Hematological Changes in Calves Exposed to a Mixture of Lipopolysaccharide and Crude Leukotoxin of Pasteurella haemolytica Terry L. Bowersock, Robert D. Walker, Jeanne M. Maddux, Dottie Fenner and Robert N. Moore
ABSTRACT The purpose of this investigation was to determine if culture supernatants of Pasteurella haemolytica containing crude leukotoxin and lipopolysaccharide (CLCL) causes disseminated intravascular coagulopathy (DIC) when injected into calves. The effect of intraduodenal (ID) exposure followed by a subsequent subcutaneous (SC) inoculation of either heat-treated or untreated CLCL was evaluated. The relative contribution of the crude leukotoxin and lipopolysaccharide (LPS) to the virulence of P. haemolytica was evaluated. One group of calves received an ID inoculation of CLCL followed two weeks later by a SC inoculation of CLCL; one group received an ID inoculation of tissue culture medium followed two weeks later by a SC inoculation of CLCL; and a third group received an ID inoculation of CLCL followed two weeks later by a SC inoculation of heat-treated CLCL. Hematological parameters used to evaluate DIC included white cell count, platelet count, neutrophil number, fibrinogen, fibrin degradation products, one stage prothrombin time (OSPT), activated partial thromboplastin time, body
groupe de veaux a requ une inoculation intraduodenale (ID) des LLNP et deux semaines plus tard, une injection sc de LLNP; un autre groupe a requ le milieu de culture sans les LLNP et deux semaines plus tard, une injection sc de LLNP; et un troisieme groupe a requ des LLNP par voie ID et deux semaines plus tard une injection sc de LLNP traites a la chaleur. Les parametres sanguines mesur's 'taient: le nombre de globules blancs, de plaquettes, et de neutrophiles, le fibrinogene, les produits de degradation de la fibrine, le temps de prothrombine (TP), le temps de thromboplastine partielle, la temperature corporelle et les signes cliniques. Chaque parametre etait mesure chez les veaux a 0, 2, 4, 6, 12 et 24 heures RESUME apres linjection sc des LLNP. Des changements au niveau de tous les Le but de cette etude etait de parametres mesures ont ete notes dans determiner si les surnageants de les trois groupes de veaux a l'exception cultures de Pasteurella haemolytica de la temperature corporelle. Les contenant des leucotoxines et des veaux du groupe ayant requ linjection lipopolysaccharides non-purifies sc des toxines traitees a la chaleur ont (LLNP) pouvaient amener une coagu- manifeste des changements moins lation intravasculaire disseminee marques comparativement aux deux (CID) lorsquinjectes a des veaux. autres groupes a l'exception du TP. Suite a une presensibilisation par les Les resultats de cette etude demonLLNP au niveau du duodenum, une trent que les leucotoxines et les injection sc des LLNP traites ou non a lipopolysaccharides de P. haemolytica la chaleur a ete realisee. Ainsi, un possedent un effet additif sur le
temperature and clinical signs. Each parameter was measured in calves at 0, 2, 4, 6, 12 and 24 h following the SC inoculation of CLCL. Each group had significant changes over time in all parameters except body temperature. Calves that received a SC inoculation of heat-treated CLCL had smaller changes in all parameters except OSPT compared to the other groups. Results suggest that the LPS and leukotoxin of P. haemolytica exert additive effects on the coagulation cascade and number of peripheral leukocytes, and that the ID inoculation of CLCL does not affect the response of calves to a SC inoculation of toxin.
Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996 (Bowersock, Moore), Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 (Maddux, Fenner) and Department of Microbiology, Michigan State University, East Lansing, Michigan (Walker). Reprint requests to Dr. T.L. Bowersock, Chief, Bacteriology Section, Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, Indiana 47907. This report represents a portion of the dissertation presented by the senior author to the graduate school of the University of Tennessee in partial fulfillment of the requirements for the PhD degree. Supported by the University of Tennessee, College of Veterinary Medicine, Institute of Agriculture, Centers of Excellence for Livestock Diseases and Human Health, Knoxville, Tennessee. Dr. Moore is the recipient of NIH Research Career Development Award AI-00657. Submitted November 30, 1989.
Can J Vet Res 1990; 54: 415-421
415
processus de coagulation et le nombre total de leucocytes, et quune presensibilisation par voie ID avec les LLNP ne modifile pas la reponse a une injection sc de la toxine. (Traduit par Dr Pascal Dubreuil).
INTRODUCTION
Pasteurella haemolytica is the primary bacterial pathogen involved in the bovine respiratory disease complex (1). Pneumonic pasteurellosis has a serious economic impact on the cattle industry, yet understanding of the virulence factors involved is incomplete. Pasteurella haemolytica produces at least two types of toxins. One is a protein exotoxin (leukotoxin) that has specific activity for ruminant leukocytes (2). Other ruminant tissues are not affected, nor are leukocytes of other animal species (3). Cattle lacking antibodies capable of neutralizing this leukotoxin are more likely to develop pulmonary disease (4). Pasteurella haemolytica also produces lipopolysaccharide (LPS). The LPS of P. haemolytica effects endotoxic reactions in laboratory animals, cardiopulmonary changes in ruminants, and changes in ruminant leukocyte function similar to those induced by other gram-negative endotoxins (5-7). Calves injected intravenously with LPS of Escherichia coli or Pseudomonas develop clinical signs of disseminated intravascular coagulopathy (DIC) (8,9). Various hematological parameters change drastically under these conditions. Postmortem changes in calves exposed to LPS include multiple ecchymoses and hemorrhages on mucosal surfaces. Experiments in laboratory animals and primates have demonstrated that intraduodenal (ID) administration of antigens can result in mucosal immunity at distant mucosal surfaces (10,1 1). We have recently shown that stimulation of gut associated lymphatic tissue (GALT) with live P. haemolytica can prime the mucosal-associated lymphatic tissue to respond to a parenteral injection of bacteria (12). Further trials to stimulate GALT with crude P. haemolytica leukotoxin preparations containing LPS (CLCL) were also successful (13). Calves inoculated ID with CLCL had increased serum and pulmonary antibodies to P. haemoly416
tica. However, when three calves inoculated ID with CLCL were given a subsequent subcutaneous inoculation of CLCL in an attempt to further increase antibody titers, two became seriously ill (unpublished observations). Calves had numerous ecchymoses on mucosal surfaces (and other lesions suggestive of disseminated intravascular coagulopathy), pulmonary neutrophil infiltrates, and exhibited clinical signs of diarrhea, dyspnea, and depression. Lesions and clinical signs similar to these have been recognized in veal calves with a hemorrhagic syndrome in which P. haemolytica was isolated (14). The cause of this adverse reaction is unclear. The ID inoculation of LPS and leukotoxin may have sensitized the calves to respond adversely to a subsequent systemic inoculation of these antigens, or it is possible that LPS and leukotoxin could have induced pathological changes due to their inherent toxic properties. The purposes of the present study were to examine the role of the endotoxin and leukotoxin of P. haemolytica in causing hematological changes in calves, and to determine if an ID inoculation of leukotoxin and endotoxin can sensitize calves to respond adversely to a subsequent parenteral inoculation of these toxins.
MATERIALS AND METHODS CALVES
Twelve mixed breed calves were purchased from several farms as previously described (12). Ages varied from 3 to 11 months of age. Ages varied within each group. Where possible, calves from the same farm were kept together to minimize stress. Animals mixed from more than one source were matched to keep age and size of calves as consistent as possible. Groups A and C had three animals each from separate farms and one animal was added to each group from another farm. Group B had four animals from the same farm. SURGICAL PLACEMENT OF INTRADUODENAL CATHETER AND INTRADUODENAL INOCULATION
Calves were inoculated ID by injection of the appropriate bacterial antigens suspended in phosphate buffered saline solution (PBSS) into
the proximal duodenum through a catheter. Intraduodenal catheterization of calves with a silastic tubing was achieved by surgical intervention as previously described (12). PLACEMENT OF INTRAVASCULAR CATHETER AND COLLECTION OF BLOOD SAMPLES
Each group of calves was housed in a separate pen in the Large Animal
Clinic of the Veterinary Teaching Hospital. The day before each calf received the SC inoculation of CLCL, an intravascular silastic catheter was placed in the right jugular vein and held securely in place by tape. A threeway stopcock was glued to the catheter and secured to the calf by gluing it to the tape. The catheter was flushed with sterile saline to ensure patency and placed in the closed position until needed for sample collection. At time 0 (immediately before the SC inoculation of CLCL was given), each calf was placed in a head catch and examined using the clinical scoring system described below. The three-way stopcock was opened, 3 mL of blood were discarded to clear the catheter of saline, and blood samples were collected and transferred to the appropriate anticoagulent. The threeway stopcock was closed without flushing and calves returned to their pen. For all other times of evaluation and collection of blood, calves were evaluated in their pens and restrained with a halter. All manipulations were performed with the minimal restraint necessary for the comfort of each calf. BACTERIAL ANTIGENS
Pasteurella haemolytica isolate 12216 was kept frozen at -70° C in skim milk and horse serum. Ten to 12 colonies of P. haemolytica were selected from an overnight culture on blood agar and inoculated into 1 L of brain heart infusion broth. This culture was incubated at 370 C in a shaker bath for 4.5 h, centrifuged at 4500 x g for 20 min and the supernatants were discarded. The bacterial pellet was resuspended in 1 L of RPMI-1640 (Gibco Laboratories) supplemented with 1% fetal bovine serum [Hyclone]. This culture was incubated at 370C in a shaker bath for 1.5 h at which time the culture was centrifuged at 1000 x g for 20 min. The supernatants were collected and filtered through a 0.2 Am filter and
found to be optimal for determination of bovine APTT (12). After this incubation 0.1 mL of calcium chloride Volume Anticoagulant Assays performed was added, mixed briefly, and the time 3.0 mL 4.5 mg EDTA CBC and for a clot to form was recorded by the differential coagulation analyzer. platelet count One stage prothrombin time 7.0 mL APTT 3.2% (OSPT) was determined using rabbit Sodium citrate OSPT brain tissue thromboplastin acquired 5.0 mL Thrombin Fibrinogen in a commercial kit (Ortho Diagnostic FDP System, Inc.). The procedure for this 5.0 mL None IgG (ELISA) test was followed as described in the EDTA = Ethylenediaminetetraacetic acid; CBC reagent insert. Briefly, 0.1 mL of test = Complete blood count; APTT = Activated partial thromboplastin time; OSPT One stage plasma was brought to 370 C for 5 min. prothrombin time; FDP = Fibrin(ogen) To the plasma was added 0.2 mL of degradation products; ELISA = Enzyme-linked brain thromboplastin. The tube was immonosorbent assay inserted into an automated photooptical coagulation analyzer and the time for a clot to form was measured. varied by more than 20% were All plasma and serum used for these repeated using a fresh Unopette®. tests were frozen at -70° C immediately Total fibrinogen was determined collection. Tests to measure using a quantitative assay (Ortho following APPT and OSPT were performed on Diagnostic System, Inc.). Briefly, a plasma allowed to thaw for no more 1:10 dilution of test plasma was added than 30 min prior to testing. to a receiving cup in a warmer to which (human) thrombin was added. The STATISTICAL ANALYSIS clotting time was determined in an The means of each parameter were automated fibrometer (Fibrosystem@ compared for each time point within fibrometer, BBL, Inc.). Fibrinogen each group and between each group by concentration was derived by compar- the Student Newman-Keul's test. ing the clotting time of the test sample to a standard curve of clotting times PROCEDURE for serial dilutions of reference Calves were divided into three standards. groups consisting of four calves each. Fibrin(ogen) degradation products Each calf had an intraduodenal (ID) (FDP) were measured using a staphy- catheter surgically implanted and was EVALUATION OF COAGULATION lococcal clumping test (Sigma Diag- allowed to recover for ten days prior to FACTORS, LEUKOCYTE AND nostics) for serum. Serial dilutions of inoculation. One group received tissue PLATELET NUMBERS test serum were compared to FDP culture medium ID and two groups Blood was collected as indicated in reference standards for their ability to were inoculated ID with CLCL. Two I for the Table determination of total clump staphylococcal cells. To calcu- weeks later each calf received a leukocyte and differential counts, late the of FDP quantity present the subcutaneous (SC) injection of either platelet count, fibrinogen and fibrin inverse of the last dilution of sample tissue culture medium, CLCL, or heat degradation product levels, one stage prothrombin and activated partial with clumping was multiplied by the treated CLCL as outlined in Table II. amount of reference FDP in the thromboplastin times. dose of standard which had clumping most Following injection of the SC Complete blood cell counts were were colblood inoculum, samples performed on an automated cell comparable to that sample. 2, at 12 24 h as lected and 4, 6, Activated partial thromboplastin counter (Model 770, Coulter Electronin Time described Table 0 blood I. ics, Inc.). Differential counts were time (APTT) was determined in an sample was collected immediately performed on Wright-Giemsa stained automated photo-optical coagulation of CLCL blood smears. A total of 200 cells were analyzer (Fibrosystem9 fibrometer, before the SC inoculation were was These given. samples assayed examined in normal samples; 50 cells BBL, Inc.). Directions were followed for activated partial thromboplastin as the described insert by reagent were examined in each leukopenic (Ortho Diagnostic System, Inc.) with time (APTT), one stage prothrombin sample. Platelet number was determined by one exception as noted. Briefly, 0.1 time (OSPT), fibrinogen, fibrin a microcollection system (Unopette@, mL test plasma was mixed with 0.1 mL degradation products (FDP), plaBecton-Dickenson and Co.). Counts of Thrombofax®, a mixture of bovine telets, total number of leukocytes were performed with a hemocytome- brain cephalin and ellagic acid. This (WBC), number of polymorphonuter using phase contrast microscopy. mixture was incubated at 370C for 2 clear cells (PMN), body temperature, Two counts were performed per min rather than 5 min as recom- and clinical evaluation score. At each sample and averaged. Counts that mended by the insert. This time was time point calves were clinically
immediately frozen at -70° C. Supernatants of P. haemolytica were later concentrated 100-fold with a Minitan ultrafiltration apparatus (Millipore Corp.) using a 30,000 molecular weight (MW) filter. Further concentration was achieved using a stirred cell concentrator (Amicon Corp.) with a 10,000 MW filter. Concentrated supernatants were frozen at -70° C in aliquots for injection. Leukotoxin activity was measured using a neutral red uptake assay (12). Primary leukotoxin supernatants contained 30 to 50 toxic units of activity. Concentrated supernatants were adjusted so that each animal received 3000 toxic units per kg body weight. The supernatant preparations were assayed for lipopolysaccharide using the limulus lysate test (Sigma Chemical Company). Each dose of concentrated supernatants also contained 1 Ag lipopolysaccharide/kg body weight. The dose used to inoculate calves was derived from experiments in which rabbits were injected with CLCL containing either 1, 3.3, or 10 ,ug LPS/ kg body weight. No adverse effects were noted in any rabbit and the immune response for each dose was similar (13). Administration of CLCL containing 1 Mg LPS/kg body weight did induce toxic responses in calves (unpublished observation).
TABLE I. Blood samples collected and assays performed
417
TABLE II. Schedule of innoculation of calves with CLCL
Introduodenal prime (Day 0) CLCL CLCL
Subcutaneous Blood sample Postbooster (0,2,4,6,12) mortem Group (Day 14) and 24 h) exam A X CLCL X B Heat treated X X CLCL C RPMI-1640 X CLCL X Each dose of CLCL contained 3000 toxic units/ kg body weight of calf as determined by neutral red uptake assay and 1 ;Lg lipopolysaccharide/ kg body weight as determined by limulus lysate assay CLCL= Crude leukotoxin containing lipopolysaccharide X = Task was performed
evaluated as previously described (16) (p < 0.05). Platelet numbers based on a scale of 0 to 10 with 0 as decreased in group C calves but not normal. Factors evaluated were significantly until 12 h. Platelet respiratory rate, respiratory effort, numbers were still decreasing in group attitude, diarrhea, coughing. and C calves at 24 h. nasal/salivary discharge making the maximum possible abnormal score 60. DIFFERENCES BETWEEN GROUPS The guidelines of the Guide to the FOR EACH PARAMETER There was no significant difference Care and Use of Experimental Animals of the Canadian Council on between any group at any time point for parameters of clinical score, body Animal Care were followed. temperature, total white cell count and total neutrophil number. For platelet numbers, group A had a greater RESULTS decrease compared to time 0 than DIFFERENCES OVER TIME group C at 6 h (p < 0.07) and again at WITHIN EACH GROUP FOR 12 h (p
ABSTRACT The purpose of this investigation was to determine if culture supernatants of Pasteurella haemolytica containing crude leukotoxin and lipopolysaccharide (CLCL) causes disseminated intravascular coagulopathy (DIC) when injected into calves. The effect of intraduodenal (ID) exposure followed by a subsequent subcutaneous (SC) inoculation of either heat-treated or untreated CLCL was evaluated. The relative contribution of the crude leukotoxin and lipopolysaccharide (LPS) to the virulence of P. haemolytica was evaluated. One group of calves received an ID inoculation of CLCL followed two weeks later by a SC inoculation of CLCL; one group received an ID inoculation of tissue culture medium followed two weeks later by a SC inoculation of CLCL; and a third group received an ID inoculation of CLCL followed two weeks later by a SC inoculation of heat-treated CLCL. Hematological parameters used to evaluate DIC included white cell count, platelet count, neutrophil number, fibrinogen, fibrin degradation products, one stage prothrombin time (OSPT), activated partial thromboplastin time, body
groupe de veaux a requ une inoculation intraduodenale (ID) des LLNP et deux semaines plus tard, une injection sc de LLNP; un autre groupe a requ le milieu de culture sans les LLNP et deux semaines plus tard, une injection sc de LLNP; et un troisieme groupe a requ des LLNP par voie ID et deux semaines plus tard une injection sc de LLNP traites a la chaleur. Les parametres sanguines mesur's 'taient: le nombre de globules blancs, de plaquettes, et de neutrophiles, le fibrinogene, les produits de degradation de la fibrine, le temps de prothrombine (TP), le temps de thromboplastine partielle, la temperature corporelle et les signes cliniques. Chaque parametre etait mesure chez les veaux a 0, 2, 4, 6, 12 et 24 heures RESUME apres linjection sc des LLNP. Des changements au niveau de tous les Le but de cette etude etait de parametres mesures ont ete notes dans determiner si les surnageants de les trois groupes de veaux a l'exception cultures de Pasteurella haemolytica de la temperature corporelle. Les contenant des leucotoxines et des veaux du groupe ayant requ linjection lipopolysaccharides non-purifies sc des toxines traitees a la chaleur ont (LLNP) pouvaient amener une coagu- manifeste des changements moins lation intravasculaire disseminee marques comparativement aux deux (CID) lorsquinjectes a des veaux. autres groupes a l'exception du TP. Suite a une presensibilisation par les Les resultats de cette etude demonLLNP au niveau du duodenum, une trent que les leucotoxines et les injection sc des LLNP traites ou non a lipopolysaccharides de P. haemolytica la chaleur a ete realisee. Ainsi, un possedent un effet additif sur le
temperature and clinical signs. Each parameter was measured in calves at 0, 2, 4, 6, 12 and 24 h following the SC inoculation of CLCL. Each group had significant changes over time in all parameters except body temperature. Calves that received a SC inoculation of heat-treated CLCL had smaller changes in all parameters except OSPT compared to the other groups. Results suggest that the LPS and leukotoxin of P. haemolytica exert additive effects on the coagulation cascade and number of peripheral leukocytes, and that the ID inoculation of CLCL does not affect the response of calves to a SC inoculation of toxin.
Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996 (Bowersock, Moore), Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 (Maddux, Fenner) and Department of Microbiology, Michigan State University, East Lansing, Michigan (Walker). Reprint requests to Dr. T.L. Bowersock, Chief, Bacteriology Section, Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, Indiana 47907. This report represents a portion of the dissertation presented by the senior author to the graduate school of the University of Tennessee in partial fulfillment of the requirements for the PhD degree. Supported by the University of Tennessee, College of Veterinary Medicine, Institute of Agriculture, Centers of Excellence for Livestock Diseases and Human Health, Knoxville, Tennessee. Dr. Moore is the recipient of NIH Research Career Development Award AI-00657. Submitted November 30, 1989.
Can J Vet Res 1990; 54: 415-421
415
processus de coagulation et le nombre total de leucocytes, et quune presensibilisation par voie ID avec les LLNP ne modifile pas la reponse a une injection sc de la toxine. (Traduit par Dr Pascal Dubreuil).
INTRODUCTION
Pasteurella haemolytica is the primary bacterial pathogen involved in the bovine respiratory disease complex (1). Pneumonic pasteurellosis has a serious economic impact on the cattle industry, yet understanding of the virulence factors involved is incomplete. Pasteurella haemolytica produces at least two types of toxins. One is a protein exotoxin (leukotoxin) that has specific activity for ruminant leukocytes (2). Other ruminant tissues are not affected, nor are leukocytes of other animal species (3). Cattle lacking antibodies capable of neutralizing this leukotoxin are more likely to develop pulmonary disease (4). Pasteurella haemolytica also produces lipopolysaccharide (LPS). The LPS of P. haemolytica effects endotoxic reactions in laboratory animals, cardiopulmonary changes in ruminants, and changes in ruminant leukocyte function similar to those induced by other gram-negative endotoxins (5-7). Calves injected intravenously with LPS of Escherichia coli or Pseudomonas develop clinical signs of disseminated intravascular coagulopathy (DIC) (8,9). Various hematological parameters change drastically under these conditions. Postmortem changes in calves exposed to LPS include multiple ecchymoses and hemorrhages on mucosal surfaces. Experiments in laboratory animals and primates have demonstrated that intraduodenal (ID) administration of antigens can result in mucosal immunity at distant mucosal surfaces (10,1 1). We have recently shown that stimulation of gut associated lymphatic tissue (GALT) with live P. haemolytica can prime the mucosal-associated lymphatic tissue to respond to a parenteral injection of bacteria (12). Further trials to stimulate GALT with crude P. haemolytica leukotoxin preparations containing LPS (CLCL) were also successful (13). Calves inoculated ID with CLCL had increased serum and pulmonary antibodies to P. haemoly416
tica. However, when three calves inoculated ID with CLCL were given a subsequent subcutaneous inoculation of CLCL in an attempt to further increase antibody titers, two became seriously ill (unpublished observations). Calves had numerous ecchymoses on mucosal surfaces (and other lesions suggestive of disseminated intravascular coagulopathy), pulmonary neutrophil infiltrates, and exhibited clinical signs of diarrhea, dyspnea, and depression. Lesions and clinical signs similar to these have been recognized in veal calves with a hemorrhagic syndrome in which P. haemolytica was isolated (14). The cause of this adverse reaction is unclear. The ID inoculation of LPS and leukotoxin may have sensitized the calves to respond adversely to a subsequent systemic inoculation of these antigens, or it is possible that LPS and leukotoxin could have induced pathological changes due to their inherent toxic properties. The purposes of the present study were to examine the role of the endotoxin and leukotoxin of P. haemolytica in causing hematological changes in calves, and to determine if an ID inoculation of leukotoxin and endotoxin can sensitize calves to respond adversely to a subsequent parenteral inoculation of these toxins.
MATERIALS AND METHODS CALVES
Twelve mixed breed calves were purchased from several farms as previously described (12). Ages varied from 3 to 11 months of age. Ages varied within each group. Where possible, calves from the same farm were kept together to minimize stress. Animals mixed from more than one source were matched to keep age and size of calves as consistent as possible. Groups A and C had three animals each from separate farms and one animal was added to each group from another farm. Group B had four animals from the same farm. SURGICAL PLACEMENT OF INTRADUODENAL CATHETER AND INTRADUODENAL INOCULATION
Calves were inoculated ID by injection of the appropriate bacterial antigens suspended in phosphate buffered saline solution (PBSS) into
the proximal duodenum through a catheter. Intraduodenal catheterization of calves with a silastic tubing was achieved by surgical intervention as previously described (12). PLACEMENT OF INTRAVASCULAR CATHETER AND COLLECTION OF BLOOD SAMPLES
Each group of calves was housed in a separate pen in the Large Animal
Clinic of the Veterinary Teaching Hospital. The day before each calf received the SC inoculation of CLCL, an intravascular silastic catheter was placed in the right jugular vein and held securely in place by tape. A threeway stopcock was glued to the catheter and secured to the calf by gluing it to the tape. The catheter was flushed with sterile saline to ensure patency and placed in the closed position until needed for sample collection. At time 0 (immediately before the SC inoculation of CLCL was given), each calf was placed in a head catch and examined using the clinical scoring system described below. The three-way stopcock was opened, 3 mL of blood were discarded to clear the catheter of saline, and blood samples were collected and transferred to the appropriate anticoagulent. The threeway stopcock was closed without flushing and calves returned to their pen. For all other times of evaluation and collection of blood, calves were evaluated in their pens and restrained with a halter. All manipulations were performed with the minimal restraint necessary for the comfort of each calf. BACTERIAL ANTIGENS
Pasteurella haemolytica isolate 12216 was kept frozen at -70° C in skim milk and horse serum. Ten to 12 colonies of P. haemolytica were selected from an overnight culture on blood agar and inoculated into 1 L of brain heart infusion broth. This culture was incubated at 370 C in a shaker bath for 4.5 h, centrifuged at 4500 x g for 20 min and the supernatants were discarded. The bacterial pellet was resuspended in 1 L of RPMI-1640 (Gibco Laboratories) supplemented with 1% fetal bovine serum [Hyclone]. This culture was incubated at 370C in a shaker bath for 1.5 h at which time the culture was centrifuged at 1000 x g for 20 min. The supernatants were collected and filtered through a 0.2 Am filter and
found to be optimal for determination of bovine APTT (12). After this incubation 0.1 mL of calcium chloride Volume Anticoagulant Assays performed was added, mixed briefly, and the time 3.0 mL 4.5 mg EDTA CBC and for a clot to form was recorded by the differential coagulation analyzer. platelet count One stage prothrombin time 7.0 mL APTT 3.2% (OSPT) was determined using rabbit Sodium citrate OSPT brain tissue thromboplastin acquired 5.0 mL Thrombin Fibrinogen in a commercial kit (Ortho Diagnostic FDP System, Inc.). The procedure for this 5.0 mL None IgG (ELISA) test was followed as described in the EDTA = Ethylenediaminetetraacetic acid; CBC reagent insert. Briefly, 0.1 mL of test = Complete blood count; APTT = Activated partial thromboplastin time; OSPT One stage plasma was brought to 370 C for 5 min. prothrombin time; FDP = Fibrin(ogen) To the plasma was added 0.2 mL of degradation products; ELISA = Enzyme-linked brain thromboplastin. The tube was immonosorbent assay inserted into an automated photooptical coagulation analyzer and the time for a clot to form was measured. varied by more than 20% were All plasma and serum used for these repeated using a fresh Unopette®. tests were frozen at -70° C immediately Total fibrinogen was determined collection. Tests to measure using a quantitative assay (Ortho following APPT and OSPT were performed on Diagnostic System, Inc.). Briefly, a plasma allowed to thaw for no more 1:10 dilution of test plasma was added than 30 min prior to testing. to a receiving cup in a warmer to which (human) thrombin was added. The STATISTICAL ANALYSIS clotting time was determined in an The means of each parameter were automated fibrometer (Fibrosystem@ compared for each time point within fibrometer, BBL, Inc.). Fibrinogen each group and between each group by concentration was derived by compar- the Student Newman-Keul's test. ing the clotting time of the test sample to a standard curve of clotting times PROCEDURE for serial dilutions of reference Calves were divided into three standards. groups consisting of four calves each. Fibrin(ogen) degradation products Each calf had an intraduodenal (ID) (FDP) were measured using a staphy- catheter surgically implanted and was EVALUATION OF COAGULATION lococcal clumping test (Sigma Diag- allowed to recover for ten days prior to FACTORS, LEUKOCYTE AND nostics) for serum. Serial dilutions of inoculation. One group received tissue PLATELET NUMBERS test serum were compared to FDP culture medium ID and two groups Blood was collected as indicated in reference standards for their ability to were inoculated ID with CLCL. Two I for the Table determination of total clump staphylococcal cells. To calcu- weeks later each calf received a leukocyte and differential counts, late the of FDP quantity present the subcutaneous (SC) injection of either platelet count, fibrinogen and fibrin inverse of the last dilution of sample tissue culture medium, CLCL, or heat degradation product levels, one stage prothrombin and activated partial with clumping was multiplied by the treated CLCL as outlined in Table II. amount of reference FDP in the thromboplastin times. dose of standard which had clumping most Following injection of the SC Complete blood cell counts were were colblood inoculum, samples performed on an automated cell comparable to that sample. 2, at 12 24 h as lected and 4, 6, Activated partial thromboplastin counter (Model 770, Coulter Electronin Time described Table 0 blood I. ics, Inc.). Differential counts were time (APTT) was determined in an sample was collected immediately performed on Wright-Giemsa stained automated photo-optical coagulation of CLCL blood smears. A total of 200 cells were analyzer (Fibrosystem9 fibrometer, before the SC inoculation were was These given. samples assayed examined in normal samples; 50 cells BBL, Inc.). Directions were followed for activated partial thromboplastin as the described insert by reagent were examined in each leukopenic (Ortho Diagnostic System, Inc.) with time (APTT), one stage prothrombin sample. Platelet number was determined by one exception as noted. Briefly, 0.1 time (OSPT), fibrinogen, fibrin a microcollection system (Unopette@, mL test plasma was mixed with 0.1 mL degradation products (FDP), plaBecton-Dickenson and Co.). Counts of Thrombofax®, a mixture of bovine telets, total number of leukocytes were performed with a hemocytome- brain cephalin and ellagic acid. This (WBC), number of polymorphonuter using phase contrast microscopy. mixture was incubated at 370C for 2 clear cells (PMN), body temperature, Two counts were performed per min rather than 5 min as recom- and clinical evaluation score. At each sample and averaged. Counts that mended by the insert. This time was time point calves were clinically
immediately frozen at -70° C. Supernatants of P. haemolytica were later concentrated 100-fold with a Minitan ultrafiltration apparatus (Millipore Corp.) using a 30,000 molecular weight (MW) filter. Further concentration was achieved using a stirred cell concentrator (Amicon Corp.) with a 10,000 MW filter. Concentrated supernatants were frozen at -70° C in aliquots for injection. Leukotoxin activity was measured using a neutral red uptake assay (12). Primary leukotoxin supernatants contained 30 to 50 toxic units of activity. Concentrated supernatants were adjusted so that each animal received 3000 toxic units per kg body weight. The supernatant preparations were assayed for lipopolysaccharide using the limulus lysate test (Sigma Chemical Company). Each dose of concentrated supernatants also contained 1 Ag lipopolysaccharide/kg body weight. The dose used to inoculate calves was derived from experiments in which rabbits were injected with CLCL containing either 1, 3.3, or 10 ,ug LPS/ kg body weight. No adverse effects were noted in any rabbit and the immune response for each dose was similar (13). Administration of CLCL containing 1 Mg LPS/kg body weight did induce toxic responses in calves (unpublished observation).
TABLE I. Blood samples collected and assays performed
417
TABLE II. Schedule of innoculation of calves with CLCL
Introduodenal prime (Day 0) CLCL CLCL
Subcutaneous Blood sample Postbooster (0,2,4,6,12) mortem Group (Day 14) and 24 h) exam A X CLCL X B Heat treated X X CLCL C RPMI-1640 X CLCL X Each dose of CLCL contained 3000 toxic units/ kg body weight of calf as determined by neutral red uptake assay and 1 ;Lg lipopolysaccharide/ kg body weight as determined by limulus lysate assay CLCL= Crude leukotoxin containing lipopolysaccharide X = Task was performed
evaluated as previously described (16) (p < 0.05). Platelet numbers based on a scale of 0 to 10 with 0 as decreased in group C calves but not normal. Factors evaluated were significantly until 12 h. Platelet respiratory rate, respiratory effort, numbers were still decreasing in group attitude, diarrhea, coughing. and C calves at 24 h. nasal/salivary discharge making the maximum possible abnormal score 60. DIFFERENCES BETWEEN GROUPS The guidelines of the Guide to the FOR EACH PARAMETER There was no significant difference Care and Use of Experimental Animals of the Canadian Council on between any group at any time point for parameters of clinical score, body Animal Care were followed. temperature, total white cell count and total neutrophil number. For platelet numbers, group A had a greater RESULTS decrease compared to time 0 than DIFFERENCES OVER TIME group C at 6 h (p < 0.07) and again at WITHIN EACH GROUP FOR 12 h (p
