Microscopic Lesions Associated with the Isolation of Haemophilus somnus from Pneumonic Bovine Lungs

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1 Vet. Pathol. 22: (1985) Microscopic Lesions Associated with the Isolation of Haemophilus somnus from Pneumonic Bovine Lungs J. J. ANDREWS, T. D. ANDERSON, L. N. SLIFE, and G. W. STEVENSON Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, Iowa Abstract. Sixty-one of 68 sets of bovine lungs from which only Haemophilus somnus was isolated had microscopic lesions of purulent bronchiolitis and bronchopneumonia. In 37 of 6 1 lungs, the bronchiolar exudates were markedly necrotic with accompanying necrosis of the adjacent bronchiolar epithelium. Bronchiolitis obliterans was prominent in 23 of 28 lungs affected with chronic lesions with abscesses present in seven. Alveolar filling with inflammatory cells (neutrophils with fewer macrophages) was limited to peribronchiolar alveoli in 25 of 6 1 lungs and was multifocal to diffuse in the other 36. Lesions in the remaining lungs (7 of 68) were classified as fibrinous pneumonia with bronchiolitis (2), fibrinous pleuritis (2), suppurative interstitial pneumonia with vasculitis (2), and diffuse congestion ( 1). The gram-negative bacterium Haemophilus sornnus is isolated frequently from cattle with infectious thromboembolic meningoencephalitis, pneumonia,. 17,20 and reproductive disorders.. 12, 16,* The lung lesions reported are fibrinous pne~monia,~ ~ fibrinous pleuri- jected to identical microbiologic procedures. Bacteriologic tis,4, suppurative bronchopneum~nia,~?~~~,~,~* I I* isolation attempts were the most standardized and were done 14, pulmonary ab~cesses,~? I5 diffuse hemorrhagic interstitial pneumonia,2* l5 vasculitis,13 thrombosis,13 and necrotizing bronchiolitis. In most reports, however, the histologic descriptions are either absent or limited. This paper describes the microscopic lung lesions associated with the isolation of H. soinnus from the lungs of 68 cattle with naturally occurring pneumonia. Materials and Methods From July 1, 1978 to June 30, 1983, all submissions of bovine tissues to the Iowa State University Veterinary Diagnostic Laboratory from which Haemophilus somnus was isolated were selected for review. In addition, 25 bovine submissions from which only Pasteurella hemolytica was isolated from pneumonic lung tissue were included in the review for comparison. Whenever parafin-embedded lung tissue was available, 6 pm sections were cut, mounted on glass slides, and stained with hematoxylin and eosin (HE) by standard methods. Lung tissues for histologic examination were selected from pneumonic cranial ventral lobes, and in a few cases, additional lung tissue was selected from less affected or nonaffected caudal lobes. Each lung section was examined, and histologic changes were described and subsequently classified according to the major lesions. Lung is used throughout to mean one or both lungs from a single animal. 131 The decision to attempt demonstration or isolation of other microbial agents was made by the diagnostician in charge of the submitted sample based on the referring veterinarian s request, the samples and their condition, and the available examination procedures. Therefore, all samples were not sub- on all lungs. All fresh lungs were swabbed with sterile cottontipped swabs, and these swabs were plated on unsupplemented 5% bovine blood agar and brain heart infusion agar supplemented with 10% bovine whole blood and 0.5% yeast extract. Inoculated 5% bovine blood agar plates were incubated at 37 C for 24 to 48 hours in normal atmosphere and duplicate plates were incubated in reduced oxygen environments. The inoculated brain heart infusion agar plates were incubated in moist chambers containing 10% CO, at 37 C for a minimum of 48 hours. Bacterial identification was done by standard procedures. Attempts to demonstrate the presence of viral and mycoplasma1 agents was by direct fluorescent antibody examination of sections of frozen lung or other appropriate tissues for infectious bovine rhinotracheitis and bovine virus diarrhea viruses and for Mycoplasma bovis and Mycoplasma dispar or by inoculation of susceptible cell culture and subsequent fluorescent antibody examination of these cells for infectious bovine rhinotracheitis, bovine virus diarrhea, bovine respiratory syncytial virus, and parainfluenza-3 viruses. Of the 68 lungs used to tabulate the lesions, bacterial isolation was attempted on all 68, virus isolation or demonstration was attempted on 58, and mycoplasma demonstration was attempted on only eight. Because viral agents are seldom isolated from or demonstrated in pneumonic bovine lungs by our laboratory (four of the 207 submissions in this study), we did not eliminate lungs from our study when virus isolation had not been attempted. Likewise, since diagnostic

2 132 Andrews et al. methods for mycoplasma infections in bovine lungs were not always available to our laboratory during the five years of this study, we did not eliminate lungs from our study when mycoplasma isolation or demonstration had not been attempted. Results H. somnus was isolated from 182 bovine submissions over the five-year period. One hundred sixty-two isolates were from pneumonic lungs (one or both lungs from a single animal), 21 from brains or cerebrospinal fluids from cattle with lesions of infectious thromboembolic meningoencephalitis, and one isolate was from a heart with myocardial infarcts. In two submissions, H. somnus was isolated from both the lung and brain of the same calf. From 70 pneumonic lung samples, other microbial agents in addition to H. somnus were isolated. The most common other bacteria were Pasteurellu multocida (37 of 70 samples) and Pasteurella hemolytica (14 of 70). Lungs with mixed infections were not used to characterize the histologic lesions associated with H. somnus isolation that are described in this paper. H. somnus was isolated from 92 pneumonic lungs in which no other bacteria, mycoplasmas, or viruses were isolated or demonstrated. In 68 of these 92 submissions, histologic sections were available for review. The location of pneumonic consolidation of lung tissue was consistently cranial ventral and involved an estimated 5 to 80% of total lung volume. Fifty-one animals from which H. somnus was isolated were necropsied at our laboratory, and the remaining 13 1 were necropsied by practicing veterinarians and the tissues were mailed to our laboratory. The most common gross description of the 68 lungs from which only H. somnus was isolated was cranial ventral, gray to red-gray, lobular consolidation with white exudate in small airways. Many lungs also contained multiple small abscesses in the consolidated lobes and occasional lungs had fibrinous exudate to fibrous connective tissue tags on the pleural surface. Mediastinal and tracheobronchial lymph nodes were mildly edematous. In approximately 13 of the lungs, the non-consolidated caudal dorsal lung had a meaty consistency with scattered regions of vesicular to bullous alveolar, septa1 and subpleural emphysema. Forty-nine of the 68 lungs from which only H. somnus was isolated were from calves of beef breeds and 17 were from dairy breeds. Thirteen of the 17 lungs from dairy cattle were from calves which weighed less than 100 kg while only three of 49 lungs from beef cattle were from calves weighing less than 100 kg. Seventeen H. sornnus isolates were from beef calves which weighed between 100 and 200 kg and 24 were from beef calves which weighed from 200 to 300 kg. One isolate was from the lungs of an adult dairy cow. No H. somnus was isolated from the lungs of adult beef cattle. The reactions in the lungs were: acute to chronic purulent bronchiolitis and bronchopneumonia with prominent necrosis of bronchiolar exudates and bronchiolar walls (37 lungs); acute to chronic bronchiolitis and bronchopneumonia with minimal bronchiolar necrosis (24 lungs); acute fibrinous pneumonia with accompanying bronchiolitis (2 lungs); acute fibrinous pleuritis (2 lungs); acute suppurative interstitial pneumonia with vasculitis and thrombosis (2 lungs); and diffuse congestion (1 lung). The reactions in the 25 lungs from which only Pasteurella hemolytica were isolated were classified as acute fibrinous pneumonia with accompanying bronchiolitis (24 lungs) and acute bronchiolitis and bronchopneumonia with minimal bronchiolar necrosis (1 lung). These lungs were included in this study for comparison purposes. Groups 1 and 2: Purulent to necrotic broncholitis and bronchopneumonia Twenty-six of the 37 lungs with necrotizing bronchiolitis (figs. 1, 2) had lesions that were classified as acute to subacute; 11 were classified as chronic. Seventeen of the 24 lungs with bronchiolitis with no necrosis (group 2) had lesions that were classified as chronic. All 28 chronically affected lungs in these two groups had marked peribronchiolar fibroplasia, and 23 of the 28 had prominent bronchiolitis obliterans (fig. 3). Filling of alveoli with inflammatory cells and condensed fibrin was limited to the immediate peribronchiolar areas in 19 of the 37 lungs in group 1 (fig. 1) and six of the 24 lungs in group 2 (25 of 61 in both groups). Remaining alveoli were often filled with various amounts of fibrinous fluid, red blood cells, and scattered neurtrophils and macrophages (figs. 1, 4)- often in a pattern of decreasing intensity from affected bronchioles (fig. 1). In the remaining 36 lungs, alveoli were packed with neutrophils, fibrin, and fewer macrophages (fig. 5) in a seemingly random multifocal to diffuse pattern of lobular involvement (fig. 6). No streaming of the inflammatory cells was apparent. Infiltration of the lamina propria of the bronchi with high numbers of plasmacytes, macrophages and neutro-

3 H. somnus Pneumonia 133 Fig. 1: Necrotic bronchiole with protein-rich fluid and inflammatory cells filling peribronchiolar alveoli. Fig. 2: Inflammatory exudates fill focally ulcerated bronchiole (arrow). Inflammatory cells in bronchiolar submucosa. HE. Fig. 3: Peribronchiolar fibroplasia and bronchiolitis obliterans. Nests of remaining bronchiolar epithelium separated from affected airway (arrows). HE. Fig. 4: Congested alveoli away from intense reactions; filled with red blood cells, a few inflammatory cells, and scanty fibrinous exudates. HE. Fig. 5: Focal alveolar exudate consists of neutrophils, macrophages, and fibrin. HE.

4 134 Andrews et al. Fig. 6: Multifocal intense alveolar filling with inflammatory cells (large arrows). Bronchiole filled with exudate (small arrow). HE. Fig. 7: Multifocal hemorrhage in lesser affected dorsal lung. HE. Fig. 8: Fibrinous exudate in an interlobular septa1 lymphatic vessel. HE. Fig. 9: Inflammatory cells and medial necrosis in arteriolar wall in lung of calf with H. somnus septicemia. HE. phils was present in most lung sections which had peribronchiolar fibrosis. Neutrophils frequently were in the bronchial mucosal epithelium. Bronchioles were plugged with necrotic debris and were often rimmed with degenerating neutrophils in the group 1 lungs (figs. 1, 2). Bronchiolar epithelium was focally to diffusely eroded and inflammatory cells were present in the bronchiolar walls (figs. 1, 2). The bronchiolar tree was unequally affected in most lungs. In those lungs in which necrosis of exudates and epithelium was present, peribronchiolar filling of the alveoli with deeply pink-staining condensed fibrin was common. This produced a striking lesion which was readily apparent at low magnifications (fig. 1). In lungs with peribronchiolar fibroplasia, organized fibrinous to fibrous plaques covered ulcerated bronchiolar surfaces. These plaques generally bulged into the lumens and caused partial to complete bronchiolar obstruction (bronchiolitis obliterans). Proliferating remnants of bronchiolar epithelium formed multiple small openings in many areas where only one bronchiole had previously been (fig. 3). Neovascularization

5 and congestion were common in the proliferating peribronchiolar connective tissue. Multifocal hemorrhage was frequent, not only in the pneumonic cranial-ventral portions of lungs but also in the lesser affected dorsal lung (fig. 7). There was no cellular inflammatory infiltration of vessel walls associated with this hemorrhage, and no vascular thrombosis was apparent. Non-consolidated lung generally had diffuse interstitial thickening that resulted from congestion, edema, and mild fibrinous exudation into the interstitial spaces with minimal fluid and fibrin leakage into alveolar spaces. In a few lungs, proliferation of type I1 pneumocytes was present. Interlobular septal lymphatics commonly were mildly distended with fibrinous to fibrinocellular exudates (fig. 8), although the extent of this change was considerably less in the more chronically affected lungs. Likewise, pleural lymphatics were occasionally distended with fibrinous exudates and fibrous plaques could be found on pleural surfaces. No fibrinous to fibrinopurulent pleuritis was observed microscopically in lungs in groups 1 and 2. Group 3: Fibrinous pneumonia In two lungs, we observed marked filling of alveoli with elongated or streaming degenerating inflammatory cells. In both lungs, this reaction was also accompanied by inflammatory fibrinocellular exudates in the bronchioles. These changes could not be distinguished from the lesions that occurred in lungs from which Pasteurella hemolytica had been isolated. Group 4: Pleuritis Acute diffuse fibrinous pleuritis was present in two lungs with no significant bronchiolar or alveolar inflammation. In both lungs, the pleuntis was extensive with the formation of large thrombi in the pleural and interlobular septal lymphatics. This was accompanied by inflammation and necrosis of arterioles and other vessels within the pleura and interlobular septa. Both the pleura and interlobular septa were markedly widened with fibrinous exudate. Group 5: Septicemia/vasculitis In two lungs there were no inflammatory changes observed except in the pulmonary arterioles where thrombosis and degenerative vasculitis (fig. 9) were present. One of these calves also had lesions of infectious thromboembolic meningoencephalitis. H. soinnus Pneumonia 135 Group 6: Diffuse congestion This one lung had only diffuse congestion and fibrinous interstitial exudation similar to the changes observed in the dorsal portions of lungs examined in groups 1 and 2. Discussion The histologic changes present in bovine lungs from which H. somnus had been isolated were generally distinct from naturally occurring and experimental Pasteurella hemolytica pneumonia.6. The H. somnus lesions did not, however, differ markedly from those associated with the isolation of Pasteurella multocida. In that previous study, however, attempts to isolate H. sumnus were done on only a small percentage of the lungs examined. It is difficult to ascertain how many of the lungs in that study, from which Pasteurella multocida was isolated, may have also been infected with H. somnus. Pasteurella multocida grows rapidly on artificial media and, when present, may prevent the isolation of H. somnus from lungs. The results of this study suggest that H. somnus is more likely to be isolated from subacute to chronic bronchopneumonias in calves rather than from acute fibrinous shipping fever -type pneumonias previously reported from the USA.2,s. 3~2 This is in agreement with European reports of isolating H. somnu3 from lungs of young calves I5 with purulent bronchopneumonia. In the present study, acute lesions were generally in cattle which weighed more than 200 kg, while chronic changes, including bronchiolitis obliterans and peribronchiolar fibrosis, were more frequent in calves under 200 kg. Younger cattle may be unable to effectively clear their lungs of H. sumnus and as a result they may develop chronic inflammatory changes. The frequency of the chronic changes of peribronchiolar fibrosis and bronchiolitis obliterans was not suggested in previous reports. The uncommon occurrence of isolating H. somnus from pneumonic lungs of animals with infectious thromboembolic mening~encephalitis ~ was further substantiated by this report. Active concurrent H. somnus pneumonia was not common in cattle with H. somnus septicemia and infectious thromboembolic meningoencephalitis. Also, previously reported respiratory lesions of necrotic to diphtheritic laryngitis2- l3 and chronic polypoid tracheitis2. l3 were not found in this study with H. somnus pneumonia. Reports of diffuse hematogenous pneumonia that include hemorrhagic interstitial pneumonia2. with

6 I36 Andrews et al. thrombus formation13 are associated with septicemic H. somnus infections. In this study, this change was infrequent (2 calves, group 5). Concurrent brain lesions of infectious thromboembolic meningoencephalitis were present in one of these two affected calves. The presence of concurrent or preceding viral infections could not be eliminated in this study. Therefore, many of the changes observed in naturally occurring H. somnus-associated pneumonia may be caused by viral infections. Lesions of acute necrotizing bronchiolitis are reported with several viral infections; these include infectious bovine rhinotracheitis,' parainfluen~a-3,~ and bovine respiratory syncytial virus.3 Experimental attempts to induce H. somnus pneumonia with and without viruses may help to determine the pathogenesis of the necrotic bronchiolitis. The histologic patterns described in groups 1 and 2 suggest that H. somnus is a cause of naturally occurring suppurative to necrotizing bronchiolitis and bronchopneumonia in cattle, particularly in younger calves. H. somnus isolation from pneumonic lungs was far more frequent than its isolation from infectious thromboembolic meningoencephalitis. This suggests that the role of H. somnus as an etiologic agent of calf pneumonia in the USA may have been underestimated and often unrecognized. Acknowledgements The authors wish to thank the faculty and staff of the Iowa State University Veterinary Diagnostic Laboratory for their professional, technical, and clerical assistance. We also wish to thank the Department of Pathology, Michigan State University, for their excellent technical and clerical assistance in preparing this manuscript. References ANDREWS, J.J.; SLIFE, L.N.; STEVENSON, G.W.: Naturally occurring Hemophilus sornnus pneumonia in cattle. Proc I11 Int Sym Vet Lab Diag, pp , 1983 BROWN, L.N.; DILLMAN, R.C.; DIERKS, R.E.: The Huemophilus somnus complex. Proc 74th Annu Meet U.S. Anim Health Assoc, pp , 1970 BRYSON, D.G.; MCNALTY, M.S.; LOGAN, E.F.; CUSH, P.F.: Respiratory syncytial virus pneumonia in young calves: Clinical and pathological findings. Am J Vet Res 44: , 1983 CORBOZ, L.; POHLENZ, J.: Experimentelle Infektionen mit sogenanntem Huemophilus somnus beim Kalb: Vergleich von Stammen mit unterschiedlicher Virulenz. Schweiz Arch Tierheilkd 118: , 1976 DIERKS, R.E.; HANNA, S.A.; DILLMAN, R.C.: Epizootiol- ogy and pathogenesis of Haemopilus somnus infection. J Am Vet Med Assoc 163: , FRIEND, S.C.; THOMSON, R.G.; WILKIE, B.N.: Pulmonary lesions induced by Pasteurellu huemolyticu in cattle. Can J Comp Med 41: , GRINER, L.A.; JENSEN, R.; BROWN, W.W.: Infectious embolic meningoencephalitis in cattle. J Am Vet Assoc 129: I, JERICHO, K.W.F.: Histologic changes in the respiratory tract of calves exposed to aerosols of bovine herpesvirus I and Pusteurellu huemolytica. J Comp Pathol 93: 73-82, JERICHO, K.W.F.; DARCEL, C.LEQ.; LANGFORD, E.V.: Respiratory disease in calves produced with aerosols of parainfluenza-3 virus and Pusteurellu huemolyticu. Can J Comp Med 46: , KLAVANO, G.G.: Observations on Haemophilus somnus infection as an agent producing reproductive disease: Infertility and abortion. Proc SOC Thenogen, pp , I LITTLE, P.B.; SORENSEN, D.K.: Bovine polioencephalomalacia, infectious embolic meningoencephalitis, and acute lead poisoning in feedlot cattle. J Am Vet Med ASSOC 155: , MILLER, R.B.; BARNUM, D.A.; MCENTEE, K.E.: Huemophilus somnus in the reproductive tracts of slaughtered cows: Location and frequency of isolations and lesions. Vet Pathol 20: I, PANCIERA, R.J.; DAHLGREN, R.R.; RINKER, H.B.: Observations on septicemia of cattle caused by a Huemophiluslike organism. Pathol Vet 5: , PRITCHARD, D.G.; MACLEOD, N.S.M.: The isolation of Huemophilus somnus following sudden deaths in suckler calves in Scotland. Vet Rec 100: , PRITCHARD, D.G.; SHREEVE, J.; BRADLEY, R.: The experimental infection of calves with a British strain of Huemophilus somnus. Res Vet Sci 26: 7-11, RUHNKE, H.L.; DOIG, P.A.; MACKAY, A.L.; GAGNON, A.; KIERSTEAD, M.: Isolation of ureaplasma from bovine granular vulvitis. Can J Comp Med 42: , SAUNDERS, J.R.; THIESSEN, W.A.; JANZEN, E.D.: Haemophilus sumnus infections. I. A ten year ( ) retrospective study of losses in cattle herds in western Canada. Can Vet J 21: , SCHIEFER, B.; WARD, G.E.; MOFFATT, R.E.: Correlation of microbiological and histological findings in bovine fibrinous pneumonia. Vet Patholl5: , STEPHENS, L.R.; LITTLE, P.B.; WILKIE, B.N.; BARNUM, D.A.: Infectious thromboembolic meningoencephalitis in cattle: A review. J Am Vet Med Assoc 178: , VAN DREUMEL, A.A.; THOMSON, G.W.; KIERSTEAD, M.: Haemophilus somnus infections in cattle in Ontario: Review with emphasis on respiratory form. Proc I Int Sym Vet Lab Diag, pp , I WEIDE, K.D.; HIBBS, C.M.; ANTHONY, H.D.: Diagnosis of bovine feedlot encephalitides. Proc US Livestock Sanit Assoc 68: , 1964 Request reprints from J.J. Andrews, Iowa State University, Veterinary Diagnostic Laboratory, Ames, Iowa (USA).