The Effect of Pasteurella haemolytica and the Leukotoxin of Pasteurella haemolytica on Bovine Lung Explants I.W. Wilkie, M.H. Fallding, P.E. Shewen and J.A. Yager

ABSTRACT Bovine lung explants were used in a study designed to compare the pathogenic effects of Pasteurella haemolytica type 1, a nonpathogenic organism Neisseria subflava, or the crude leukotoxin ofP. haemolytica on alveolar macrophages and lung parenchymal cells. Concentrated, purified peripheral blood neutrophil suspensions were added with the bacteria to some explants. Duplicate pairs of cultures from each treatment group were fixed at regular intervals up to 24 hours after seeding and morphological changes were assessed by light and electron microscopy. Pasteurella haemolytica caused deterioration of alveolar macrophages within one hour but did not affect parenchymal cells for more than 12 hours. Neisseria subflava did not affect alveolar macrophages initially, but caused an accelerated deterioration after four hours. After 24 hours, bacterial overgrowth caused similar deterioration of all cells in explants seeded with either bacterium. Alveolar macrophages phagocytosed large numbers of N. subflava but rarely ingested P. haemolytica. Added neutrophils did not have any discernible effect on any of the explants and did not potentiate bacterial effects. Addition of crude leukotoxin of P. haemolytica to the culture medium significantly accelerated alveolar macrophage deterioration without apparent effect on

haemolytica. L'addition de neutrophiles aux milieux n'a eu aucun effet. De son cote, la leucotoxine de P. haemolytica a augmente la vitesse de deterioration des macrophages mais n'a pas affecte les cellules du parenchyme. Ces resultants supportent RESUME l'hypothese que les lesions observees lors de pasteurellose ne sont pas Cette etude avait pour but de necessairement le resultant direct de comparer la pathogenicite de Pasteur- l'infection bacterienne. ella haemolytica type 1, de sa leucotoxine et d'un organisme nonINTRODUCTION pathogene (Neisseria subflava) sur les macrophages alveolaires ou les celWhile the lesions of bovine pneulules du parenchyme de poumons de bovins. Des suspensions de neutro- monic pasteurellosis have been well philes circulants ont ete ajoutees dans described (1,2), their pathogenesis quelques cas a ces milieux. Apres remains less than completely underensemencement des bacteries et a stood. In particular, the origin of the intervalles reguliers, deux milieux par typical foci of coagulative necrosis, traitement ont ete fixes pour verifier which occur in most acute cases, has les changements morphologiques des not been satisfactorily explained (2). cellules pulmonaires par microscopie Although vascular lesions and thromoptique ou electronique. Pasteurella bosis have been described in the lungs haemolytica a provoque une altera- of some cattle with fibrinous pneumotion des macrophages en moins d'une nias (3,4), infarction is not generally heure mais n'a pas affecte les cellules believed to be the main cause of the du parenchyme en 12 heures. Neisseria necrotic foci (5). Pasteurella haemolytica secretes a subflava n'a pas affecte les macrophages au debuts mais a accelere leur substance during its lag and exponendeterioration apres quatre heures. tial growth phases which is toxic to Apres 24 heures de culture et indepen- bovine macrophages (6,8) and neutrodamment du traitement, on retrouvait phils (7) but not to kidney cells (8). The les memes alterations a la suite d'une effect of this leukotoxin on lung trop grande croissance bacterienne. parenchymal cells is not known, but it Les macrophages alveolaires ont does appear to be an important demontre une forte phagocytose des virulence factor and antileukotoxin N. subflava mais tres peu des P. antibody titers have been correlated parenchymal cell survival. These results support the hypothesis that the severe tissue destruction of fulminant pneumonic pasteurellosis is not a direct result of bacterial infection.

Veterinary Laboratory Services, Ontario Ministry of Agriculture and Food, P.O. Box 3612, Guelph, Ontario NIH 6R8 (Wilkie) and Department of Biomedical Sciences (Fallding), Department of Veterinary Microbiology and Immunology (Shewen), Department of Pathology (Yager), Ontario Veterinary College, University of Guelph, Guelph, Ontario N 1G 2W I (Yager). Reprint requests to Dr. J.A. Yager. This research project was supported by the Ontario Ministry of Agriculture and Food and the Ontario Cattleman's Association. Submitted June 28, 1988.

Can J Vet Res 1990; 54: 151-156


with immunity to P. haemolytica challenge in calves (9). It is increasingly recognized that host inflammatory responses make a major contribution to tissue injury in many lung diseases (10). Both humoral immune mechanisms (11) and neutrophils (12) have been shown to be important in mediating parenchymal damage. Calves pretreated to remove circulating neutrophils developed no physiological or morphological lung changes when challenged with a dose of P. haemolytica which caused hypoxemia and necrotizing bronchopneumonia in normal calves (13). The difficulties of separating the contributions of bacteria and host inflammatory responses to the development of fulminant pneumonic pasteurellosis led to the use of cultured lung explants, in order to examine the effect of the bacteria in isolation from the intact inflammatory response. Cultured, mature, organ tissues have not been used extensively in the study of infectious disease although simplified methods for maintaining cultures have been available for a long time (14). Recently, cultured lung slices have been used to study the pathogenesis of pulmonary injury mediated by oxidants (15) and neutrophils (16). In the following experiments we compared the morphological effects of adding P. haemolytica, Neisseria subflava, with or without added neutrophils, or the crude leukotoxin of P. haemolytica to calf lung slices to determine the relative contributions of bacteria and their products to tissue damage. MATERIALS AND METHODS LUNG EXPLANTS

Six Holstein calves 3-4 wk old were anesthetized to the point of apnea with intravenous sodium pentobarbital (Somnotol, MTC Pharmaceuticals, Montreal, Quebec). The chest wall was clipped and scrubbed with an aqueous iodophore solution (Wescodyne, West Chemical Products Ltd., Montreal, Quebec). A large flap of skin and three or four ribs were reflected to expose the anterior half of the lung. A piece of the middle lobe approximately 3 x 3 x 2 cm was removed aseptically, and placed in a 152

sterile petri dish with sufficient sterile phosphate-buffered saline (PBS) to keep it moist. Under a laminar-flow hood, the tissue was washed twice with PBS. Smaller pieces were transferred to a clean petri dish for cutting into approximately 1 mm x 1 mm x 0.75 mm pieces, keeping the tissue moist with small amounts of culture medium. The lung slices were placed on sterile lens-paper squares, supported by metal grids (Falcon Grids, Oxnard, California), in the wells of 60 mm organ-culture dishes (Falcon organ-culture dishes #3037, Oxnard, California). The center wells were filled with culture medium to saturate the paper, and the outer wells were filled with 5 mL sterile water. Cultures were incubated at 370C in humidified air with 5% CO2. For all explants, Eagles' minimal essential medium (EMEM) (Grand Island Biological Co., New York) with 10% heatinactivated fetal bovine serum (FBS) (Grand Island Biological Co., New York) was used. BACTERIA

One 18 h colony from a blood-agar plate of either Pasteurella haemolytica type 1 (ATCC #43270), or Neisseria subflava (ATCC #19243) was added to 50 mL of heart brain infusion broth (HBIB) (Difco Laboratories, Detroit, Michigan) in 100 mL conical flasks and incubated at 37°C for 16 h in a shaker-bath. The bacterial suspensions were then washed by centrifugation at 7000 g for 15 min and resuspended in fresh HBIB (three times). On the final resuspension, sufficient HBIB was added to bring the bacterial concentration to approximately 2 x 108/ mL by comparing the optical density of the suspension with optical density curves previously prepared for each bacterial species. The final inoculum of 5 ,uL therefore contained approximately 106 bacteria. This number was found in pilot experiments to cause morphological changes without excessive bacterial overgrowth. The actual number of colony-forming units in the suspensions was determined using the Spiral Plater (Spiral Systems Inc., Cincinnati, Ohio) system. To inoculate cultures, 25 g disposable needles were attached to an automatic, airdisplacement pipette (Pipetteman,

Gilson Medical Electronics, France) by pressing the needles onto cut-off disposable tips. Crude, lyophilized, P. haemolytica leukotoxin prepared as previously described (8) and stored at -700 C, was diluted in EMEM immediately before addition to cultures. The heat-inactivated leukotoxin was prepared one day before use, heated in a water bath at 56°C for 1 h, then refrigerated at 40 C overnight. NEUTROPHIL SEPARATION

Approximately 50 mL venous blood was drawn into heparinized tubes from each calf prior to anesthesia, centrifuged in 10 mL aliquots at 270 g for 12 min and the plasma and platelets discarded. The packed cells were diluted 1:1 (v/v) with PBS and layered over 10 mL Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) in 50 mL conical tubes, then centrifuged at 1050 g for 30 min. All layers above the red cells and neutrophils were removed by aspiration and the red cells lysed with 35 mL chilled, distilled water. Isotonicity was restored after 20 s by adding 10 mL of 5.4% sodium chloride solution. The neutrophils were pelleted by centrifugation, the supernatant discarded and the pellet resuspended in a small amount of culture medium for counting and viability check by trypan blue exclusion. The final suspension was adjusted to contain approximately 107 viable neutrophils per mL. ELECTRON MICROSCOPY

For each treatment and for each time interval, duplicate cultures from the six calves were fixed in 2.5% phosphate-buffered glutaraldehyde and postfixed in 1% osmium tetroxide. They were dehydrated by standard techniques for Epon (Marivac Ltd., Halifax, Nova Scotia) embedding. A single 1 ,u section was cut from the center of each block and stained with methylene blue for macrophage counting. Four blocks were chosen from each time and treatment group for ultrathin sectioning. Ultrathin sections were stained with lead citrate/ uranyl acetate. EXPERIMENTAL DESIGN

1. To evaluate the effects of bacteria or bacteria plus neutrophils on bovine lung explants, pairs of lung

Fig. 1. Bovine lung explant (untreated) after 24 h in culture. Interalveolar septa are condensed, epithelial cells vacuolated and the nuclei of some parenchymal cells are pyknotic. Two alveolar macrophages appear relatively intact (arrows). Toluidine blue. X320.

counted and classed as live or dead using the following morphological criteria for irreversible cell damage: 1) karyolysis, karyorrhexis or pyknosis of the nucleus, and 2) marked cytoplasmic vacuolation or disruption of the cytoplasmic membrane. A total of 144 cultures from three calves were used for the macrophage viability counts. 2. To evaluate the effect of crude P. haemolytica leukotoxin on bovine lung explants, pairs of lung slices were placed in culture dishes and allotted to groups of 12. Each group was treated as follows: 1. No further treatment 2. 3.0 mg/ mL crude leukotoxin in culture medium 3. 0.3 mg/ mL crude leukotoxin in culture medium 4. 3.0 mg/ mL heat-inactivated leukotoxin added to culture medium Two sets of cultures from each group were fixed at 1, 2, 4, 8, 12 and 24 h after treatment. Fixed tissues were processed and examined as for experiment 1.

Fig. 3. Bovine lung explants (untreated) after 4 h in culture. An alveolar macrophage (arrow) has developed numerous cytoplasmic vacuoles but is otherwise normal. Toluidine blue. X600.

Scattered pyknotic nuclei were probably those of endothelial cells. The slices were placed in the center wells presence of bacteria did not affect the of 48 culture dishes and alloted apparent survival of epithelial cells randomly to six groups of eight, until 24 h when there was a marked each of which received 5 IAL of one drop in pH of the culture medium of the following: followed by rapid necrosis of the entire 1. HBIB (control) explants. This was associated with 2. HBIB plus 106 neutrophils very heavy growth of bacteria and was 3. 106 P. haemolytica in HBIB similar in both Pasteurella and 4. 106 P. haemolytica in HBIB plus Neisseria-seeded cultures. 106 neutrophils STATISTICAL ANALYSIS There were marked between5. 106 N. subflava in HBIB treatment differences in the appearwere evaluated by Treatment effects 6. 106 N. subflava in HBIB plus 106 analysis of variance. Individual ance of alveolar macrophages. In neutrophils control cultures, most macrophages Two sets of cultures from each treatment means were also compared developed a few cytoplasmic vacuoles to controls t-test. by Student's group were fixed 1, 2, 4 and 24 h after and dense cytoplasmic inclusions (Fig. treatment. One micron sections from Statistical significance was attributed 3). After 12 h, alveolar macrophages if p

The effect of Pasteurella haemolytica and the leukotoxin of Pasteurella haemolytica on bovine lung explants.

Bovine lung explants were used in a study designed to compare the pathogenic effects of Pasteurella haemolytica type 1, a nonpathogenic organism Neiss...
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