5/797The online version of this article can be found at:

 DOI: 10.1177/104063871002200528

2010 22: 797J VET Diagn InvestKyathanahalli S. Janardhan, Mike Hays, Neil Dyer, Richard D. Oberst and Brad M. DeBey

)Bison Bison Outbreak in a Herd of North American Bison (Mycoplasma Bovis  

Published by:

http://www.sagepublications.com

On behalf of: 

  Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

can be found at:Journal of Veterinary Diagnostic InvestigationAdditional services and information for    

  http://vdi.sagepub.com/cgi/alertsEmail Alerts:

 

http://vdi.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

What is This? 

- Sep 1, 2010Version of Record >>

by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from by guest on October 11, 2013vdi.sagepub.comDownloaded from

J Vet Diagn Invest 22:797–801 (2010)

Mycoplasma bovis outbreak in a herd of North American bison (Bison bison)

Kyathanahalli S. Janardhan, Mike Hays, Neil Dyer, Richard D. Oberst, Brad M. DeBey1

Abstract. A disease outbreak of high morbidity and high mortality in bison (Bison bison) was investigated.Clinical signs included lameness, swollen joints, respiratory distress, and lethargy. Fifty-three of 194 animalsdied. Cows between 5 and 10 years of age were the most affected group, in which 40 of 88 animals died.Necropsies were performed on several animals. There were abscesses in the lung and liver, as well asfibrinosuppurative pleuritis, polyarthritis, and disseminated microabscesses in various organs. No significantbacteria were isolated by routine aerobic cultures of lung and liver from 2 representative cases. However,Mycoplasma cultures were positive. Polymerase chain reaction tests on the isolated bacteria were positive forMycoplasma bovis. Histologically, the abscesses were characterized by areas of necrosis with variablemineralization rimmed by granulomatous inflammation and fibrous tissue. No new animals had beenintroduced into the herd, but a cattle herd was present adjacent to the affected bison herd. Two restrictionfragment length polymorphism techniques were used to compare the bison isolate and another bison isolatefrom an outbreak in North Dakota with a field isolate of M. bovis from cattle and with a laboratory controlstrain of M. bovis; the isolates and control strain were found to be similar. The isolates and the control weresequenced and compared with sequences in GenBank. Bison isolates were more than 99% homologous to M.bovis sequences in GenBank. It was concluded that M. bovis in bison can cause disseminated infection with ahigh morbidity and mortality and that bison isolates are similar to bovine M. bovis isolates.

Key words: Bison; gene; Mycoplasma bovis; pathology; polymerase chain reaction; restriction fragmentlength polymorphism.

<!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff|./author-grp[1]/dept-list)>Mycoplasma bovis in bovines is known to cause

pneumonia, mastitis, arthritis, abortion, and keratocon-junctivitis.7 Although there are several reports on Myco-plasma-induced diseases in bovines,3,10 only 1 report wasfound on Mycoplasma infection in bison.2 Mycoplasmabovis infection causes significant economic loss to the cattleindustry.6,7 Once on the verge of extinction, bison numbershave substantially increased from approximately 1,000 inthe late 1800s to approximately 500,000 in North America.4

Increasing demand for bison meat, primarily because of itsnutritional value, has led to the growth of the commercialbison industry (Firmage-O’Brien K: 2008, Bison on thecomeback trail. Canadian Agriculture at a Glance.Statistics Canada Catalogue no. 96-325-XIE2007000.Available at http://www.statcan.gc.ca/pub/96-325-x/2007000/article/10504-eng.pdf. Accessed January 10,2010). Because of such growth, it has become importantto understand the diseases that affect bison. Although thereare publications describing the importance of such diseasesas malignant catarrhal fever,8 anthrax,11 and brucellosis9 inbison, there is only a single publication reporting myco-plasmosis in bison.2 The current report investigates anoutbreak of disease in bison characterized by highmorbidity and mortality, in which M. bovis was identifiedas the cause.

The disease outbreak occurred from mid-September 2006to January 2007 in Kansas. The herd contained a total of194 bison, along with a few elk. Among the bison, 97 wereadults (5–10 years old), 50 were in the range of 1–2 years,and 47 were calves. There had been no introduction of newanimals in the 4 years before the outbreak. A cattle herdwas present adjacent to the bison herd. The affected bisonwere weak, with swollen joints, lameness, respiratorydistress, and mastitis. Most of the animals died naturally,and a few were euthanized because of the poor clinicalcondition. Fifty-three bison died during the diseaseoutbreak. The percentage of mortality in different agegroups is shown in Table 1. Necropsies were performed onseveral animals by the submitting veterinarian, withsamples from 2 bison submitted to Kansas State VeterinaryDiagnostic Laboratory (KSVDL case nos. 06-57154 and07-03804). The gross lesions included mucopurulenteffusion in multiple joints; disseminated microabscesses inmany organs, including spleen, mesentery, lungs (Fig. 1A),liver (Fig. 1B), uterus, and intestine; pericardial effusion;fibrinopurulent pleuritis; and mastitis. The abscesses in thelung (present in all lobes and up to 40 cm in diameter) andliver were very large.

Microscopically, there were multifocal areas of necro-granulomatous inflammation in the lung. These werecharacterized by central areas of necrosis, which wereoccasionally mineralized, surrounded by a rim of degener-ate neutrophils, lymphocytes, plasma cells, and macro-phages, which were further surrounded by layers of fibrousconnective tissue (Fig. 1C). Similar lesions were present inthe liver (Fig. 1D). The microscopic changes are similar toM. bovis–induced microscopic lesions in cattle.3,10

From the Department of Diagnostic Medicine Pathobiology,Kansas State University, Manhattan, KS (Janardhan, Hays, Oberst,DeBey), and the Veterinary Diagnostic Laboratory, North DakotaState University, Fargo, ND (Dyer).

1 Corresponding Author: Brad M. DeBey, Department ofDiagnostic Medicine Pathobiology, Kansas State University,Manhattan, KS 66502. debey@vet.k-state.edu

Case Reports 797

In 1 bison (KSVDL 06-57154), Mycoplasma culture wasperformed on synovial fluid, and aerobic bacterial culturewas performed on liver, lung, and spleen. In the other bison(KSVDL 07-03804), aerobic and Mycoplasma cultures wereperformed using lung and liver. No significant bacteriawere isolated by routine aerobic cultures. Mycoplasma sp. wasisolated in Mycoplasma-specific cultures. Mycoplasma isolatedfrom the lung and liver of KSVDL 07-03804 was used forfurther tests (hereafter, Kansas [KS] isolate). In addition tothese specimens, an isolate of M. bovis from another outbreak

of mycoplasmosis in bison (hereafter, North Dakota [ND]isolate) was compared with the KS isolate.2

To identify the species of Mycoplasma, the isolates (NDand KS) were compared with known M. bovis (KSVDL 06-11241) and laboratory control (KSU-TY-1) using M. bovis–specific polymerase chain reaction (PCR) and 2 differentrestriction fragment length polymorphism (RFLP) tech-niques. The ND and KS isolates streaked on Mycoplasmaagar platesa were inoculated into Mycoplasma broth. Onemilliliter of culture fluid was centrifuged at 14,000 3 g, andthe cell pellet was used to extract nucleic acid using acommercial kit.b The nucleic acid was used for furthercharacterization of the bacteria.

The PCR was performed using M. bovis–specific (MBV-F 59-TGATAGCAATATCATAGCGGC-39; MBV-R 59-GTAGCATCATTTCCTATGCTAC-39) primers at a250 nM concentration in a 25-ml reaction mixture containing12.5 ml of 23 SYBR Green supermix,c sterile water to avolume of 22.5, and 2.5 ml of sample nucleic acid.Amplification was performed by an initial denaturing/

Table 1. Details of the bison herd affected with Mycoplasmabovis during the outbreak.

Age No. of bison in herd No. of deaths % mortality

Calves 47 5 10.61–2 years 50 5 105–10 years 97 43 44.3Total 194 53 27.3

Figure 1. Gross and microscopic changes in Mycoplasma bovis–infected bison (Bison bison). Lung (A) and liver (B) containingmultifocal abscesses. Necrogranulomatous inflammation in the hematoxylin and eosin–stained lung (C; bar 5 100 mm) and liver (D; bar5 100 mm) sections.

798 Case Reports

activation step of 95uC for 5 min, followed by 45 cycles of95uC for 10 sec, 55uC for 15 sec, and 72uC for 30 sec with theSYBR Green fluorescence recorded at the 72uC extensionstep of each cycle. The bacteria were positive for M. bovis.

For RFLP, 2 different methods were used. First, a methoddescribed previously (Lauerman L: 1998, Nucleic acidamplification assays for diagnosis of animal diseases.American Association of Veterinary Laboratory Diagnosti-cians) was used. A Mycoplasma spp. forward primer (CGP1-F 59-ACACCATGGGAGCTGGTAAT-39) and reverseprimer (CGP1-R 59-CTTCATCGACTTTCAGACC-CAAGGCAT-39) were used at a 250 nM concentration ina 100-ml reaction mixture containing 12.5 ml of 23 iQSupermix,d sterile water to a volume of 90, and 10 ml ofsample nucleic acid. Ten microliters of each reaction wasutilized to demonstrate the presence of an approximately490-bp fragment on electrophoresis in a 2% agarose gel (datanot shown). Fifteen microliters of each (KS, ND, and bovineisolates and laboratory control) amplicon from Mycoplasmaspp. The PCR fragments were digested separately in a 30-mlfinal reaction volume using restriction enzymes (Mse I,e

TSP509 I,f Dra I,g and Rsa Ih) following manufacturer-recommended reaction conditions and restriction buffers.After digestion, 63 blue orange loading dye was added to therestriction reaction tube, mixed well, and loaded onto a 5%

Tris–borate–ethylenediamine tetra-acetic acid (EDTA) pre-made polyacrylamide geli for electrophoresis.

A second 16S RFLP was performed according to themethod describe previously1 for Mycoplasma spp. Fifteenmicroliters of each amplicon generated by the 16S PCRwere digested in separate reactions with restriction enzymes(EcoR I,j Mlu I,k and BamH Il) following manufacturer-recommended reaction conditions and restriction buffers.After digestion, 63 blue orange loading dye was added tothe restriction reaction tube, mixed well, and loaded onto a5% Tris–borate–EDTA precast polyacrylamide geli forelectrophoresis.

The RFLP patterns were the same between M. bovisisolates from bison (KS and ND isolates) and bovine(KSVDL 06-11241) and laboratory control (KSU-TY-1).The RFLP performed according to the method previouslycited (Lauerman L: 1998, Nucleic acid amplification assaysfor diagnosis of animal diseases) yielded a pattern (data notshown) consistent with the expected pattern for M. bovis. Thefragment sizes for the 16S RFLP patterns were all consistentwith the sizes suggested previously1 for M. bovis, with EcoR Idigestion resulting in bands of 340 and 661 bp; Mlu I digestionresulting in bands of 297, 332, and 372 bp; and BamH Idigestion resulting in bands of 414 and 587 bp (Fig. 2).

Further, the sequences of bison isolates were comparedwith the bovine isolate and the laboratory control. The 16Sribosomal DNA gene of each M. bovis isolate from either abovine or a bison source was amplified using previouslydescribed primers.1 Presence of an approximate 1,000-bpfragment was verified by 1.5% agarose electrophoresis andthen cloned into the pCR8/GW/TOPO TA plasmid vectorm

according to the manufacturer’s instructions. The 16Sribosomal DNA gene plasmid clones were sequenced at theUniversity of Arkansas Medical Sciences DNA SequencingLaboratory (Little Rock, Arkansas). Sequences were ana-lyzed and aligned using the MEGA 4.0 software package.13

The 16S sequences of KS (GenBank accessionno. GU993858) and ND (GenBank accession no. GU993861)M. bovis isolates were more than 99% homologous to thesequence of M. bovis bovine isolate (KSVDL 06-11241,GenBank accession no. GU993859) and M. bovis control(KSU-TY-1, GenBank accession no. GU993860; Fig. 3). Allof these isolates are 99% homologous to M. bovis sequence inGenBank (GenBank accession no. U02968).

The present investigation implicates M. bovis as an agentcausing high morbidity and mortality in bison. Mycoplasmabovis infection in cattle results in pneumonia, arthritis, otitismedia, keratoconjunctivitis, and mastitis. Calves developpneumonia, arthritis, and otitis media. Reports in adult dairy

Figure 2. Restriction fragment length polymorphism analysis of Kansas (KS) and North Dakota (ND) isolates and theircomparison with a bovine Mycoplasma bovis isolate (KSVDL 06-11241) and a M. bovis laboratory control (KSU-TY-1).

Case Reports 799

Figure 3. Sequence data for Kansas isolate (KSBison), North Dakota isolate (NDBison), Mycoplasma bovis isolate from cattle(BovineMbovis), and laboratory control (Mboviscontrol). The KS and ND isolates are more than 99% homologous to the M. bovisisolate from cattle (KSVDL 06-11241) and the laboratory control (KSU-TY-1).

800 Case Reports

cattle describe mastitis, with feeder and stocker cattleexperiencing pneumonia and arthritis.6 In comparison, thedisease occurrence in bison appears to vary slightly. In the KSoutbreak, the majority of affected bison were in the 5–10-yearage group, whereas calves and 1–2-year-old bison wereaffected to a lesser degree. Mycoplasma bovis induced adisseminated disease resulting in abscesses or infection ofmultiple organs, including lung, liver, mesentery, intestine,uterus, mammary gland, and joints. The ND outbreak2

reported primary involvement of lungs and joints. It isdifficult to speculate on the variation in lesion patternsbetween the KS and ND outbreaks. In calves experimentallyinfected with M. bovis, bacteria was isolated from liver,spleen, and kidneys at 21 days postinfection.12 Mycoplasmabovis was immunohistochemically detected in liver andkidney in an additional report of naturally infected calves.5

These findings highlight the ability of M. bovis to disseminatehematogenously to various organs. In the KS outbreak, thedisease process was very likely chronic, leading to septicemiaand seeding of organisms into different tissues.

To identify the pathogen, bacterial isolates from KS andND were sequenced and compared using BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Both the isolateswere 99% homologous to M. bovis sequences in GenBankand were also 99% homologous to sequences of Mycoplas-ma agalactiae. However, the RFLP pattern was consistentwith a previously described M. bovis pattern (Lauerman L:1998, Nucleic acid amplification assays for diagnosis ofanimal diseases). Also, bison samples were positive by PCRfor M. bovis when M. bovis–specific primers were used.Based on the findings of the current study, routinediagnostic tests, such as culture, PCR, and RFLP, usedfor diagnosis of M. bovis in bovine samples will be useful indiagnosing M. bovis infection in bison.

The present investigation highlights the importance of M.bovis infection in bison. The source of pathogen remainsunknown; however, it is reasonable to speculate that thecattle adjacent to the affected bison herd are the probablecandidates. In view of recent reports describing the severity ofmycoplasmosis in bison, it is important for producers toadopt preventive measures avoiding M. bovis exposure andinfection.

Acknowledgements. The authors thank the Director ofKansas State Veterinary Diagnostic Laboratory, Dr. GaryAnderson, for the financial support for this project. Theauthors also thank Dr. Byron Bachman, Lindsborg Veter-inary Hospital, Lindsborg, Kansas, for performing thenecropsies, submitting the appropriate samples, and provid-ing a detailed history; and Mr. Cliff Peterson, MaxwellRefuge manager, for providing details on the bison herd.

Sources and manufacturers

a. Catalog no. G102, Hardy Diagnostics, Santa Maria, CA.b. Qiagen Viral RNA Kit (catalog no. 52906), Qiagen Corp.,

Valencia, CA.

c. Catalog no. 170-8880, Bio-Rad Laboratories, Hercules, CA.

d. Catalog no. 170-8860, Bio-Rad Laboratories, Hercules, CA.

e. Catalog no. R0525S, New England Biolabs Inc., Ipswich, MA.

f. Catalog no. R0576S, New England Biolabs Inc., Ipswich, MA.

g. Catalog no. R0129S, New England Biolabs Inc., Ipswich, MA.

h. Catalog no. R0167S, New England Biolabs Inc., Ipswich, MA.

i. Catalog no. 1161-1127, Bio-Rad Laboratories, Hercules, CA.

j. Catalog no. R6011, Promega Corp., Madison, WI.

k. Catalog no. R6381, Promega Corp., Madison, WI.

l. Catalog no. R6021, Promega Corp., Madison, WI.

m. Catalog no. K2500-20, Invitrogen Corp., Carlsbad, CA.

References

1. Alberti A, Addis MF, Chessa B, et al.: 2006, Molecular and

antigenic characterization of a Mycoplasma bovis strain

causing an outbreak of infectious keratoconjunctivitis. J VetDiagn Invest 18:41–51.

2. Dyer N, Hansen-Lardy L, Krogh D, et al.: 2008, An outbreak

of chronic pneumonia and polyarthritis syndrome caused by

Mycoplasma bovis in feedlot bison (Bison bison). J Vet Diagn

Invest 20:369–371.

3. Gagea MI, Bateman KG, Shanahan RA, et al.: 2006,

Naturally occurring Mycoplasma bovis-associated pneumonia

and polyarthritis in feedlot beef calves. J Vet Diagn Invest

18:29–40.

4. Hedrick PW: 2009, Conservation genetics and North Amer-

ican bison (Bison bison). J Hered 100:411–420.

5. Maeda T, Shibahara T, Kimura K, et al.: 2003, Mycoplasma

bovis–associated suppurative otitis media and pneumonia inbull calves. J Comp Pathol 129:100–110.

6. Maunsell FP, Donovan GA: 2009, Mycoplasma bovis infec-

tions in young calves. Vet Clin North Am Food Anim Pract

25:139–177, vii.

7. Nicholas RA, Ayling RD: 2003, Mycoplasma bovis: disease,diagnosis, and control. Res Vet Sci 74:105–112.

8. O’Toole D, Li H, Sourk C, et al.: 2002, Malignant catarrhal

fever in a bison (Bison bison) feedlot, 1993–2000. J Vet Diagn

Invest 14:183–193.

9. Rhyan JC, Aune K, Roffe T, et al.: 2009, Pathogenesis and

epidemiology of brucellosis in Yellowstone bison: serologic

and culture results from adult females and their progeny. J

Wildl Dis 45:729–739.

10. Rodriguez F, Bryson DG, Ball HJ, Forster F: 1996,

Pathological and immunohistochemical studies of natural

and experimental Mycoplasma bovis pneumonia in calves. J

Comp Pathol 115:151–162.

11. Shury TK, Frandsen D, O’Brodovich L: 2009, Anthrax infree-ranging bison in the Prince Albert National Park area of

Saskatchewan in 2008. Can Vet J 50:152–154.

12. Stipkovits L, Ripley PH, Tenk M, et al.: 2005, The efficacy of

valnemulin (Econor) in the control of disease caused byexperimental infection of calves with Mycoplasma bovis. Res

Vet Sci 78:207–215.

13. Tamura K, Dudley J, Nei M, Kumar S: 2007, MEGA4:

Molecular Evolutionary Genetics Analysis (MEGA) softwareversion 4.0. Mol Biol Evol 24:1596–1599.

Case Reports 801