Journal of Veterinary Diagnostic Investigation

http://vdi.sagepub.com/ Bronchopneumonia in two dairy calves associated with Mannheimia species cluster V infection
Ann P. Britton and Erin N. Zabek J VET Diagn Invest 2012 24: 1043 originally published online 5 September 2012 DOI: 10.1177/1040638712457930 The online version of this article can be found at: http://vdi.sagepub.com/content/24/6/1043

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on and ZabekMannheimia spp. cluster V bronchopneumonia in calves

JVDXXX10.1177/1040638712457930Britt

Bronchopneumonia in two dairy calves associated with Mannheimia species cluster V infection
Ann P. Britton,1 Erin N. Zabek

Journal of Veterinary Diagnostic Investigation 24(6) 10431046 2012 The Author(s) Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638712457930 http://jvdi.sagepub.com

Abstract. The pathological, bacteriological, and molecular findings of two 3-week-old Holstein calves with bronchopneumonia are presented. Heavy pure growth of a Mannheimia species most closely aligned with the unnamed cluster V strains on the basis of 16S ribosomal RNA sequencing was detected in the lungs of both calves in association with Bovine respiratory syncytial virus infection. While Mannheimia species closely related to cluster V strains have occasionally been reported in association with pneumonia, meningitis, and abortion in cattle, the current report provides a description of the gross and histopathological lesions produced by a cluster V strain of Mannheimia species. Lesions in the lung were found to be typical of those described for Mannheimia haemolytica with the absence of areas of coagulation necrosis rimmed by leukocytes and more pronounced intra-alveolar hemorrhage. Lesions were linked to the presence of leukotoxin A based on phenotypic hemolysis and molecular demonstration of the leukotoxin A gene. Key words: Bronchopneumonia; calves; cluster V strains; enzootic pneumonia; Mannheimia spp. Mexican slaughter calf.13 While M. haemolytica has been extensively studied and much is understood about the bacterium and its pathogenicity, little is known about the pathogenicity of other Mannheimia spp. in cattle.9 Mannheimia haemolytica is a commensal of the bovine nasopharynx, which, under normal circumstances, is readily cleared from the lung. When respiratory clearance mechanisms are impaired and/or increased numbers of bacteria enter the lung, pneumonia may develop.6 Mannheimia spp. other than M. haemolytica have also been isolated from the nasal passages of cattle, including the unnamed cluster V strains. Evaluation of nasal swabs from healthy Belgian calves using 16S rRNA sequencing analysis revealed that 37% were colonized by Pasteurella multocida, whereas 6% were carrying Mannheimia spp. including M. haemolytica, M. varigena, M. glucosida, and 1 isolate exhibiting >99% sequence similarity to a rumenal cluster V strain of Mannheimia.8 Eight Mannheimia-like isolates from cattle (4 from pneumonic lung, 1 from meningitis, and 3 from the nose or trachea of animals with respiratory disease) were determined to be closely related to strains within the unnamed 16S rRNA cluster V of Mannheimia.2,3 Although M. haemolytica remains the predominant bovine pathogen, previous studies illustrate that other species of Mannheimia can occasionally be responsible for disease, and isolates with similarity to those in cluster V have been implicated. In the
From the Animal Health Centre, BC Ministry of Agriculture, Abbotsford, British Columbia, Canada. Corresponding Author: Ann P. Britton, 1767 Angus Campbell Road, Abbotsford, BC, Canada V3G 2M3. Ann.P.Britton@gov.bc.ca
1

Pasteurella haemolytica, a member of the proteobacterial family Pasteurellaceae, has long been recognized as a complex of bacterial pathogens in ruminants. Until 1999, this bacterium was comprised of 2 biotypes, A and T, based on the ability to ferment either L-arabinose or trehalose, of which there were 17 serotypes and 9 untypeable strains.4 In 1999, polyphasic analysis of the trehalose-negative P. haemolytica complex lead to demonstration of distinct genetic and phenotypic groups prompting reclassification of these bacteria in a new genus, Mannheimia.4 Five distinct 16S ribosomal RNA (rRNA) clusters were identified (clusters IV) representing 6 new species of which 5 were named: Mannheimia haemolytica and M. glucosida (cluster I), M. ruminalis (cluster II), M. granulomatis (cluster III), and M. varigena (cluster IV).4 Speciation of the unnamed cluster V strains, comprised of P. haemolytica biogroups 7 and 10 from the bovine rumen plus P. haemolytica untypeable group 5 from a bovine abortion, awaits further molecular characterization.4,10 Despite this new classification system, identification of isolates is difficult and laborious, and routine bacteriological investigations may not be sufficient for accurate identification in all cases, which complicates the understanding of the role that species and strains other than M. haemolytica play in bovine diseases.3,9 Mannheimia haemolytica is a commonly diagnosed respiratory pathogen of cattle, which has a large economic impact on the global cattle industry.20 Mannheimia species other than M. haemolytica are occasionally associated with bovine disease. For example, M. varigena was isolated from cattle with pneumonia, abortion, septicemia, mastitis, and meningitis,3,9 and M. glucosida was isolated from a pneumonic

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Figure 1. Lung. A, interlobular lymphatics are distended with fibrin and inflammatory cells (asterisks). Multifocal areas of hemorrhage and leukocyte infiltration are present in alveolar spaces and airways. Hematoxylin and eosin (HE). Bar = 300 m. B, higher power micrograph of panel A showing clumps of necrotic leukocytes with nuclear streaming (arrowheads) intermixed with areas of alveolar hemorrhage. HE. Bar = 50 m. C, consolidation of parenchyma is generalized in lobules. HE. Bar = 300 m. D, hemorrhage is widespread with early inflammation at the bronchioloalveolar junction. HE. Bar = 300 m.

current report, bronchopneumonia in two 3-week-old Holstein calves associated with Mannheimia spp. cluster V infection is described. In January 2009, two 3-week-old Holstein calves, 1 male and 1 female, were submitted to the Animal Health Centre in Abbotsford, British Columbia, Canada for necropsy examination. The calves had been given 34 liters of colostrum at birth and were housed in individual calf pens in a calf barn with 14 other calves. These 2 calves were part of a group of 7 calves that developed diarrhea at 710 days of age. The 2 calves had been treated with florfenicola and were tube fed with milk and electrolytes for the diarrhea. The calves subsequently developed labored breathing, became progressively weaker, and died. At necropsy, both calves exhibited sunken eyes indicative of dehydration. Unclotted milk was observed in the reticulorumen and abomasum. The intermediate, anteroventral aspect of the diaphragmatic and ventral aspect of the anteroventral lung lobes were mottled tan, dark red, purple, and

reddish-orange with lobular depression. The discolored lobes were firm and, on cut surface, tan-colored exudate could be expressed from airways. The female calf exhibited fibrin over the pleural surface and fibrinous adhesions of the discolored lobes to the pericardial sac. Microscopically, interlobular lymphatics and interstitium were dilated with edema fluid, fibrin strands, and variable numbers of macrophages and neutrophils (Fig. 1A). Affected lobules exhibited variable accumulation of edema fluid, fibrin, erythrocytes, macrophages, neutrophils, and occasional coccobacillary bacteria in alveolar spaces with multifocal clumps of necrotic leukocytes exhibiting nuclear streaming (Fig. 1B). Airways contained exudate similar to adjacent alveolar spaces resulting in a pattern of lobular consolidation, which varied from patchy (Fig. 1A) to generalized (Fig. 1C). Alveolar hemorrhage varied from multifocal (Fig. 1A) to widespread (Fig. 1D) and was observed with both early and more advanced consolidated inflammation. Foci of atelectasis were occasionally noted, and fibrin was

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 Mannheimia spp. cluster V bronchopneumonia in calves focally observed on the pleural surface and in peribronchial lymphatics. Bronchiolar epithelial necrosis and attenuation, often associated with intraluminal syncytial cells, were occasionally observed. Using a monoclonal antiBovine respiratory syncytial virus (BRSV) antibody,b immunohistochemistry revealed the presence of BRSV in syncytial cells, alveolar macrophages, and bronchiolar epithelium. Heavy pure growth of a small, smooth, clear betahemolytic colony was noted in lung samples from both calves after 24 hr of aerobic incubation. Following subculture, the organism was identified as an oxidase and indolepositive, Gram-negative coccobacillus. Initial identification of the organism was made using a commercial identification system,c which produced an identification of M. granulomatis with a 99% probability. To confirm the identification, 16S rRNA sequencing was performed. Utilizing a single, well-isolated bacterial colony, DNA was extracted using a commercial kitd according to the manufacturers protocol. The 16S rDNA gene was amplified using primers 27f and 519r.14 The 529-bp polymerase chain reaction (PCR) product was sequenced and compared to known sequences in GenBank using the National Center for Biotechnology Information BLAST program (http://www. ncbi.nlm.nih.gov/blast/Blast.cgi). The product matched a cluster of different Mannheimia spp. including M. haemolytica, M. glucosida, M. ruminalis, M. granulomatis, and M. varigena. Further analysis of the sequenced product using a commercial software packagee revealed >99% identity with Mannheimia spp. R19.2 and HPA121, bovine rumenal strains within the cluster V group of the Mannheimia complex.4 In addition to the 16S rRNA sequencing, PCR was performed on the isolate for the detection of the leukotoxin A (lktA) gene. Primers specific for the leukotoxin pore-forming repeats in toxin domain11 amplified a 1,145base pair fragment from the isolate, which matched the positive control, a M. haemolytica isolate from a diagnostic case of bovine fibrinous bronchopneumonia. Both calves were diagnosed with marked bronchopneumonia associated with Mannheimia spp. cluster V and BRSV infection. Bacterial bronchopneumonia caused by bacteria of the Pasteurellaceae family is a common, economically important disease of cattle, which most frequently presents as shipping fever in feedlot cattle and enzootic pneumonia in calves.6 In dairy herds, enzootic pneumonia is a multifactorial disease associated with primary viral or mycoplasmal infection and secondary bacterial infection.20 Risk factors include failure of passive transfer of immunoglobulin, poor ventilation, crowding, stress, and concurrent disease.6 Bovine respiratory syncytial virus is the most common viral etiology associated with pneumonia in dairy calves,5,20,21 and demonstration of BRSV in the 2 calves reported herein most likely represents primary infection. Pasteurella multocida is the most common secondary bacterial pathogen associated with enzootic pneumonia

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followed by M. haemolytica,5,6 reflecting nasopharyngeal commensalism of healthy calves.7,8 Isolation of neither P. multocida nor M. haemolytica from the 2 calves in the current report is thus unusual. Initial identification of the isolate using phenotypic and biochemical criteria alone revealed 99% probability of M. granulomatis, an indole-negative organism most often associated with a granulomatous skin condition in Brazilian cattle.4 Molecular testing subsequently demonstrated the isolate to be most closely related to the unnamed 16S rRNA cluster V strains, illustrating well the difficulty and exhaustive analysis needed to accurately identify some Mannheimia isolates.2,3 Cluster V strains are rumen commensals that have occasionally been isolated from the nasal passages of cattle.2,3,8 In the present case, the calves were being tube fed, suggesting that contamination of the oropharynx during extubation may have occurred. Alternatively, the Mannheimia cluster V strain may have been a nasopharyngeal commensal. The isolate from the present case was hemolytic, which is highly correlated with the lktA genotype in Mannheimia spp.17,18 A member of the Escherichia coli subfamily of cytotoxic, pore-forming repeats in toxin proteins, lktA is reported to be the primary virulence factor of M. haemolytica.15 At low concentration, lktA activates the oxidative capacity of macrophages and neutrophils and release of inflammatory mediators and chemoattractants for neutrophils, thus exacerbating the early inflammatory response.15,18 Increasing lktA concentration stimulates leukocyte apoptosis followed by pore formation and necrosis of leukocytes due to membrane damage.15,18 Mutant strains of M. haemolytica expressing either altered or inactivated lktA are associated with decreased necrosis or absence of leukocyte degeneration resulting in less severe lung lesions and lower morbidity and mortality.12,18,19 The dense multifocal infiltrates of apoptotic and necrotic leukocytes observed in the 2 calves in the current report were typical of those described for M. haemolytica with lktA activity6 compatible with the PCR demonstration of the lktA genotype in the Mannheimia cluster V bacterium cultured from the lungs. The lesion differed somewhat from that classically described for M. haemolytica in that areas of parenchymal coagulation necrosis surrounded by a dense rim of leukocytes were lacking in the 2 calves, and intra-alveolar hemorrhage was a prominent feature.6 Intra-alveolar hemorrhage is not reported to be a feature of BRSV pneumonia in cattle,6 and this lesion was attributed to the Mannheimia spp. cluster V infection. Full expression of virulence of M. haemolytica is dependent upon the interactive effects of lktA, endotoxin, and immune-modulating proteins produced by the bacterium.16 LktA has no direct effect on endothelium, whereas the combined effects of lktA and endotoxin on leukocyte activity are thought to cause endothelial damage.1 Parenchymal coagulation necrosis surrounded by a rim of leukocytes, a feature of M. haemolytica infection, is thought to be mediated by endotoxin-induced procoagulant activity

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7. Catry B, Decostere A, Schwarz S, et al.: 2006, Detection of tetracycline-resistant and susceptible Pasteurellaceae in the nasopharynx of loose group-housed calves. Vet Res Commun 30:707715. 8. Catry B, Haesebrouck F, De Vliegher S, et al.: 2005, Variability in acquired resistance of Pasteurella and Mannheimia isolates from the nasopharynx of calves, with particular reference to different herd types. Microb Drug Resist 11:387394. 9. Catry B, Opsomer G, Decostere A, et al.: 2004, Fatal meningitis in a calf caused by Mannheimia varigena. Res Vet Sci 77:187188. 10. Davies RL, Paster BJ, Dewhirst FE: 1996, Phylogenetic rela tionships and diversity within the Pasteurella haemolytica complex based on 16S rRNA sequence comparison and outer membrane protein and lipopolysaccharide analysis. Int J Syst Bacteriol 46:736744. 11. Fisher MA, Weiser GC, Hunter DL, Ward AC: 1999, Use of a polymerase chain reaction method to detect the leukotoxin gene lktA in biogroup and biovariant isolates of Pasteurella haemolytica and P. trehalosi. Am J Vet Res 60:14021406. 12. Highlander SK, Fedorova ND, Dusek DM, et al.: 2000, Inac tivation of Pasteurella (Mannheimia) haemolytica leukotoxin causes partial attenuation of virulence in a calf challenge model. Infect Immun 68:39163922. 13. Jaramillo-Arango CJ, Hernandez-Castro R, CampuzanoOcampo V, et al.: 2007, Characterisation of Mannheimia sp. and P. multocida strains isolated from bovine pneumonic lungs in two slaughterhouses in Mexico. J Anim Vet Adv 6:13981404. 14. Lane, DJ: 1991, 16S/23S rRNA sequencing. In: Nucleic acid techniques in bacterial systematics, ed. Stackebrandt E, Goodfellow M, pp. 115175. Wiley, New York, NY. 15. Larsen J, Pedersen AG, Christensen H, et al.: 2007, Evidence for vertical inheritance and loss of the leukotoxin operon in genus Mannheimia. J Mol Evol 64:423437. 16. Lawrence PK, Kittichotirat W, McDermott JE, Bumgar ner RE: 2010, A three-way comparative genomic analysis of Mannheimia haemolytica isolates. BMC Genomics 11:535. 17. Murphy GL, Whitworth LC, Clinkenbeard KD, Clinkenbeard PA: 1995, Hemolytic activity of the Pasteurella haemolytica leukotoxin. Infect Immun 63:32093212. 18. Narayanan SK, Nagaraja TG, Chengappa MM, Stewart GC: 2002, Leukotoxins of gram-negative bacteria. Vet Microbiol 84:337356. 19. Petras SF, Chidambaram M, Illyes EF, et al.: 1995, Antigenic and virulence properties of Pasteurella haemolytica leukotoxin mutants. Infect Immun 63:10331039. 20. Radostits OM, Gay CC, Hinchcliff KW, Constable PD: 2007, Enzootic pneumonia of calves. In: Veterinary medicine. A textbook of the diseases of cattle, horses, sheep, pigs, and goats, 10th ed., pp. 13361339. Saunders Elsevier, New York, NY. 21. Uttenthal A, Jensen NP, Blom JY: 1996, Viral aetiology of enzootic pneumonia in Danish dairy herds: diagnostic tools and epidemiology. Vet Rec 139:114117.

and infarction plus the combined effects of endotoxin and lktA on leukocyte oxidative activity.1,6 Thus, the prominent hemorrhage and lack of large areas of coagulation necrosis rimmed by leukocytes observed in the calves may reflect synergistic effects of lktA and endotoxin unique to this cluster V strain. Acknowledgements
The authors would like to gratefully acknowledge the assistance and technical expertise of Dr. Gary Marty, Sandra Etheridge, and Joanne Taylor.

Sources and manufacturers

a. Nuflor, Intervet, Kirkland, Quebec, Canada. b. Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA. c. Biolog Inc., Hayward, CA. d. QIAamp DNA Mini Kit, Qiagen Inc., Toronto, Ontario, Canada. e. ClustalW, MegAlign Program, DNASTAR Inc., Madison, WI.

Declaration of conflicting interests

The author(s) declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

References
1. Ackermann MR, Brogden KA: 2000, Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica. Microbes Infect 2:10791088. 2. Angen , Aalbaek B, Falsen E, et al.: 1997, Relationships among strains classified with the ruminant Pasteurella haemolytica-complex using quantitative evaluation of phenotypic data. Zentralbl Bakteriol 285:459479. 3. Angen , Ahrens P, Bisgaard M: 2002, Phenotypic and genotypic characterization of Mannheimia (Pasteurella) haemolyticalike strains isolated from diseased animals in Denmark. Vet Microbiol 84:103114. 4. Angen , Mutters R, Caugant DA, et al.: 1999, Taxonomic relationships of the [Pasteurella] haemolytica complex as evaluated by DNA-DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb. nov., Mannheimia granulomatis comb. nov., Mannheimia glucosida sp. nov., Mannheimia ruminalis sp. nov. and Mannheimia varigena sp. nov. Int J Syst Bacteriol 49:6786. 5. Baker JC, Werdin RE, Ames TR, et al.: 1986, Study on the etiologic role of bovine respiratory syncytial virus in pneumonia of dairy calves. J Am Vet Med Assoc 189:6670. 6. Caswell JL, Williams KJ: 2007, Respiratory system. In: Jubb, Kennedy, and Palmers pathology of domestic animals, ed. Maxie MG, 5th ed., pp. 601602. Saunders Elsevier, Philadelphia, PA.

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