”HM—0*- PEYSEOWGEC AM} PM‘EOLOGIC CHANGES ER CARES GIVER ESCHERECEEA {30%.} ER: mmm GR MS‘E’EURELLA ML'EGCEEA 1: Emu {raw 45120 {lemma (:5 M‘ 3‘ MCI‘EEQAK SHE URWEESETY Hawker RE Hag Mama; 3??? 3 1293 01009 1035 llHIWllllHIII}”WINIHIWIHHIIIIHIlllilllillllUll 1/ L1 U—niversitY I 1 ABSTRACT PHYSIOLOGIC AND PATHOLOGIC CHANGES IN CALVES GIVEN ESCHERICHIA COLI ENDOTOXIN OR PASTEURELLA MULTOCIDA BY Babiker El Hag Musa Ten healthy Holstein bull calves, ranging in age from 8 to 36 days, were used in this experiment. Three animals were inoculated subcu- taneously with 4.75 x 105 viable Pasteurella multocida, strain 656, organisms, and a fourth animal received half of the above dose. Another four animals were injected intravenously with Escherichia coli endotoxin. (026:36) at the rate of 0.03-0.088 mg./1b. body weight. The remaining two animals were the control animals. Clinical observations and blood collections were made at regular intervals. The blood was used for bacteriologic examinations and for determinations of routine hemograms. Serum was separated from.the blood samples and was used for the following determinations: glucose, serum glutamic-oxaloacetic transaminiase, serum glutamic-pyruvic transaminase, and cholesterol. Sera from the control animals and from the animals inoculated with thehPasteureZZa organisms were used to inoculate 11-day- old chick embryos of the white leghorn strain. Serum (0.1 ml.) was inoculated into the embryonated eggs intravenously. Babiker E1 Hag Muss Following death of the calves, or termination of the experiment, a complete necropsy was conducted. Specimens from various organs were collected for pathologic and bacteriologic examinations. This study showed that the physical signs, hematologic findings, and glucose and cholesterol levels followed more or less a similar trend whether the calves were given Pasteurella multocida organisms or Escherichia coli endotoxin. Levels of serum glutamic—oxaloacetic trans- aminase were characteristically elevated as a result of tissue damage accompanying Escherichia coli endotoxin, whereas serum glutamic-pyruvic transaminase levels were more characteristically elevated during Pasteurella multocida infection. The lung had the most prominent changes in both PasteureZZa-infected calves and Escherichia coli endo- toxemic calves. Macroscopic and microscopic lesions were strikingly similar for the two groups of calves. Pasteurella multocida organisms were isolated from blood and tissues of animals inoculated with that organism. The primary effect of endotoxin was on the blood'and blood vascular system, but whether this was a direct effect or mediated through other substances was not determined. Based on the similarities of the physical, chemical, and necr0psy findings for the two groups of calves, there was the suggestion that an endotoxin identical to, or nearly identical to, Escherichia coli endo- toxin, was released into the animal tissues during the course of pas- teurellosis (PasteureZZa muZtocida). The detection of such an endotoxin in the blood by a bioassay method employing ll-day-old chick embryos was discussed. PHYSIOLOGIC AND PATHOLOGIC CHANGES IN CALVES GIVEN ESCHERICHIA COLI ENDOTOXIN OR PMSTEURELLA MULTOCIDA By Babiker El Hag Musa A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Surgery and Medicine 1971 66:73.35 ACKNOWLEDGMENTS The author wishes to express his appreciation and gratitude to. Dr. G. H. Conner of the Department of Large Animal Surgery and Medicine for his generous help, guidance, and constructive criticism in the, writing of this thesis. Special acknowledgment is made to Dr. G. R. Carter of the Depart- ment of Microbiology and Public Health for his help and encouragement in the bacteriologic and bioassay aspects of this project, and to Dr. Bhola N. Gupta of the Department of Large Animal Surgery and Medicine for his assistance in microscopic interpretation and photography. Sincerest feelings of gratitude are directed to Dr. K. K. Keahey of the Department of Pathology, and Dr. F. H. Oberst, Chairman of the Department of Large Animal Surgery and Medicine for providing facili- ties invaluable in the performance of the experimental work. The author is deeply indebted to Miss Roberta Milar and Miss Judy Lee for their help with the hematologic and biochemical determinations, and Dr. Esther Roege for her efforts in sectioning and staining the various tissues which made this project complete. Finally, I would like to thank the Research Committee of the University of Khartoum, Sudan, for making this study possible. 11 TABLE OF CONTENTS Page LITERATURE REVIEW I I I I I I I I I I I I I I I I I I I I I I I I I 1 Definition I I I I I I I I I I I I I I I I I I I I I I I I I 1 . Species Susceptibility to Endotoxin . . . . . . . . . . . . 1 General I I I I I I I I I I I I . I I I I I I I I I I I I I I 2 Endotoxin in Laboratory Animals. . . . . . . . . . . 2 Physical signs Hemodynamic and other tissue reactions to endotoxin Endotoxin in Large Animals . . . . . . . . . . . . . 5 Physical signs Hemodynamic and other tissue reactions to endotoxin Spec1f1c I I I I I I I I I I I I I I I I I I I I I I I I I I 9 Endotoxin from Eacherichia coZi. . . . . . . . . . . 9 Hemodynamic and other tissue reactions Mode of Action of Endotoxin . . . . . . . . . . . . . . . . 11 Lysosome Release Hypothesis. . . . . . . . . . . . . 11 Catecholamine Release Hypothesis . . . . . . . . . . 11 Histamine Release Hypothesis . . . . . . . . . . . . 12 Miscellaneous Less Popular Hypotheses. . . . . . . . 12 Antigen-antibody reaction Generalized cell poison, key cells poison or central nervous system effect Cessation of production of mucus Bioassay of Endotoxin . . . . . . . . . . . . . . . . . . . 13 PasteureZZa multocidh . . . . . . . . . . . . . . . . . . . 14 iii Page History. . . . . . . . . . . . . . . . . . . . . . . 15 Incidence. . . . . . . . . . . . . . . . . . . . . . 15 Physical Signs . . . . . . . . . . . . . . . . . . . 15 Hemodynamic and Other Tissue Reactions . . . . . . . 16 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . 18 Pasteurella multocidh Organism. . . . . . . . . . . . . . . 18 Escherichia coli Endotoxin. . . . . . . . . . . . . . . . . 18 Chick Embryo Assay. . . . . . . . . . . . . . . . . .'. . . 19 Experimental Animals. . . . . .g. . . . . . . . . . . . . . 19 Clinical Observations . . . . . . . . . . . . . . . . . . . 21 Necropsy and Histopathology . . . . . . . . . . . . . . . . 21 Bacteriologic Examination . . . . . . . . . . . . . . . . . 22 Hematologic Determinations. . . . . . . . . . . . . . . . . 22 Biochemical Determinations. . . . . . . . . . . . . . . . . 23 Sugar Determinations . . . . . . . . . . . . . . . . 23 Enzyme Determinations. . . . . . . . . . . . . . . . 23 Cholesterol Determination. . . . . . . . . . . . . . 23 Statistical Analysis. . . . . . . . . . . . . . . . . . . . 24. RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Physical Signs. . . . . . . . . . . . . . . . . . . . . . . 25 Pasteurella multocida, Group P . . . . . . . . . . . 25 Escherichia coli, Group E. . . . . . . . . . . . . . 25 Bacteriologic Examination of Jugular Blood. . . . s . . . . 29 Pasteurella multocida, Group P . . . . . . . . . . . 29 Escherichia coli Endotoxin, Group E. . . . . . . . . 29 Control Group C. . . . . . . . . . . . . . . . . . . 29 Necropsy Findings . . . . . . . . . . . . . . . . . . . . . 31 PasteureZZa muZtocida, Group P . . . . . . . . . . . 31_ iv Page Escherichia coli, Group E. . . . . . . . . . . . . . 31 Control Group C. . . . . . . . . . . . . . . . . . . 31 Bacteriologic Examination at Time of Necropsy . . . . . . . 31 Histopathologic Examination . . . . . . . . . . . . . . . . 33 Pasteurella multocida, Group P . . . . . . . . . . . 33 Escherichia coli, Group E. . . . . . . . . . . . . . 36 Hematologic Examination . . . . . . . . . . . . . . . . . . 39 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . 60 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62' MPENDIX I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 69 Table LIST OF TABLES Mean values for body temperature and heart rate for animals inoculated with Pasteurella multocida organisms (Group P) as compared to the control animal . . . . . . Mean values for body temperature and heart rate for animals receiving.Escherichia coZi endotoxin (Group E) as compared to the control animal . . . . . . . . . . . Mean hematologic and biochemical values for the various groups of animals during the period of experimentation. Hematologic and blood chemical values for animals in the Pasteurella multocida group (P) . . . . . . . . . . Hematologic and blood chemical values for animals in the endotoxin group (E) . . . . . . . . . . . . . . . . Summary of results of the chick embryo bioassays. . . . vi Page 27 3O 40 41 44 51 Figure 10 LIST OF FIGURES Prostration and twisting of the neck prior to death in PasteureZZa-inoculated calf . . . . . . . . . . . . Mean body temperature values for experimental calves . Subcapsular ecchymotic hemorrhage in the spleen of PasteureZZa-inoculated calf (dorsal view). . . . . . . Subcapsular petechial hemorrhage in the spleen of endotoxin-treated calf (dorsal view) . . . . . . . . . Photomicrograph of the lung of PasteureZZa-inoculated calf. Congestion and hemorrhage (arrow) in the a1v301i I H & E stain I x 190 I I I I I I I I I I I I I Photomicrograph of the lung of PasteureZZa-inoculated calf. Congestion of the blood vessels. H & E_stain.- X 1900 o a o a o o o a o o o o a o o o o o o o o '0 o o Photomicrograph of the lung of a calf 6 hours after endotoxin inoculation showing pneumonic areas. H & E stain. x 190 a o o o o o o o o o o o o o 'o o o o o o Photomicrograph of the lung of a calf 6 hours after- endotoxin inoculation. Notice the infiltration of serous fluid into the alveoli. H & E stain. x 190 . . Mean blood platelet counts for experimental calves . . Mean values for absolute neutrophil counts for experimental calves. . . . . . . . . . . . . . . . . . vii Page 26 28 32 32 34 35 37 38 47 49 LIST OF APPENDICES Appendix Page 1 Hematologic and blood chemistry data used for analy- sis of variance between Group P and Group E. . . . . . . 69 viii LITERATURE REVIEW Definition The present concept (Ribi et al., 1964) is that endotoxin is a form of a lipOpolysaccharide complex which could be released on disruption of bacterial cells or could be extracted from intact bacteria. Schalm (1965) reported that the endotoxin is located on or in the cell wall of gramrnegative bacteria and is released on autolysis. However, there is much difference of opinion among investigators as to what part of. this macromolecule is responsible for the biological activity. Knox and Bain (1960) isolated from Pasteurella multocida, type I, a polysaccharide fraction which is a biologically active endotoxin. Ribi et al. (1961) claimed that the biological activity is not related to the lipid frac- tion but to the large polysaccharide molecule. Osborne (1965), Schalm (1965), and Alaupovic et a1. (1968) related the toxin to a lipopolysac- charide-protein complex rather than to polysaccharide alone. Species Susceptibility to Endotoxin Berczi et al. (1966), testing the effect of Escherichia coli endo— toxin on 11 species of animals, found that there is a certain correlation of endotoxin sensitivity with the phylogenetic maturity of the species. Fish and frogs displayed an extreme resistance to endotoxin while the mammalian species displayed a wide range of sensitivity to endotoxin: for instance, calves were extremely seasiiivey-whereaa rats.and mice‘ were rather resistant. Cruchley et a1. (1967) observed that endotoxins 2 extracted from different sources have similar biological responses. These include toxicity, pyrogenicity, the ability to induce nonspecific resistance to infection, diarrhea, tumor necrosis and a local and general Shwartzman reaction. General Endotoxin in Laboratory Animals Extensive research has been performed on laboratory animals in con- trast to research on large animals. Consequently, much of our present knowledge about endotoxin was derived primarily from.the smaller animal species. Physical signs, A lot of variations exist between these species in clinical manifestations, but the current clinical concepts as given by Osborne (1965) include chill, biphasic fever, respiratory distress, depression, hypotension, cold extremities, anorexia, oliguria and, occasionally, anuria. Zahi and Hutner (1944) and Berry et aZ.-(1959) noticed hypothermia in mice following the administration of endotoxin. Berczi et a2. (1966) reported vomition in the cat 1 to 2 hours after endotoxin was administered as being characteristic. Sheagren et a1. (1967) mentioned that the Rhesus monkey during endotoxic shock may show hypothermia if left uncovered during the period of shock. Hemodynamic and other tissue reactions-to endotoxin. The-presence of disseminated vascular lesions in dogs,acats, rabbits, rats and mice, resulting in congestion, hemorrhage, thrombosis and necrosis in dif- ferent organs as well as changes in blood, were well documented by many investigators. Bennett and Beeson (1953) found leukopenia in rabbits following the administration of endotoxin and Thomas and Jones (1959) 3 discovered that endotoxin could cure leukemia in rats. Berenheimer and Schwartz (1964) examined 17 bacterial toxins for their capacity to dis— rupt rabbit leukocyte lysosomes and liver lysosomes. They found that there was a correlation between the hemolytic property of-a toxin and its capacity to disrupt lysosomes. Berry et al. (1959) determined blood sugar, liver and muscle glycogen, and total body carbohydrate in mice receiving Salmonella endotoxin. They found complete depletion of the carbohydrates. Hinshaw et al. (1960 and 1961) reported a rise in plasma histamine in the dog after injection of endotoxin. Cohn and Morse et al. (1960) noted that endotoxin stimulates the phagocytic activity of the leukocytes in rabbits. Kuida et al. (1961) found little variation in reactions to endotoxins in species such as the cat, rabbit, and monkey. Nykiel and Glaviano (1961) demonstrated in the dog that endotoxin stimu- lates the medulla of the adrenal gland and hence increases epinephrine in the adrenal venous blood. Palmerio et al. (1962) observed increased catecholamines in the blood of the rabbit as well as injury to the myocardiumnwhen a bacterial endotoxin was administered. Ushiba et al. (1962) found that gram-negative bacilli in the intestine of mice multi- plied rapidly following intraperitoneal injection of bacterial endo- toxin, while bacteria such as Lactobacilli,which normally exist in a larger number than gramrnegative bacilli, remained constant in number. Hinshaw and Nelson (1962) noted splanchnic pooling of blood following the injection of an endotoxin. Hemodynamic reactions have been studied in the dog more than in any other specieSwaccording to Alican (1962), who cited from the work of others the following responses to endotoxin; hemoconcentration, plasma loss, increased plasma hemoglobin, bloody diarrhea, hemorrhagic necrosis of the bowel mucosa, marked lymph flow in the intestine, and adrenal stimulation. Herring et al. (1963) 4 observed that circulating endotoxin in rabbits was distributed between plasma and platelets. Muller and Smith (1963) showed that there was a rise in hematocrit during endotoxic shock in the dog. They also noted an immediate fall in blood pressure, in cardiac output, and in central venous pressure. Petersdorf and Shulman (1964) found that injected- bacterial endotoxin in rabbits and dogs was taken by the reticuloendo- thelial system. Schaedler and Dubos (1964) reported that NCS mice, which were free of ordinary mouse pathogens as well as intestinal Escherichia coli, were highly resistant to lethal effects of endotoxin. Osborne (1965) observed that microembolism, thrombosis, pulmonary edema, ischemia, and anoxia of many organs, notably the kidneys and lungs, were among the most common findings in bacterial endotoxic shock. SchaLm (1965) gave the following sequence of events after administration of endotoxin: arteriolar constriction, fever, hypoglycemia, and leuk0penia followed by leukosis. Berczi et al. (1966) reported extensive edema and hemor- rhage in the cat after administration of endotoxin. -Bounous et al. (1966) observed that the intestinal mucous barrier was reduced during shock in the dog and that the intestine was.accessible to proteolytic digestion. Dellenback et al. (1966) noted that the dog's central blood volume decreased while splanchnic blood increased during endotoxic shock. Lemperle (1966) found that injected endotoxin in mice is apparently taken up almost exclusively by the reticuloendothelial system and that death occurs after failure of this system to remove circulating endotoxin. McKay et al. (1966) gave an excellent microscopic study of the effects of the bacterial endotoxin on the blood vascular system of rats. They found that the first change was agglutination and sequestration of plate- lets in the pulmonary capillaries associated with a sequestration of polymorphonuclear leukocytes. One hour after injection, some of the 5 leukocytes were disrupted and then discharged their cytoplasmic and nuclear components into the plasma.- Fibrin deposits appeared in the vicinity of platelets 4 hours later. They concluded that bacterial endo- toxin induces damage by triggering the clotting mechanism in viva. Tikoff et al. (1966) reported that the target organ for endotoxin in cats and sheep is the lung. The reaction of dog's liver to endotoxin was increased fibrillar material, then depletion of glycogen and pooling of blood (Boler and Bibighaus, 1967). Cortell and Conrad (1967) claimed that intestinal histology and the life span of mucosal cells is not changed by injecting Escherichia coli endotoxin in albino rats. Hinshaw et al. (1967) in their study with the coyote found a progressive increase in serum glutamic-oxaloacetic transaminase, constant blood urea nitrogen, hemoconcentration, systemic hypotension, depressed liver function, hepatic-renal and adrenal congestion, intestinal necrosis, and gall- bladder edema. The liver was the primary target organ. Levy et al. (1967) described the histologic changes in mouse liver after a single dose of endotoxin. Rebers et al. (1967) showed that mice and rabbits react similarly to Pasteurella multocida endotoxin. Granulocytopenia, followed by granulocytosis in the Rhesus monkey were noted by Sheagren et al. (1967). The chicken reacts to endotoxin by the development of yellowish, necrotic, and irregular foci of necrosis in the liver, as reported by Trauscott and Innis (1967). Endotoxin in Large Animals Little work has been done with endotoxin in large animals in con- trast to the extensive work in small laboratory animals. 6 Physical signs. Schalm (1965) found that following the administra- tion of endotoxin to many of the large animals there were evident responses characterized first by arteriolar constriction, fever, hypo- glycemia, and leukopenia followed by leukosis. With a lethal-dose, the animals became anorectic, progressively weak, ataxic, and highly feverish, with respiratory distress and blood-tinged foam at the nostrils. Vomi- tion, diarrhea, convulsions, and death in 24 hours represented the terminal stages. Carroll et al. (1965) indicated that the immunologic, phsyio— logic, and pathologic responses of animals to administration of endotoxin were the same regardless of the species of gramrnegative organism from which the endotoxin was obtained. Ruminants. Halmagyi et al. (1963) claimed that in sheep respiratory arrest is an important feature of endotoxic shock. Carroll et al. (1964) administered endotoxin into the udder of the cow and found the rectal temperature 7 hours postinoculation to be 106-107 F. Mullenax et al. (1966) reported a decrease in rumen motility, momentary apnea, and increased respiratory frequency in ruminants following the administration of endotoxin via the intravenous, subcutaneous, and intraruminal-routes. Horses. Schalm (1965) showed that after intraperitoneal injection of endotoxin in the horse, collapse resulted within an hour. Hemodynamic and other tissue reactions to endotoxin. Lillehei and Maclean (1959) observed that during endotoxic shock in man, there was plasma loss, increase in hematocrit, increase in plasma hemoglobin, and necrosis of the bowel. 7 Ruminants. Halmagyi et al. (1963) indicated that in sheep the lung is the main target organ and that the response to endotoxic shock consists mainly of a marked rise in pulmonary arterial pressure and a fall in both cardiac output and in systemic arterial pressure. Davis and Simbert (1964) described the events in swine after Escherichia coli endotoxin injection as follows: glucose level initially dr0pped, then increased, but finally decreased below preinoculation levels. There was also a decrease in the number of white blood cells. Carroll et al. (1964) induced maximal local response in lactating mammary glands of the cow by intramammary inoculation of Aerobacter aerogenes endotoxin. Local‘ edematous swelling was observed within 2 hours. After 3 hours, serum albumin was detected in milk and temporary hyp0proteinemia resulted. The number of leukocytes in milk peaked at 6 hours. Hansen (1964) investigated an outbreak of toxic liver injury in ruminants.’ He found that the glutamic-pyruvic transaminase test gave the least correlation with liver injury, while the glutamic-oxaloacetic transaminase test was, closely correlated, very sensitive, and may have been elevated before clinical symptOms were detectable. Schalm (1965) characterized anaphy- lactic shock in the cow by leukOpenia and increased packed cell volume. Tikoff and Kuida (1965) stated: '"Pulmonary vasoconstriction is a prominent feature of the hemodynamic responses to endotoxin in the calf." Mullenax et al. (1966) reported leukopenia in cattle in l to 4 hours, followed by leukocytosis about 24 hours after inoculation of an endotoxin prepared from rumen bacteria. They also found hyperglycemia in l.to 3 hours, followed by hypoglycemia at 5 to 7 hours, and a decrease in serum beta- globulin 2 to 3 days later. Rebers et al. (1967) isolated from virulent encapsulated Pasteurella multocida a lipOpolysaccharide-protein antigenic complex, which is a heat stable particulate. This, when injected in 8 fractional milligram amounts into mice, rabbits, and calves, produced toxic reactions which frequently resulted in death of the host. Surviving animals developed immunity to live organisms. In calves (13 to 18 weeks of age), after intravenous administration of this endotoxin, the sequence of events was described as: rapid shallow breathing within 5 to 15 minutes, depression, increased salivation, and lacrimation; 30 minutes later there was diarrhea which was mucoid and then watery. The rate of salivation was increased as the terminal phase of the shock approached and a nasal discharge appeared, followed by recumbency. One calf showed coma and died within 4 to 5 hours. They found also that in calves the signs following an injection of endotoxin resembled those which appear after challenge with the whole organism. Heddleston et al. (1967) reported that calves injected with Pasteurella multocida develOped septi- cemia and died in 24-48 hours. They stated that "If the clinical signs and death are the result of an endotoxin, than sufficient toxin was released from infected lungs to produce clinical manifesta- tions." Rhoades et al. (1967) studied the gross and the microsc0pic lesions resulting from acute infections and from administration of endotoxin of Pasteurella multocida in 3 calves and 2 pigs. Pasteurella multocida endotoxemia is characterized by widely distributed hemorrhages, edema, general hyperemia, and pneumonia. These lesions were similar to those detected at necropsy in calves infected with Pasteurella multccida organisms. The calves which received endotoxin intravenously died within 4-1/2 hours, while those injected with the whole organisms died within 40 hours. The lungs of the endotoxemic calves were markedly hyperemic, hemorrhagic, and edematous, and a moderate degree of alveolar emphysema existed.’ There was slight hyperemia of the digestive tract, liver, 9 spleen, and pancreas, and moderate hyperemia of the kidneys. A slight degree of cloudy swelling in the liver and interlobular edema in the pancreas were also observed. Lymph nodes were hyperemic and edematous. Horses. Rooney et al. (1963) reported a disease in the horse which they called Colitis "X". The etilogy of the disease was not clear, but its clinical and pathologic manifestations indicated that it was related to endotoxemia. Endotoxins prepared from several strains of Escherichia coli gave an identical disease syndrome when injected intravenously. Schalm (1965) described the effects of endotoxin in horses. Leukopenia was followed by an increased number of leukocytes. Packed cell volume initially rose, then drapped, only to increase again near completion of the experiment. Plasma protein concentration paralleled the packed cell volume. Petechial hemorrhages were observed in the lungs, heart, serosa of the gastrointestinal tract, mesentery, and cortex of the kidney. The small intestine was dilated and contained a yellowish material. Intestinal mucosa was congested and edematous, while the changes in the large intestine and cecum were not as marked. Carroll et al. (1965) indicated that the peracute form of endotoxemia in the horse resembles the general- ized Shwartzman reaction. Specific Endotoxin from Escherichia coli For many years, the endotoxin of Escherichia coli has been the pre- paration of choice in the study of the endotoxic shock in different species of animals. Extensive studies have been done with Escherichia coli endo- toxin in laboratory animals, but very little work has been done in labora- tory animals. Among those who used Escherichia coli endotoxin to 10 elucidate certain aspects of endotoxic shock are: Horwitz et al. (1962), studying the effect of endotoxin on platelets in rabbits; Harley and Johnson (1963), studying the clotting mechanism in endotoxic shock in dogs; Lawferd and White (1964), studying abnormal serum components after administration of Escherichia coli endotoxin in rats; Forbes (1965), studying induction of mitosis in macrOphages by endotoxin in mice; and Starzecki et al. (1967), studying distribution of endotoxin 51Cr after its administration in dogs. Hemodynamic and other tissue reactions. Tikoff et al. (1966) studied the hemodynamic effects of endotoxin from Escherichia coli in anesthetized calves, ranging in age from 2 to 4 weeks. The dose of endo— toxin administered was either 0.25 or 0.5 mg./kg. body weight, with most animals receiving the latter dose. Nine of 14 calves died 12 hours fol- lowing receipt of endotoxin and 2 more died later (2nd and 4th day). They found the most striking effects to be pulmonary arterial hyper- tension and systemic arterial hypotension. Hemoconcentration, hepato- splanchnic pooling, and metabolic acidosis were either absent or minimal. The lung was the major target organ. At the time of necropsy, they found mild generalized visceral congestion which was easily visible in the liver but minimal in the bowel. Small endocardial hemorrhages were also observed. The lung was edematous with widespread areas of hemorrhage and atelectasis. Microscopically, the lesions were severe capillary engorgement, focal edema, and hemorrhage. Also, edema fluid and blood were found in the alveoli and bronchioles. 'Aside from congestion, all other tissues did not show significant changes. 11 Mode of Action of Endotoxin The endotoxins, when administered in either animal or man, have a‘ very wide spectrum of biological activity. But not all of_the activities of endotoxin are related to each other. There are still many differences of opinion about the mode of action of the endotoxin. The following hypotheses seem to be quite well documented. Lysosome Release Hypothesis Weissmann and Thomas (1964) postulated that endotoxin affects the stability of the lysosomes with the subsequent release of acid hydrolase enzymes into cell sap or surrounding tissue, thus causing damage in the tissues. This gives rise to lactate, which lowers the intracellular pH and causes either the membrane of the lysosome or the autOphagic vacuoles to become fragile. This hypothesis links together many pieces of information presented by numerous investigators. Among these are: Berry et al. (1959), Nykiel and Galviano (1961), Atwood and Kass (1964), and Berry (1964). Catecholamine Release Hypothesis Fine and Minton (1966) observed that endotoxin is capable of acting directly on certain tissues like granulocytes and platelets. From the lethal effects of the endotoxin, they postulated that "The endotoxin increases the release of catechol- amine in splanchnic tissues produced by the splanchnic sympathetic nerves, and this results in loss of the integrity of blood vessels in the splanchnic area and hence the lethal effect is produced." This hypothesis also confirms scattered information presented by other investigators, among whom are: Thomas (1956), Nykiel and Glaviano (1961), and Palmerio et al. (1962). 12 Histamine Release Hypothesis Hinshaw et al. (1960 and 1961) observed an increase in blood hista— mine and in the histamine-histidine ratio (H/Hd). Also, the activity of histidine decarboxylase enzyme is increased. They then postulated that "histamine plays an active role in endotoxic shock." Miscellaneous Less Popular Hypotheses Antigen-antibody reaction. Suter (1964) stated that "there are some observations that some of the biological manifestations of.endotoxin action may be based on antigen—antibody reaction." However, Landy and weidanz (1964) found natural antibodies against a variety of gramrnegative organisms in the sera of different laboratory and large animals, which might play a part in the antigen-antibody reaction. Generalized cell poison, key cells poison, or central nervous system effect. Kass et al. (1964) stated that "The wide variety of recorded physiological effects suggests either that endotoxin is a generalized cellular poison or that it has widespread effects on key cells that are widely distributed throughout the body (such as the vascular endothelium) or that there is a pri— mary locus as in a major communications center such as the central nervous system that is specifically affected." This hypothesis also agrees with the work of Alper et al. (1967). Cessation of production of mucus. Bounous et al. (1966) observed that endotoxin administration results in cessation of the production of mucus by intestinal mucosa, and thus the intestine is accessible to proteolytic digestion. l3 Bioassay of Endotoxin Braude (1964) suggested that the bioassay of endotoxin in tissues or body fluid should be based either on its toxicity, i.e., its ability to produce fever, abortion, dermal necrosis, and death, or on its antie genic properties. He found also that the body fluids may react with-the endotoxin to reduce or accentuate their toxicity. The route of administra- tion affects absorption. The lethality depends also on the persistence of the endotoxin in the blood. Many investigators started looking at the possibility of using some laboratory animals for carrying out the bioassay of endotoxin. Smith and Thomas (1956) indicated that the inoculation of.chorioallantoic mem- brane of a 10-day-old chick embryo with endotoxin of a gramrnegative microorganism resulted in multiple hemorrhages and death of the embryo within a few hours. They noticed also that the susceptibility to the endotoxin was maximal in 10-day-old embryo, while younger or older embryos showed little or no response. The optimal inoculation tempera- ture for effect of endotoxin was 39.5 C. Hook et al. (1961) gave intra- venous injections of Escherichia coli endotoxin (0.111g.) to lS-day-old chick embryos and found extensive brain hemorrhages, hydrocephalus, and necrosis of some nerves. Injections of.a sublethal dose increased the- chick embryo's resistance. They concluded that the response of chicken embryo to bacterial endotoxin is determined by the route, dose, and age of the embryo. Finkelstein (1964) stated "The chick embryo may be regarded as essentially germ-free and immunologically virgin thus better in bioassay than animal hosts used." He indicated that death of the embryo occurs within a matter of hours in susceptible embryos and that the chick embryo becomes refractory to intravenous administration of endotoxin during the 11th to 15th day of 14 of incubation. The magnitude of change of susceptibility was greater than 10,000-fold. Berczi et al. (1966) found the lethal dose for a chick weighing 1 gram to be greater than 50 mg./kg. body weight.< Milner and Finkelstein (1966), comparing the bioassay of endotoxin in the chick to that in rabbits, concluded that the chick embryo test gives satisfactory reproducible results and is both cheaper and easier to perform than the pyrogen test in rabbits. Trauscott and Inniss (1967) explored the possi- bility of using chicks rather than embryos for the bioassay. They found that chicks generally demand a high dose of endotoxin, but 2dweek-old chicks were susceptible to endotoxin provided it was prepared by sodium chloride extraction. The test is considered positive when 2 to 3 yellow- ish, necrotic, and irregular foci are found on the liver of these birds. They noted also that meat strain chicks were more susceptible than egg strain chicks. Keiss et al. (1963) tested the potency of Pasteurella hemolytica endotoxin in sheep by using both the rabbit dermal test and the hemodynamic effect test. Spink and Starzecki (1967) performed a bioassay of endotoxin by what they called the rabbit-epinephrine-skin test which employs an injection of 5 to 6 ml. of plasma (to be tested) intravenously and simultaneously with the intradermal injection of epinephrine. The test is considered positive if an area of hemorrhagic necrosis of 20 mm. or more is observed within 24 hours at the site 'where the epinephrine was injected. Pasteurella Multccida The subject of_pasteurellosis in domestic animals has been extensively aatudied by many workers due to its economic implications in many countries. 15 History Bain (1963) mentioned that pasteurellosis of cattle was first described in 1878 by Bollinger, and the causative agent was isolated by Kitt in 1885. A German pathologist (Hueppe) was.the first to use the term hemorrhagic septicemia (HS). Incidence Hemorrhagic septicemia occurs in Southern Europe, including the U.S.S.R., North, Central, and East Africa, the Near East, and Southern and Southeast Asia. V Robert's (1947) serotype I or Carter's (1957) type B has caused hemorrhagic septicemia in bison in the U.S.A., and cultures of these still exist in various collections. Wilson and Miles (1964) identified the Pasteurella multocida organism as a small, gram-negative, ovoid bacillus showing bipolar stain- ing. The organism is aerobic and facultatively anaerobic, slightly ferments carbohydrate, is indol positive, but neither produces gas nor liquifies gelatin. The genus Pasteurella was.fu11y described by Merchant and Packer (1967). Carter (1967) has reviewed comprehensively the whole subject of pasteurellosis and hemorrhagic septicemia. Rhoades et al. (1967) induced hemorrhagic septicemia experimentally in 3 calves and 2 pigs. They reported widely distributed hemorrhage, edema, and general hyperemia. Physical Signs Blood and Henderson (1968) stated that the disease is characterized by a sudden onset of fever (106—107 F.), profuse salivation, severe depression, and death in 24 hours. Localization may occur in subcutaneous tissues resulting in hot, painful swellings about the throat, dewlap, l6 brisket, or perineum, and severe dyspnea if respiration is obstructed. In the.later stages of an outbreak of hemorrhagic septicemia there may be signs of pulmonary or alimentary involvement. Hemodynamic and Other Tissue Reactions Runnells et al. (1965) described pasteurellosis in cattle as an acute infectious disease caused by Pasteurella multocida and occasionally by Pasteurella hemclytica. These organisms are normal inhabitants of the bovine lung and are, on occasion, capable of invading the tissues and producing pneumonia. Usually the disease is associated with shipping of cattle, or the introduction of new cattle into a herd of animals. It is especially common in feedlot cattle. In most instances, Pas- teurella, by themselves,do not produce pneumonia unless associated with or preceded by a viral infection. On the other hand, the viral mani- festation by itself produces a very mild transitory disease. The prin- cipal lesions are those of an acute fibrinous pneumonia with generalized passive hyperemia, multiple hemorrhages in serous and mucous membranes, and degenerative alterations in the parenchymatous organs. In some animals, there is general edema due to passive hyperemia, hypoproteinemia, and shock. Smith and Jones (1966) stated that "Gross lesions in fatal cases of pasteurellosis are diverse and hardly specific. In cattle, losses following shipping ('shipping fever,‘ 'shipping pneumonia') are usually the result of pneumonia, consolidation of the lung being most frequent in the apical portions, less often extending to the diaphragmatic lobes. Interlobular edema may be present but is seldom prominent, nor are hemor- rhages outstanding in the bovine disease. Organisms are reportedly numerous in the pneumonic areas of‘ the lung . " Blood and Henderson (1968) described the lesions of hemorrhagic septi- cemia as: generalized petechial hemorrhage particularly under the l7 serosa and edema of the lungs and lymph nodes. Subcutaneous infiltra- tion of gelatinous fluid may be present in a few animals. Lesions of early pneumonia and hemorrhagic gastroenteritis are also seen. MATERIALS AND METHODS Pasteurella multccidb Organism Pasteurella multocida strain 656 was used as one of the experi- mental inocula. The original culture was isolated from a buffalo suf- fering from an-acute case of hemorrhagic septicemia in the Yellowstone National Park in 1922. The organism.was grown in brain heart infusion broth for 18 hours at 98.6 F. A laboratory mouse (Webster strain) was inoculated subcu- taneously with 0.1 m1. of the broth culture. Eighteen hours later in the agonal stage of the infection blood was drawn aseptically from the heart and 0.5 ml. was inoculated subcutaneously into a male white New Zealand rabbit. The latter died in about 20 hours; immediately the liver was exposed aseptically and a small portion was removed which ‘weighed 7.75 grams. It was cut into small pieces and then placed in a Ten Broeck tissue grinder and ground finely in human blood. Four milliliters of human blood (500 ml. blood in 40 ml. Alsever's solution) were used for each gram of liver substance. Immediately after grinding, 0.5 ml. portions were placed in small sterile test tubes and quickly frozen, using dry ice in alcohol. The frozen material was stored in a freezer (-75 F.) until used to inoculate animals in Group P. Escherichia coli Endotoxin Escherichia coli (E. coli) lipopolysaccharide (E. coli [026:B6J) was supplied by Difco Laboratories, Detroit, Michigan, U.S.A. It was 18 19 dissolved in sterile distilled water to make a final concentration of l mg./ml. and was stored at 37.4 F. (refrigerator) until used. Chick Embryo Assgy, White leghorn eggs were incubated in a humidified, self-turning incubator at a controlled temperature of 100.4 i_l.8 F. At 11 days, a large vein in the chorioallantoic membrane of the chick embryo was iden— tified by candling. A small triangular window was cut over the vein using a small carborundum disc. The soft shell membrane was then cleared by swabbing with sterile mineral oil, and an intravenous injection of: serum (0.1 ml.) to be tested was made using a 27-gauge needle. Eggs observed to bleed significantly from the injected vein were discarded. A.layer of melted paraffin was placed over the window and the eggs were returned to a non-turning tray in the incubator. Observations were con- ducted, by candling, at the let, 24th, and 40th hour postinoculation for death of the chick embryo. Prior to intravenous injection the serum 1was filtered through a 200 mu membrane filter. Routine bacteriologic examinations of dead embryos included cultures on blood agar and smears for detection of bacterial contamination. Five embryos were used per serum sample to be assayed. Chick embryo assays were conducted for ani- mals in Groups P and C. Experimental Animals Ten healthy Holstein bull calves, varying in age from 8 to 36 days and in weight from 64 to 140 pounds, were used. The animals were divided into 3 groups, P, E, and C, with the various ages and weights being represented in each group. 20 1. Group P was composed of 4 animals, P1, P3, P4, and P5. All the animals except P3 were inoculated subcutaneously with 1 ml. of rabbit liver material (described before) containing approximately 4.75-x4105 viable Pasteurella multccida strain 656 organisms. Animal P3 received. 0.5 m1. of the same material. Blood samples were drawn from animals P11 and P3 by jugular venipuncture using suitable needles and syringes. From animals P4 and PS, the blood samples were drawn through an indwelling polyethylene catheter which had been fixed in the jugular vein 1 day before dosing the animal. Blood clotting inside the catheter was pre- vented by filling it with sodium heparin solution after each blood col- lection. Before each blood collection, heparin solution was withdrawn and discarded. Physical observations and sample collections were made according to the following schedule: Animals P1 and P3: Every 12 hours until each died. Animals P4 and P At 6, 12, 14. 16, 18, 20, 22, 22., 25, 26, 5: 28, and 30 hours postinoculation. 2. Group E was composed of 4 animals, E1, E2, E3, and E4. They were all dosed intravenously with Escherichia coli endotoxin as follows: E1 injected at the rate of 0.030 mg./lb. body weight, total' dose being 3 mg. E2 injected at the rate of 0.036 mg./lb. body weight, total dose being 4 mg. E3 injected at the rate of 0.088 mg./1b. body weight, total dose being 8 mg. E4 injected at the rate of 0.057 mg./lb. body weight, total dose being 4 mg. 21 The blood samples from.the animals in this group were drawn through an indwelling polyethylene catheter which had been fixed in the jugular vein 1 day before dosing the animal. Blood clotting inside the catheter was prevented by filling it with sodium heparin solution after each blood collection. The heparin solution was withdrawn and discarded before each sample collection. Clinical observations and blood collections (postinoculation) for animals in Group E were as follows: at 5, 10, 15, 30, 45, 60 and 90 minutes, 3 hours, and at hourly intervals thereafter until the first signs of recovery. 3. Group C was the control group, composed of 2 animals, C1 and C2. Animal C1 was treated in all respects as animals in Group E, except for the endotoxin injection. Animal C2 was treated the same as animals P4 and P5 except that Pasteurella multocida organisms were not administered. Clinical Observations The clinical signs, rectal temperature, and heart rate, determined by a stethoscope over the heart area, were recorded in all the animals at the time of blood sampling. Necropsy and Hist0pathology_ Animals in Group C were euthanatized with an electric shock (110 voltage) at the termination of the experiment. Two animals in Group E, (E2, E4) were likewise euthanatized shortly after recovery from endotoxic shock. Animal E3 died as a result of the endotoxin. All animals in Group P died as a result of the Pasteurella infection. 22 A complete necropsy was conducted on all the animals except E1, and specimens from the brain, kidney, adrenals, liver, intestine, lymph nodes, and lungs were taken for histopathologic and bacteriologic exami- nations. The specimens for histopathologic examination were fixed in 10% buffered neutral formalin, paraffin* embedded, and cut at 6 u. The sections were stained with hematoxylin and eosin, as described by the Armed Forces Institute of Pathology (1960). Bacteriologic,Examination The-blood samples, drawn aseptically, and the specimens taken at necropsy referred to above, were subjected to routine bacteriologic examination. Blood was inoculated into diphasic blood culture bottles and organs and tissues were inoculated onto blood agar. Routine pro- cedures were employed for the identification of those bacteria isolated. Hematologic Determinations, Approximately 2.5 ml. of blood were collected in a small test tube containing 0.05 ml. of 7.5% tripotassiumethylenediaminetetraacetate. (KBEDTA) for the hematologic determinations. Total erythrocyte and total leukocyte counts were performed on an electronic cell counter.** Packed cell volume (PCV) was performed using the microhematocrit method. Hemoglobin (Hb.) was determined by the cyanmethemoglobin method using a Coleman Junior spectrophotometer. *Paraplast, Scientific Products, Evanston, Illinois. **Electronic Coulter Counter, Model A. 23 Platelets were counted using the method described by Schweizer and Gerarde (1964). Smears for differential leukocyte counts were made from the test tube blood soon after collection, promptly dried, stained with Wright's stain, and 100 cells differentiated. Biochemical Determinations About 20 ml. of blood were drawn at the time of each observation. This blood was placed in 2 test tubes and was centrifuged in a refrigerated IEC international centrifuge at 19 x 102 rpm for 20 to 30 minutes. The serum was then transferred, using a Pasteur pipette, to a plastic vial ‘which was stoppered and stored in a freezer (-76 F.) until the tests were run . Sugar Determination The sugar content of the serum was determined using the Folin-Wu ‘method (Folin and Wu, 1919). Enzyme Determinations Serum glutamic-oxaloacetic transaminase (SGOT) and_serum glutamic- pyruvic transaminase (SGPT) were determined using the standard method described by MW." Cholesterol Determination Serum cholesterol was determined by the Ferro and Ham modified pro- cedure (Ferro and Ham, 1960). *SIGMA Technical Bulletin No. 505, Reissued September, 1967. 24 Statistical Agalysis Where appropriate, the mean, the standard error of the mean, and, the level of significance were determined. Analysis of variance was conducted on the hematologic and biochemical findings of both Groups P and E. h. be 0‘ la: n,’ lcri RESULTS Physical Siggg_ Pasteurella multccidh, Group P Within 7 to 16 hours after inoculation, there were signs.of depres- sion accompanied by respiratory distress, salivation and infrequent nasal discharges. The degree of depression progressed continually throughout the course of the disease, to the point where the animals 'were unable to eat and remained recumbent 18 to 20 hours before death occurred. Painful swellings around the throat, dewlap, and the brisket developed about 24 to 26 hours postinoculation. During the terminal stages of the disease, there was mild diarrhea, the animal rested its ‘head more or less permanently on its shoulder, and the tongue, which became progressively bluish in color, partially protruded from the mouth (Figure l). The rectal temperature (F.) and the heart rate (per minute) are given in Table l and Figure 2. The temperature rose from 101.2 F. at 6 hours postinoculation to 104.4 F. at the 18th hour and remained within less than one degree of that peak until death. The heart rate was elevated throughout the course of the disease with peaks reached at the 14th, 24th, and 30th hours after inoculation. .Escherichiagccli, Group E Within 5 minutes after the injections of the endotoxin, all animals ‘were lying down and were experiencing difficulty in respiration. Animal 25 26 H-\.—a‘*_> ‘_7- _ _‘, Figure l. Prostration and twisting of the neck prior to death in Pasteurella—inoculated calf. 27 Table 1. Mean values for body temperature and heart rate for animals inoculated with Pasteurella multccida organisms (Group P) as compared to the control animal AL _. Time Group P 4 Control (02) (hrs.) Temp. (F.) Heart ratefmin. Temp. (F.) Heart rate/min. 6 102.8 79 101.2 68 12 103.8 86 101.2 68 14 104.1 96 102.0 76 16 104.3 76 101.2 68 18 104.4 84 101.2 68 20 104.1 ' 93 101.2 64 22 104.7 120 101.2 64 24 104.5 140 102.0 64 25 103.8 85 102.0 64 26 104.9 137 102.0 64 28 104.7 132 102.0 60 30 104.4 167 102.0 64 32 104.6 164 --- --_ l05.0 i04.5 l04.0 l03.5 l03.0 l02.5 O N O l0l.0 Body Tempe retu re (F) § U'I .§ 28 Group(P) *—‘ Group(E) '- ------ ‘ Control ‘-“‘ ,/\, /‘"“"‘/ ._._/ mm_~_/ 61(11ch I2 14 16 16 20 22 24 25 26 28 30 (hours) P Group5 lO i5 30 45 60 90 no lBO 240 300 360 (minutes) it) Post Inoculation Periods Fig.2, --- Mean Body Temperature Values for Experimental Calves. 29 E3 showed the most difficulty and, in the next 5 minutes, began to strug- gle and then died 15 minutes postinoculation. Respiratory distress con- tinued to progress in the other 3 animals and, in about 45 minutes after the endotoxin administration, the animals were "panting" with their tongues partially protruding. They frequently rested their heads on the shoulder region or on the floor. It was at approximately this time that soft feces were passed.’ These signs continued during the period of endotoxic shock, after which the character and rate of respiration gradually returned to a normal pattern.l Passing of feces ceased and the animal resumed an alert attitude.- For animals E1 and E4, recovery occurred at 6 hours postinoculation and, for animal 32, at 7 hours postinoculation. Body temperature and heart rate remained elevated throughout the course of shock (Table 2 and Figure 2). For each determination, there *was a sharp rise within the first 5 minutes. The highest readings were reached at 3 hours postinoculation and then, after 5 hours, they gradually declined. BacteriologIc Examination of Jugular Blood Pasteurella multccida, Group P Pasteurella multccida was recovered from the 6th hour after inocu- lation until all animals died. No other organisms were detected. Escherichia coli Endotoxin, Group E No organisms were isolated from the blood. Control Group C No organisms were isolated from the blood. 30 Table 2. Mean values for body temperature and heart rate for animals receiving Escherichia coli endotoxin (Group E) as compared to the control animal Time Group E 4, Control (C1) (min.) Temp. (F.) Heart rate/min. Temp. (F.) Heart rate7min. 5 102.7 61 100.6 96 10 103.1 67 100.6 96 15 103.0 69 100.6 96 30 103.0 60 . 100.6 96 45 103.4 60 100.6 96 60 103.5 72 100.6 96 90 103.4 94 100.6 96 120 103 . 5 110 100 . 6 96 180 103.6 124 101.7 60 240 103 . 6 112 101 . 7 60 300 102.9 110 101.2 60 360 102 .5 94 101. 2 76 31 Necropsy Findings Gross Lesions Pasteurella multocida, Group P. Petechial and ecchymotic hemorrhages were widely distributed throughout the pectoral and eternal muscles and on the outer surface of the trachea, thymus, pericardium, epicardium, endo- cardium, spleen (Figure 3), forestomach, and the mesentery. Congestion was evident in the lungs, small intestine, lymph nodes, the medulla of the kidneys, the adrenals, and the brain. The liver was pale: Edema was detected in the ventral portion of the neck and around the thoracic inlet. The ruminal contents were dry and both the_urinary bladder and gallbladder were full. Calf number P5 showed, in addition to these findings, hemorrhage in the tricuspid and bicuspid valves of the heart. Escherichia coli, Group E. The organs in the abdominal and thoracic cavities were congested in all the animals. In all calves, except E3, there was petechial hemorrhage on the outer surface of the trachea, lung, and the spleen (Figure 4). The adrenals were hemorrhagic. The trachea and the bronchi contained some frothy material. Both the urinary bladder and the gallbladder were full. Control Group C. No gross lesions were observed in the control animals. Bacteriologic Examination at Time of Necropsy From animals in Group P, Pasteurella multccida was isolated in pure culture from the liver, lung, spleen, kidney, and lymph nodes, but not from the bile or urine. Escherichia coli was isolated from.the intestines. 32 Figure 3. Subcapsular ecchymotic hemorrhage in the spleen of Pasteurella-inoculated calf (dorsal view). Figure 4. Subcapsular petechial hemorrhage in the spleen of endotoxin-treated calf (dorsal view). 33 From animals in Group E, no organisms were isolated from.the liver, spleen, kidneys, and lungs. Escherichia coli was isolated from the intestines. From animals in Group C, Escherichia coli was isolated from the intestines. No organisms were cultured from the liver, spleen, kidneys, or lungs. Histopathologic Examination Pasteurella multccida,,Group_P Examination of the lung revealed widespread hyperemia, edema, and interstitial pneumonia, which was characterized by proliferation of septal cells and infiltration with many lymphocytes and a few neutro- phils (Figures 5 and 6). The lumen of some of the bronchioles contained an inflammatory exudate. There was a mild degree of hemorrhage in the epicardium and the endocardium. In addition, there was myocardial hemorrhage in animal P1. The intestinal submucosa was edematous, the villi were hyperemic, and goblet cells were very few in number. The liver was hyperemic with a slight degree of cloudy swelling and fatty degeneration. There was hyaline degeneration of the capsule of the spleen along with subcapsular hemorrhage, hyperemia, and a mild depletion of Malpighian corpuscles. The lymph nodes were hyperemic. There was a moderate degree of cloudy swelling in the renal tubular epithelium.- 34 Figure 5. Photomicrograph of_the lung of Pasteurella-inoculated calf. Congestion and hemor- rhage (arrow) in the alveoli. H & E stain. x 190. 35 Figure 6. Photomicrograph of the lung of. Pasteurella-inoculated calf. Congestion of the blood vessels. H 8 E stain. x 190. re1 the tra Wei Pigh Will 7&23: ‘L. N: I '2‘. uqu 36 The dura mater of the brain was hemorrhagic and infiltrated with leukocytes and neutrophils. The cerebrum, medulla, and cerebellum revealed hyperemia and a slight degree of hemorrhage. Escherichia coli, Group E The lung parenchyma was hyperemic and infiltrated with neutrOphils, and the capillary bed of the lung contained numerous leukocytes, mainly neutrophils. Some of the alveoli contained erythrocytes, neutrOphils, and fibrin clots. The interlobular and intralobular tissues were edema- tous. A large portion of the bronchioles were collapsed, the tunica adventitia and tunica media of the blood vessels were frequently hemor- rhagic, and some thrombi were observed in them. The pleura of animal E4 was also hemorrhagic and infiltrated with neutrophils (Figures 7 and 8). The heart was moderately hyperemic. Examination of the intestine revealed a high degree of congestion in the submucosa and, in the villi, there was moderate congestion, occasional hemorrhage, edema, and infil- tration of neutrophils. The liver was slightly congested with a moderate degree of cloudy swelling and occasional hemorrhage. The spleen was hyperemic with subserosal hemorrhage, and the Mal- pighian corpuscles were surrounded by some neutrophils and macrophages. The rate of mitosis was increased in the lymph nodes, and the medulla was slightly hyperemic. There-was slight hyperemia, subcapsular edema, cloudy swelling, and vacuolar degeneration, especially in the cortical convoluted tubules of the kidney. The adrenal glands were moderately congested and slightly hemorrhagic. The pia mater, medulla, and the cerebellum of the brain 37 J . I ' .5, U"!- 3.15:». --___. ~ _ ---_ %“_~..—- —‘ Figure 7. Photomicrograph of the lung of a calf 6 hours after endotoxin inoculation showing pneumonic areas. H & E stain. x 190. 38 ~'- 9. .o ‘1 \. é a. o .2. r... o Notice the infil- tration of serous fluid into the alveoli. x 190. Photomicrograph of the lung of a calf Figure 8. 6 hours after endotoxin inoculation. 39 were slightly hyperemic. Small patches of hemorrhage were detected in the medulla and the cerebellum. Hematolggic Examination The mean and standard error of the mean values for the various hematologic studies (glucose, cholesterol, serum glutamic-oxaloacetic transaminase, and serum glutamic-pyruvic transaminase) for animals in Groups P, E, and C are presented in Tables 3, 4, and S-and Appendix 1. Erythrocyte counts for animals in Group P were essentially normal; however, the number of erythrocytes was elevated (P < 0.01) for animals in Group E throughout the shock period (Tables 4 and 5). The packed cell volume drapped (P < 0.01) in Group P during the first 16 hours and was also low on the 22nd and 25th hours (Table 4). In Group E the packed cell volume was elevated (P < 0.01) during the first 45 minutes of the shock period (Table 5). For mean corpuscular volumes there were no significant changes between control and inoculated animals. For animals in Group P, the mean hemoglobin values were signifi- cantly decreased (P < 0.01) from the mean value for the control animal (Table 4) during the first 14 postinoculation hours and periodically thereafter, whereas for Group E there was a significant increase (P < 0.01) over the value for the control animal (Table 5) during the first 45 minutes and at several readings thereafter. Starting with the 18th postinjection hour, there was a definite trend for the platelet count to decrease (P < 0.01) as the disease pro- gressed (Group P, Table 4 and Figure 9). A similar trend (P < 0.01) was noted for the endotoxin group (E) starting 60 minutes after dosing and lasting throughout the shock period (Table 5 and Figure 9). 40 Table 3. Mean hematologic and biochemical values for the various groups of animals during the period of experimentation Mean and Standard Error of the Mean Factors C1.(control) Group E ficz (control) Group P use at 106 m3 7.3:0.0 7.9:0.2** 9.8:0.9 9.2:0.3 PCV z 30.5:0.2 33.5:0.1** 44.2:0.5 38.2:0.5** MCVu3 41.4:0.4 42.2:0.4 42.2:0.1 40.6:0.5 Hb. gm.Z 9.5:0.0 10.5:0.0** 14.8:0.2 11.2:0.3** ucnc z 31.3:0.2 31.5:0.0 33.8:0.1 31.0:0.2** Platelet/m3 570454: 349895**: 729318: 428845»: 44920 28298 32550 70580 WBC/mm3 5529:818 2761:420** 6045:386 7370:796 Lymphocyte A 2175:317 2455:412 2866:255 2662:280 Neutrophil A 2878:504 2:36:62» 3004:200 4837:665** Eosinophil 0.0:0.0 30:9 5:? 111 0106636 .mg.z 113:7 100:13 107:7 135:11* Cholesterol mg./d1. 134.5:15 114.2:7 104:10 95:11 scor 52:5 80:5“ 52:4 65:6 son 17.1:8 14.1:1 8:1 18:1 A - Absolute number SGOT - Serum glutamic-oxaloacetic transaminase expressed as Sigma-Frankel units/m1. SGPT - Serum glutamic-pyruvic transaminase expressed as Sigma- Frankel units/ml. C1 - Control for endotoxin Group E. C2 - Control for PasteureZZa multocidh Group P. *Significant (P < 0.05). **Highly significant (P < 0.01). 41 Table 4. Hematologic and blood chemical values1 for animals in the Pasteurella multocidh group (P) Control Mean value of 12 house- cutive deter- mination for Hours After Injection of Orggnisms Factors each factor 6 12 14 16 mac x 105/m3 9.8:0.9 8.4:1 7.8:1 8.1:1 8.7:2 PCV Z 44.210.5 35.213 35.214 35.214 36.214 ucv u 3 42.4:0.1 39.0:2 41.0:9 40.3:2 40.0:3 Hb. gm.Z 14.8:0.2 10.1:1 10.3:1 10.3:1 12.0:1 MCHC z 33.6:0.1 31.2:0.4 32.4:0.7 32.0:0.8 31.3:1 Platelet/m3 729318: 722250: 795750: 702000: 645750: 32550 215250 416750 323000- 166750 WBC/mm3 6045: 5816: 10766-1 9133: 86661 386 1994 2730 I483 2602 Lymphocyte A 2866: 3126: 3187: 4371: 2458: 255 1226 639 1671 895 Neutrophil A 3004: 2655: 7525: 6870: 6208: 200 801 2059 967 1741 Eosinophil A 515 [12117 0.010 0.010 0.010 Glucose mg.Z 10217 17019 166133 173126 169124 Cholesterol mg./d1. 104:10 73:17 93:3 104:3 146-:31 $001 52:4 48:4 51:4 55:10 52:1 scp'r 8:1 13:2 15:3 13:2 10:3 Table 4--(Cont'd.) fhours After Injection of Ogganisms Factors 18 20 22 '24 25 R30 x 106/mm3 10.2:2 10.1:2 11.4:6 9.3:2 7.6:0.8 rev 2 40.8:5 41.5:5 37.7:9 39.6:4 36.1:3 ncv113 39.8:3 42.3:4 39.1:11 42.8:5 43.1:1 Kb. 38.2 12.5:2 10.8:3 11.6:3 11.7:1 10.1:1 none 2 31.5:7 30.9:1 30.2:2 31.4:0.1 30.8:0.5 Platelet/mm3 317500: 239400: 92500: 545000:’ 286000: 32500 45600 0.0 155000 104000 WBC/mm3 5183:_ 7783: 6025: 7850: 12800: 1708 4538 3875 2853 1200 Lymphocyte A 2458: 1679: 1783: 1219: 2915: 895 154 276 328 1395 Neutrophil A 62001 35031. 56501 48021: 49321 1741 1642 4011 4203~ 2357 Eosinophil A 0.0:0.0 0.0:0.0 0.0:0.0 0.0:0.0 o.0:0.0 Glucose mg.Z 97:27* 9710.6** 191:76 141:45 188112** Cholesterol mg./d1. 95:40 109:88 132:26 85118 130:13 scor 51:8 46:2 51:21 53:20 70:25 8091 14:2 16:4 12:9 17:3 22:8 Table 4--(Cont'd.) 43 L4 4 Hours After Injection of Organisms Factors 26 28 0 RBC x 105mm3 10.0:2 10.7:2 8.5:p.0 PCV z 39.1:3 41.0:3 38.213 ucv u 3 43.2:1 38.816 38.3:3 Hb. gm.z 11.2:1. 13.1:2 10.1:0.0 MCHC z 30.7:0.6 w 31.3:0.0 28.910.0- P1atelet/mm3 207000: 163000: 430000: 183000 0.0 0.0 mac/um3 7150: 3075: 4200: 2741 425 0.0 Lymphocyte A 30201 16721 24701 1327 587 0.0 Neutrophil A 40601 14101 16301 1776 995 0.0 Eosinophil A 0.010.0 0.010.0 0.010.0 Glucose mg.2 128119 76148* 80152 Cholesterol mg./d1. 82119 53131 13219.0 sco'r 99:48 120:75 8310.0 scpr 28:8 30:14 20:0.0 A - Absolute number. SGOT - Serum glutamic—oxaloacetic transaminase expressed as Sigma-Frankel units/m1. SGPT - Serum glutamic-pyruvic transaminase expressed as Sigma-. Frankel units/m1. *Significant (P < 0.05). **High1y significant (P < 0.01). 1Expressed as the mean value and standard error of the mean. 44 Table 5. Hematologic and blood chemical values1 for animals in the endotoxin group (E) Factors Control Mean value of 12 conse- cutive deter- mination for each factor Minutes After Injectionlof Endotoxin 5 10 15 30 RBC x 106/mm3 PCV Z MCV 03 Hb. gm.2 MCHC z Platelet/mm3 mac/m3 Lymphocyte A Neutrophil A Eosinophil A Glucose mg.Z Cholesterol mg./d1. SGOT SGPT 7.3:0.0 30.5:0.2 41.4:0.4 9.5:0.o 31.3:0.2 570454: 44920 5220: 818 2175: 217 2878:_ 504 0.0:0.0 113:7 134:15 52:; 17:8 8.211 7.9:0.8 8.1:0.6 5.6:2 34:1 33.6:0.4 33.6:0.5 34.3:0.4 43.1:4 43.6:4 42.1:3 44.2:5 10.6:0.3 10.7:0.2 10.3:0.1 10.2:0.1 31.3:0.5 31.9:0.3 31.4:0.1 31.3:0.7 545000:_ 471250: 452500:_ 400000: 60449 88867 100778 108282 6212+ 4025+ 3887+ 3250+ 1303' 818' 444’ 236‘ 5630:_ 36331. 3729:_ 3153: 1120 698 358 262 582: 373: 145: 81: 238 129 92 40 0.0:0.0 68:35 17:17 16:16 109:11 156:47 180:58 170:41 121:14 100:21 121:24 133:25 59:8 72:11 63:4 66:19 12:0.4 10.7:1 12:1 11:2 Table 5—(Cont'd.) Minutes After Injection of Endotoxin Factors 60 90 120 180 mac 1: 106/mm3 8.1:1 7.7:1 7.9:1 8.6:1 Pcvz 33.1:0.6 33.1:0.1 33.3:0.1 34.3:0.7 ucv u 3 42 .2:6 44.4:5 43.5:6 40.9:4 Hb. 3111.2 10.3:0.2 10.2:0.2 10.6:0.3 10.3:0.1 14080 x 31.3:0.1 31.4:1 31.610.5 31.7:0.7 Platelet/m3 326666*1 358333*1 338333*1 313333*: 60850 40034 32446 26822 NBC/m3 27161 24831 1600: 1366: 356 224 702 667 Lymphocyte A 25271 22501 14921 12841 410 159 682 627 Neutrophil A 1811 ~ 2151 871 831 57 74 0.5 40 Eosinophil A 2510.0 5610.0 15115 0.0-10.0 Glucose 613.: 99:16 127:0.0 76:10 52:19* Cholesterol 155146 7410 . 0 108127 105133 mg./dl. $001 64:11 99:0.0 82:15 77:5 3011 15:4 20:0.0 15:2 11:0.0 Table 5--(Cont'd.) 46 Minutes After Injection of Endotoxin Factors 240 300 360 RBC x 106/mm3 8.7:0.7 8.110.? 8.210.9 PCV z 33.3:0.7 33.3:1 32.5:1- MCV113 38.214 41.413 40.1:4 Hb. gm.z 10.0:0.5 10.3:0.5 10.3:0.4 14080 x 31.5:0.7 3110.6 31.7:0.4 P1ate1et/mm3 231666*0: 255000*8:_ 231666*:: 32188 35000 31797 WEC/mm3 1483: 1216: 1833: 712 669 845 Lymphocyte A 12321 7101 10631 645 318 313 Neutrophil A 1821_ 4831 7521 55 358 550 EosinOphil A 41123 13112 12112 Glucose mg.Z 62115* 6212* 6413* Cholesterol mg./dl. 130111 78117 104133 3001 93:13 92118 128:30* SGPT 15:2 18:2* 14:3 A - Absolute number. SGOT - Serum glutamic-oxaloacetic transaminase expressed as SigmarFrankel units/m1. SGPT - Serum glutamic-pyruvic transaminase expressed as Sigma- Frankel units/m1. *Significant (P < 0.05). **Highly significant (P < 0.01). 1 Expressed as the mean value and standard error of the mean. Platelets x l03/m3 I000 8 300 200 l00 47 Group P) '—* Group E) .' """" ° Control ""'"" ./ ./ / Group 6 (P) Group 5 (E) l2 l4 l6 l8 20 no IS 30 45 60 22 24 25 26 28 30 (hours) 90 I20 l80 240 300 360 (nlnutes) Post Inoculation Perlods Fig. q--- Mean Blood Platelet Counts for Experlnontal Calves. 48 The total leukocytic counts for Group P were more or less increased during the first 16 hours following inoculation and decreased thereafter (Table 4), but a significant reduction in the leukocytic counts (P < 0.01) was observed in Group E (Table 5). This reduction in leukocytes was observed about 60 minutes following the administration of the endotoxin and persisted throughout the period of shock. The total absolute numbers of lymphocytes for Group P were somewhat reduced during the middle phase and near the terminal stage of the‘ disease, while in Group E the lymphocytes were elevated during the first 90 minutes following dosing of the animals but were reduced thereafter. There was nearly a complete absence of eosinophils throughout the period of the disease in Group P, but their level remained fluctuating following endotoxin injections (Tables 4 and 5). 4 The differential leukocytic count revealed a significant neutro- philia (P < 0.01) between the 12th and 25th postinjection hours in Group P (Table 4 and Figure 10). Within the first 15 minutes of the shock period, neutropenia was evident in Group E. Neutropenia persisted in_this group throughout the shock period (Table 5 and Figure 10). Blood glucose levels were significantly higher (P < 0.05) than- those for the control in Group P during the first 16 hours and between the 22nd and 25th hours. The amount of glucose in the blood was very low just prior to death (Table 4). An elevation in blood glucose was observed between 10 and 30 minutes; thereafter, a significant reduction (P < 0.05) in blood glucose was Observed to begin 3 hours following endotoxin injection (Group E, Table 5) and to continue until the end of the shock period. Cholesterol levels in both Groups P and E indicated periodic fluc- tuations (Tables 4 and 5). However, when compared with the control 49 Group(P) """‘ Group(E) 0'"---4 Control ~—-v-v l0000 8000 6000 4000 CONTROL 2000 :F "‘ IOOO E J! “'800 3 x .3 , 600 :3 ea /' \\ (I \“ 200 - "xx. .54. .- '1 \‘-. "I \..-' \“‘ .“.. "‘-" 7. ------ ' Group 6 l2 l4 l6 I8 20 22 24 25 26 28 30 (hours) (P) 6201):.) 5 l0 l 5 30 ‘05 60 90 I20 '80 2“ 300 E 360 (nlnutes) Post Inoculatlon Perlede Flg.|O--- Mean Value: for Absolute Neutrophll Counts for Experimental Calves: 50 animals, levels of cholesterol were especially lower at the beginning and near the end of the experiment. There was no significant change in the serum level of the enzyme glutamic-oxaloacetic transaminase in Group P. In Group E the enzyme was increased in the blood (P < 0.01) starting 1.5 hours following administration of the endotoxin until the end of the shock period. The glutamic-pyruvic transaminase was significantly elevated in the blood of animals in Group P (P < 0.01) from the 25th hour postinocula- tion until the animals died; but in Group E the enzyme showed a transi- tory rise (P < 0.05) at the 5th hour. In comparing all the parameters for the Pasteurella multocida group and the Escherichia coli endotoxin group, an analysis of variance indicated that there was no significant difference between the 2 groups, except in a very few instances which are indicated by asterisks (Appendix 1). The results of the chick embryo bioassays are given in Table 6. Sera collected from the PasteureZZa group at the 6th and 32nd postinocu- lation_hours, as well as sera from the control animals, were not lethal to chick embryos. Sera collected 26 hours postinoculation resulted in the death of only 1 of the 5 embryos. .nunov.wo.omooo on» venouwmooo one: noana .mamaoowuo afieooaN3E1uNNehxoem§m nous pounnflaeuooo monuments .uooawueeuo unu mo ooaueflofiow hound .maouuooo.onu no undo on» ad .uo Headed ecu mo ooaoeflooooa nuance .mamanewuo.nfimooaN3£1GNNmfiamamum saga voueHouooH managed mums mm vow .em .Hm .manaaoo Houuooo.o:u ouoz No one Ho 51 H o e H o 3 H o e ..ue Nm no N «cm o N . N H u n u .6: Na mm H «4H m H H m H H m .6: on me e H o w, H o o H o .6: oN mm H H m H o o H o e .0; o mm H H N H H m H H m .6: oN cm H o .o H o o H o e .0: c He N o 1m N o m N o m .6: Nm No N o N N o n N o m .oHa m Ho 7 v0” Hu Uflmn— was”: vmflfiu VGOQ Ohm—"H4 Gun flu UQMQ 0>HH< «5 mafia... Hfin—E—HZ lagoons Ioaonua . - teenage . coauooaaou Hesac< oe , 6N ‘ HN oHeaom omwuoim on» no coauoaaoocH noum< muoom mhummoofin choose xUHno can no muaomou mo muqaaum .o manna DISCUSSION The physical signs shown by the calves inoculated with Pasteurella multocida organisms, Group P, were nearly identical to those described by Blood and Henderson (1968) for bovine pasteurellosis. The signs observed in the group injected with Eacherichia coli endotoxin, Group E, were more or less similar to those in Group P, but they differed in the time of onset and severity. Both groups had elevated rectal temperatures, depression, respira- tory distress, alimentary involvement, and recumbency. These signs, except for the alimentary involvement, were seen 7 to 16 hours after inoculation for calves in Group P, and 5 minutes after injection for calves in Group E. Diarrhea was observed in Group P during the terminal stages of the disease, while in Group E it was observed 45 minutes after injection and was not as severe as described by Schalm (1965). The severity of diarrhea (Group E) may be dependent upon amount of endotoxin given. Animals in Group E did not have the local painful swellings as seen for Group P. This might be due to the relatively short period of stress characteristic of endotoxic shock. The heart rate decreased during the first hour following administration of endotoxin (Group E). Whether this was due to a direct effect of endotoxin on the cardiovascular system or was mediated through other substances, like catecholamines (Fine and Minton, 1966), is not certain. Higher thermal reactions were attained for animals in Group P than for those in Group E. It is probable that the thermal reaction is dependent upon the dose of 52 53 endotoxin.- The physical signs observed in the calves during endotoxic shock differed from those described in small laboratory animals inasmuch as there was no biphasic fever (Osborne, 1965), no hypothermia (zahi and Hutner, 1944; Berry et al., 1959; Sheagren et al., 1967), and no vomition (Berczi et al., 1966). Pasteurella multocida organisms were recovered from the blood of calves in Group P from about 6 hours after inoculation until death occurred. At necropsy, PasteureZZa multocida‘was present in the liver, lung, spleen, kidney, and lymph nodes, but not in the bile or urine. Escherichia coli was isolated from the intestine.‘ No organisms were found in the blood of calves that received endotoxin, and cultures after necropsy did not reveal the presence of bacteria from the liver, spleen, kidneys, and lungs.- Coliform organisms were isolated from the intestine of all calves in Group E. It is known that Escherichia coli organisms are normal inhabitants of the digestive tract of calves. Usually these organisms multiply rapidly in the digestive tract of mice following endotoxin administration (Ushiba et al., 1962). Probably Escherichia coli organisms in the digestive tract play an important~ role in aggravating the existing endotoxic shock, as it has been found that mice free from ordinary pathogens as well as Escherichia coli were highly resistant to the lethal effects of endotoxin (Schaedler and Dubos, 1964). The gross lesions observed in calves of both Group.P and Group E included congestion and widespread hemorrhage of visceral organs. The 'hemorrhage was in general more widespread in Group P than in Group E. The edema detected in the ventral portion of the neck and around the thoracic inlet (Group P) was absent from animals in Group E. It is 54 possible that the lack of edema was due to the relatively short period of shock for animals in Group E. Microscopic examination indicated that for both groups, the lung had the most prominent changes. Pulmonary lesions in Group P calves. were widespread hyperemia, edema, and interstitial pneumonia, while for Group E calves they were hyperemia, leukocytic infiltration, petechial hemorrhage, edema, and thrombosis. In both groups there were varying degrees of petechial hemorrhage and hyperemia in the heart, intestine, liver, spleen, lymph nodes, and the brain. Early cellular degeneration was also detected in the liver and the kidney of both groups. Histopathologic findings in Group P animals indicated that this organism was capable of producing pneumonia somewhat similar to pee— teurellosis described by Runnells et al. (1965) and Smith and Jones (1966). However, the lung was also edematous, a finding which was a prominent feature of hemorrhagic septicemia as reported by Blood and Henderson (1968). Those in Group E were in partial agreement with Tikoff.et al. (1966), in that the lung was the major target organ after administration of Escherichia coli endotoxin. In this respect calves. are similar to sheep (Halmagyi at al., 1963), cats (Tikoff et al., 1966), and most small laboratory animals (Osborne, 1965) but differ from dogs (Alican, 1962), chickens (Trauscott and Inniss, 1967), and coyotes (Hinshaw et al., 1967). The gross and microscopic lesions (Group E) observed in this study were similar to those described by Rhoades et.aZ. (1967) in calves following Pasteurella multocida endo- toxin injections. This striking similarity suggests that endotoxin .from.PasteureZZa multocida organisms or Escherichia coli organisms behaves similarly in the body systems. Such a suggestion confirms the earlier observation of Carroll et al. (1965) that the pathologic 55 responses of animals to administration of endotoxin were the same regard- less of the species of gramrnegative organisms from which the_endotoxin is obtained. Furthermore, the histopathologic findings for both groups in the present study were more or less similar suggesting that the fatal outcome in calves with hemorrhagic septicemia might be due to an endo- toxin released sometime during the course of the disease. Although the lung was the major target organ in both groups, other important organs. (for example, the brain, liver, kidney, and spleen) were also involved and might contribute to the final outcome of hemorrhagic.septicemia or endotoxic shock. Published reports for erythrocytic counts and indexes, for normal young calves, usually show a marked disparity (Holman, 1956; Greatorex, 1954 and 1957). To minimize the day—to—day variations in blood counts, and possibly in other determinations, the figures reported herein for each of the 2 control animals (Tables 3, 4, and 5) represent an average (mean) of 12 consecutive observations made on samples collected through- out the course of the experiment. The red cell count did not change in Group P but increased follow- ing administration of Escherichia coli endotoxin. Whether endotoxin has a stimulating effect on the erythropoietic system is not known, nor is there an explanation for the mechanisms involved. The packed cell volume dropped in Group P during the first 16 hours. As the disease progressed, there was a mild degree of hemoconcentration and a rise in the packed cell volume. This might be due to the slowly developing edema in the subcutaneous tissues of the neck region. In Group E there was an initial rise in the packed cell volume, a finding in.common agreement with that of Tikoff et al. (1966). In this respect endotoxemic calves were similar to endotoxemic horses (Schalm, 1965), 56 cows (Schalm, 1965), man (Lillehei and Maclean, 1959), and dogs (Alican, 1962; Muller and Smith, 1963). The mean corpuscular volume was not altered following injection of either Pasteurella multocida organisms or Escherichia coli endotoxin. No explanation is offered for the initial drop in hemoglobin concen- tration (Group P). Animals in Group E had an initial rise in hemoglobin. In this respect, endotoxemic calves were similar to endotoxemic man (Lillehei and MacLean, 1959) and dogs (Alican, 1962). The mean corpuscu- lar hemoglobin, on the other hand, was slightly increased in Group P, particularly during the terminal stages of the disease, while it remained unchanged in Group E throughout the shock period.' The rapid decrease in numbers of blood platelets following Pas- teureZZa multocida inoculation or Escherichia coli endotoxin administra- tion may be a result of platelet sequestration in the capillaries of some of the body's organs (McKay et al., 1966), or it might be a result of other mechanisms. However, the widespread hemorrhage observed in both groups (P and E) probably is due to injury of vascular endothelium (Kass et al., 1964), coupled with failure in the-clotting mechanisms as a result of the removal of platelets from the circulation. The total leukocytic counts of the calves in Group P were initially elevated but, as the disease progressed, they were decreased. Calves in Group E showed a reduction in the leukocytic counts 1 hour following the administration of the endotoxin and persisting until the end of the shock period. The differential leukocytic count revealed a reduction in the lymphocytic counts of the animals in Group P and-E during the terminal stages of either hemorrhagic septicemia or Escherichia coli endotoxic shock. 57 The neutrophil counts of the calves in Group P followed more or less the same pattern as observed with the total leukocytic counts of these animals. The neutrOpenia detected in Group E, in contrast to that observed in Group P, persisted throughout the shock period and was accompanied by a concurrent leukopenia. Endotoxemic calves in this respect were similar to endotoxemic cows (Schalm, 1965; Mullenax et al.. 1966), horses (Schalm, 1965), and rabbits (Bennett and Beeson, 1953). Eosinophils were almost absent in calves with hemorrhagic septicemia, but they were present, in normal numbers, during endotoxic shock. If eosinophils_played a role in either of the 2 conditions, it was not- reflected by any significant changes in numbers circulating in peripheral blood. Their absence during PasteureZZa infection is perhaps noteworthy. The normal level of blood glucose in young calves is more or less similar to that of the monogastric animals and represents the main source of energy during calfhood. However, following inoculation of PasteureZZa multccida organisms, the level of glucose was observed to increase ini- tially, but during the second half of the disease period, blood glucose values were somewhat reduced. Since the animals were unable to feed during this time, the source of glucose may have been endogenous or from the body reserves; hence, it is probable that the terminal drop in. the blood glucose observed in Group P may be.due to lack of mobilization of these reserves or to their depletion. In Group E a drop in blood glucose was detected during the terminal stage of the shock period. Endotoxemic calves in this respect were similar to endotoxemicmice (Berry et al., 1959) and dogs (Boler and Bibighaus, 1967). It is known that cholesterol is the precursor of the steroid com- pounds produced in the animal's body. These steroid compounds include a variety of substances, such as vitamin D, gonadal hormones, and 58 adrenal cortical hormones. In the present study, the levels of choles- terol were initially and terminally lower (in both Groups P and E), than_ the control levels. This might be due to a depressed steroidogenesis or rapid removal of cholesterol from the circulation following either of the 2 treatments adopted. Glutamic-oxaloacetic transaminase (SGOT) enzyme is widely distributed in many tissues of normal animals, being especially abundant in the‘ liver, myocardium, and skeletal muscles. When these tissues are damaged, the enzymes diffuse across the damaged cell wall and enter the blood stream. Glutamic-pyruvic transaminase is not as widely distributed in normal calves as is SGOT. The blood of healthy animals usually contains minimal amounts of these enzymes. The level of SGOT was not changed in Group P but was elevated in Group E from 1.5 hours after endotoxin administration until the end of the shock period. Serum glutamic- pyruvic transaminase was elevated in Group P during the last third of the disease but showed only a transitory rise in Grouplion the 5th hour following the administration of the endotoxin. These results indicate that injury accompanying PasteureZZa multocidh infection characteris- tically elevates levels of serum glutamic-pyruvic transaminase while injury due to Escherichia coli endotoxin characteristically elevated levels of serum glutamic-oxaloacetic transaminase. Hence, endotoxemic calves were similar to endotoxemia in other ruminants (Hansen, 1964) and the coyote (Hinshaw et al., 1967). There were a few significant differences between Group P and Group E in total leukocyte, neutrophil, or lymphocyte counts, and-in blood glucose concentrations. The significant differences observed in leuko- cytes, neutrOphils, and in lymphocytes between the 2 groups were accounted for by a more marked neutropenia and initially elevated lymphocytes in 59 Group E than in Group P. The preinjection levels of blood glucose were higher in Group E than in Group P. This may, in part, account for the few significant differences between the 2 groups during the course of experimentation. Apart from these, all the other hematologic findings, as well as glucose, cholesterol, serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase levels, were not significantly different between Groups P and E, indicating that physiological responses were more or less similar, following either Pasteurella Multccida inoculation or Escherichia coli endotoxin injection.- Further, this may indicate that during the course of hemorrhagic-septicemia, an endotoxin similar to Escherichia coli endotoxin may be released. No lethal effect was produced in the ll-day-old chick embryos fol- lowing intravenous administration of serum from either the control animals or.from Group P animals. Hook et al. (1961) and Berczi et al. (1966) described the lethal dose of endotoxin for the chick embryo, which may be higher than the amount of endotoxin present in the 0.1 ml. of serum from animals in Group P, if such an endotoxin was present.‘ On the other hand, the chick embryo might be refractory to intravenous administration of the endotoxin at this particular age (Smith and Thomas, 1956; Finkelstein, 1964). It is apparent that in performing chick embryo bioassays for endo- toxin, it would be advisable to concentrate the amount of serum.that can be inoculated or possibly to inject these embryos at other ages which might be more suitable. SUMMARY AND CONCLUSIONS 1. Inoculation of PasteureZZa multccida organisms or Escherichia coli endotoxin into experimental animals gave the same physiologic and- pathologic responses. This possibly indicates that in hemorrhagic septicemia an endotoxin similar to Escherichia coli endotoxin is released. 2. Although the lung had the most prominent changes during either PasteureZZa muZtocida infection or Escherichia coli endotoxemia, other important organs in the body were affected as well. 'Hence, the fatal~ outcome following either treatment may be due to a smm of multiple factors rather_than a single one. 3. The primary effect of Escherichia coli endotoxin seems'to be on the blood platelets, neutrOphils, and the vascular system. This may be a direct effect of the lipOpolysaccharide or may be mediated through other substances and systems. 4. The marked reduction in number of blood platelets may be an important factor in the widespread hemorrhage seen in both hemorrhagic septicemia and Escherichia coli endotoxemia. 5. Hypoglycemia was observed during the terminal stages of either Pasteurella multocida infectiOn or Escherichia coli endotoxemia. 6. Blood cholesterol levels were initially and terminally lowered in inoculated animals. 7. Tissue injury accompanying PasteureZZa multocidh infection characteristically elevated levels of the serum glutamic-pyruvic 6O 61 transaminase, while injury accompanying Escherichia coli endotoxin char- acteristically elevated the level of serum glutamic-oxaloacetic, transaminase. 8. The bioassay for endotoxin using chick embryos produced negative results for animals in the hemorrhagic septicemia group. Suggested improvements in this test include a method for concentration of the; endotoxin in serum or the injection of chick embryos at ages that might be more sensitive than at 11 days. REFERENCES REFERENCES Alaupovic, P., Solomon, L. A., Olson, A. 0., Jordan, M5 M., and Hinshaw, L. 8.: Relationship of Chemical Structure to Hemodynamic Proper- ties of Endotoxins. Proc. Soc. Exp. Biol. Med. N.Y., 127, (1968): 253-260. Alican, F.: Pathophysiology of Endotoxin Shock. Am. J. Med. Sci., 244, (1962): 237-257. 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Weissmann, G., and Thomas, L.: On a Mechanism of Tissue Damage by Bac- terial Endotoxin, in Bacterial Endotoxins, Landy, M., and Braun, W., editors. Rutgers, The State University, New Brunswick, N.J., 1964. Wilson, G. S., and Miles, A. A.: Topley and Wilson’s Principles of' Bacteriology and Inmunity, Vol. I, 5th ed., The Williams and Wilkins Co., Baltimore, 1964. Zahi, P. A., and Hutner, H.: Temperature Factors in the Action of Cer- tain Bacterial Endotoxin. Proc. Soc. Exp. Biol. Med. N.Y., 56, (1944): 156-158. APPENDIX 69 NuoH mum HHNH NHwH HHoH nHeH e.omNH NHWH Boom NHHoo HHNm ouwo cauwm HHHHN omen pawn euwo soon mwanH HnuooH oNHoNH onuon Hmfloo HHHon eHHoNH NdwNN .Ho\.Ne. Honoumoaonu HeuoNH NHHNNH mnHNNH oNHoNH Neume mnHooH ccHHuooH NuoNH N.oa ooooeHo NHHNH o.ouo.o NHHNH o.ouo.o oHHNH o.on5.o o.ouo.o «HHWH « HHeoooHocu coeuoo,,NoNHuoeNo eanHWoH eoNHoNoo ccoNHHwNm mmoNHonN temNuNNm NoouwmoN 4 HHoooooooz NoNHNNHn moouomoN onnHonNn NNoHuNNnc enouwmom omoHoNHm NNNHHHmom HNHHHoNHn <.ooeooeoaNH NNNHoon‘ NocNuoooo ooouwoom oNHnHwnHo «NHonNoo omNNHooNoH momHHNHNo oooHHonn naa\um= .mmNmoH .meooN meooH chNNn nieces .pmNoHo noooeo .mmNnHN +oooooo +¢mNmoo +eonNmo +oooNoN +omNHNo +onNmoN +ooonom +¢mNNNN naa\ooHoooHe N.ean HHHN H.oan N.QHNN n.eHNm N.QHNN n.oHHm o.eHHm .N can: N.QHHH HHHH‘ H-ouHH HHoH H.ouHH HHoH n.QHHH HHoH . N.:» .om oueo «Hon nHNo NHeo cueo NHHo nuwo pawn melee: 6.8Hon m.quom oHNm nHNN n.9flon eHNm Hues nuwn N sue Huo.m NHN.N o.oHH.N HHH.» m.quo.N Hue.N How.» duo.» .nas\ooH_u one 8 E so so 8 E E E _. 832: .oHa 0N .uc oH . .oHa mH .oc oH .oHa oH .oc NH .cHa m .0; o coauedooooH nouu< mafia m moose vac m macaw nooeuoo oooofiuo> mo mammamno you mom: some zuumfiaoco ocean one ofiwoaoualom .H Navooeo< H 7O NHNH mHoH odHcN oHNH eHeH HHNH NHHH $2 88 mHHNN ONHNm o. 9.8 HNHHm HHHS NHoo 6H8 ‘ anon 88 NNHSH NHHNN so“: oNHHnH oonmH 8H2: SHNoH SH? 43.»... Honoumoaoeu. oHHNN SHSH o.o.+.NNH NNHoNH oHHoo NHH8H NHHNNH NHH8 N48 88:3 oNHoN canoe 3H3 canoe NH» o.oHo.o 8H8 o.oHo.o 4 28888 3H3 saunas. oNNHN 83.886 RHHNH NHooHonom NHHHNNN nooHHoonm .4 388882 NSHNEH memHHmHoN «SHHomNN NNNHNHNH oHoHNNNN oNNHmNNH «HNNHNNNN SHHoNcH 54 3:88.83 NEHoooH NSNHomoN .HNNHNNeN nNNnHNNoo 82:33 8388: 28828 NoNHHNNHN N538 18on 882 I88 I o.o I88... 188.. I988 I821” +888 +888 +888 +82% +8...on +8.32 1882.. +8mNHn moatoHoooHe sang HSHHm HHHN NHon HHHm HHHN .2ng NSHNm N one: NSHHH NHNH tong eHNH NSHoH NHNH odHHH 32 N.8 .8 once mu? 8H3 NHHom NH? NHNo mHNo «H8 n e 82 How? at». H603 3H8 con? NHHe cone». None. N >8 Hue; NHNN HHNN onHH HHHN NHHSH ASHH.» NHNSH mafiooH .8. one , as $3 as EV A5 EV A8 A3 333m .5... oNH do .N .52. 8 .2 NN . .58 8 do 8 .58 me. do NH cofiuuasoooa uoum< mafia A.v.uaooyllfl Navdomq< 71 o.qwo~ nHwH “Had «fiuom ~H+H uuwN quad ‘muwu snow omHRH o.oH~m 8H: 2H8: 2H8 8H8 mu: 2H8 .88 I. I. I. I. I. I. I. I. .Hv\.wa mm+¢oa o.o+amH na+s~ Hm+~m Ha+oma aa+am mm+mofl na+¢mfl Houuumwaono nu«o o.QmeH ‘uuao miuwn mdfiao anuwoa aduqm mauxafl n.wa amouaao Iofluwa o.quo.o hfiuwa o.qua.o nauwc o.qud.o o.qua.o o.quo.o _¢.Hfinaoafimom HmnHWmh o.auwmefl annuwm« ommfidm¢a onuama omhduaoo¢ «Heuw8 ,Nnmauwohm < Hflsaouusmz «HnHWoOH o.quwnqw manuwah manuahea .meqHWmma “unfiuamom humuemufl ~me¢Hflmoq ¢_ou»ao:gaau m¢muwmma o.quo~+ acquwflua nuiuwhom Nauuwmqa thmuamflfi ++~ouuwemfl oomduoommfl naa\oms Ixmnam I. o.o Iboomm I. o.o ImmaNm .moomma Iwumou .mooeoa +¢ooflm~ +¢ooomq +ooomm~ +ooomea +¢eoan~ +oooNom +mmmmam +oooom~ maa\uuaouuam «.ouam o.QHm~ e.qH~m o.qfiqm ~.qH~n 8.8HHm 5.8Ham n.8H4n N one: ¢.qwoa o.quoa m.qudH Nuwa «.quqa &H~H «.quafl dud” N.am .nm {How o.quaq «Haw mac; {Ham {Hag {awe aflnq mq+>gz dHWm o.onm auwm “awe 5.8me “awn “.8H8m nuwm N >um ¢.qun.m o.qu.m n.qua.m mH~.oH h.qH~.m uuo.oa III m.QHm.~ maa\ooa x 0mm Amv Amy Amy Amy Amv Amv Amy Amy muououm .afia com .us on .afia con .un QN .afia oqm .us om .afia and .H; mm coauuasooaH uuum< mafia A.v.unouvIIH wavaomg4 72 .cmma mnu mo uouum vuuvamum was duos ma vommmunxma .Aao.o v mv_muauuoHMHu unuofimaauam kagwflm++ .Amo.o v my monouomuflv unaufiMflamee. .cflx0uovam mNoo uwxovkmxcmm saws vuuoonaw manawn< Amv .mamaauwuo ufivooust UNNmaxmumBm nuaa woumaauoafi manafia< u Amy .Ha\mufiq= Hoxnaumlnawam ma vmmmwumxw mmuafiadmcouu 0H>5uhmluaaou5Hw aauom .Hmwm .Ha\mqua wacwumlmawwm ma vmmmounxm mmdcfiaamamuu uwuouuoamNOIuaaauaam abuom I 300m .uonana.uuaaomn< I 4 HICHIGGN STQTE UNIV. 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